Expression of Cholera Toxin B–Proinsulin Fusion Protein in Lettuce and Tobacco Chloroplasts – Oral Administration Protects Against Development of Insulitis in Non-Obese Diabetic Mice

Lettuce and tobacco chloroplast transgenic lines expressing the cholera toxin B subunit–human proinsulin (CTB-Pins) fusion protein were generated. CTB-Pins accumulated up to ~16% of total soluble protein (TSP) in tobacco and up to ~2.5% of TSP in lettuce. Eight milligrams of powdered tobacco leaf material expressing CTB-Pins or, as negative controls, CTB–green fluorescent protein (CTB-GFP) or interferon–GFP (IFN-GFP), or untransformed leaf, were administered orally, each week for 7 weeks, to 5-week-old female non-obese diabetic (NOD) mice. The pancreas of CTB-Pins-treated mice showed decreased infiltration of cells characteristic of lymphocytes (insulitis); insulin-producing β-cells in the pancreatic islets of CTB-Pins-treated mice were significantly preserved, with lower blood or urine glucose levels, by contrast with the few β-cells remaining in the pancreatic islets of the negative controls. Increased expression of immunosuppressive cytokines, such as interleukin-4 and interleukin-10 (IL-4 and IL-10), was observed in the pancreas of CTB-Pins-treated NOD mice. Serum levels of immunoglobulin G1 (IgG1), but not IgG2a, were elevated in CTB-Pins-treated mice. Taken together, T-helper 2 (Th2) lymphocyte-mediated oral tolerance is a likely mechanism for the prevention of pancreatic insulitis and the preservation of insulin-producing β-cells. This is the first report of expression of a therapeutic protein in transgenic chloroplasts of an edible crop. Transplastomic lettuce plants expressing CTB-Pins grew normally and transgenes were maternally inherited in T1 progeny. This opens up the possibility for the low-cost production and delivery of human therapeutic proteins, and a strategy for the treatment of various other autoimmune diseases

Synchronised TeraHertz radiation and soft X-rays produced in a FEL oscillator

In this paper, we present the generalities of the Compton interaction process; we analyse the different paradigms of Inverse Compton Sources, implemented or in commissioning phase at various facilities, or proposed as future projects. We present an overview of the state of the art, with a discussion of the most demanding challenges

State of the art of high-flux Compton/Thomson X-rays sources

In this paper, we present the generalities of the Compton interaction process; we analyse the different paradigms of Inverse Compton Sources, implemented or in commissioning phase at various facilities, or proposed as future projects. We present an overview of the state of the art, with a discussion of the most demanding challenges

Coherent, high repetition rate tender x-ray free-electron laser seeded by an extreme ultra-violet free-electron laser oscillator

A seeded FEL driven by a linac based on super conducting cavities, generating 108-1010 coherent photons per shot at 2-5 keV with 0.2-1 MHz of repetition rate, can address the need of a source devoted to fine analysis of matter using the linear spectroscopy technique. The seeding scheme described hereafter is a multi-stage cascade upshifting the radiation frequency by a factor 20-40. The x-ray range can be achieved with a seed constituted by a coherent flash in the extreme ultraviolet range provided by an FEL oscillator operating at 12-14 nm. The whole chain of x-ray generation is described by means of start-to-end three-dimensional simulations

Downregulation of CYP17A1 by 20-hydroxyecdysone: plasma progesterone and its vasodilatory properties

Aim: To investigate the effect of 20-hydroxyecdysone on steroidogenic pathway genes and plasma progesterone, and its potential impact on vascular functions. Methods: Chimeric mice with humanized liver were treated with 20-hydroxyecdysone for 3 days, and hepatic steroidogenic pathway genes and plasma progesterone were measured by transcriptomics and GC–MS/MS, respectively. Direct effects on muscle and mesenteric arterioles were assessed by myography. Results: CYP17A1 was downregulated in 20-hydroxyecdysone-treated mice compared with untreated group (p = 0.04), with an insignificant increase in plasma progesterone. Progesterone caused vasorelaxation which was blocked by 60 mM KCl, but unaffected by nitric oxide synthase inhibition. Conclusion: In the short term, 20-hydroxyecdysone mediates CYP17A1 downregulation without a significant increase in plasma progesterone, which has a vasodilatory effect involving inhibition of voltage-dependent calcium channels, and the potential to enhance 20-hydroxyecdysone vasorelaxation

High Repetition Rate and Coherent Free-Electron Laser in the X-Rays Range Tailored for Linear Spectroscopy

Fine time-resolved analysis of matter\u2014i.e., spectroscopy and photon scattering\u2014in the linear response regime requires fs-scale pulsed, high repetition rate, fully coherent X-ray sources. A seeded Free Electron Laser (FEL) driven by a Linac based on Super Conducting cavities, generating 108 \u2013 1010 coherent photons at 2\u20135 keV with 0.2\u20131 MHz of repetition rate, can address this need. Three different seeding schemes, reaching the X-ray range, are described hereafter. The first two are multi-stage cascades upshifting the radiation frequency by a factor of 10\u201330 starting from a seed represented by a coherent flash of extreme ultraviolet light. This radiation can be provided either by the High Harmonic Generation of an optical laser or by an FEL Oscillator operating at 12\u201314 nm. The third scheme is a regenerative amplifier working with X-ray mirrors. The whole chain of the X-ray generation is here described by means of start-to-end simulations

Molecular correlates of axonal and synaptic pathology in mouse models of Batten disease

Neuronal ceroid lipofuscinoses (NCLs; Batten disease) are collectively the most frequent autosomal-recessive neurodegenerative disease of childhood, but the underlying cellular and molecular mechanisms remain unclear. Several lines of evidence have highlighted the important role that non-somatic compartments of neurons (axons and synapses) play in the instigation and progression of NCL pathogenesis. Here, we report a progressive breakdown of axons and synapses in the brains of two different mouse models of NCL: Ppt1−/− model of infantile NCL and Cln6nclf model of variant late-infantile NCL. Synaptic pathology was evident in the thalamus and cortex of these mice, but occurred much earlier within the thalamus. Quantitative comparisons of expression levels for a subset of proteins previously implicated in regulation of axonal and synaptic vulnerability revealed changes in proteins involved with synaptic function/stability and cell-cycle regulation in both strains of NCL mice. Protein expression changes were present at pre/early-symptomatic stages, occurring in advance of morphologically detectable synaptic or axonal pathology and again displayed regional selectivity, occurring first within the thalamus and only later in the cortex. Although significant differences in individual protein expression profiles existed between the two NCL models studied, 2 of the 15 proteins examined (VDAC1 and Pttg1) displayed robust and significant changes at pre/early-symptomatic time-points in both models. Our study demonstrates that synapses and axons are important early pathological targets in the NCLs and has identified two proteins, VDAC1 and Pttg1, with the potential for use as in vivo biomarkers of pre/early-symptomatic axonal and synaptic vulnerability in the NCLs

Brixsino High-Flux Dual X-Ray and THz Radiation Source Based on Energy Recovery Linacs

We present the conceptual design of a compact light source named BriXSinO. BriXSinO was born as demonstrator of the Marix project, but it is also a dual high flux radiation source Inverse Compton Source (ICS) of X-ray and Free-Electron Laser of THz spectral range radiation conceived for medical applications and general applied research. The accelerator is a push-pull CW-SC Energy Recovery Linac (ERL) based on superconducting cavities technology and allows to sustain MW-class beam power with almost just one hundred kW active power dissipation/consumption. ICS line produces 33 keV monochromatic X-Rays via Compton scattering of the electron beam with a laser system in Fabry-Pérot cavity at a repetition rate of 100 MHz. The THz FEL oscillator is based on an undulator imbedded in optical cavity and generates THz wavelengths from 15 to 50 micron

MariX, an advanced MHz-class repetition rate X-ray source for linear regime time-resolved spectroscopy and photon scattering

The need of a fs-scale pulsed, high repetition rate, X-ray source for time-resolved fine analysis of matter (spectroscopy and photon scattering) in the linear response regime is addressed by the conceptual design of a facility called MariX (Multi-disciplinary Advanced Research Infrastructure for the generation and application of X-rays) outperforming current X-ray sources for the declared scope. MariX is based on the original design of a two-pass two-way superconducting linear electron accelerator, equipped with an arc compressor, to be operated in CW mode (1 MHz). MariX provides FEL emission in the range 0.2–8 keV with 108 photons per pulse ideally suited for photoelectric effect and inelastic X-ray scattering experiments. The accelerator complex includes an early stage that supports an advanced inverse Compton source of very high-flux hard X-rays of energies up to 180 keV that is well adapted for large area radiological imaging, realizing a broad science programme and serving a multidisciplinary user community, covering fundamental science of matter and application to life sciences, including health at preclinical and clinical level

Central Action of Peripherally Applied Botulinum Toxin Type A on Pain and Dural Protein Extravasation in Rat Model of Trigeminal Neuropathy

BACKGROUND: Infraorbital nerve constriction (IoNC) is an experimental model of trigeminal neuropathy. We investigated if IoNC is accompanied by dural extravasation and if botulinum toxin type A (BoNT/A) can reduce pain and dural extravasation in this model. ----- METHODOLOGY/PRINCIPAL FINDINGS: Rats which developed mechanical allodynia 14 days after the IoNC were injected with BoNT/A (3.5 U/kg) into vibrissal pad. Allodynia was tested by von Frey filaments and dural extravasation was measured as colorimetric absorbance of Evans blue - plasma protein complexes. Presence of dural extravasation was also examined in orofacial formalin-induced pain. Unilateral IoNC, as well as formalin injection, produced bilateral dural extravasation. Single unilateral BoNT/A injection bilaterally reduced IoNC induced dural extravasation, as well as allodynia (lasting more than 2 weeks). Similarly, BoNT/A reduced formalin-induced pain and dural extravasation. Effects of BoNT/A on pain and dural extravasation in IoNC model were dependent on axonal transport through sensory neurons, as evidenced by colchicine injections (5 mM, 2 µl) into the trigeminal ganglion completely preventing BoNT/A effects. ----- CONCLUSIONS/SIGNIFICANCE: Two different types of pain, IoNC and formalin, are accompanied by dural extravasation. The lasting effect of a unilateral injection of BoNT/A in experimental animals suggests that BoNT/A might have a long-term beneficial effect in craniofacial pain associated with dural neurogenic inflammation. Bilateral effects of BoNT/A and dependence on retrograde axonal transport suggest a central site of its action