Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells

Background

The photorespiratory nitrogen cycle in C₃ plants involves an extensive diversion of carbon and nitrogen away from the direct pathways of assimilation. The liberated ammonia is re-assimilated, but up to 25% of the carbon may be released into the atmosphere as CO₂. Because of the loss of CO₂ and high energy costs, there has been considerable interest in attempts to decrease the flux through the cycle in C₃ plants. Transgenic tobacco plants were generated that contained the genes gcl and hyi from E. coli encoding glyoxylate carboligase (EC 4.1.1.47) and hydroxypyruvate isomerase (EC 5.3.1.22) respectively, targeted to the peroxisomes. It was presumed that the two enzymes could work together and compete with the aminotransferases that convert glyoxylate to glycine, thus avoiding ammonia production in the photorespiratory nitrogen cycle.

Results

When grown in ambient air, but not in elevated CO₂, the transgenic tobacco lines had a distinctive phenotype of necrotic lesions on the leaves. Three of the six lines chosen for a detailed study contained single copies of the gcl gene, two contained single copies of both the gcl and hyi genes and one line contained multiple copies of both gcl and hyi genes. The gcl protein was detected in the five transgenic lines containing single copies of the gcl gene but hyi protein was not detected in any of the transgenic lines. The content of soluble amino acids including glycine and serine, was generally increased in the transgenic lines growing in air, when compared to the wild type. The content of soluble sugars, glucose, fructose and sucrose in the shoot was decreased in transgenic lines growing in air, consistent with decreased carbon assimilation.

Conclusions

Tobacco plants have been generated that produce bacterial glyoxylate carboligase but not hydroxypyruvate isomerase. The transgenic plants exhibit a stress response when exposed to air, suggesting that some glyoxylate is diverted away from conversion to glycine in a deleterious short-circuit of the photorespiratory nitrogen cycle. This diversion in metabolism gave rise to increased concentrations of amino acids, in particular glutamine and asparagine in the leaves and a decrease of soluble sugars.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">ex vivo</Emphasis> effect of hyaluronic acid on erythrocyte flow properties

Background  

Hyaluronic acid (HA) is present in many tissues; its presence in serum may be related to certain inflammatory conditions, tissue damage, sepsis, liver malfunction and some malignancies. In the present work, our goal was to investigate the significance of hyaluronic acid effect on erythrocyte flow properties. Therefore we performed in vitro experiments incubating red blood cells (RBCs) with several HA concentrations. Afterwards, in order to corroborate the pathophysiological significance of the results obtained, we replicated the in vitro experiment with ex vivo RBCs from diagnosed rheumatoid arthritis (RA) patients, a serum HA-increasing pathology.     

Development of Lactococcus lactis encoding fluorescent proteins,GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system

Fluorescent proteins are useful reporter molecules for a variety of biological systems. We present an alternative strategy for cloning reporter genes that are regulated by the nisin-controlled gene expression (NICE) system. Lactoccocus lactis was genetically engineered to express green fluorescent protein (GFP), mCherry or near-infrared fluorescent protein (iRFP). The reporter gene sequences were optimized to be expressed by L. lactis using inducible promoter pNis within the pNZ8048 vector. Expression of constructions that carry mCherry or GFP was observed by fluorescence microscopy 2 h after induction with nisin. Expression of iRFP was evaluated at 700 nm using an infrared scanner; cultures induced for 6 h showed greater iRFP expression than non-induced cultures or those expressing GFP. We demonstrated that L. lactis can express efficiently GFP, mCherry and iRFP fluorescent proteins using an inducible expression system. These strains will be useful for live cell imaging studies in vitro or for imaging studies in vivo in the case of iRFP.     

T-helper 17 cell cytokines and interferon type I: partners in crime in systemic lupus erythematosus?

Introduction

A hallmark of systemic autoimmune diseases like systemic lupus erythematosus (SLE) is the increased expression of interferon (IFN) type I inducible genes, so-called IFN type I signature. Recently, T-helper 17 subset (Th17 cells), which produces IL-17A, IL-17F, IL-21, and IL-22, has been implicated in SLE. As CCR6 enriches for Th17 cells, we used this approach to investigate whether CCR6⁺ memory T-helper cells producing IL-17A, IL-17F, IL-21, and/or IL-22 are increased in SLE patients and whether this increase is related to the presence of IFN type I signature.

Methods

In total, 25 SLE patients and 15 healthy controls (HCs) were included. SLE patients were divided into IFN type I signature-positive (IFN⁺) (n = 16) and negative (IFN^-) (n = 9) patients, as assessed by mRNA expression of IFN-inducible genes (IFIGs) in monocytes. Expression of IL-17A, IL-17F, IL-21, and IL-22 by CD4⁺CD45RO⁺CCR6⁺ T cells (CCR6⁺ cells) was measured with flow cytometry and compared between IFN⁺, IFN^- patients and HCs.

Results

Increased percentages of IL-17A and IL-17A/IL-17F double-producing CCR6⁺ cells were observed in IFN⁺ patients compared with IFN^- patients and HCs. IL-17A and IL-17F expression within CCR6⁺ cells correlated significantly with IFIG expression. In addition, we found significant correlation between B-cell activating factor of the tumor necrosis family (BAFF)–a factor strongly correlating with IFN type I - and IL-21 producing CCR6⁺ cells.

Conclusions

We show for the first time higher percentages of IL-17A and IL-17A/IL-17F double-producing CCR6⁺ memory T-helper cells in IFN⁺ SLE patients, supporting the hypothesis that IFN type I co-acts with Th17 cytokines in SLE pathogenesis.     

Visual field maps in human cortex

Much of the visual cortex is organized into visual field maps: nearby neurons have receptive fields at nearby locations in the image. Mammalian species generally have multiple visual field maps with each species having similar, but not identical, maps. The introduction of functional magnetic resonance imaging made it possible to identify visual field maps in human cortex, including several near (1) medial occipital (V1,V2,V3), (2) lateral occipital (LO-1,LO-2, hMT+), (3) ventral occipital (hV4, VO-1, VO-2), (4) dorsal occipital (V3A, V3B), and (5) posterior parietal cortex (IPS-0 to IPS-4). Evidence is accumulating for additional maps, including some in the frontal lobe. Cortical maps are arranged into clusters in which several maps have parallel eccentricity representations, while the angular representations within a cluster alternate in visual field sign. Visual field maps have been linked to functional and perceptual properties of the visual system at various spatial scales, ranging from the level of individual maps to map clusters to dorsal-ventral streams. We survey recent measurements of human visual field maps, describe hypotheses about the function and relationships between maps, and consider methods to improve map measurements and characterize the response properties of neurons comprising these maps.     

Adaptation of blood sugar regulation during muscular activity. Effects of training]

Our aim was to study the adaptation of energetic metabolism to muscular activity and the effects of training upon these adaptations. So we realized swimming and running tests on young healthy adults, selected in "trained" and "untrained". The effects of muscular activity were reflected by a raise of serum F.F.A., clearly noted in "untrained" subjects ; the levels of glucose and growth hormone both raised, but especially in "trained" subjects. Cortisol level raised in varying degrees while insulinemia presented little changes.     

In vitro zygotic embryo culture of wild Musa acuminata ssp.malaccensis and factors affecting germination and seedling growth

In vitro zygotic embryo culture of wild banana significantly increased the germination compared to greenhouse grown seeds. Embryo orientation and BAP concentration significantly affected germination rate. These factors together with gelling agent, dark and light conditions and coconut water, also showed variable effects on the number of roots per plant, root length, shoot length, number of days to root emergence and number of days to shoot emergence.     

Role of HLA DRB1*15 and HLA DRB1*16alleles in the genetic susceptibility to develop systemic lupus erythematosus (SLE) after Chikungunya and Zika viruses infection in México

Systemic lupus erythematosus (SLE) is a clinically and genetically heterogeneous disease particularly prevalent in Mexico. Althoughits etiology is unknown, genetic factors strongly influence its presenceas well as triggering factors, such as viral infections, including Cytomegalovirus and Epstein-Barr virus. Here,the study presents the appearance of de novoSLE (patients who did not present SLE before de virus infection, corroborated by serological analysis and negative for antinuclear antibodies) cases in Mexicans who live near the southern border of Mexico, who presented clinical symptoms of arthritic, hematological, mucocutaneous and renal SLE, after Zika and/ or Chikungunya virus infection. Low resolution class Ⅱ HLA typing was performed, which found a significantly increased frequency of HLA DRB1*02 (15 and 16)when compared to a group of 99 healthy individuals (P =0.001, OR=4.5, IC95% 1.8~11.0). All the patients were diagnosed with SLE 1 to 3 years after being confirmed with the Zika, and/or Chikungunya infection. At the point of acute viral infection, none of the patients presented clinical signs or symptoms of autoimmunity or were negative for antinuclear antibodies. In genetically susceptible individuals, Zika and Chikungunya viral infection can trigger SLE.