A genome-scale metabolic model of Cupriavidus necator H16 integrated with TraDIS and transcriptomic data reveals metabolic insights for biotechnological applications

Exploiting biological processes to recycle renewable carbon into high value platform chemicals provides a sustainable and greener alternative to current reliance on petrochemicals. In this regard Cupriavidus necator H16 represents a particularly promising microbial chassis due to its ability to grow on a wide range of low-cost feedstocks, including the waste gas carbon dioxide, whilst also naturally producing large quantities of polyhydroxybutyrate (PHB) during nutrient-limited conditions. Understanding the complex metabolic behaviour of this bacterium is a prerequisite for the design of successful engineering strategies for optimising product yields. We present a genome-scale metabolic model (GSM) of C. necator H16 (denoted iCN1361), which is directly constructed from the BioCyc database to improve the readability and reusability of the model. After the initial automated construction, we have performed extensive curation and both theoretical and experimental validation. By carrying out a genome-wide essentiality screening using a Transposon-directed Insertion site Sequencing (TraDIS) approach, we showed that the model could predict gene knockout phenotypes with a high level of accuracy. Importantly, we indicate how experimental and computational predictions can be used to improve model structure and, thus, model accuracy as well as to evaluate potential false positives identified in the experiments. Finally, by integrating transcriptomics data with iCN1361 we create a condition-specific model, which, importantly, better reflects PHB production in C. necator H16. Observed changes in the omics data and in-silico-estimated alterations in fluxes were then used to predict the regulatory control of key cellular processes. The results presented demonstrate that iCN1361 is a valuable tool for unravelling the system-level metabolic behaviour of C. necator H16 and can provide useful insights for designing metabolic engineering strategies

Refraction and Strabismus Evaluation in Preterm Infants With and Without Retinopathy of Prematurity

Abstract Purpose:To investigate cycloplegic refraction and ocular alignment in a population of preterm infants at one and six years old with regard to birth weight (BW) and gestational age (GA). Methods:We considered 261 preterm infants with BW of 1500gr or less and/or GA of 32 weeks or less. We evaluated visual refraction and ocular alignment records on each patient at the age of one and six years. We considered retinopathy of prematurity (ROP) stage I and II as mild ROP and higher ROP stages as severe ROP. We identified three groups of patients: 217 infants (434 eyes) without evidence of ROP (Group 1: control group), 28 infants (56 eyes) with mild ROP (Group 2) and 16 infants (32 eyes) affected by severe ROP (Group 3). T Student test was performed at the end of the study to compare data. Results:The incidence of visual refraction and ocular alignment at the age of one year old in Group 2 and 3 compared to control Group 1 was respectively: emmetropia 1.9% (Group 2) and 5% (Group 3) vs 4% (Group 1); myopia 17.3% (p=0.001) and 39% (p=0.002) vs 5%; hypermetropia 27.8% and 19% vs 37%; astigmatism 45% and 37% vs 54%; strabismus 11.5% (p=0.001) and 38% (p=0.002) vs 5.3%. The incidence of visual refraction and ocular alignment at the age of six years old was respectively: emmetropia 2% and 6% vs 4.5%; myopia 10.7% (p=0.003) and 25% (p=0.001) vs 5.5%; hypermetropia 46.4% and 40.6% vs 60.08%; astigmatism 39.2% and 28.1% vs 29% and strabismus 25% (p=0.003) and 56.25% (p=0.003) vs 11.5%. Conclusions:Myopia and strabismus had a significative high incidence at one and six years follow up Groups 2 and 3 compared to control Group 1. The risk of these disorders increases significantly (p<0.05) with the severity of ROP (Group 2 and Group 3 vs Group 1). These data underline the importance of a correct diagnosis and follow up of myopia and strabismus to prevent amblyopia and visual impairment in adulthood. Our data confirm and better underline at six years follow up what we have already showed in our previous study (ARVO 2006, 5299-B714) at one year follow u

Visualising the concept of metabolic regulation in bacteria: a simple laboratory experiment on polyhydroxybutyrate storage

Regulation is a key concept for understanding the dynamics of metabolism in bacteria. This report outlines a simple laboratory experiment aimed at studying a key form of regulation in bacterial metabolism, exemplifying how microbes switch the flux of carbon towards alternative metabolic fates as a function of nutrient availability and according to their physiological status. The experiment involves the use of a Sinorhizobium meliloti wild-type strain and its isogenic derivative carrying a null mutation in the gene encoding the synthase that generates the storage polymer polyhydroxybutyrate (PHB). These strains serve to characterise the effects of growth phase and carbon availability at the end of balanced growth on the extent of PHB accumulation by rhizobia. The experimental set-up can be completed in a main 4-h laboratory session, followed by an additional 1-h session after an initial incubation for subsequent bacterial-colony counting (48 h), and requires equipment usually available at any university. The protocol described here is performed by the undergraduates of our biochemistry course. We consider this exercise to be a useful complementary tool for spotlighting the concept of metabolic regulation in bacteria and for promoting scientific thinking along with the development of students’ wet-bench laboratory skills.Fil: Lagares, Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; ArgentinaFil: Garavaglia, Matías Javier. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; ArgentinaFil: Robledo, Natalia Belén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; ArgentinaFil: Valverde, Claudio Fabián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; ArgentinaFil: Goñi, Sandra Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ingeniería Genética y Biología Molecular y Celular; ArgentinaFil: Lozano, Mario Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ingeniería Genética y Biología Molecular y Celular; Argentin

Performance of encryption schemes in chaotic optical communication: a multifractal approach

From the estimation of the Hurst exponent and the multifractality degree we discriminate the security levels of two typical encoding schemes usually applied in chaos-based communication systems. We also analyze the effects that the sampling period and the message amplitude have on the goodness of these techniques. We compare our results with those obtained by considering an information theory approach. The Hurst exponent seems to be a sensitive and powerful tool for discriminating the presence of a message embedded in a chaotic carrier

Human T cells engineered with a leukemia lipid-specific TCR enables donor-unrestricted recognition of CD1c-expressing leukemia

Acute leukemia relapsing after chemotherapy plus allogeneic hematopoietic stem cell transplantation can be treated with donor-derived T cells, but this is hampered by the need for donor/recipient MHC-matching and often results in graft-versus-host disease, prompting the search for new donor-unrestricted strategies targeting malignant cells. Leukemia blasts express CD1c antigen-presenting molecules, which are identical in all individuals and expressed only by mature leukocytes, and are recognized by T cell clones specific for the CD1c-restricted leukemia-associated methyl-lysophosphatidic acid (mLPA) lipid antigen. Here, we show that human T cells engineered to express an mLPA-specific TCR, target diverse CD1c-expressing leukemia blasts in vitro and significantly delay the progression of three models of leukemia xenograft in NSG mice, an effect that is boosted by mLPA-cellular immunization. These results highlight a strategy to redirect T cells against leukemia via transfer of a lipid-specific TCR that could be used across MHC barriers with reduced risk of graft-versus-host disease. Leukaemia therapy may benefit from the use of antigens that are less restricted to individual donors. Here the authors engineered T cells with a TCR specific for a CD1c restricted lipid leukaemia antigen and show that they can protect against disease progression in mouse leukaemia xenograft models

Emergence of antitumor cytolytic T cells is associated with maintenance of hematologic remission in children with acute myeloid leukemia

Although the graft-versus-leukemia effect of allogeneic bone marrow transplantation (BMT) is of paramount importance in the maintenance of disease remission, the role played by the autologous T-cell response in antitumor immune surveillance is less defined. We evaluated the emergence of antileukemia cytotoxic T-lymphocyte precursors (CTLp's) and the correlation of this phenomenon with maintenance of hematologic remission in 16 children with acute myeloid leukemia (AML), treated with either chemotherapy alone (5 patients) or with autologous BMT (A-BMT, 11 patients). Antileukemia CTLp's were detectable in 8 patients in remission after induction chemotherapy; none of them subsequently had a relapse. Of the 8 patients who did not show detectable CTLp frequency while in remission after induction chemotherapy, 7 subsequently experienced leukemia relapse. In patients undergoing A-BMT, molecular fingerprinting of the TCR-Vbeta repertoire, performed on antileukemia lines, demonstrated that selected antileukemia T-cell clonotypes, detectable in bone marrow before transplantation, survived ex vivo pharmacologic purging and were found in the recipient after A-BMT. These data provide evidence for an active role of autologous T cells in the maintenance of hematologic remission and also suggest that quantification of antileukemia CTLp frequency may be a useful tool to identify patients at high risk for relapse, thus potentially benefiting from an allogeneic antitumor effect

Tristetraprolin promotes hepatic inflammation and tumor initiation but restrains cancer progression to malignancy

Tristetraprolin (TTP) is a key post-transcriptional regulator of inflammatory and oncogenic transcripts. Accordingly, TTP was reported to act as a tumor suppressor in specific cancers. Herein, we investigated how TTP contributes to the development of liver inflammation and fibrosis, which are key drivers of hepatocarcinogenesis, as well as to the onset and progression of hepatocellular carcinoma (HCC)