Generation of an Fsp1 (fibroblast‐specific protein 1)‐Flpo transgenic mouse strain

Recombination systems represent a major breakthrough in the field of genetic model engineering. The Flp recombinases (Flp, Flpe, and Flpo) bind and cleave DNA Frt sites. We created a transgenic mouse strain ([Fsp1‐Flpo]) expressing the Flpo recombinase in fibroblasts. This strain was obtained by random insertion inside mouse zygotes after pronuclear injection. Flpo expression was placed under the control of the promoter of Fsp1 (fibroblast‐specific protein 1) gene, whose expression starts after gastrulation at Day 8.5 in cells of mesenchymal origin. We verified the correct expression and function of the Flpo enzyme by several ex vivo and in vivo approaches. The [Fsp1‐Flpo] strain represents a genuine tool to further target the recombination of transgenes with Frt sites specifically in cells of mesenchymal origin or with a fibroblastic phenotype.     

Association of CAT polymorphisms with catalase activity and exposure to environmental oxidative stimuli

We tested the hypotheses that catalase activity is modified by CAT single nucleotide polymorphisms (SNPs) (-262;-844), and by their interactions with oxidant exposures (coal dusts, smoking), lymphotoxin alpha (LTA, NcoI) and tumor necrosis factor (TNF, -308) in 196 miners. Erythrocyte catalase, superoxide dismutase, and glutathione peroxidase activities were measured. The CAT -262 SNP was related to lower catalase activity (104, 87 and 72 k/g hemoglobin for CC, CT and TT, respectively, p < 0.0001). Regardless of CAT SNPs, the LTA NcoI but not the TNF-308 SNP was associated with catalase activity (p = 0.04 and p = 0.8). CAT -262 T carriers were less frequent in highly exposed miners (OR = 0.39 [0.20–0.78], p = 0.007). In CAT -262 T carriers only, catalase activity decreased with high dust exposure (p = 0.01). Haplotype analyses (combined CAT SNPs) confirm these results. Results show that CAT -262 and LTA NcoI SNPs, and interaction with coal dust exposure, influenced catalase activity.     

Cyclooxygenase-2 positively regulates Akt signalling and enhances survival of erythroleukemia cells exposed to anticancer agents

Cyclooxygenase-2 (COX-2) has been found to be highly expressed in many types of cancers and to contribute to tumorigenesis via the inhibition of apoptosis, increased angiogenesis and invasiveness. In hematological malignancies, COX-2 expression was found to correlate with poor patient prognosis. However, the exact role of COX-2 expression in these malignancies, and particularly in erythroleukemias, remains unclear. The aim of this work was to describe and understand the relationships between COX-2 expression and apoptosis rate in erythroleukemia cells after apoptosis induction by several anticancer agents. We used three different erythroleukemia cell lines in which COX-2 expression was modulated by transfection with either COX-2 siRNA or COX-2 cDNA. These cellular models were then treated with apoptosis inducers and apoptosis onset and intensity was followed. Cell signalling was evaluated in unstimulated transfected cells or after apoptosis induction. We found that COX-2 inhibition rendered erythroleukemia cells more sensitive to apoptosis induction and that in cells overexpressing COX-2 apoptosis induction was reduced. We demonstrated that COX-2 inhibition decreased the pro-survival Akt signalling and activated the negative regulator of Akt signalling, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Conversely, in COX-2 overexpressing cells, Akt signalling was activated and PTEN was inhibited. In these last cells, inhibition of casein kinase 2 or Akt signalling restored sensitivity to apoptotic agents. Our findings highlighted that COX-2 can positively regulate Akt signalling mostly through PTEN inhibition, partly via casein kinase 2 activation, and enhances survival of erythroleukemia cells exposed to anticancer agents.     

Subthalamic Nucleus High-Frequency Stimulation Restores Altered Electrophysiological Properties of Cortical Neurons in Parkinsonian Rat

Electrophysiological recordings performed in parkinsonian patients and animal models have confirmed the occurrence of alterations in firing rate and pattern of basal ganglia neurons, but the outcome of these changes in thalamo-cortical networks remains unclear. Using rats rendered parkinsonian, we investigated, at a cellular level in vivo, the electrophysiological changes induced in the pyramidal cells of the motor cortex by the dopaminergic transmission interruption and further characterized the impact of high-frequency electrical stimulation of the subthalamic nucleus, a procedure alleviating parkinsonian symptoms. We provided evidence that a lesion restricted to the substantia nigra pars compacta resulted in a marked increase in the mean firing rate and bursting pattern of pyramidal neurons of the motor cortex. These alterations were underlain by changes of the electrical membranes properties of pyramidal cells including depolarized resting membrane potential and increased input resistance. The modifications induced by the dopaminergic loss were more pronounced in cortico-striatal than in cortico-subthalamic neurons. Furthermore, subthalamic nucleus high-frequency stimulation applied at parameters alleviating parkinsonian signs regularized the firing pattern of pyramidal cells and restored their electrical membrane properties.     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies     

The Effect of Three-Monthly Albendazole Treatment on Malarial Parasitemia and Allergy: A Household-Based Cluster-Randomized,Double-Blind,Placebo-Controlled Trial

Background

Helminth infections are proposed to have immunomodulatory activities affecting health outcomes either detrimentally or beneficially. We evaluated the effects of albendazole treatment, every three months for 21 months, on STH, malarial parasitemia and allergy.

Methods and Findings

A household-based cluster-randomized, double-blind, placebo-controlled trial was conducted in an area in Indonesia endemic for STH. Using computer-aided block randomization, 481 households (2022 subjects) and 473 households (1982 subjects) were assigned to receive placebo and albendazole, respectively, every three months. The treatment code was concealed from trial investigators and participants. Malarial parasitemia and malaria-like symptoms were assessed in participants older than four years of age while skin prick test (SPT) to allergens as well as reported symptoms of allergy in children aged 5–15 years. The general impact of treatment on STH prevalence and body mass index (BMI) was evaluated. Primary outcomes were prevalence of malarial parasitemia and SPT to any allergen. Analysis was by intention to treat. At 9 and 21 months post-treatment 80.8% and 80.1% of the study subjects were retained, respectively. The intensive treatment regiment resulted in a reduction in the prevalence of STH by 48% in albendazole and 9% in placebo group. Albendazole treatment led to a transient increase in malarial parasitemia at 6 months post treatment (OR 4.16(1.35–12.80)) and no statistically significant increase in SPT reactivity (OR 1.18(0.74–1.86) at 9 months or 1.37 (0.93–2.01) 21 months). No effect of anthelminthic treatment was found on BMI, reported malaria-like- and allergy symptoms. No adverse effects were reported.

Conclusions

The study indicates that intensive community treatment of 3 monthly albendazole administration for 21 months over two years leads to a reduction in STH. This degree of reduction appears safe without any increased risk of malaria or allergies.

Trial Registration

Controlled-Trials.com ISRCTN83830814     

Basal Ganglia Neuronal Activity during Scanning Eye Movements in Parkinson’s Disease

The oculomotor role of the basal ganglia has been supported by extensive evidence, although their role in scanning eye movements is poorly understood. Nineteen Parkinsońs disease patients, which underwent implantation of deep brain stimulation electrodes, were investigated with simultaneous intraoperative microelectrode recordings and single channel electrooculography in a scanning eye movement task by viewing a series of colored pictures selected from the International Affective Picture System. Four patients additionally underwent a visually guided saccade task. Microelectrode recordings were analyzed selectively from the subthalamic nucleus, substantia nigra pars reticulata and from the globus pallidus by the WaveClus program which allowed for detection and sorting of individual neurons. The relationship between neuronal firing rate and eye movements was studied by crosscorrelation analysis. Out of 183 neurons that were detected, 130 were found in the subthalamic nucleus, 30 in the substantia nigra and 23 in the globus pallidus. Twenty percent of the neurons in each of these structures showed eye movement-related activity. Neurons related to scanning eye movements were mostly unrelated to the visually guided saccades. We conclude that a relatively large number of basal ganglia neurons are involved in eye motion control. Surprisingly, neurons related to scanning eye movements differed from neurons activated during saccades suggesting functional specialization and segregation of both systems for eye movement control.     

SRAP Polymorphisms Associated to Cell Wall Degradability in Lignified Stems of Alfalfa

The identification of DNA polymorphisms associated to increased cell wall (CW) degradability could accelerate the development of alfalfa (Medicago sativa L.) cultivars with superior ethanol conversion yields. Genotypes with high (D+) or low (D?) CW degradability were recently identified within a biomass-type and three winter-hardy-type populations using near-infrared reflectance spectroscopy (NIRS) prediction of CW glucose released by enzyme saccharification. In this report, we used sequence-related amplified polymorphism to search for DNA variations associated to differences in enzyme-released glucose. A bulk segregant analysis (BSA) of pooled DNA samples (20 plants/bulk) from D+, D?, and randomly chosen genotypes uncovered polymorphisms associated to CW degradability. Polymorphisms that increase or decrease in intensity between D+ and D? bulks indicated the presence of genomic regions with either positive or negative effects on CW degradability. A primer pair (Me4-R14) generated a fragment, which increased in intensity in the D+ bulk of the biomass-type population. Conversely, the amplification of that fragment declined in the D+ bulks of the winter-hardy-type populations. Interestingly, these populations differ in their degradability. Assessment of the genotypic occurrence of this fragment confirmed that polymorphism detected with BSA reflects changes in the frequency of occurrence within populations. Sequence analysis of the Me4-R14 fragment revealed homologies with sequences from Medicago truncatula, a model species for legumes with documented synteny with M. sativa. Our results show that genomic regions associated to CW degradability can be identified using the combination of BSA of genotypes with contrasted degradability and a PCR-based amplification technique.     

Evaluating the Potential for the Environmentally Sustainable Control of Foot and Mouth Disease in Sub-Saharan Africa

Strategies to control transboundary diseases have in the past generated unintended negative consequences for both the environment and local human populations. Integrating perspectives from across disciplines, including livestock, veterinary and conservation sectors, is necessary for identifying disease control strategies that optimise environmental goods and services at the wildlife-livestock interface. Prompted by the recent development of a global strategy for the control and elimination of foot-and-mouth disease (FMD), this paper seeks insight into the consequences of, and rational options for potential FMD control measures in relation to environmental, conservation and human poverty considerations in Africa. We suggest a more environmentally nuanced process of FMD control that safe-guards the integrity of wild populations and the ecosystem dynamics on which human livelihoods depend while simultaneously improving socio-economic conditions of rural people. In particular, we outline five major issues that need to be considered: 1) improved understanding of the different FMD viral strains and how they circulate between domestic and wildlife populations; 2) an appreciation for the economic value of wildlife for many African countries whose presence might preclude the country from ever achieving an FMD-free status; 3) exploring ways in which livestock production can be improved without compromising wildlife such as implementing commodity-based trading schemes; 4) introducing a participatory approach involving local farmers and the national veterinary services in the control of FMD; and 5) finally the possibility that transfrontier conservation might offer new hope of integrating decision-making at the wildlife-livestock interface.     

No Evidence That Soluble TACI Induces Signalling via Membrane-Expressed BAFF and APRIL in Myeloid Cells

Myeloid cells express the TNF family ligands BAFF/BLyS and APRIL, which exert their effects on B cells at different stages of differentiation via the receptors BAFFR, TACI (Transmembrane Activator and CAML-Interactor) and/or BCMA (B Cell Maturation Antigen). BAFF and APRIL are proteins expressed at the cell membrane, with both extracellular and intracellular domains. Therefore, receptor/ligand engagement may also result in signals in ligand-expressing cells via so-called “reverse signalling”. In order to understand how TACI-Fc (atacicept) technically may mediate immune stimulation instead of suppression, we investigated its potential to activate reverse signalling through BAFF and APRIL. BAFFR-Fc and TACI-Fc, but not Fn14-Fc, reproducibly stimulated the ERK and other signalling pathways in bone marrow-derived mouse macrophages. However, these effects were independent of BAFF or APRIL since the same activation profile was observed with BAFF- or APRIL-deficient cells. Instead, cell activation correlated with the presence of high molecular mass forms of BAFFR-Fc and TACI-Fc and was strongly impaired in macrophages deficient for Fc receptor gamma chain. Moreover, a TACI-Fc defective for Fc receptor binding elicited no detectable signal. Although these results do not formally rule out the existence of BAFF or APRIL reverse signalling (via pathways not tested in this study), they provide no evidence in support of reverse signalling and point to the importance of using appropriate specificity controls when working with Fc receptor-expressing myeloid cells.