A CAG repeat threshold for therapeutics targeting somatic instability in Huntington's disease.

The Huntington's disease mutation is a CAG repeat expansion in the huntingtin gene that results in an expanded polyglutamine tract in the huntingtin protein. The CAG repeat is unstable and expansions of hundreds of CAGs have been detected in Huntington's disease post-mortem brains. The age of disease onset can be predicted partially from the length of the CAG repeat as measured in blood. Onset age is also determined by genetic modifiers, which in six cases involve variation in DNA mismatch repair pathways genes. Knocking-out specific mismatch repair genes in mouse models of Huntington's disease prevents somatic CAG repeat expansion. Taken together, these results have led to the hypothesis that somatic CAG repeat expansion in Huntington's disease brains is required for pathogenesis. Therefore, the pathogenic repeat threshold in brain is longer than (CAG)40, as measured in blood, and is currently unknown. The mismatch repair gene MSH3 has become a major focus for therapeutic development, as unlike other mismatch repair genes, nullizygosity for MSH3 does not cause malignancies associated with mismatch repair deficiency. Potential treatments targeting MSH3 currently under development include gene therapy, biologics and small molecules, which will be assessed for efficacy in mouse models of Huntington's disease. The zQ175 knock-in model carries a mutation of approximately (CAG)185 and develops early molecular and pathological phenotypes that have been extensively characterized. Therefore, we crossed the mutant huntingtin allele onto heterozygous and homozygous Msh3 knockout backgrounds to determine the maximum benefit of targeting Msh3 in this model. Ablation of Msh3 prevented somatic expansion throughout the brain and periphery, and reduction of Msh3 by 50% decreased the rate of expansion. This had no effect on the deposition of huntingtin aggregation in the nuclei of striatal neurons, nor on the dysregulated striatal transcriptional profile. This contrasts with ablating Msh3 in knock-in models with shorter CAG repeat expansions. Therefore, further expansion of a (CAG)185 repeat in striatal neurons does not accelerate the onset of molecular and neuropathological phenotypes. It is striking that highly expanded CAG repeats of a similar size in humans cause disease onset before 2 years of age, indicating that somatic CAG repeat expansion in the brain is not required for pathogenesis. Given that the trajectory for somatic CAG expansion in the brains of Huntington's disease mutation carriers is unknown, our study underlines the importance of administering treatments targeting somatic instability as early as possible.

Alternative processing of human HTT mRNA with implications for Huntington's disease therapeutics.

Huntington disease is caused by a CAG repeat expansion in exon 1 of the huntingtin gene (HTT) that is translated into a polyglutamine stretch in the huntingtin protein (HTT). We previously showed that HTT mRNA carrying an expanded CAG repeat was incompletely spliced to generate HTT1a, an exon 1 only transcript, which was translated to produce the highly aggregation-prone and pathogenic exon 1 HTT protein. This occurred in all knock-in mouse models of Huntington's disease and could be detected in patient cell lines and post-mortem brains. To extend these findings to a model system expressing human HTT, we took advantage of YAC128 mice that are transgenic for a yeast artificial chromosome carrying human HTT with an expanded CAG repeat. We discovered that the HTT1a transcript could be detected throughout the brains of YAC128 mice. We implemented RNAscope to visualize HTT transcripts at the single molecule level and found that full-length HTT and HTT1a were retained together in large nuclear RNA clusters, as well as being present as single transcripts in the cytoplasm. Homogeneous time-resolved fluorescence analysis demonstrated that the HTT1a transcript had been translated to produce the exon 1 HTT protein. The levels of exon 1 HTT in YAC128 mice, correlated with HTT aggregation, supportive of the hypothesis that exon 1 HTT initiates the aggregation process. Huntingtin-lowering strategies are a major focus of therapeutic development for Huntington's disease. These approaches often target full-length HTT alone and would not be expected to reduce pathogenic exon 1 HTT levels. We have established YAC128 mouse embryonic fibroblast lines and shown that, together with our QuantiGene multiplex assay, these provide an effective screening tool for agents that target HTT transcripts. The effects of current targeting strategies on nuclear RNA clusters are unknown, structures that may have a pathogenic role or alternatively could be protective by retaining HTT1a in the nucleus and preventing it from being translated. In light of recently halted antisense oligonucleotide trials, it is vital that agents targeting HTT1a are developed, and that the effects of HTT-lowering strategies on the subcellular levels of all HTT transcripts and their various HTT protein isoforms are understood.

Expression of mutant exon 1 huntingtin fragments in human neural stem cells and neurons causes inclusion formation and mitochondrial dysfunction.

Robust cellular models are key in determining pathological mechanisms that lead to neurotoxicity in Huntington's disease (HD) and for high throughput pre-clinical screening of potential therapeutic compounds. Such models exist but mostly comprise non-human or non-neuronal cells that may not recapitulate the correct biochemical milieu involved in pathology. We have developed a new human neuronal cell model of HD, using neural stem cells (ReNcell VM NSCs) stably transduced to express exon 1 huntingtin (HTT) fragments with variable length polyglutamine (polyQ) tracts. Using a system with matched expression levels of exon 1 HTT fragments, we investigated the effect of increasing polyQ repeat length on HTT inclusion formation, location, neuronal survival, and mitochondrial function with a view to creating an in vitro screening platform for therapeutic screening. We found that expression of exon 1 HTT fragments with longer polyQ tracts led to the formation of intra-nuclear inclusions in a polyQ length-dependent manner during neurogenesis. There was no overt effect on neuronal viability, but defects of mitochondrial function were found in the pathogenic lines. Thus, we have a human neuronal cell model of HD that may recapitulate some of the earliest stages of HD pathogenesis, namely inclusion formation and mitochondrial dysfunction.

Live-cell super-resolution microscopy reveals a primary role for diffusion in polyglutamine-driven aggresome assembly.

The mechanisms leading to self-assembly of misfolded proteins into amyloid aggregates have been studied extensively in the test tube under well-controlled conditions. However, to what extent these processes are representative of those in the cellular environment remains unclear. Using super-resolution imaging of live cells, we show here that an amyloidogenic polyglutamine-containing protein first forms small, amorphous aggregate clusters in the cytosol, chiefly by diffusion. Dynamic interactions among these clusters limited their elongation and led to structures with a branched morphology, differing from the predominantly linear fibrils observed in vitro Some of these clusters then assembled via active transport at the microtubule-organizing center and thereby initiated the formation of perinuclear aggresomes. Although it is widely believed that aggresome formation is entirely governed by active transport along microtubules, here we demonstrate, using a combined approach of advanced imaging and mathematical modeling, that diffusion is the principal mechanism driving aggresome expansion. We found that the increasing surface area of the expanding aggresome increases the rate of accretion caused by diffusion of cytosolic aggregates and that this pathway soon dominates aggresome assembly. Our findings lead to a different view of aggresome formation than that proposed previously. We also show that aggresomes mature over time, becoming more compacted as the structure grows. The presence of large perinuclear aggregates profoundly affects the behavior and health of the cell, and our super-resolution imaging results indicate that aggresome formation and development are governed by highly dynamic processes that could be important for the design of potential therapeutic strategies.

Nutritional Considerations for Bouldering.

Bouldering competitions are held up to International level and governed by the International Federation of Sport Climbing. Bouldering has been selected to feature at the 2020 Olympic Games in Tokyo, however, physiological qualities and nutritional requirements to optimize performance remain inadequately defined due to large gaps in the literature. The primary goals of training include optimizing the capacity of the anaerobic energy systems and developing sport-specific strength, with emphasis on the isometric function of the forearm flexors responsible for grip. Bouldering athletes typically possess a lean physique, similar to the characteristics of sport climbers with reported body fat values of 6-12%. Athletes strive for a low body weight to improve power to weight ratio and limit the load on the extremities. Specialized nutritional support is uncommon and poor nutritional practices such as chronic carbohydrate restriction are prevalent, compromising the health of the athletes. The high intensity nature of bouldering demands a focus on adequate carbohydrate availability. Protein intake and timing should be structured to maximize muscle protein synthesis and recovery, with the literature suggesting 0.25-0.3 g/kg in 3-4 hr intervals. Supplementing with creatine and b-alanine may provide some benefit by augmenting the capacity of the anaerobic systems. Boulderers are encouraged to seek advice from nutrition experts to enhance performance, particularly important when weight loss is the desired outcome. Further research is warranted across all nutritional aspects of bouldering which is summarized in this review.

Screening for genes involved in antibody response to sheep red blood cells in the chicken, Gallus gallus.

Antibody response, an important trait in both agriculture and biomedicine, plays a part in protecting animals from infection. Dissecting molecular basis of antibody response may improve artificial selection for natural disease resistance in livestock and poultry. A number of genetic markers associated with antibody response have been identified in the chicken and mouse by linkage-based association studies, which only define genomic regions by genetic markers but do not pinpoint genes for antibody response. In contrast, global expression profiling has been applied to define the molecular bases of a variety of biological traits through identification of differentially expressed genes (DEGs). Here, we employed Affimetrix GeneChip Chicken Genome Arrays to identify differentially expressed genes for antibody response to sheep red blood cells (SRBC) using chickens challenged with and without SRBC or chickens with high and low anti-SRBC titers. The DEGs include those with known (i.e., MHC class I and IgH genes) or unknown function in antibody response. Classification test of these genes suggested that the response of the chicken to intravenous injection of SRBC involved multiple biological processes, including response to stress or other different stimuli, sugar, carbohydrate or protein binding, and cell or soluble fraction, in addition to antibody response. This preliminary study thus provides an insight into molecular basis of antibody response to SRBC in the chicken.

A Multi-Institutional Description of Processes and Outcomes of Postbaccalaureate Research Education Programs in the Mid-Atlantic Region.

ABSTRACT: Outcome data from 6 National Institutes of Health-funded Postbaccalaureate Research Education Programs (PREPs) in the Mid-Atlantic region were combined to give a multi-institutional perspective on their scholars' characteristics and progress through biomedical research training. The institutions hosting these programs were Johns Hopkins University School of Medicine, the Medical University of South Carolina, the University of Maryland School of Medicine, the University of North Carolina at Chapel Hill, Virginia Commonwealth University, and Virginia Polytechnic Institute and State University. The authors summarize the institutional pathways, demographics, undergraduate institutions, and graduate institutions for a total of 384 PREP scholars who completed the programs by June 2021. A total of 228 (59.4%) of these PREP scholars identified as Black or African American, 116 (30.2%) as Hispanic or Latinx, and 269 (70.0%) as female. The authors found that 376 of 384 scholars (97.9%) who started PREP finished their program, 319 of 376 (84.8%) who finished PREP matriculated into PhD or MD/PhD programs, and 284 of 319 (89.0%) who matriculated have obtained their PhD or are successfully making progress toward their PhD.

Implantation site and lesion topology determine efficacy of a human neural stem cell line in a rat model of chronic stroke.

Stroke remains one of the most promising targets for cell therapy. Thorough preclinical efficacy testing of human neural stem cell (hNSC) lines in a rat model of stroke (transient middle cerebral artery occlusion) is, however, required for translation into a clinical setting. Magnetic resonance imaging (MRI) here confirmed stroke damage and allowed the targeted injection of 450,000 hNSCs (CTX0E03) into peri-infarct tissue, rather than the lesion cyst. Intraparenchymal cell implants improved sensorimotor dysfunctions (bilateral asymmetry test) and motor deficits (footfault test and rotameter). Importantly, analyses based on lesion topology (striatal vs. striatal + cortical damage) revealed a more significant improvement in animals with a stroke confined to the striatum. However, no improvement in learning and memory (water maze) was evident. An intracerebroventricular injection of cells did not result in any improvement. MRI-based lesion, striatal and cortical volumes were unchanged in treated animals compared to those with stroke that received an intraparenchymal injection of suspension vehicle. Grafted cells only survived after intraparenchymal injection with a striatal + cortical topology resulting in better graft survival (16,026 cells) than in animals with smaller striatal lesions (2,374 cells). Almost 20% of cells differentiated into glial fibrillary acidic protein+ astrocytes, but <2% turned into FOX3+ neurons. These results indicate that CTX0E03 implants robustly recover behavioral dysfunction over a 3-month time frame and that this effect is specific to their site of implantation. Lesion topology is potentially an important factor in the recovery, with a stroke confined to the striatum showing a better outcome compared to a larger area of damage.

Early detection of exon 1 huntingtin aggregation in zQ175 brains by molecular and histological approaches.

Huntingtin-lowering approaches that target huntingtin expression are a major focus for therapeutic intervention for Huntington's disease. When the cytosine, adenine and guanine repeat is expanded, the huntingtin pre-mRNA is alternatively processed to generate the full-length huntingtin and HTT1a transcripts. HTT1a encodes the aggregation-prone and highly pathogenic exon 1 huntingtin protein. In evaluating huntingtin-lowering approaches, understanding how the targeting strategy modulates levels of both transcripts and the huntingtin protein isoforms that they encode will be essential. Given the aggregation-propensity of exon 1 huntingtin, the impact of a given strategy on the levels and subcellular location of aggregated huntingtin will need to be determined. We have developed and applied sensitive molecular approaches to monitor the levels of aggregated and soluble huntingtin isoforms in tissue lysates. We have used these, in combination with immunohistochemistry, to map the appearance and accumulation of aggregated huntingtin throughout the CNS of zQ175 mice, a model of Huntington's disease frequently chosen for preclinical studies. Aggregation analyses were performed on tissues from zQ175 and wild-type mice at monthly intervals from 1 to 6 months of age. We developed three homogeneous time-resolved fluorescence assays to track the accumulation of aggregated huntingtin and showed that two of these were specific for the exon 1 huntingtin protein. Collectively, the homogeneous time-resolved fluorescence assays detected huntingtin aggregation in the 10 zQ175 CNS regions by 1-2 months of age. Immunohistochemistry with the polyclonal S830 anti-huntingtin antibody showed that nuclear huntingtin aggregation, in the form of a diffuse nuclear immunostain, could be visualized in the striatum, hippocampal CA1 region and layer IV of the somatosensory cortex by 2 months. That this diffuse nuclear immunostain represented aggregated huntingtin was confirmed by immunohistochemistry with a polyglutamine-specific antibody, which required formic acid antigen retrieval to expose its epitope. By 6 months of age, nuclear and cytoplasmic inclusions were widely distributed throughout the brain. Homogeneous time-resolved fluorescence analysis showed that the comparative levels of soluble exon 1 huntingtin between CNS regions correlated with those for huntingtin aggregation. We found that soluble exon 1 huntingtin levels decreased over the 6-month period, whilst those of soluble full-length mutant huntingtin remained unchanged, data that were confirmed for the cortex by immunoprecipitation and western blotting. These data support the hypothesis that exon 1 huntingtin initiates the aggregation process in knock-in mouse models and pave the way for a detailed analysis of huntingtin aggregation in response to huntingtin-lowering treatments.

Use of high-content imaging to quantify transduction of AAV-PHP viruses in the brain following systemic delivery.

The engineering of the AAV-PHP capsids was an important development for CNS research and the modulation of gene expression in the brain. They cross the blood brain barrier and transduce brain cells after intravenous systemic delivery, a property dependent on the genotype of Ly6a, the AAV-PHP capsid receptor. It is important to determine the transduction efficiency of a given viral preparation, as well as the comparative tropism for different brain cells; however, manual estimation of adeno-associated viral transduction efficiencies can be biased and time consuming. Therefore, we have used the Opera Phenix high-content screening system, equipped with the Harmony processing and analysis software, to reduce bias and develop an automated approach to determining transduction efficiency in the mouse brain. We used R Studio and 'gatepoints' to segment the data captured from coronal brain sections into brain regions of interest. C57BL/6J and CBA/Ca mice were injected with an AAV-PHP.B virus containing a green fluorescent protein reporter with a nuclear localization signal. Coronal sections at 600 µm intervals throughout the entire brain were stained with Hoechst dye, combined with immunofluorescence to NeuN and green fluorescent protein to identify all cell nuclei, neurons and transduced cells, respectively. Automated data analysis was applied to give an estimate of neuronal percentages and transduction efficiencies throughout the entire brain as well as for the cortex, striatum and hippocampus. The data from each coronal section from a given mouse were highly comparable. The percentage of neurons in the C57BL/6J and CBA/Ca brains was approximately 40% and this was higher in the cortex than striatum and hippocampus. The systemic injection of AAV-PHP.B resulted in similar transduction rates across the entire brain for C57BL/6J mice. Approximately 10-15% of all cells were transduced, with neuronal transduction efficiencies ranging from 5% to 15%, estimates that were similar across brain regions, and were in contrast to the much more localized transduction efficiencies achieved through intracerebral injection. We confirmed that the delivery of the AAV-PHP.B viruses to the brain from the vasculature resulted in widespread transduction. Our methodology allows the rapid comparison of transduction rates between brain regions producing comparable data to more time-consuming approaches. The methodology developed here can be applied to the automated quantification of any parameter of interest that can be captured as a fluorescent signal.

Correlative light and electron microscopy suggests that mutant huntingtin dysregulates the endolysosomal pathway in presymptomatic Huntington's disease.

Huntington's disease (HD) is a late onset, inherited neurodegenerative disorder for which early pathogenic events remain poorly understood. Here we show that mutant exon 1 HTT proteins are recruited to a subset of cytoplasmic aggregates in the cell bodies of neurons in brain sections from presymptomatic HD, but not wild-type, mice. This occurred in a disease stage and polyglutamine-length dependent manner. We successfully adapted a high-resolution correlative light and electron microscopy methodology, originally developed for mammalian and yeast cells, to allow us to correlate light microscopy and electron microscopy images on the same brain section within an accuracy of 100 nm. Using this approach, we identified these recruitment sites as single membrane bound, vesicle-rich endolysosomal organelles, specifically as (1) multivesicular bodies (MVBs), or amphisomes and (2) autolysosomes or residual bodies. The organelles were often found in close-proximity to phagophore-like structures. Immunogold labeling localized mutant HTT to non-fibrillar, electron lucent structures within the lumen of these organelles. In presymptomatic HD, the recruitment organelles were predominantly MVBs/amphisomes, whereas in late-stage HD, there were more autolysosomes or residual bodies. Electron tomograms indicated the fusion of small vesicles with the vacuole within the lumen, suggesting that MVBs develop into residual bodies. We found that markers of MVB-related exocytosis were depleted in presymptomatic mice and throughout the disease course. This suggests that endolysosomal homeostasis has moved away from exocytosis toward lysosome fusion and degradation, in response to the need to clear the chronically aggregating mutant HTT protein, and that this occurs at an early stage in HD pathogenesis.

Subcellular Localization And Formation Of Huntingtin Aggregates Correlates With Symptom Onset And Progression In A Huntington'S Disease Model.

Huntington's disease is caused by the expansion of a CAG repeat within exon 1 of the HTT gene, which is unstable, leading to further expansion, the extent of which is brain region and peripheral tissue specific. The identification of DNA repair genes as genetic modifiers of Huntington's disease, that were known to abrogate somatic instability in Huntington's disease mouse models, demonstrated that somatic CAG expansion is central to disease pathogenesis, and that the CAG repeat threshold for pathogenesis in specific brain cells might not be known. We have previously shown that the HTT gene is incompletely spliced generating a small transcript that encodes the highly pathogenic exon 1 HTT protein. The longer the CAG repeat, the more of this toxic fragment is generated, providing a pathogenic consequence for somatic expansion. Here, we have used the R6/2 mouse model to investigate the molecular and behavioural consequences of expressing exon 1 HTT with 90 CAGs, a mutation that causes juvenile Huntington's disease, compared to R6/2 mice carrying ∼200 CAGs, a repeat expansion of a size rarely found in Huntington's disease patient's blood, but which has been detected in post-mortem brains as a consequence of somatic CAG repeat expansion. We show that nuclear aggregation occurred earlier in R6/2(CAG)90 mice and that this correlated with the onset of transcriptional dysregulation. Whereas in R6/2(CAG)200 mice, cytoplasmic aggregates accumulated rapidly and closely tracked with the progression of behavioural phenotypes and with end-stage disease. We find that aggregate species formed in the R6/2(CAG)90 brains have different properties to those in the R6/2(CAG)200 mice. Within the nucleus, they retain a diffuse punctate appearance throughout the course of the disease, can be partially solubilized by detergents and have a greater seeding potential in young mice. In contrast, aggregates from R6/2(CAG)200 brains polymerize into larger structures that appear as inclusion bodies. These data emphasize that a subcellular analysis, using multiple complementary approaches, must be undertaken in order to draw any conclusions about the relationship between HTT aggregation and the onset and progression of disease phenotypes.

Correlations of Behavioral Deficits with Brain Pathology Assessed through Longitudinal MRI and Histopathology in the HdhQ150/Q150 Mouse Model of Huntington's Disease.

A variety of mouse models have been developed that express mutant huntingtin (mHTT) leading to aggregates and inclusions that model the molecular pathology observed in Huntington's disease. Here we show that although homozygous HdhQ150 knock-in mice developed motor impairments (rotarod, locomotor activity, grip strength) by 36 weeks of age, cognitive dysfunction (swimming T maze, fear conditioning, odor discrimination, social interaction) was not evident by 94 weeks. Concomitant to behavioral assessments, T2-weighted MRI volume measurements indicated a slower striatal growth with a significant difference between wild type (WT) and HdhQ150 mice being present even at 15 weeks. Indeed, MRI indicated significant volumetric changes prior to the emergence of the "clinical horizon" of motor impairments at 36 weeks of age. A striatal decrease of 27% was observed over 94 weeks with cortex (12%) and hippocampus (21%) also indicating significant atrophy. A hypothesis-free analysis using tensor-based morphometry highlighted further regions undergoing atrophy by contrasting brain growth and regional neurodegeneration. Histology revealed the widespread presence of mHTT aggregates and cellular inclusions. However, there was little evidence of correlations between these outcome measures, potentially indicating that other factors are important in the causal cascade linking the molecular pathology to the emergence of behavioral impairments. In conclusion, the HdhQ150 mouse model replicates many aspects of the human condition, including an extended pre-manifest period prior to the emergence of motor impairments.

Simulated Leaching (Migration) Study for a Model Container-Closure System Applicable to Parenteral and Ophthalmic Drug Products.

A simulating leaching (migration) study was performed on a model container-closure system relevant to parenteral and ophthalmic drug products. This container-closure system consisted of a linear low-density polyethylene bottle (primary container), a polypropylene cap and an elastomeric cap liner (closure), an adhesive label (labeling), and a foil overpouch (secondary container). The bottles were filled with simulating solvents (aqueous salt/acid mixture at pH 2.5, aqueous buffer at pH 9.5, and 1/1 v/v isopropanol/water), a label was affixed to the filled and capped bottles, the filled bottles were placed into the foil overpouch, and the filled and pouched units were stored either upright or inverted for up to 6 months at 40 °C. After storage, the leaching solutions were tested for leached substances using multiple complementary analytical techniques to address volatile, semi-volatile, and non-volatile organic and inorganic extractables as potential leachables.The leaching data generated supported several conclusions, including that (1) the extractables (leachables) profile revealed by a simulating leaching study can qualitatively be correlated with compositional information for materials of construction, (2) the chemical nature of both the extracting medium and the individual extractables (leachables) can markedly affect the resulting profile, and (3) while direct contact between a drug product and a system's material of construction may exacerbate the leaching of substances from that material by the drug product, direct contact is not a prerequisite for migration and leaching to occur.LAY ABSTRACT: The migration of container-related extractables from a model pharmaceutical container-closure system and into simulated drug product solutions was studied, focusing on circumstances relevant to parenteral and ophthalmic drug products. The model system was constructed specifically to address the migration of extractables from labels applied to the outside of the primary container. The study demonstrated that (1) the extractables that do migrate can be correlated to the composition of the materials used to construct the container-closure systems, (2) the extent of migration is affected by the chemical nature of the simulating solutions and the extractables themselves, and (3) even though labels may not be in direct contact with a contained solution, label-related extractables can accumulate as leachables in those solutions.

Myostatin inhibition prevents skeletal muscle pathophysiology in Huntington's disease mice.

Huntington's disease (HD) is an inherited neurodegenerative disorder of which skeletal muscle atrophy is a common feature, and multiple lines of evidence support a muscle-based pathophysiology in HD mouse models. Inhibition of myostatin signaling increases muscle mass, and therapeutic approaches based on this are in clinical development. We have used a soluble ActRIIB decoy receptor (ACVR2B/Fc) to test the effects of myostatin/activin A inhibition in the R6/2 mouse model of HD. Weekly administration from 5 to 11 weeks of age prevented body weight loss, skeletal muscle atrophy, muscle weakness, contractile abnormalities, the loss of functional motor units in EDL muscles and delayed end-stage disease. Inhibition of myostatin/activin A signaling activated transcriptional profiles to increase muscle mass in wild type and R6/2 mice but did little to modulate the extensive Huntington's disease-associated transcriptional dysregulation, consistent with treatment having little impact on HTT aggregation levels. Modalities that inhibit myostatin signaling are currently in clinical trials for a variety of indications, the outcomes of which will present the opportunity to assess the potential benefits of targeting this pathway in HD patients.

Uptake of veterinary medicines from soils into plants.

Medicines play an important role in the treatment and prevention of disease. Whereas the side effects on human and animal health resulting directly from treatment have been widely documented, only recently have the occurrence and fate of medicines in the environment and the potential consequences for human health been recognized as an issue warranting consideration. Medicines have been shown to be released to soils and to persist in the environment. This study was performed to investigate the potential for a range of veterinary medicines to be taken up from soil by plants used for human consumption and to assess the potential significance of this exposure route in terms of human health. Soil analyses indicated that, for selected substances, measurable residues of these are likely to occur in soils for at least 5 months following application of manure containing these compounds. Experimental studies on the uptake of veterinary medicines into carrot roots (tubers) and lettuce leaves showed that only florfenicol, levamisole, and trimethoprim were taken up by lettuces, whereas diazinon, enrofloxacin, florfenicol, and trimethoprim were detected in carrot roots. Measured concentrations in plant material were used to model potential adult human exposure to these compounds. Although exposure concentrations were appreciable in a few instances, accounting for approximately 10% of the acceptable daily intake values (ADI), all were lower than the ADI values, indicating that, at least for compounds with properties similar to those considered here, there is little evidence of an appreciable risk. This exposure route may, however, be important when veterinary medicines have a very low ADI, at which they elicit subtle effects over prolonged periods, or when exposure is occurring via a number of routes at once. Although degradation products (produced in the soil or the plant) were not measured, it is possible for some substances that these could increase the risks to consumers.

A web-based genetic polymorphism learning approach for high school students and science teachers*.

Variation and polymorphism are concepts that are central to genetics and genomics, primary biological disciplines in which high school students and undergraduates require a solid foundation. From 1998 through 2002, a web-based genetics education program was developed for high school teachers and students. The program included an exercise on using freely available bioinformatics tools on the Internet to detect single nucleotide polymorphisms in genomic DNA and gene-based sequences to evaluate variation or polymorphism. Similar tools were also used to show the functional effect, if any, of the single nucleotide polymorphisms. A total of 25 science teachers and 60 students from high schools in Alabama and Virginia participated in the program that ranged from 2 to 4 weeks. Seventy percent of the teachers have now developed a web-based module to teach at least two lessons involving DNA variation and how it influences other disciplines, including evolution. Among former high school students, five are in Ph.D. programs in genetics or related subjects, and 80% are in medical school or in college in a biology or pre-med major. The exercise is simple to implement, and the cost is relatively low, requiring only a computer with an Internet connection. It also provides a foundation for introducing students to the theory of evolution, a concept that remains controversial in high school science curricula. Similar programs, if properly implemented, may result in fostering more interest in the biological sciences among prospective college students and ensure a good foundation in the pipeline for career biologists and scientists.

The complete mitochondrial genome sequence of the budgerigar, Melopsittacus undulatus.

Here, we describe the budgie's mitochondrial genome sequence, a resource that can facilitate this parrot's use as a model organism as well as for determining its phylogenetic relatedness to other parrots/Psittaciformes. The estimated total length of the sequence was 18,193 bp. In addition to the to the 13 protein and tRNA and rRNA coding regions, the sequence also includes a duplicated hypervariable region, a feature unique to only a few birds. The two hypervariable regions shared a sequence identity of about 86%.

3D reconstruction of 2D fluorescence histology images and registration with in vivo MR images: application in a rodent stroke model.

To validate and add value to non-invasive imaging techniques, the corresponding histology is required to establish biological correlates. We present an efficient, semi-automated image-processing pipeline that uses immunohistochemically stained sections to reconstruct a 3D brain volume from 2D histological images before registering these with the corresponding 3D in vivo magnetic resonance images (MRI). A multistep registration procedure that first aligns the "global" volume by using the centre of mass and then applies a rigid and affine alignment based on signal intensities is described. This technique was applied to a training set of three rat brain volumes before being validated on three normal brains. Application of the approach to register "abnormal" images from a rat model of stroke allowed the neurobiological correlates of the variations in the hyper-intense MRI signal intensity caused by infarction to be investigated. For evaluation, the corresponding anatomical landmarks in MR and histology were defined to measure the registration accuracy. A registration error of 0.249 mm (approximately one in-plane voxel dimension) was evident in healthy rat brains and of 0.323 mm in a rodent model of stroke. The proposed reconstruction and registration pipeline allowed for the precise analysis of non-invasive MRI and corresponding microstructural histological features in 3D. We were thus able to interrogate histology to deduce the cause of MRI signal variations in the lesion cavity and the peri-infarct area.