Comparative pulmonary toxicities of lunar dusts and terrestrial dusts (TiO2 & SiO2) in rats and an assessment of the impact of particle-generated oxidants on the dusts' toxicities.

Humans will set foot on the Moon again soon. The lunar dust (LD) is potentially reactive and could pose an inhalation hazard to lunar explorers. We elucidated LD toxicity and investigated the toxicological impact of particle surface reactivity (SR) using three LDs, quartz, and TiO2. We first isolated the respirable-size-fraction of an Apollo-14 regolith and ground two coarser samples to produce fine LDs with increased SR. SR measurements of these five respirable-sized dusts, determined by their in-vitro ability to generate hydroxyl radicals (•OH), showed that ground LDs > unground LD ≥ TiO2 ≥ quartz. Rats were each intratracheally instilled with 0, 1, 2.5, or 7.5 mg of a test dust. Toxicity biomarkers and histopathology were assessed up to 13 weeks after the bolus instillation. All dusts caused dose-dependent-increases in pulmonary lesions and toxicity biomarkers. The three LDs, which possessed mineral compositions/properties similar to Arizona volcanic ash, were moderately toxic. Despite a 14-fold •OH difference among these three LDs, their toxicities were indistinguishable. Quartz produced the lowest •OH amount but showed the greatest toxicity. Our results showed no correlation between the toxicity of mineral dusts and their ability to generate free radicals. We also showed that the amounts of oxidants per neutrophil increased with doses, time and the cytotoxicity of the dusts in the lung, which supports our postulation that dust-elicited neutrophilia is the major persistent source of oxidative stress. These results and the discussion of the crucial roles of the short-lived, continuously replenished neutrophils in dust-induced pathogenesis are presented.

Macaque retinal ganglion cell responses to visual patterns: harmonic composition, noise, and psychophysical detectability.

The goal of these experiments was to test how well cell responses to visual patterns can be predicted from the sinewave tuning curve. Magnocellular (MC) and parvocellular (PC) ganglion cell responses to different spatial waveforms (sinewave, squarewave, and ramp waveforms) were measured across a range of spatial frequencies. Sinewave spatial tuning curves were fit with standard Gaussian models. From these fits, waveforms and spatial tuning of a cell's responses to the other waveforms were predicted for different harmonics by scaling in amplitude for the power in the waveform's Fourier expansion series over spatial frequency. Since higher spatial harmonics move at a higher temporal frequency, an additional scaling for each harmonic by the MC (bandpass) or PC (lowpass) temporal response was included, together with response phase. Finally, the model included a rectifying nonlinearity. This provided a largely satisfactory estimation of MC and PC cell responses to complex waveforms. As a consequence of their transient responses, MC responses to complex waveforms were found to have significantly more energy in higher spatial harmonic components than PC responses. Response variance (noise) was also quantified as a function of harmonic component. Noise increased to some degree for the higher harmonics. The data are relevant for psychophysical detection or discrimination of visual patterns, and we discuss the results in this context.

Independence and interaction of luminance and chromatic contributions to spatial hyperacuity performance.

Here we test interactions of luminance and chromatic input to spatial hyperacuity mechanisms. First, we tested alignment of luminance and chromatic gratings matched or mismatched in contrast polarity or grating type. Thresholds with matched gratings were low while all mismatched pairs were elevated. Second, we determined alignment acuity as a function of luminance or chromatic contrast alone or in the presence of constant contrast components of the other type. For in-phase components, performance followed the envelope of the more sensitive mechanism. However, polarity reversals revealed an asymmetric effect for luminance and chromatic conditions, which suggested that luminance can override chromatic mechanisms in hyperacuity; we interpret these findings in the context of spatial mechanisms.

A cellular model for sporadic ALS using patient-derived induced pluripotent stem cells.

Development of therapeutics for genetically complex neurodegenerative diseases such as sporadic amyotrophic lateral sclerosis (ALS) has largely been hampered by lack of relevant disease models. Reprogramming of sporadic ALS patients' fibroblasts into induced pluripotent stem cells (iPSC) and differentiation into affected neurons that show a disease phenotype could provide a cellular model for disease mechanism studies and drug discovery. Here we report the reprogramming to pluripotency of fibroblasts from a large cohort of healthy controls and ALS patients and their differentiation into motor neurons. We demonstrate that motor neurons derived from three sALS patients show de novo TDP-43 aggregation and that the aggregates recapitulate pathology in postmortem tissue from one of the same patients from which the iPSC were derived. We configured a high-content chemical screen using the TDP-43 aggregate endpoint both in lower motor neurons and upper motor neuron like cells and identified FDA-approved small molecule modulators including Digoxin demonstrating the feasibility of patient-derived iPSC-based disease modeling for drug screening.

Toxicity of lunar dust assessed in inhalation-exposed rats.

Humans will again set foot on the moon. The moon is covered by a layer of fine dust, which can pose a respiratory hazard. We investigated the pulmonary toxicity of lunar dust in rats exposed to 0, 2.1, 6.8, 20.8 and 60.6 mg/m(3) of respirable-size lunar dust for 4 weeks (6 h/day, 5 days/week); the aerosols in the nose-only exposure chambers were generated from a jet-mill ground preparation of a lunar soil collected during the Apollo 14 mission. After 4 weeks of exposure to air or lunar dust, groups of five rats were euthanized 1 day, 1 week, 4 weeks or 13 weeks after the last exposure for assessment of pulmonary toxicity. Biomarkers of toxicity assessed in bronchoalveolar fluids showed concentration-dependent changes; biomarkers that showed treatment effects were total cell and neutrophil counts, total protein concentrations and cellular enzymes (lactate dehydrogenase, glutamyl transferase and aspartate transaminase). No statistically significant differences in these biomarkers were detected between rats exposed to air and those exposed to the two low concentrations of lunar dust. Dose-dependent histopathology, including inflammation, septal thickening, fibrosis and granulomas, in the lung was observed at the two higher exposure concentrations. No lesions were detected in rats exposed to ≤6.8 mg/m(3). This 4-week exposure study in rats showed that 6.8 mg/m(3) was the highest no-observable-adverse-effect level (NOAEL). These results will be useful for assessing the health risk to humans of exposure to lunar dust, establishing human exposure limits and guiding the design of dust mitigation systems in lunar landers or habitats.

The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors.

Pancreas development begins with the formation of buds at specific sites in the embryonic foregut endoderm. We used recombination-based lineage tracing in vivo to show that Ptf1a (also known as PTF1-p48) is expressed at these early stages in the progenitors of pancreatic ducts, exocrine and endocrine cells, rather than being an exocrine-specific gene as previously described. Moreover, inactivation of Ptf1a switches the character of pancreatic progenitors such that their progeny proliferate in and adopt the normal fates of duodenal epithelium, including its stem-cell compartment. Consistent with the proposal that Ptf1a supports the specification of precursors of all three pancreatic cell types, transgene-based expression of Pdx1, a gene essential to pancreas formation, from Ptf1a cis-regulatory sequences restores pancreas tissue to Pdx1-null mice that otherwise lack mature exocrine and endocrine cells because of an early arrest in organogenesis. These experiments provide evidence that Ptf1a expression is specifically connected to the acquisition of pancreatic fate by undifferentiated foregut endoderm.

Psychophysical and physiological responses to gratings with luminance and chromatic components of different spatial frequencies.

Gratings that contain luminance and chromatic components of different spatial frequencies were used to study the segregation of signals in luminance and chromatic pathways. Psychophysical detection and discrimination thresholds to these compound gratings, with luminance and chromatic components of the one either half or double the spatial frequency of the other, were measured in human observers. Spatial frequency tuning curves for detection of compound gratings followed the envelope of those for luminance and chromatic gratings. Different grating types were discriminable at detection threshold. Fourier analysis of physiological responses of macaque retinal ganglion cells to compound waveforms showed chromatic information to be restricted to the parvocellular pathway and luminance information to the magnocellular pathway. Taken together, the human psychophysical and macaque physiological data support the strict segregation of luminance and chromatic information in independent channels, with the magnocellular and parvocellular pathways, respectively, serving as likely the physiological substrates.

Ocular toxicity of authentic lunar dust.

BACKGROUND: Dust exposure is a well-known occupational hazard for terrestrial workers and astronauts alike and will continue to be a concern as humankind pursues exploration and habitation of objects beyond Earth. Humankind's limited exploration experience with the Apollo Program indicates that exposure to dust will be unavoidable. Therefore, NASA must assess potential toxicity and recommend appropriate mitigation measures to ensure that explorers are adequately protected. Visual acuity is critical during exploration activities and operations aboard spacecraft. Therefore, the present research was performed to ascertain the ocular toxicity of authentic lunar dust. METHODS: Small (mean particle diameter = 2.9 ± 1.0 µm), reactive lunar dust particles were produced by grinding bulk dust under ultrapure nitrogen conditions. Chemical reactivity and cytotoxicity testing were performed using the commercially available EpiOcularTM assay. Subsequent in vivo Draize testing utilized a larger size fraction of unground lunar dust that is more relevant to ocular exposures (particles <120 µm; median particle diameter = 50.9 ± 19.8 µm). RESULTS: In vitro testing indicated minimal irritancy potential based on the time required to reduce cell viability by 50% (ET50). Follow-up testing using the Draize standard protocol confirmed that the lunar dust was minimally irritating. Minor irritation of the upper eyelids was noted at the 1-hour observation point, but these effects resolved within 24 hours. In addition, no corneal scratching was observed using fluorescein stain. CONCLUSIONS: Low-titanium mare lunar dust is minimally irritating to the eyes and is considered a nuisance dust for ocular exposure. No special precautions are recommended to protect against ocular exposures, but fully shielded goggles may be used if dust becomes a nuisance.

Role of the Superior Colliculus in Guiding Movements Not Made by the Eyes.

The superior colliculus (SC) has long been associated with the neural control of eye movements. Over seventy years ago, the orderly topography of saccade vectors and corresponding visual field locations were discovered in the cat SC. Since then, numerous high-impact studies have investigated and manipulated the relationship between visuotopic space and saccade vector across this topography to better understand the physiological underpinnings of the sensorimotor signal transformation. However, less attention has been paid to the other motor responses that may be associated with SC activity, ranging in complexity from concerted movements of skeletomotor muscle groups, such as arm-reaching movements, to behaviors that involve whole-body movement sequences, such as fight-or-flight responses in murine models. This review surveys these more complex movements associated with SC (optic tectum in nonmammalian species) activity and, where possible, provides phylogenetic and ethological perspective.

The spatial structure of cone-opponent receptive fields in macaque retina.

The receptive field structure of long (L) to middle (M) wavelength (L/M) cone-opponent ganglion cells of the parafoveal macaque retina was investigated using drifting gratings. Gratings were luminance, chromatic or selective for the L- or M-cones. Based on these spatial tuning curves, receptive field profiles for the individual cones were derived. Receptive field profiles were coarse compared to single cones, and often could not be described by a simple Gaussian, having shallower flanks. There was a continuum of spatial properties, which blurred any systematic distinction between Type I and Type II receptive fields. Opponent center-surround organization within a single cone was rare. Usually, responses to all four grating types could be described based on the cone receptive field profiles. An exception was a few cells that showed irregularities of amplitude and phase at high spatial frequencies for one or other of the cone isolating conditions. The data are related to standard models of M/L opponent receptive fields and implications for central processing are considered.

Robotic cloning and Protein Production Platform of the Northeast Structural Genomics Consortium.

In this chapter we describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples using Escherichia coli host vectors. The platform is centered on 6X-His affinity-tagged protein constructs, allowing for a similar purification procedure for most targets, and the implementation of high-throughput parallel methods. In most cases, these affinity-purified proteins are sufficiently homogeneous that a single subsequent gel filtration chromatography step is adequate to produce protein preparations that are greater than 98% pure. Using this platform, over 1000 different proteins have been cloned, expressed, and purified in tens of milligram quantities over the last 36-month period (see Summary Statistics for All Targets, ). Our experience using a hierarchical multiplex expression and purification strategy, also described in this chapter, has allowed us to achieve success in producing not only protein samples but also many three-dimensional structures. As of December 2004, the NESG Consortium has deposited over 145 new protein structures to the Protein Data Bank (PDB); about two-thirds of these protein samples were produced by the NESG Protein Production Facility described here. The methods described here have proven effective in producing quality samples of both eukaryotic and prokaryotic proteins. These improved robotic and?or parallel cloning, expression, protein production, and biophysical screening technologies will be of broad value to the structural biology, functional proteomics, and structural genomics communities.

Human secreted tau increases amyloid-beta production.

The interaction of amyloid-beta (Aß) and tau in the pathogenesis of Alzheimer's disease is a subject of intense inquiry, with the bulk of evidence indicating that changes in tau are downstream of Aß. It has been shown however, that human tau overexpression in amyloid precursor protein transgenic mice increases Aß plaque deposition. Here, we confirm that human tau increases Aß levels. To determine if the observed changes in Aß levels were because of intracellular or extracellular secreted tau (eTau for extracellular tau), we affinity purified secreted tau from Alzheimer's disease patient-derived cortical neuron conditioned media and analyzed it by liquid chromatography-mass spectrometry. We found the extracellular species to be composed predominantly of a series of N-terminal fragments of tau, with no evidence of C-terminal tau fragments. We characterized a subset of high affinity tau antibodies, each capable of engaging and neutralizing eTau. We found that neutralizing eTau reduces Aß levels in vitro in primary human cortical neurons where exogenously adding eTau increases Aß levels. In vivo, neutralizing human tau in 2 human tau transgenic models also reduced Aß levels. We show that the human tau insert sequence is sufficient to cause the observed increase in Aß levels. Our data furthermore suggest that neuronal hyperactivity may be the mechanism by which this regulation occurs. We show that neuronal hyperactivity regulates both eTau secretion and Aß production. Electrophysiological analysis shows for the first time that secreted eTau causes neuronal hyperactivity. Its induction of hyperactivity may be the mechanism by which eTau regulates Aß production. Together with previous findings, these data posit a novel connection between tau and Aß, suggesting a dynamic mechanism of positive feed forward regulation. Aß drives the disease pathway through tau, with eTau further increasing Aß levels, perpetuating a destructive cycle.

Luminance and chromatic contributions to a hyperacuity task: isolation by contrast polarity and target separation.

Vernier thresholds are known to be elevated when a target pair has opposite contrast polarity. Polarity reversal is used to assess the role of luminance and chromatic pathways in hyperacuity performance. Psychophysical hyperacuity thresholds were measured for pairs of gratings of various combinations of luminance (Lum) and chromatic (Chr) contrast polarities, at different ratios of luminance to chromatic contrast. With two red-green gratings of matched luminance and chromatic polarity (+Lum+Chr), there was an elevation of threshold at isoluminance. When both luminance and chromatic polarity were mismatched (-Lum-Chr), thresholds were substantially elevated under all conditions. With the same luminance contrast polarity and opposite chromatic polarity (+Lum-Chr) thresholds were only elevated close to isoluminance; in the reverse condition (-Lum+Chr), thresholds were elevated as in the -Lum-Chr condition except close to equiluminance. Similar data were obtained for gratings isolating the short-wavelength cone mechanism. Further psychophysical measurements assessed the role of target separation with matched or mismatched contrast polarity; similar results were found for luminance and chromatic gratings. Comparison physiological data were collected from parafoveal ganglion cells of the macaque retina. Positional precision of ganglion cell signals was assessed under conditions related to the psychophysical measurements. On the basis of these combined observations, it is argued that both magnocellular, parvocellular, and koniocellular pathways have access to cortical positional mechanisms associated with vernier acuity.

The 2.35 A structure of the TenA homolog from Pyrococcus furiosus supports an enzymatic function in thiamine metabolism.

TenA (transcriptional enhancer A) has been proposed to function as a transcriptional regulator based on observed changes in gene-expression patterns when overexpressed in Bacillus subtilis. However, studies of the distribution of proteins involved in thiamine biosynthesis in different fully sequenced genomes have suggested that TenA may be an enzyme involved in thiamine biosynthesis, with a function related to that of the ThiC protein. The crystal structure of PF1337, the TenA homolog from Pyrococcus furiosus, is presented here. The protomer comprises a bundle of alpha-helices with a similar tertiary structure and topology to that of human heme oxygenase-1, even though there is no significant sequence homology. A solvent-sequestered cavity lined by phylogenetically conserved residues is found at the core of this bundle in PF1337 and this cavity is observed to contain electron density for 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate, the product of the ThiC enzyme. In contrast, the modestly acidic surface of PF1337 shows minimal levels of sequence conservation and a dearth of the basic residues that are typically involved in DNA binding in transcription factors. Without significant conservation of its surface properties, TenA is unlikely to mediate functionally important protein-protein or protein-DNA interactions. Therefore, the crystal structure of PF1337 supports the hypothesis that TenA homologs have an indirect effect in altering gene-expression patterns and function instead as enzymes involved in thiamine metabolism.