Abstract Over-expression of mutant copper, zinc superoxide dismutase (SOD) in mice induces ALS and has become the most widely used model of neurodegeneration. However, no pharmaceutical agent in 20 ...years has extended lifespan by more than a few weeks. The Copper-Chaperone-for-SOD (CCS) protein completes the maturation of SOD by inserting copper, but paradoxically human CCS causes mice co-expressing mutant SOD to die within two weeks of birth. Hypothesizing that co-expression of CCS created copper deficiency in spinal cord, we treated these pups with the PET-imaging agent CuATSM, which is known to deliver copper into the CNS within minutes. CuATSM prevented the early mortality of CCSxSOD mice, while markedly increasing Cu, Zn SOD protein in their ventral spinal cord. Remarkably, continued treatment with CuATSM extended the survival of these mice by an average of 18 months. When CuATSM treatment was stopped, these mice developed ALS-related symptoms and died within 3 months. Restoring CuATSM treatment could rescue these mice after they became symptomatic, providing a means to start and stop disease progression. All ALS patients also express human CCS, raising the hope that familial SOD ALS patients could respond to CuATSM treatment similarly to the CCSxSOD mice.
Our objective was to determine the effect of concentration factor (CF) on the removal of serum protein (SP) from skim milk during microfiltration (MF) at 50°C using a 0.3-μm-pore-size spiral-wound ...(SW) polymeric polyvinylidene fluoride (PVDF) membrane. Pasteurized (72°C for 16 s) skim milk was MF (50°C) at 3 CF (1.50, 2.25, and 3.00×), each on a separate day of processing starting with skim milk. Two phases of MF were used at each CF, with an initial startup-stabilization phase (40min in full recycle mode) to achieve the desired CF, followed by a steady-state phase (90-min feed-and-bleed with recycle) where data was collected. The experiment was replicated 3 times, and SP removal from skim milk was quantified at each CF. System pressures, flow rates, CF, and fluxes were monitored during the 90-min run. Permeate flux increased (12.8, 15.3, and 19.0 kg/m² per hour) with decreasing CF from 3.00 to 1.50×, whereas fouled water flux did not differ among CF, indicating that the effect of membrane fouling on hydraulic resistance of the membrane was similar at all CF. However, the CF used when microfiltering skim milk (50°C) with a 0.3-μm polymeric SW PVDF membrane did affect the percentage of SP removed. As CF increased from 1.50 to 3.00×, the percentage of SP removed from skim milk increased from 10.56 to 35.57%, in a single stage bleed-and-feed MF system. Percentage SP removal from skim milk was lower than the theoretical value. Rejection of SP during MF of skim milk with SW PVDF membranes was caused by fouling of the membrane, not by the membrane itself and differences in the foulant characteristic among CF influenced SP rejection more than it influenced hydraulic resistance. We hypothesize that differences in the conditions near the surface of the membrane and within the pores during the first few minutes of processing, when casein micelles pass through the membrane, influenced the rejection of SP because more pore size narrowing and plugging occurred at low CF than at high CF due to a slower rate of gel layer formation at low CF. It is possible that percentage removal of SP from skim milk at 50°C could be improved by optimization of the membrane pore size, feed solution composition and concentration, and controlling the rate of formation of the concentration polarization-derived gel layer at the surface of the membrane during the first few minutes of processing.
Most current research has focused on using ceramic microfiltration (MF) membranes for micellar casein concentrate production, but little research has focused on the use of polymeric spiral-wound (SW) ...MF membranes. A method for the production of a serum protein (SP)–reduced micellar casein concentrate using SW MF was compared with a ceramic MF membrane. Pasteurized (79°C, 18s) skim milk (1,100 kg) was microfiltered at 50°C about 3 × concentration using a 0.3-μm polyvinylidene fluoride spiral-wound membrane, bleed-and-feed, 3-stage process, using 2 diafiltration stages, where the retentate was diluted 1:2 with reverse osmosis water. Skim milk, permeate, and retentate were analyzed for SP content, and the reduction of SP from skim milk was determined. Theoretically, 68% of the SP content of skim milk can be removed using a single-stage 3× MF. If 2 subsequent water diafiltration stages are used, an additional 22% and 7% of the SP can be removed, respectively, giving a total SP removal of 97%. Removal of SP greater than 95% has been achieved using a 0.1-μm pore size ceramic uniform transmembrane pressure (UTP) MF membrane after a 3-stage MF with diafiltration process. One stage of MF plus 2 stages of diafiltration of 50°C skim milk using a polyvinylidene fluoride polymeric SW 0.3-μm membrane yielded a total SP reduction of only 70.3% (stages 1, 2, and 3: 38.6, 20.8, and 10.9%, respectively). The SP removal rate for the polymeric SW MF membrane was lower in all 3 stages of processing (stages 1, 2, and 3: 0.05, 0.04, and 0.03 kg/m2 per hour, respectively) than that of the comparable ceramic UTP MF membrane (stages 1, 2, and 3: 0.30, 0.11, and 0.06 kg/m2 per hour, respectively), indicating that SW MF is less efficient at removing SP from 50°C skim milk than the ceramic UTP system. To estimate the number of steps required for the SW system to reach 95% SP removal, the third-stage SP removal rate (27.4% of the starting material SP content) was used to extrapolate that an additional 5 water diafiltration stages would be necessary, for a total of 8 stages, to remove 95% of the SP from skim milk. The 8-plus stages necessary to remove >95% SP for the SW MF membrane would create more permeate and a lengthier process than required with ceramic membranes.
During pathology of the nervous system, increased extracellular ATP acts both as a cytotoxic factor and pro-inflammatory mediator through P2X(7) receptors. In animal models of amyotrophic lateral ...sclerosis (ALS), astrocytes expressing superoxide dismutase 1 (SOD1G93A) mutations display a neuroinflammatory phenotype and contribute to disease progression and motor neuron death. Here we studied the role of extracellular ATP acting through P2X(7) receptors as an initiator of a neurotoxic phenotype that leads to astrocyte-mediated motor neuron death in non-transgenic and SOD1G93A astrocytes.
We evaluated motor neuron survival after co-culture with SOD1G93A or non-transgenic astrocytes pretreated with agents known to modulate ATP release or P2X(7) receptor. We also characterized astrocyte proliferation and extracellular ATP degradation.
Repeated stimulation by ATP or the P2X(7)-selective agonist BzATP caused astrocytes to become neurotoxic, inducing death of motor neurons. Involvement of P2X(7) receptor was further confirmed by Brilliant blue G inhibition of ATP and BzATP effects. In SOD1G93A astrocyte cultures, pharmacological inhibition of P2X(7) receptor or increased extracellular ATP degradation with the enzyme apyrase was sufficient to completely abolish their toxicity towards motor neurons. SOD1G93A astrocytes also displayed increased ATP-dependent proliferation and a basal increase in extracellular ATP degradation.
Here we found that P2X(7) receptor activation in spinal cord astrocytes initiated a neurotoxic phenotype that leads to motor neuron death. Remarkably, the neurotoxic phenotype of SOD1G93A astrocytes depended upon basal activation the P2X(7) receptor. Thus, pharmacological inhibition of P2X(7) receptor might reduce neuroinflammation in ALS through astrocytes.
The P2X7 receptor/channel responds to extracellular ATP and is associated with neuronal death and neuroinflammation in spinal cord injury and amyotrophic lateral sclerosis. Whether activation of P2X7 ...directly causes motor neuron death is unknown. We found that cultured motor neurons isolated from embryonic rat spinal cord express P2X7 and underwent caspase‐dependent apoptosis when exposed to exceptionally low concentrations of the P2X7 agonist 2′(3′)‐O‐(4‐Benzoylbenzoyl)‐ATP. The P2X7 inhibitors BBG, oATP, and KN‐62 prevented 2′(3′)‐O‐(4‐Benzoylbenzoyl)‐ATP‐induced motor neuron death. The endogenous P2X7 agonist ATP induced motor neuron death at low concentrations (1‐100 μM). High concentrations of ATP (1 mM) paradoxically became protective due to degradation in the culture media to produce adenosine and activate adenosine receptors. P2X7‐induced motor neuron death was dependent on neuronal nitric oxide synthase‐mediated production of peroxynitrite, p38 activation, and autocrine FAS signaling. Taken together, our results indicate that motor neurons are highly sensitive to P2X7 activation, which triggers apoptosis by activation of the well‐established peroxynitrite/FAS death pathway in motor neurons.
The extracellular ATP receptor P2X7 is present in microglia and astrocytes. Here, P2X7 is shown to be expressed and functional in embryonic motor neurons. Motor neurons are unusually sensitive to ATP, with low micromolar concentrations of ATP inducing death through a mechanism involving endogenous peroxynitrite generation and FAS. These results support a direct role of P2X7 in motor neuron death as well as promoting neuroinflammation in spinal cord injury and amyotrophic lateral sclerosis (ALS).
Read the Editorial Highlight for this article on doi: 10.1111/jnc.12321.
•Model of diffusion into cells encapsulated in protective polymer coating.•Dimensionless Fickian diffusion model over realistic range of physical parameters.•Two regimes identified: one limited by ...diffusion and the other limited by consumption.•Examined oxygen diffusion into pancreatic islets encapsulated in alginate.•Parameter allowing cell viability determined.
Fickian diffusion into a core-shell geometry is modeled. The interior core mimics pancreatic Langerhan islets and the exterior shell acts as inert protection. The consumption of oxygen diffusing into the cells is approximated using Michaelis-Menten kinetics. The problem is transformed to dimensionless units and solved numerically. Two regimes are identified, one that is diffusion limited and the other consumption limited. A regression is fit that describes the concentration at the center of the cells as a function of the relevant physical parameters. It is determined that, in a cell culture environment, the cells will remain viable as long as the islet has a radius of around 142 µm or less and the encapsulating shell has a radius of less than approximately 283 µm. When the islet is on the order of 100 µm it is possible for the cells to remain viable in environments with as little as 4.6×10−2 mol/m−3 O2. These results indicate such an encapsulation scheme may be used to prepare artificial pancreas to treat diabetes.
Abstract
Ubiquitous expression of mutant Cu/Zn-superoxide dismutase (SOD1) selectively affects motor neurons in the central nervous system (CNS), causing the adult-onset degenerative disease ...amyotrophic lateral sclerosis (ALS). The CNS-specific impact of ubiquitous mutant SOD1 expression is recapitulated in transgenic mouse models of the disease. Here we present outcomes for the metallo-complex Cu
II
(atsm) tested for therapeutic efficacy in mice expressing SOD1
G93A
on a mixed genetic background. Oral administration of Cu
II
(atsm) delayed the onset of neurological symptoms, improved locomotive capacity and extended overall survival. Although the ALS-like phenotype of SOD1
G93A
mice is instigated by expression of the mutant SOD1, we show the improved phenotype of the Cu
II
(atsm)-treated animals involves an increase in mature mutant SOD1 protein in the disease-affected spinal cord, where concomitant increases in copper and SOD1 activity are also evident. In contrast to these effects in the spinal cord, treating with Cu
II
(atsm) had no effect in liver on either mutant SOD1 protein levels or its activity, indicating a CNS-selective SOD1 response to the drug. These data provide support for Cu
II
(atsm) as a treatment option for ALS as well as insight to the CNS-selective effects of mutant SOD1.
Age is a recognized risk factor for amyotrophic lateral sclerosis (ALS), a paralytic disease characterized by progressive loss of motor neurons and neuroinflammation. A hallmark of aging is the ...accumulation of senescent cells. Yet, the pathogenic role of cellular senescence in ALS remains poorly understood. In rats bearing the ALS-linked SOD1
mutation, microgliosis contribute to motor neuron death, and its pharmacologic downregulation results in increased survival. Here, we have explored whether gliosis and motor neuron loss were associated with cellular senescence in the spinal cord during paralysis progression. In the lumbar spinal cord of symptomatic SOD1
rats, numerous cells displayed nuclear p16
as well as loss of nuclear Lamin B1 expression, two recognized senescence-associated markers. The number of p16
-positive nuclei increased by four-fold while Lamin B1-negative nuclei increased by 1,2-fold, respect to non-transgenic or asymptomatic transgenic rats. p16
-positive nuclei and Lamin B1-negative nuclei were typically localized in a subset of hypertrophic Iba1-positive microglia, occasionally exhibiting nuclear giant multinucleated cell aggregates and abnormal nuclear morphology. Next, we analyzed senescence markers in cell cultures of microglia obtained from the spinal cord of symptomatic SOD1
rats. Although microglia actively proliferated in cultures, a subset of them developed senescence markers after few days
and subsequent passages. Senescent SOD1
microglia in culture conditions were characterized by large and flat morphology, senescence-associated beta-Galactosidase (SA-β-Gal) activity as well as positive labeling for p16
, p53, matrix metalloproteinase-1 (MMP-1) and nitrotyrosine, suggesting a senescent-associated secretory phenotype (SASP). Remarkably, in the degenerating lumbar spinal cord other cell types, including ChAT-positive motor neurons and GFAP-expressing astrocytes, also displayed nuclear p16
staining. These results suggest that cellular senescence is closely associated with inflammation and motor neuron loss occurring after paralysis onset in SOD1
rats. The emergence of senescent cells could mediate key pathogenic mechanisms in ALS.
Recent developments of transition-edge sensors (TESs), based on extensive experience in ground-based experiments, have been making the sensor techniques mature enough for their application on future ...satellite cosmic microwave background (CMB) polarization experiments. LiteBIRD is in the most advanced phase among such future satellites, targeting its launch in Japanese Fiscal Year 2027 (2027FY) with JAXA’s H3 rocket. It will accommodate more than 4000 TESs in focal planes of reflective low-frequency and refractive medium-and-high-frequency telescopes in order to detect a signature imprinted on the CMB by the primordial gravitational waves predicted in cosmic inflation. The total wide frequency coverage between 34 and 448 GHz enables us to extract such weak spiral polarization patterns through the precise subtraction of our Galaxy’s foreground emission by using spectral differences among CMB and foreground signals. Telescopes are cooled down to 5 K for suppressing thermal noise and contain polarization modulators with transmissive half-wave plates at individual apertures for separating sky polarization signals from artificial polarization and for mitigating from instrumental 1/
f
noise. Passive cooling by using V-grooves supports active cooling with mechanical coolers as well as adiabatic demagnetization refrigerators. Sky observations from the second Sun–Earth Lagrangian point, L2, are planned for 3 years. An international collaboration between Japan, the USA, Canada, and Europe is sharing various roles. In May 2019, the Institute of Space and Astronautical Science, JAXA, selected LiteBIRD as the strategic large mission No. 2.
Lenslet-coupled antenna arrays have been used in CMB experiments and are the baseline technology for the next-generation satellite missions such as LiteBIRD and PICO. Lenslets are small hemispherical ...lenses mounted on the focal plane that couple light to the detectors and are typically made of silicon or alumina due to their high focusing power and low absorption loss. To minimize reflection at the vacuum-dielectric interface, lenslets require anti-reflection (AR) coatings. Metamaterials have been used in large microwave optical components because they avoid any mismatch on the thermal expansion between the lens and its coating, but so far they have only been machined on surfaces of comparatively large radius of curvature. As a first step to understand the feasibility of machining metamaterial AR layers in lenslets through laser-etching for the LiteBIRD mission, a model in ANSYS HFSS was developed. The goal of the simulation was to optimize transmission in three frequency bands while meeting assumed laser machinability constraints and optical requirements. Simulation results from flat silicon show that an AR metamaterial coating made under the assumed conditions is feasible, and the baseline parameters for further curved-surface studies are provided.
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