Production of transgenic dwarf surfclams, Mulinia lateralis, with pantropic retroviral vectors.

A pantropic pseudotyped retroviral vector containing the envelope protein of vesicular stomatitis virus was used as a gene transfer vector in the dwarf surfclam, Mulinia lateralis. These pantropic retroviral vectors have an extremely broad host cell range and can infect many nonmammalian species. Newly fertilized dwarf surfclam eggs were electroporated at 700 V in the presence of 1 x 10(4) colony-forming units of pantropic pseudotyped retroviral particles. Infection was well tolerated and did not affect the survival rate of the embryos. Gametes collected from P1 presumptive transgenic animals were analyzed for the presence of provirus by PCR, and in different experiments 13-33% of the gamete pools were positive for the transgene. Dot blot hybridization of DNA samples from the F1 offspring of two different crosses between infected P1 and wild-type individuals revealed that 28% and 31% of F1 offspring were transgenic, respectively. Southern blot analysis of DNA isolated from PCR-positive F1 animals confirmed integration of a single copy of the provirus into the host genome. Thus, the germ lines of these two P1 transgenic animals were mosaic for the transgene. Expression of beta-galactosidase encoded by the provirus was detected in transgenic but not control surfclam embryos. Pantropic pseudotyped retroviral vectors provide a useful method for the stable introduction of foreign genetic information into surfclams and may facilitate the introduction of desirable genetic traits into commercially important shellfish and crustaceans.

Genetic engineering of carbohydrate biosynthetic pathways in transgenic mice demonstrates cell cycle-associated regulation of glycoconjugate production in small intestinal epithelial cells.

Proliferation, migration-associated differentiation, and cell death occur continuously and in a spatially well-organized fashion along the crypt-villus axis of the mouse small intestine, making it an attractive system for studying how these processes are regulated and interrelated. A pathway for producing glycoconjugates was engineered in adult FVB/N transgenic mice by expressing a human alpha 1,3/4-fucosyltransferase (alpha 1,3/4-FT; EC 2.4.1.65) along the length of this crypt-villus axis. The alpha 1,3/4-FT can use lacto-N-tetraose or lacto-neo-N-tetraose core chains to generate Lewis (Le) blood group antigens Le(a) or Le(x), respectively, and H type 1 or H type 2 core chains to produce Leb and Le(y). Single- and multilabel immunohistochemical studies revealed that expression of the alpha 1,3/4-FT results in production of Le(a) and Leb antigens in both undifferentiated proliferated crypt cells and in differentiated postmitotic villus-associated epithelial cells. In contrast, Le(x) antigens were restricted to crypt cells. Villus enterocytes can be induced to reenter the cell cycle by expression of simian virus 40 tumor antigen under the control of a promoter that only functions in differentiated members of this lineage. Bitransgenic animals, generated from a cross of FVB/N alpha 1,3/4-FT with FVB/N simian virus 40 tumor antigen mice, expand the range of Le(x) expression to include villus-associated enterocytes that have reentered the cell cycle. Thus, the fucosylations unveil a proliferation-dependent switch in oligosaccharide production, as defined by a monoclonal antibody specific for the Le(x) epitope. These findings show that genetic engineering of oligosaccharide biosynthetic pathways can be used to define markers for entry into, or progression through, the cell cycle and to identify changes in endogenous carbohydrate metabolism that occur when proliferative status is altered in a manner that is not deleterious to the system under study.

Control of cell division in Escherichia coli: regulation of transcription of ftsQA involves both rpoS and SdiA-mediated autoinduction.

The conditioning of culture medium by the production of growth-regulatory substances is a well-established phenomenon with eukaryotic cells. It has recently been shown that many prokaryotes are also capable of modulating growth, and in some cases sensing cell density, by production of extracellular signaling molecules, thereby allowing single celled prokaryotes to function in some respects as multicellular organisms. As Escherichia coli shifts from exponential growth to stationary growth, many changes occur, including cell division leading to formation of short minicells and expression of numerous genes not expressed in exponential phase. An understanding of the coordination between the morphological changes associated with cell division and the physiological and metabolic changes is of fundamental importance to understanding regulation of the prokaryotic cell cycle. The ftsQA genes, which encode functions required for cell division in E. coli, are regulated by promoters P1 and P2, located upstream of the ftsQ gene. The P1 promoter is rpoS-stimulated and the second, P2, is regulated by a member of the LuxR subfamily of transcriptional activators, SdiA, exhibiting features characteristic of an autoinduction (quorum sensing) mechanism. The activity of SdiA is potentiated by N-acyl-homoserine lactones, which are the autoinducers of luciferase synthesis in luminous marine bacteria as well as of pathogenesis functions in several pathogenic bacteria. A compound(s) produced by E. coli itself during growth in Luria Broth stimulates transcription from P2 in an SdiA-dependent process. Another substance(s) enhances transcription of rpoS and (perhaps indirectly) of ftsQA via promoter P1. It appears that this bimodal control mechanism may comprise a fail-safe system, such that transcription of the ftsQA genes may be properly regulated under a variety of different environmental and physiological conditions.

Anti-nuclear antibody production and immune-complex glomerulonephritis in BALB/c mice treated with pristane.

The pathogenesis of systemic lupus erythematosus is thought to be primarily under genetic control, with environmental factors playing a secondary role. However, it has been shown recently that intraperitoneal injection of pristane (2,6,10,14-tetramethylpentadecane) induces autoantibodies typical of lupus in BALB/c mice, a strain not usually considered to be genetically susceptible to the disease. In this study, the induction of autoimmune disease by pristane was investigated. BALB/c mice receiving pristane were tested for autoantibody production and histopathological evidence of glomerulonephritis. Six of 11 mice developed IgM anti-single-stranded DNA antibodies shortly after receiving pristane and 4 developed IgM anti-histone antibodies, but anti-double-stranded DNA antibodies were absent. IgG anti-DNA and anti-histone antibodies were absent. In contrast, the lupus-associated anti-nuclear ribonucleoprotein/Sm and anti-Su autoantibodies produced by these mice were predominantly IgG. In addition to autoantibodies, most of the mice developed significant proteinuria. Light microscopy of the kidney showed segmental or diffuse proliferative glomerulonephritis. Electron microscopy showed subepithelial and mesangial immune-complex deposits and epithelial foot process effacement. Immunofluorescence revealed striking glomerular deposition of IgM, IgG, and C3 with a mesangial or mesangiocapillary distribution. Thus, pristane induces immune-complex glomerulonephritis in association with autoantibodies typical of lupus in BALB/c mice. These data support the idea that lupus is produced by an interplay of genetic and environmental factors and that unlike the MRL or (NZB x W)F1 mouse models, in which genetic susceptibility factors are of primary importance, environmental factors are of considerable importance in the autoimmune disease of pristane-treated BALB/c mice.

Local nitric oxide production in viral and autoimmune diseases of the central nervous system.

Because of the short half-life of NO, previous studies implicating NO in central nervous system pathology during infection had to rely on the demonstration of elevated levels of NO synthase mRNA or enzyme expression or NO metabolites such as nitrate and nitrite in the infected brain. To more definitively investigate the potential causative role of NO in lesions of the central nervous system in animals infected with neurotropic viruses or suffering from experimental allergic encephalitis, we have determined directly the levels of NO present in the central nervous system of such animals. Using spin trapping of NO and electron paramagnetic resonance spectroscopy, we confirm here that copious amounts of NO (up to 30-fold more than control) are elaborated in the brains of rats infected with rabies virus or borna disease virus, as well as in the spinal cords of rats that had received myelin basic protein-specific T cells.

Use of yeast artificial chromosomes (YACs) in studies of mammalian development: production of beta-globin locus YAC mice carrying human globin developmental mutants.

To test whether yeast artificial chromosomes (YACs) can be used in the investigation of mammalian development, we analyzed the phenotypes of transgenic mice carrying two types of beta-globin locus YAC developmental mutants: (i) mice carrying a G-->A transition at position -117 of the A gamma gene, which is responsible for the Greek A gamma form of hereditary persistence of fetal hemoglobin (HPFH), and (ii) beta-globin locus YAC transgenic lines carrying delta- and beta-globin gene deletions with 5' breakpoints similar to those of deletional HPFH and delta beta-thalassemia syndromes. The mice carrying the -117 A gamma G-->A mutation displayed a delayed gamma- to beta-globin gene switch and continued to express A gamma-globin chains in the adult stage of development as expected for carriers of Greek HPFH, indicating that the YAC/transgenic mouse system allows the analysis of the developmental role of cis-acting motifs. The analysis of mice carrying 3' deletions first provided evidence in support of the hypothesis that imported enhancers are responsible for the phenotypes of deletional HPFH and second indicated that autonomous silencing is the primary mechanism for turning off the gamma-globin genes in the adult. Collectively, our results suggest that transgenic mice carrying YAC mutations provide a useful model for the analysis of the control of gene expression during development.

Inhibitory Control is a Rate-Limiting Factor to Preschoolers' Use of Irregular Inflection

We investigated whether children’s inhibitory control is associated with their ability to produce irregular verb forms as well as learn from corrective feedback following their use of an over-regularized form. Forty-eight 3.5 to 4.5 year old children were tested on the irregular past tense and provided with adult corrective input via models of correct use or recasts of errors following ungrammatical responses. Inhibitory control was assessed with a three-item battery of tasks that required suppressing a prepotent response in favor of a non-canonical one. Results showed that inhibitory control was predictive of children’s initial production of irregular forms and not associated with their post-feedback production of irregulars. These findings show that children’s executive functioning skills may be a rate-limiting factor on their ability to produce correct forms, but might not interact with their ability to learn from input in this domain. Findings are discussed in terms of current theories of past-tense acquisition and learning from input more broadly.

European antitrust control and standard setting. Bruegel Working Paper 2013/01, February 2012

Standards reduce production costs and increase products’ value to consumers. Standards however entail risks of anti-competitive abuse. After the adoption of a standard, the chosen technology normally lacks credible substitutes. The owner of the patented technology might thus have additional market power relative to locked-in licensees, and might exploit this power to charge higher access rates. In the economic literature this phenomenon is referred to as ‘hold-up’. To reduce the risk of hold-up, standard-setting organisations often require patent holders to disclose their standard-essential patents before the adoption of the standard and to commit to license on fair, reasonable and non-discriminatory (FRAND) terms. The European Commission normally investigates unfair pricing abuse in a standard-setting context if a patent holder who committed to FRAND ex-ante is suspected not to abide to it ex-post. However, this approach risks ignoring a number of potential abuses which are likely harmful for welfare. That can happen if, for example, ex-post a licensee is able to impose excessively low access rates (‘reverse hold-up’) or if a patent holder acquires additional market power thanks to the standard but its essential patents are not encumbered by FRAND commitments, for instance because the patent holder did not directly participate to the standard setting process and was therefore not required by the standard-setting organisations to commit to FRAND ex-ante. A consistent policy by the Commission capable of tackling all sources of harm should be enforced regardless of whether FRAND commitments are given. Antitrust enforcement should hinge on the identification of a distortion in the bargaining process around technology access prices, which is determined by the adoption of the standard and is not attributable to pro-competitive merits of any of the involved players.

Standard-setting abuse: the case for antitrust control. Bruegel Policy Brief 2013/01, February 2013

Standards reduce production costs and increase the value of products to consumers; ultimately they significantly contribute to economic development. Standards however entail risks of anti-competitive abuse. After the adoption of a standard, the elimination of competition between technologies can lead to consumer harm. Fair, reasonable, nondiscriminatory (FRAND) commitments made by patent holders have been used to mitigate that risk. The European Commission recognises the importance of standards, but European Union competition policy is still seeking to identify well-targeted and efficient enforcement rules.

Fuzzy control of a water pump for an agricultural plant growth system

At the present time there is a high pressure toward the improvement of all the production processes. Those improvements can be sensed in several directions in particular those that involve energy efficiency. The definition of tight energy efficiency improvement policies is transversal to several operational areas ranging from industry to public services. As can be expected, agricultural processes are not immune to this tendency. This statement takes more severe contours when dealing with indoor productions where it is required to artificially control the climate inside the building or a partial growing zone. Regarding the latter, this paper presents an innovative system that improves energy efficiency of a trees growing platform. This new system requires the control of both a water pump and a gas heating system based on information provided by an array of sensors. In order to do this, a multi-input, multi-output regulator was implemented by means of a Fuzzy logic control strategy. Presented results show that it is possible to simultaneously keep track of the desired growing temperature set-point while maintaining actuators stress within an acceptable range.

Regulatory T Cells Accumulate in the Lung Allergic Inflammation and Efficiently Suppress T-Cell Proliferation but Not Th2 Cytokine Production

Foxp3(+)CD25(+)CD4(+) regulatory T cells are vital for peripheral tolerance and control of tissue inflammation. In this study, we characterized the phenotype and monitored the migration and activity of regulatory T cells present in the airways of allergic or tolerant mice after allergen challenge. To induce lung allergic inflammation, mice were sensitized twice with ovalbumin/aluminum hydroxide gel and challenged twice with intranasal ovalbumin. Tolerance was induced by oral administration of ovalbumin for 5 consecutive days prior to OVA sensitization and challenge. We detected regulatory T cells (Foxp3(+)CD25(+)CD4(+) T cells) in the airways of allergic and tolerant mice; however, the number of regulatory T cells was more than 40-fold higher in allergic mice than in tolerant mice. Lung regulatory T cells expressed an effector/memory phenotype (CCR4(high)CD62L(low)CD44(high)CD54(high)CD69(+)) that distinguished them from naive regulatory T cells (CCR4(int)CD62L(high)CD44(int)CD54(int)CD69(-)). These regulatory T cells efficiently suppressed pulmonary T-cell proliferation but not Th2 cytokine production.

Impaired renal endothelial nitric oxide synthase and reticulocyte production as modulators of hypertension induced by recombinant human erythropoietin in the rat

INTRODUCTION AND AIMS: Hypertension is a common side effect of recombinant human erythropoietin (rHuEPO) therapy; however, the exact pathways remain to be elucidated. The discovery of non-hematopoietic actions of rHuEPO increased the number of patients that could putatively benefit from this therapy; however, to achieve those effects higher doses are usually needed, which increase the risk and incidence of adverse events. Our aim was to study the effect of a broad range of rHuEPO doses on hematological and biochemical parameters, blood pressure and renal function and damage in the rat, focusing on endothelial nitric oxide synthase (eNOS) and hypoxia-inducible factors (HIFs). METHODS: Male Wistar rats were divided in 5 groups receiving different doses of rHuEPO (100, 200, 400 and 600 IU/kg body weight (BW)/week) and saline solution (control), during 3 weeks. Blood and 24h urine were collected to perform hematological and biochemical analysis. Blood pressure (BP) was measured by the tail-cuff method. The kidney tissue was collected to mRNA and protein expression assays and to characterize renal lesions. RESULTS: A dose-dependent increase in red blood cells count, hematocrit and hemoglobin levels was found with rHuEPO therapy, in rHuEPO200, rHuEPO400 and rHuEPO600 groups. Increased reticulocyte count was found in the rHuEPO400 and rHuEPO600 groups. BP raised in all groups receiving rHuEPO. The rHuEPO200 and rHuEPO600 groups presented increased kidney protein levels of HIF2α and a reduction in kidney protein levels of eNOS, along with the highest grade of vascular and tubular renal lesions. CONCLUSIONS: Our study showed that rHuEPO-induced hypertension might involve indirect (hematological) and direct (renal) effects which varies according to the dose used. Thus, rHuEPO therapy should be performed rationally and under adequate surveillance, as hypertension develops even with lower doses. Especial caution with higher doses should be taken, as rHuEPO-induced hypertension leads to early renal damage without alterations in traditional markers of renal function, thus masking the serious adverse effects and risks.

Intraperitoneal bevacizumab for control of malignant ascites due to advanced-stage gastrointestinal cancers: A multicentre double-blind, placebo-controlled phase II study - AIO SUP-0108

PURPOSE: Malignant ascites is debilitating for patients with advanced cancer. As shown previously, tumour cell production of vascular endothelial growth factor might be a major cause of the formation of malignant ascites. Intraperitoneal bevacizumab could therefore be an option for symptom control in refractory ascites. PATIENTS AND METHODS: Patients with advanced gastrointestinal cancer and malignant ascites who had undergone paracentesis at least twice within the past 4 weeks were randomly assigned in a 2:1 ratio to intraperitoneal bevacizumab (400 mg absolute) or placebo after paracentesis. During the 8-week treatment period, a minimum interval of 14 d was kept between the applications of the study drug. Primary end-point was paracentesis-free survival (ParFS). RESULTS: Fifty-three patients (median age 63 years) were randomised. Forty-nine patients received at least one study drug application and qualified for the main analysis. The proportion of patients with at least one common toxicity criteria grade III-V event was similar with 20/33 (61%) on bevacizumab and 11/16 (69%) on placebo. Median ParFS was 14 d (95% confidence interval [CI]: 11-17) in the bevacizumab arm and 10.5 d (95% CI: 7-21) on placebo (hazard ratio 0.74, 95% CI: 0.40-1.37; P = 0.16). The longest paracentesis-free period was 19 d on bevacizumab (range 6-66 d) and 17.5 d in the placebo arm (range 4-42) (P = 0.85). Median overall survival was 64 d (95% CI: 45-103) on bevacizumab compared to 31.5 d (95% CI: 20-117) on placebo (P = 0.31). CONCLUSION: Intraperitoneal bevacizumab was well tolerated. Overall, treatment did not result in a significantly better symptom control of malignant ascites. However, patients defined by specific immune characteristics may benefit.

Opposing RA and FGF signals control proximodistal vertebrate limb development through regulation of Meis genes.

Vertebrate limbs develop in a temporal proximodistal sequence, with proximal regions specified and generated earlier than distal ones. Whereas considerable information is available on the mechanisms promoting limb growth, those involved in determining the proximodistal identity of limb parts remain largely unknown. We show here that retinoic acid (RA) is an upstream activator of the proximal determinant genes Meis1 and Meis2. RA promotes proximalization of limb cells and endogenous RA signaling is required to maintain the proximal Meis domain in the limb. RA synthesis and signaling range, which initially span the entire lateral plate mesoderm, become restricted to proximal limb domains by the apical ectodermal ridge (AER) activity following limb initiation. We identify fibroblast growth factor (FGF) as the main molecule responsible for this AER activity and propose a model integrating the role of FGF in limb cell proliferation, with a specific function in promoting distalization through inhibition of RA production and signaling.

Electric Submersible Pumps in Oil Production and Their Efficiency Analysis

The paper describes the structure of electricity consumption of electric submersible pumps. Analyzed the loss of all elements of the pumping unit: the motor, the cable line,transformer, control stations and pump. Discusses how to optimize power consumption of oil production.