Harnessing natural variation to identify cis regulators of sex-biased gene expression in a multi-strain mouse liver model.

Sex differences in gene expression are widespread in the liver, where many autosomal factors act in tandem with growth hormone signaling to regulate individual variability of sex differences in liver metabolism and disease. Here, we compare hepatic transcriptomic and epigenetic profiles of mouse strains C57BL/6J and CAST/EiJ, representing two subspecies separated by 0.5-1 million years of evolution, to elucidate the actions of genetic factors regulating liver sex differences. We identify 144 protein coding genes and 78 lncRNAs showing strain-conserved sex bias; many have gene ontologies relevant to liver function, are more highly liver-specific and show greater sex bias, and are more proximally regulated than genes whose sex bias is strain-dependent. The strain-conserved genes include key growth hormone-dependent transcriptional regulators of liver sex bias; however, three other transcription factors, Trim24, Tox, and Zfp809, lose their sex-biased expression in CAST/EiJ mouse liver. To elucidate the observed strain specificities in expression, we characterized the strain-dependence of sex-biased chromatin opening and enhancer marks at cis regulatory elements (CREs) within expression quantitative trait loci (eQTL) regulating liver sex-biased genes. Strikingly, 208 of 286 eQTLs with strain-specific, sex-differential effects on expression were associated with a complete gain, loss, or reversal of the sex differences in expression between strains. Moreover, 166 of the 286 eQTLs were linked to the strain-dependent gain or loss of localized sex-biased CREs. Remarkably, a subset of these CREs apparently lacked strain-specific genetic variants yet showed coordinated, strain-dependent sex-biased epigenetic regulation. Thus, we directly link hundreds of strain-specific genetic variants to the high variability in CRE activity and expression of sex-biased genes and uncover underlying genetically-determined epigenetic states controlling liver sex bias in genetically diverse mouse populations.

Sea anemone model has a single Toll-like receptor that can function in pathogen detection, NF-κB signal transduction, and development.

In organisms from insects to vertebrates, Toll-like receptors (TLRs) are primary pathogen detectors that activate downstream pathways, specifically those that direct expression of innate immune effector genes. TLRs also have roles in development in many species. The sea anemone Nematostella vectensis is a useful cnidarian model to study the origins of TLR signaling because its genome encodes a single TLR and homologs of many downstream signaling components, including the NF-κB pathway. We have characterized the single N. vectensis TLR (Nv-TLR) and demonstrated that it can activate canonical NF-κB signaling in human cells. Furthermore, we show that the intracellular Toll/IL-1 receptor (TIR) domain of Nv-TLR can interact with the human TLR adapter proteins MAL and MYD88. We demonstrate that the coral pathogen Vibrio coralliilyticus causes a rapidly lethal disease in N. vectensis and that heat-inactivated V. coralliilyticus and bacterial flagellin can activate a reconstituted Nv-TLR-to-NF-κB pathway in human cells. By immunostaining of anemones, we show that Nv-TLR is expressed in a subset of cnidocytes and that many of these Nv-TLR-expressing cells also express Nv-NF-κB. Additionally, the nematosome, which is a Nematostella-specific multicellular structure, expresses Nv-TLR and many innate immune pathway homologs and can engulf V. coralliilyticus Morpholino knockdown indicates that Nv-TLR also has an essential role during early embryonic development. Our characterization of this primitive TLR and identification of a bacterial pathogen for N. vectensis reveal ancient TLR functions and provide a model for studying the molecular basis of cnidarian disease and immunity.

Extensive changes in DNA methylation are associated with expression of mutant huntingtin.

The earliest stages of Huntington disease are marked by changes in gene expression that are caused in an indirect and poorly understood manner by polyglutamine expansions in the huntingtin (HTT) protein. To explore the hypothesis that DNA methylation may be altered in cells expressing mutated HTT, we use reduced representation bisulfite sequencing (RRBS) to map sites of DNA methylation in cells carrying either wild-type or mutant HTT. We find that a large fraction of the genes that change in expression in the presence of mutant huntingtin demonstrate significant changes in DNA methylation. Regions with low CpG content, which have previously been shown to undergo methylation changes in response to neuronal activity, are disproportionately affected. On the basis of the sequence of regions that change in methylation, we identify AP-1 and SOX2 as transcriptional regulators associated with DNA methylation changes, and we confirm these hypotheses using genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq). Our findings suggest new mechanisms for the effects of polyglutamine-expanded HTT. These results also raise important questions about the potential effects of changes in DNA methylation on neurogenesis and cognitive decline in patients with Huntington disease.

Country-Wide Ecological Health Assessment Methodology for Air Toxics: Bridging Gaps in Ecosystem Impact Understanding and Policy Foundations.

Amid the growing concerns about air toxics from pollution sources, much emphasis has been placed on their impacts on human health. However, there has been limited research conducted to assess the cumulative country-wide impact of air toxics on both terrestrial and aquatic ecosystems, as well as the complex interactions within food webs. Traditional approaches, including those of the United States Environmental Protection Agency (US EPA), lack versatility in addressing diverse emission sources and their distinct ecological repercussions. This study addresses these gaps by introducing the Ecological Health Assessment Methodology (EHAM), a novel approach that transcends traditional methods by enabling both comprehensive country-wide and detailed regional ecological risk assessments across terrestrial and aquatic ecosystems. EHAM also advances the field by developing new food-chain multipliers (magnification factors) for localized ecosystem food web models. Employing traditional ecological multimedia risk assessment of toxics' fate and transport techniques as its foundation, this study extends US EPA methodologies to a broader range of emission sources. The quantification of risk estimation employs the quotient method, which yields an ecological screening quotient (ESQ). Utilizing Kuwait as a case study for the application of this methodology, this study's findings for data from 2017 indicate a substantial ecological risk in Kuwait's coastal zone, with cumulative ESQ values reaching as high as 3.12 × 103 for carnivorous shorebirds, contrasted by negligible risks in the inland and production zones, where ESQ values for all groups are consistently below 1.0. By analyzing the toxicity reference value (TRV) against the expected daily exposure of receptors to air toxics, the proposed methodology provides valuable insights into the potential ecological risks and their subsequent impacts on ecological populations. The present contribution aims to deepen the understanding of the ecological health implications of air toxics and lay the foundation for informed, ecology-driven policymaking, underscoring the need for measures to mitigate these impacts.

Transcriptional characterization of iPSC-derived microglia as a model for therapeutic development in neurodegeneration.

Microglia are the resident immune cells in the brain that play a key role in driving neuroinflammation, a hallmark of neurodegenerative disorders. Inducible microglia-like cells have been developed as an in vitro platform for molecular and therapeutic hypothesis generation and testing. However, there has been no systematic assessment of similarity of these cells to primary human microglia along with their responsiveness to external cues expected of primary cells in the brain. In this study, we performed transcriptional characterization of commercially available human inducible pluripotent stem cell (iPSC)-derived microglia-like (iMGL) cells by bulk and single cell RNA sequencing to assess their similarity with primary human microglia. To evaluate their stimulation responsiveness, iMGL cells were treated with Liver X Receptor (LXR) pathway agonists and their transcriptional responses characterized by bulk and single cell RNA sequencing. Bulk transcriptome analyses demonstrate that iMGL cells have a similar overall expression profile to freshly isolated human primary microglia and express many key microglial transcription factors and functional and disease-associated genes. Notably, at the single-cell level, iMGL cells exhibit distinct transcriptional subpopulations, representing both homeostatic and activated states present in normal and diseased primary microglia. Treatment of iMGL cells with LXR pathway agonists induces robust transcriptional changes in lipid metabolism and cell cycle at the bulk level. At the single cell level, we observe heterogeneity in responses between cell subpopulations in homeostatic and activated states and deconvolute bulk expression changes into their corresponding single cell states. In summary, our results demonstrate that iMGL cells exhibit a complex transcriptional profile and responsiveness, reminiscent of in vivo microglia, and thus represent a promising model system for therapeutic development in neurodegeneration.

New sensors and techniques for the structural health monitoring of propulsion systems.

The ability to monitor the structural health of the rotating components, especially in the hot sections of turbine engines, is of major interest to aero community in improving engine safety and reliability. The use of instrumentation for these applications remains very challenging. It requires sensors and techniques that are highly accurate, are able to operate in a high temperature environment, and can detect minute changes and hidden flaws before catastrophic events occur. The National Aeronautics and Space Administration (NASA), through the Aviation Safety Program (AVSP), has taken a lead role in the development of new sensor technologies and techniques for the in situ structural health monitoring of gas turbine engines. This paper presents a summary of key results and findings obtained from three different structural health monitoring approaches that have been investigated. This includes evaluating the performance of a novel microwave blade tip clearance sensor; a vibration based crack detection technique using an externally mounted capacitive blade tip clearance sensor; and lastly the results of using data driven anomaly detection algorithms for detecting cracks in a rotating disk.

Isotocin regulates paternal care in a monogamous cichlid fish.

While the survival value of paternal care is well understood, little is known about its physiological basis. Here we investigate the neuroendocrine contributions to paternal care in the monogamous cichlid, Amatitlania nigrofasciata. We first explored the dynamic range of paternal care in three experimental groups: biparental males (control fathers housed with their mate), single fathers (mate removed), or lone males (mate and offspring removed). We found that control males gradually increase paternal care over time, whereas single fathers increased care immediately after mate removal. Males with offspring present had lower levels of circulating 11-ketotestosterone (11-KT) yet still maintained aggressive displays toward brood predators. To determine what brain regions may contribute to paternal care, we quantified induction of the immediate early gene c-Fos, and found that single fathers have more c-Fos induction in the forebrain area Vv (putative lateral septum homologue), but not in the central pallium (area Dc). While overall preoptic area c-Fos induction was similar between groups, we found that parvocellular preoptic isotocin (IST) neurons in single fathers showed increased c-Fos induction, suggesting IST may facilitate the increase of paternal care after mate removal. To functionally test the role of IST in regulating paternal care, we treated biparental males with an IST receptor antagonist, which blocked paternal care. Our results indicate that isotocin plays a significant role in promoting paternal care, and more broadly suggest that the convergent evolution of paternal care across vertebrates may have recruited similar neuroendocrine mechanisms.

ER-α agonist induces conversion of fibroblasts into myofibroblasts, while ER-ß agonist increases ECM production and wound tensile strength of healing skin wounds in ovariectomised rats.

Oestrogen deprivation is one of the major factors responsible for many age-related processes, including poor wound healing in women. Previously, it has been shown that oestrogens have a modulatory effect in different wound-healing models. Therefore, in this study, the effect of selective oestrogen receptor (ER) agonists (PPT - ER-α agonist, DPN - ER-ß agonist) on excisional and incisional wound-healing models was compared in ovariectomised rats in vivo as well as on human dermal fibroblasts (HDF) and human umbilical endothelial cells (HUVEC) in vitro. In the in vivo study, 4 months after either ovariectomy or sham ovariectomy, Sprague-Dawley rats were randomly divided into four groups and subjected to two incisional and excisional wounds: (i) control - sham operated, vehicle-treated; (ii) ovariectomised, vehicle-treated; (iii) ovariectomised, PPT treated; (iv) ovariectomised, DPN treated. In the in vitro study, HDFs and HUVECs were used. After treatment with ER agonists, cells were processed for immunocytochemistry and gelatin zymography. Our study shows that stimulation of ER-α leads to the differentiation of fibroblasts into myofibroblasts both in vivo and in vitro. On the other hand, the formation of extracellular matrix was more prominent, and wound tensile strength (TS) was increased when ER-ß was stimulated. In contrast, stimulation of ER-α led to a more prominent increase in the expression of MMP-2 and decrease in wound TS. New information is presented in this investigation concerning oestrogen replacement therapy (ERT) in different wound-healing models. This study demonstrates that the ERT should be both wound and receptor-type specific.

Molecular characterization and brain distribution of the progesterone receptor in whiptail lizards.

Progesterone and its nuclear receptor are critical in modulating reproductive physiology and behavior in female and male vertebrates. Whiptail lizards (genus Cnemidophorus) are an excellent model system in which to study the evolution of sexual behavior, as both the ancestral and descendent species exist. Male-typical sexual behavior is mediated by progesterone in both the ancestral species and the descendant all-female species, although the molecular characterization and distribution of the progesterone receptor protein throughout the reptilian brain is not well understood. To better understand the gene targets and ligand binding properties of the progesterone receptor in whiptails, we cloned the promoter and coding sequence of the progesterone receptor and analyzed the predicted protein structure. We next determined the distribution of the progesterone receptor protein and mRNA throughout the brain of Cnemidophorus inornatus and Cnemidophorus uniparens by immunohistochemistry and in situ hybridization. We found the progesterone receptor to be present in many brain regions known to regulate social behavior and processing of stimulus salience across many vertebrates, including the ventral tegmental area, amygdala, nucleus accumbens and several hypothalamic nuclei. Additionally, we quantified immunoreactive cells in the preoptic area and ventromedial hypothalamus in females of both species and males of the ancestral species. We found differences between both species and across ovarian states. Our results significantly extend our understanding of progesterone modulation in the reptilian brain and support the important role of the nuclear progesterone receptor in modulating sexual behavior in reptiles and across vertebrates.

ORAL RABIES VACCINATION STRATEGIES TOWARD RACCOON (PROCYON LOTOR) RABIES ELIMINATION ON SUBURBAN LONG ISLAND, NEW YORK, USA.

Approximately 1.86 million baits containing a vaccinia-rabies glycoprotein recombinant vaccine were distributed with helicopters, vehicles, and bait stations during 2006-10. A bait density of 250 baits/km2 effectively controlled rabies cases in enzootic and preepizootic areas. However, a cluster of 11 rabid raccoons at the eastern edge of infection resulted in the initiation of semiannual, high-density (500 baits/km2) vaccination campaigns in approximately 20% of the oral rabies vaccination zone during July and September (2007-09). Bait success (i.e., chewed sachets or removed baits) at bait stations was negatively associated with station distances from water. Conversely, bait success improved with increasing distances from roads. Bait stations deployed significantly more baits in developed open space when compared to low- and medium- to high-intensity developed areas. However, a difference was not detected between developed open space and forest habitats. Rabies was confined to 86 raccoons within 317 km2 (10%) of a 3,133 km2 suburban landscape, with a disproportionate number of rabid raccoons (n=74) in developed areas, when compared to 10 cases in forest-wetland habitats. Two rabid raccoons did not fall within either general land-use classification. Rabies advanced 15.1 km eastward at a rate of 6.4 km/yr during a 28-mo interval (2004-06).

VIRUS NEUTRALIZING ANTIBODY FOLLOWING ORAL RABIES VACCINATION OF RACCOONS (PROCYON LOTOR) ON SUBURBAN LONG ISLAND, NEW YORK, USA.

Vaccine-laden baits were distributed to interrupt and halt raccoon (Procyon lotor) rabies transmission in suburban Nassau and Suffolk counties on Long Island, New York, US. Fishmeal polymer baits containing the RABORAL V-RG® vaccine were deployed with helicopters, bait stations, and vehicles at a target density of 250 baits/km2 during annual September campaigns (2006-10). Semiannual campaigns (500 baits/km2) were also initiated in a portion of the treatment zone (2007-09) in response to a persistent focus of rabid raccoons. The last enzootic case was reported in January 2009. The final vaccination campaign was completed in 2010. The raccoon variant of rabies virus is no longer circulating in Nassau or Suffolk counties. Significantly greater probabilities of raccoon seroconversion were observed in helicopter-deployed bait zones. The lowest probabilities of seroconversion were identified in vehicle and bait station-deployment bait zones, with a marginal advantage associated with bait-station deployment. Seroconversion was negatively associated with developed, medium-intensity areas and increasing human population density. Significantly higher rabies virus neutralizing antibody endpoint titrations were detected in helicopter and bait station-deployment zones.

Characterization of the dopamine system in the brain of the túngara frog, Physalaemus pustulosus.

Dopamine is an evolutionarily ancient neurotransmitter that plays an essential role in mediating behavior. In vertebrates, dopamine is central to the mesolimbic reward system, a neural network concerned with the valuation of stimulus salience, and to the nigrostriatal motor system and hypothalamic nuclei involved in the regulation of locomotion and social behavior. In amphibians, dopaminergic neurons have been mapped out in several species, yet the distribution of dopaminoreceptive cells is unknown. The túngara frog, Physalaemus pustulosus, is an excellent model system for the study of neural mechanisms by which valuations of stimuli salience and social decisions are made, especially in the context of mate choice. In order to better understand where dopamine acts to regulate social decisions in this species, we have determined the distribution of putative dopaminergic cells (using tyrosine hydroxylase immunohistochemistry) and cells receptive to dopaminergic signaling (using DARPP-32 immunohistochemistry) throughout the brain of P. pustulosus. The distribution of dopaminergic cells was comparable to other anurans. DARPP-32 immunoreactivity was identified in key brain regions known to modulate social behavior in other vertebrates including the proposed anuran homologues of the mammalian amygdalar complex, nucleus accumbens, hippocampus, striatum, preoptic area, anterior hypothalamus, ventromedial hypothalamus, and ventral tegmental area/substantia nigra pars compacta. Due to its widespread distribution, DARPP-32 likely also plays many roles in non-limbic brain regions that mediate non-social information processing. These results significantly extend our understanding of the distribution of the dopaminergic system in the anuran brain and beyond.

Impact of 3D genome organization, guided by cohesin and CTCF looping, on sex-biased chromatin interactions and gene expression in mouse liver.

Several thousand sex-differential distal enhancers have been identified in mouse liver; however, their links to sex-biased genes and the impact of any sex-differences in nuclear organization and chromatin interactions are unknown. To address these issues, we first characterized 1847 mouse liver genomic regions showing significant sex differential occupancy by cohesin and CTCF, two key 3D nuclear organizing factors. These sex-differential binding sites were primarily distal to sex-biased genes but rarely generated sex-differential TAD (topologically associating domain) or intra-TAD loop anchors, and were sometimes found in TADs without sex-biased genes. A substantial subset of sex-biased cohesin-non-CTCF binding sites, but not sex-biased cohesin-and-CTCF binding sites, overlapped sex-biased enhancers. Cohesin depletion reduced the expression of male-biased genes with distal, but not proximal, sex-biased enhancers by >10-fold, implicating cohesin in long-range enhancer interactions regulating sex-biased genes. Using circularized chromosome conformation capture-based sequencing (4C-seq), we showed that sex differences in distal sex-biased enhancer-promoter interactions are common. Intra-TAD loops with sex-independent cohesin-and-CTCF anchors conferred sex specificity to chromatin interactions indirectly, by insulating sex-biased enhancer-promoter contacts and by bringing sex-biased genes into closer proximity to sex-biased enhancers. Furthermore, sex-differential chromatin interactions involving sex-biased gene promoters, enhancers, and lncRNAs were associated with sex-biased binding of cohesin and/or CTCF. These studies elucidate how 3D genome organization impacts sex-biased gene expression in a non-reproductive tissue through both direct and indirect effects of cohesin and CTCF looping on distal enhancer interactions with sex-differentially expressed genes.

Computational prediction of CTCF/cohesin-based intra-TAD loops that insulate chromatin contacts and gene expression in mouse liver.

CTCF and cohesin are key drivers of 3D-nuclear organization, anchoring the megabase-scale Topologically Associating Domains (TADs) that segment the genome. Here, we present and validate a computational method to predict cohesin-and-CTCF binding sites that form intra-TAD DNA loops. The intra-TAD loop anchors identified are structurally indistinguishable from TAD anchors regarding binding partners, sequence conservation, and resistance to cohesin knockdown; further, the intra-TAD loops retain key functional features of TADs, including chromatin contact insulation, blockage of repressive histone mark spread, and ubiquity across tissues. We propose that intra-TAD loops form by the same loop extrusion mechanism as the larger TAD loops, and that their shorter length enables finer regulatory control in restricting enhancer-promoter interactions, which enables selective, high-level expression of gene targets of super-enhancers and genes located within repressive nuclear compartments. These findings elucidate the role of intra-TAD cohesin-and-CTCF binding in nuclear organization associated with widespread insulation of distal enhancer activity.

CHORIORETINAL BIOPSY FOR THE DIAGNOSIS OF ENDOGENOUS ENDOPHTHALMITIS DUE TO ESCHERICHIA COLI.

PURPOSE: To describe the novel use of a chorioretinal biopsy technique to confirm the microbiological diagnosis of endogenous Escherichia coli (E. coli) endophthalmitis, when other investigations have been proven nondiagnostic. METHODS: Case report of an 82-year-old white man with endogenous endophthalmitis without a clearly identifiable source of infection. RESULTS: After systemic cultures and multiple aqueous and vitreous samples were unable to identify a causative organism, chorioretinal biopsy of a subretinal abscess was used to confirm the microbiological diagnosis. This ensured appropriate ophthalmic and systemic treatment of infection. CONCLUSION: Endogenous E. coli endophthalmitis is a rare and aggressive condition usually seen in patients with insulin-dependent diabetes with concurrent urinary tract infection. This case demonstrates chorioretinal biopsy to be a viable and effective method of establishing a firm microbiological diagnosis in cases of culture-negative endophthalmitis.

Hyper- and hypo- nutrition studies of the hepatic transcriptome and epigenome suggest that PPARα regulates anaerobic glycolysis.

Diet plays a crucial role in shaping human health and disease. Diets promoting obesity and insulin resistance can lead to severe metabolic diseases, while calorie-restricted (CR) diets can improve health and extend lifespan. In this work, we fed mice either a chow diet (CD), a 16 week high-fat diet (HFD), or a CR diet to compare and contrast the effects of these diets on mouse liver biology. We collected transcriptomic and epigenomic datasets from these mice using RNA-Seq and DNase-Seq. We found that both CR and HFD induce extensive transcriptional changes, in some cases altering the same genes in the same direction. We used our epigenomic data to infer transcriptional regulatory proteins bound near these genes that likely influence their expression levels. In particular, we found evidence for critical roles played by PPARα and RXRα. We used ChIP-Seq to profile the binding locations for these factors in HFD and CR livers. We found extensive binding of PPARα near genes involved in glycolysis/gluconeogenesis and uncovered a role for this factor in regulating anaerobic glycolysis. Overall, we generated extensive transcriptional and epigenomic datasets from livers of mice fed these diets and uncovered new functions and gene targets for PPARα.

Hepatic Dysfunction Caused by Consumption of a High-Fat Diet.

Obesity is a major human health crisis that promotes insulin resistance and, ultimately, type 2 diabetes. The molecular mechanisms that mediate this response occur across many highly complex biological regulatory levels that are incompletely understood. Here, we present a comprehensive molecular systems biology study of hepatic responses to high-fat feeding in mice. We interrogated diet-induced epigenomic, transcriptomic, proteomic, and metabolomic alterations using high-throughput omic methods and used a network modeling approach to integrate these diverse molecular signals. Our model indicated that disruption of hepatic architecture and enhanced hepatocyte apoptosis are among the numerous biological processes that contribute to early liver dysfunction and low-grade inflammation during the development of diet-induced metabolic syndrome. We validated these model findings with additional experiments on mouse liver sections. In total, we present an integrative systems biology study of diet-induced hepatic insulin resistance that uncovered molecular features promoting the development and maintenance of metabolic disease.

Brain-computer interfaces for 1-D and 2-D cursor control: designs using volitional control of the EEG spectrum or steady-state visual evoked potentials.

We have developed and tested two electroencephalogram (EEG)-based brain-computer interfaces (BCI) for users to control a cursor on a computer display. Our system uses an adaptive algorithm, based on kernel partial least squares classification (KPLS), to associate patterns in multichannel EEG frequency spectra with cursor controls. Our first BCI, Target Practice, is a system for one-dimensional device control, in which participants use biofeedback to learn voluntary control of their EEG spectra. Target Practice uses a KPLS classifier to map power spectra of 62-electrode EEG signals to rightward or leftward position of a moving cursor on a computer display. Three subjects learned to control motion of a cursor on a video display in multiple blocks of 60 trials over periods of up to six weeks. The best subject's average skill in correct selection of the cursor direction grew from 58% to 88% after 13 training sessions. Target Practice also implements online control of two artifact sources: 1) removal of ocular artifact by linear subtraction of wavelet-smoothed vertical and horizontal electrooculograms (EOG) signals, 2) control of muscle artifact by inhibition of BCI training during periods of relatively high power in the 40-64 Hz band. The second BCI, Think Pointer, is a system for two-dimensional cursor control. Steady-state visual evoked potentials (SSVEP) are triggered by four flickering checkerboard stimuli located in narrow strips at each edge of the display. The user attends to one of the four beacons to initiate motion in the desired direction. The SSVEP signals are recorded from 12 electrodes located over the occipital region. A KPLS classifier is individually calibrated to map multichannel frequency bands of the SSVEP signals to right-left or up-down motion of a cursor on a computer display. The display stops moving when the user attends to a central fixation point. As for Target Practice, Think Pointer also implements wavelet-based online removal of ocular artifact; however, in Think Pointer muscle artifact is controlled via adaptive normalization of the SSVEP. Training of the classifier requires about 3 min. We have tested our system in real-time operation in three human subjects. Across subjects and sessions, control accuracy ranged from 80% to 100% correct with lags of 1-5 s for movement initiation and turning. We have also developed a realistic demonstration of our system for control of a moving map display (http://ti.arc.nasa.gov/).