Probing the energetic particle environment near the Sun.

NASA's Parker Solar Probe mission1 recently plunged through the inner heliosphere of the Sun to its perihelia, about 24 million kilometres from the Sun. Previous studies farther from the Sun (performed mostly at a distance of 1 astronomical unit) indicate that solar energetic particles are accelerated from a few kiloelectronvolts up to near-relativistic energies via at least two processes: 'impulsive' events, which are usually associated with magnetic reconnection in solar flares and are typically enriched in electrons, helium-3 and heavier ions2, and 'gradual' events3,4, which are typically associated with large coronal-mass-ejection-driven shocks and compressions moving through the corona and inner solar wind and are the dominant source of protons with energies between 1 and 10 megaelectronvolts. However, some events show aspects of both processes and the electron-proton ratio is not bimodally distributed, as would be expected if there were only two possible processes5. These processes have been very difficult to resolve from prior observations, owing to the various transport effects that affect the energetic particle population en route to more distant spacecraft6. Here we report observations of the near-Sun energetic particle radiation environment over the first two orbits of the probe. We find a variety of energetic particle events accelerated both locally and remotely including by corotating interaction regions, impulsive events driven by acceleration near the Sun, and an event related to a coronal mass ejection. We provide direct observations of the energetic particle radiation environment in the region just above the corona of the Sun and directly explore the physics of particle acceleration and transport.

Measurements of the neutron spectrum in transit to Mars on the Mars Science Laboratory.

The Mars Science Laboratory spacecraft, containing the Curiosity rover, was launched to Mars on 26 November 2011. Although designed for measuring the radiation on the surface of Mars, the Radiation Assessment Detector (RAD) measured the radiation environment inside the spacecraft during most of the 253-day, 560-million-kilometer cruise to Mars. An important factor for determining the biological impact of the radiation environment inside the spacecraft is the specific contribution of neutrons with their high biological effectiveness. We apply an inversion method (based on a maximum-likelihood estimation) to calculate the neutron and gamma spectra from the RAD neutral particle measurements. The measured neutron spectrum (12-436 MeV) translates into a radiation dose rate of 3.8±1.2 µGy/day and a dose equivalent of 19±5 µSv/day. Extrapolating the measured spectrum (0.1-1000 MeV), we find that the total neutron-induced dose rate is 6±2 µGy/day and the dose equivalent rate is 30±10 µSv/day. For a 360 day round-trip from Earth to Mars with comparable shielding, this translates into a neutron induced dose equivalent of about 11±4 mSv.

The main pillar: Assessment of space weather observational asset performance supporting nowcasting, forecasting, and research to operations.

Space weather forecasting critically depends upon availability of timely and reliable observational data. It is therefore particularly important to understand how existing and newly planned observational assets perform during periods of severe space weather. Extreme space weather creates challenging conditions under which instrumentation and spacecraft may be impeded or in which parameters reach values that are outside the nominal observational range. This paper analyzes existing and upcoming observational capabilities for forecasting, and discusses how the findings may impact space weather research and its transition to operations. A single limitation to the assessment is lack of information provided to us on radiation monitor performance, which caused us not to fully assess (i.e., not assess short term) radiation storm forecasting. The assessment finds that at least two widely spaced coronagraphs including L4 would provide reliability for Earth-bound CMEs. Furthermore, all magnetic field measurements assessed fully meet requirements. However, with current or even with near term new assets in place, in the worst-case scenario there could be a near-complete lack of key near-real-time solar wind plasma data of severe disturbances heading toward and impacting Earth's magnetosphere. Models that attempt to simulate the effects of these disturbances in near real time or with archival data require solar wind plasma observations as input. Moreover, the study finds that near-future observational assets will be less capable of advancing the understanding of extreme geomagnetic disturbances at Earth, which might make the resulting space weather models unsuitable for transition to operations. KEY POINTS: Manuscript assesses current and near-future space weather assetsCurrent assets unreliable for forecasting of severe geomagnetic stormsNear-future assets will not improve the situation.

Measurements of energetic particle radiation in transit to Mars on the Mars Science Laboratory.

The Mars Science Laboratory spacecraft, containing the Curiosity rover, was launched to Mars on 26 November 2011, and for most of the 253-day, 560-million-kilometer cruise to Mars, the Radiation Assessment Detector made detailed measurements of the energetic particle radiation environment inside the spacecraft. These data provide insights into the radiation hazards that would be associated with a human mission to Mars. We report measurements of the radiation dose, dose equivalent, and linear energy transfer spectra. The dose equivalent for even the shortest round-trip with current propulsion systems and comparable shielding is found to be 0.66 ± 0.12 sievert.

Modulated binding of SATB1, a matrix attachment region protein, to the AT-rich sequence flanking the major breakpoint region of BCL2.

The t(14,18) chromosomal translocation that occurs in human follicular lymphoma constitutively activates the BCL2 gene and disrupts control of apoptosis. Interestingly, 70% of the t(14,18) translocations are confined to three 15-bp clusters positioned within a 150-bp region (major breakpoint region or [MBR]) in the untranslated portion of terminal exon 3. We analyzed DNA-protein interactions in the MBR, as these may play some role in targeting the translocation to this region. An 87-bp segment (87MBR) immediately 3' to breakpoint cluster 3 was essential for DNA-protein interaction monitored with mobility shift assays. We further delineated a core binding region within 87MBR: a 33-bp, very AT-rich sequence highly conserved between the human and mouse BCL2 gene (37MBR). We have purified and identified one of the core factors as the matrix attachment region (MAR) binding protein, SATB1, which is known to bind to AT-rich sequences with a high propensity to unwind. Additional factors in nuclear extracts, which we have not yet characterized further, increased SATB1 affinity for the 37MBR target four- to fivefold. Specific binding activity within 37MBR displayed cell cycle regulation in Jurkat T cells, while levels of SATB1 remained constant throughout the cell cycle. Finally, we demonstrated in vivo binding of SATB1 to the MBR, strongly suggesting the BCL2 major breakpoint region is a MAR. We discuss the potential consequences of our observations for both MBR fragility and regulatory function.

Sac excision is essential to adequate laparoscopic repair of paraesophageal hernia.

BACKGROUND: We compared the incidence of early hernia recurrence in nonrandomized but consecutive patients undergoing laparoscopic repair of paraesophageal hernia (LRPH) without and with excision of the hernia sac. METHODS: LRPH was completed in 55 of 58 patients. In the first 25 patients, the sac was not excised. Total sac excision was performed in the subsequent 30 patients. All patients had crural repair with or without fundoplication, or gastropexy. RESULTS: Mean age of patients was 68 years (range, 34-95). There were three conversions; one patient died postoperatively. Mean operative time was 225 min in the first group and 190 min in the sac excision group. Median length of stay was 2 days (range, 1-15) for both groups. CONCLUSIONS: A precise method of total sac excision simplified dissection. It also ensured complete reduction of the hernia and availability of adequate esophageal length. Operative time was not increased, and no subsequent early recurrences were observed (p < 0.05).

Non-erythroid alpha-spectrin breakdown by calpain and interleukin 1 beta-converting-enzyme-like protease(s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis.

The cytoskeletal protein non-erythroid alpha-spectrin is well documented as an endogenous calpain substrate, especially under pathophysiological conditions. In cell necrosis (e.g. maitotoxin-treated neuroblastoma SH-SY5Y cells), alpha-spectrin breakdown products (SBDPs) of 150 kDa and 145 kDa were produced by cellular calpains. In contrast, in neuronal cells undergoing apoptosis (cerebellar granule neurons subjected to low potassium and SH-SY5Y cells treated with staurosporine), an additional SBDP of 120 kDa was also observed. The formation of the 120 kDa SBDP was insensitive to calpain inhibitors but was completely blocked by an interleukin 1 beta-converting-enzyme (ICE)-like protease inhibitor, Z-Asp-CH2OC(O)-2,6-dichlorobenzene. Autolytic activation of both calpain and the ICE homologue CPP32 was also observed in apoptotic cells. alpha-Spectrin can also be cleaved in vitro by purified calpains to produce the SBDP doublet of 150/145 kDa and by ICE and ICE homologues [ICH-1, ICH-2 and CPP32(beta)] to produce a 150 kDa SBDP. In addition, CPP32 and ICE also produced a 120 kDa SBDP. Furthermore inhibition of either ICE-like protease(s) or calpain protects both granule neurons and SH-SY5Y cells against apoptosis. Our results suggest that both protease families participate in the expression of neuronal apoptosis.

Phenytoin pretreatment prevents hypoxic-ischemic brain damage in neonatal rats.

This study was performed to investigate whether the anticonvulsant phenytoin has neuroprotective effect in a model of hypoxia-ischemia with neonatal rats. The left carotid artery of each rat was ligated, followed by 3 h of hypoxic exposure (8% O2) in a temperature-regulated environment (36 degrees C). Two weeks later, brain damage was assessed by measuring loss of brain hemisphere weight. Phenytoin had no effect on body temperature or plasma glucose, but attenuated brain damage in a dose-dependent manner (3, 10, and 30 mg/kg i.p.) when administered before the hypoxic episode. Phenytoin administered during or after hypoxia did not alter hypoxic brain damage significantly. A parallel experiment using histological examination of frozen brain sections demonstrated less brain infarction after phenytoin treatment (30 mg/kg i.p.). In an additional experiment measuring breakdown of an endogenous brain calpain substrate, spectrin, phenytoin treatment reduced this measure of early cellular damage. Our results indicate that pretreatment with phenytoin is neuroprotective at a plasma phenytoin concentration of approximately 12 micrograms/ml. These results are consistent with the hypothesis that blockade of voltage-dependent sodium channels reduces brain damage following ischemia.

An alpha-mercaptoacrylic acid derivative is a selective nonpeptide cell-permeable calpain inhibitor and is neuroprotective.

Overactivation of calcium-activated neutral protease (calpain) has been implicated in the pathophysiology of several degenerative conditions, including stroke, myocardial ischemia, neuromuscular degeneration, and cataract formation. Alpha-mercaptoacrylate derivatives (exemplified by PD150606), with potent and selective inhibitory actions against calpain, have been identified. PD150606 exhibits the following characteristics: (i) Ki values for mu- and m-calpains of 0.21 microM and 0.37 microM, respectively, (ii) high specificity for calpains relative to other proteases, (iii) uncompetitive inhibition with respect to substrate, and (iv) it does not shield calpain against inactivation by the active-site inhibitor trans-(epoxysuccinyl)-L-leucyl-amido-3-methylbutane, suggesting a nonactive site action for PD150606. The recombinant calcium-binding domain from each of the large or small subunits of mu-calpain was found to interact with PD150606. In low micromolar range, PD15O6O6 inhibited calpain activity in two intact cell systems. The neuroprotective effects of this class of compound were also demonstrated by the ability of PD150606 to attenuate hypoxic/hypoglycemic injury to cerebrocortical neurons in culture and excitotoxic injury to Purkinje cells in cerebellar slices.

Total protein extraction from cultured cells for use in electrophoresis and western blotting.

Experimentation with cultured cells often requires analyzing cellular protein extract by gel electrophoresis and immunoblotting. Traditional methods for extracting cellular proteins by homogenization or detergent solubilization usually produce protein samples that are viscous (due to the presence of DNA) and prone to degradation due to the presence of endogenous protease activity. We have developed a method that involves solubilization of cells with sodium dodecyl sulfate (SDS), precipitation of proteins with trichloroacetic acid (TCA) with special physical exclusion of DNA aggregate and reconstitution of precipitated proteins with Tris base. Protein samples prepared by this method contain little DNA, making them ideal for long-term storage. The solubilized total protein extracts are fully compatible with protein assay, gel electrophoresis and Western blotting. When compared to protein extracts from a homogenization method, those from the TCA method showed an identical total protein staining pattern on SDS polyacrylamide gel electrophoresis and contained distinct cellular proteins recognized by many monoclonal and polyclonal antibodies tested (including anti-actin, spectrin, protein kinase C (alpha), talin and spectrin) on Western blots.

Aurintricarboxylic acid is an inhibitor of mu- and m-calpain.

Aurintricarboxylic acid (ATA) is an endonuclease inhibitor which has been shown to block apoptotic cell death. We have now demonstrated that ATA is also an inhibitor of the Ca(2+)-activated neutral protease (calpain), a class of cytosolic enzyme that may also be activated during apoptosis. The two major calpain isoforms (mu- and m-calpain) were both inhibited by ATA with IC50's of 22 microM and 10 microM, respectively. The autolysis of purified mu-calpain was prevented by ATA in a concentration-dependent manner. Using casein zymography, it was found that the inhibition of mu-calpain by ATA was reversible. Finally, in a fetal rat cerebrocortical culture model of excitotoxicity, pre- and post-treatment of ATA (50 microM) reduced N-methyl-D-aspartate (NMDA)-induced spectrin breakdown and neuronal death, while application of ATA concurrent to NMDA challenge alone had no effect. This pattern of protection could not be explained by simple NMDA receptor antagonism. We thus propose that the neuroprotective effect of ATA could be in part due to its ability to inhibit calpain.

Casein zymography: a method to study mu-calpain, m-calpain, and their inhibitory agents.

A zymographic assay for calpains in nondenaturing casein-containing polyacrylamide gels was developed. Calpain samples were run into the polyacrylamide gels by electrophoresis using a Tris-glycine buffer containing 1 mM EGTA to stabilize calpains. Upon completion of the electrophoresis, the gels were washed and incubated in a calpain activation buffer containing 1-4 mM calcium and 10 mM dithiothreitol for 20-24 h. After staining of the casein gels with Coomassie blue G250, both mu-calpain and m-calpain showed up as clearing bands. The amount of calpain loaded was proportional to the brightness of the clearing band. m-calpain can be easily distinguished from mu-calpain due to its higher mobility in the gel. Irreversible inhibitor (e.g., E64c) or tight-binding calmidazolium-treated mu-calpain remained inactive in the casein zymogram, whereas reversible inhibitor (e.g., calpain inhibitor I) was released from the protease by migration and dilution, lifting its inhibition. Crude homogenate of cultured cells (erythrocytes, Molt-4 and cerebrocortical neurons) or tissue (rat brain) can be directly analyzed for the presence of calpain isoforms despite the presence of endogenous calpastatin. Using this technique, mu-calpain activity in Molt-4 cells was found to decrease progressively with A23187 treatment, as a reflection of autolytic inactivation.

Problems in formulating method-insensitive proficiency testing materials.

Significant problems exist in formulating method-insensitive proficiency materials. Many steps are required in processing human plasma, and difficult choices are involved in the selection of appropriate materials to be added to the processed plasma. Additionally, analytes may vary widely in their recovery from method to method. To enhance understanding of the procedures and problems involved in the development and manufacture of proficiency materials, a number of constituents and their method-specific recoveries are reviewed.

The effect of glass-ceramic bone implant materials on the in vitro formation of hydroxyapatite.

The extracts of four glass-ceramic bone implant materials were investigated for dissolved material, for effects on in vitro formation of hydroxyapatite, and for surface morphology of glass-ceramic particles in scanning electron microscopy. In vitro leaching released substances that affected in vitro formation of hydroxyapatite, i.e., initiation time and growth of crystals. Leaching also changed the surface morphology of the materials. The ability of the materials to bond to bone did not correlate with the inhibition of hydroxyapatite formation by the released substances. Surface morphology and other factors at present not yet known are probably involved in controlling the bonding to bone of these ceramics.

The mineral of bone.

This is a review of the chemistry and structure of synthetic, mineral, and biologic hydroxyapatites. Bone apatite has a large, reactive specific-surface and is characterized by its crystal imperfection and non-stoichiometry. Precipitated and bone hydroxyapatites are in the submicroscopic size range where their solubility decreases rapidly with a small increment of crystal growth. A discussion is given of the various mechanisms proposed for tissue mineralization. The body seems to contain a number of nucleating and inhibiting mechanisms which seem to work in concert, possibly providing redundant pathways to the mineralization of tissue.

The structure of bone apatite surfaces.

This is a review of the surface chemistry of bone mineral and its synthetic counterpart hydroxyapatite. Small-angle x-ray scattering and low-temperature nitrogen adsorption measurements show bone mineral surfaces range from 100 to 200 m2/g. The heats of adsorption of small molecules on bone and apatite surfaces show that these materials have polarizing surfaces which form strong bonds with polar and polarizable molecules. Water is hydrogen bonded to these surfaces with energies ranging from 23 Kcal/mol, for low coverage, to 11 Kcal/mol after two full layers; the latter value shows that after two monolayers the water is bonded as strongly to the solution as it is to the apatite surface. Stearic acid in cyclohexane adsorbs on bone and apatite surfaces in a closed-packed manner with the straight-chain molecules in parallel array with the end carboxyl groups hydrogen bonded to surface electronegative ions. Synthetic hydroxyapatite has long been used in chromatography because of the bonding capacity apatite surface has for certain proteins and polynucleotides. The metabolic interplay between bone mineral and the body results from the high magnitude and high reactivity of the mineral surface.

Mineral parameters in early fracture repair.

In an effort to define and characterize the initial mineralization product of fracture-healing, we studied the mineral components within a model of endochondral osseous repair. Fracture calluses from the tibiae of rats and rabbits undergoing endochondral fracture-healing were analyzed, in toto and following density fractionation, by physicochemical and crystallographic techniques. Significant changes in mineral composition, crystal size, and density occurred in the early phases of fracture repair. In the rat, two weeks after fracture, the calcium-to-phosphorus ratio was higher than that of the mineral component, possibly due to calcium-binding to some of the macromolecules known to be present. The earliest mineral was poorly crystallized hydroxyapatite with a high carbonate content. Crystal perfection improved rapidly and approached that of normal diaphyseal bone within eight weeks after endochondral fracture in both the rabbit and the rat.