Steven Williams

Draft Genome of the Filarial Nematode Parasite Brugia malayi

Parasitic nematodes that cause elephantiasis and river blindness threaten hundreds of millions of people in the developing world. We have sequenced the ∼90 megabase (Mb) genome of the human filarial parasite Brugia malayi and predict ∼11,500 protein coding genes in 71 Mb of robustly assembled sequence. Comparative analysis with the free-living, model nematode Caenorhabditis elegans revealed that, despite these genes having maintained little conservation of local synteny during ∼350 million years of evolution, they largely remain in linkage on chromosomal units. More than 100 conserved operons were identified. Analysis of the predicted proteome provides evidence for adaptations of B. malayi to niches in its human and vector hosts and insights into the molecular basis of a mutualistic relationship with its Wolbachia endosymbiont. These findings offer a foundation for rational drug design.

Generic brain activation mapping in functional magnetic resonance imaging : A nonparametric approach

We report a novel method to identify brain regions generically activated by periodic experimental design in functional magnetic resonance imaging data. This involves: 1) registering each of N individual functional magnetic resonance imaging datasets in a standard space; 2) computing the median standardised power of response to the experimental design; 3) testing median standardised power at each voxel against its nonparametrically ascertained distribution under the null hypothesis of no experimental effect; and 4) constructing a generic brain activation map. The method is validated by analysis of 6 null images, acquired under conditions when the null hypothesis was known to be true; 8 images acquired during periodic auditory-verbal stimulation; and 6 images acquired during periodic performance of a covert verbal fluency task.

Methods for Diagnosis and Treatment of Stimulus-Correlated Motion in Generic Brain Activation Studies Using fMRI

Movement‐related effects in realigned fMRI timeseries can be corrected by regression on linear functions of estimated positional displacements of an individual subjects head during image acquisition. However, this entails biased (under)estimation of the experimental effect whenever subject motion is not independent of the experimental input function. Methods for diagnosing such stimulus‐correlated motion (SCM) are illustrated by application to fMRI data acquired from 5 schizophrenics and 5 normal controls during periodic performance of a verbal fluency task. The schizophrenic group data were more severely affected by SCM than the control group data. Analysis of covariance (ANCOVA) was used, with a voxelwise measure of SCM as a covariate, to estimate between‐group differences in power of periodic signal change while controlling for variability in SCM across groups. Failure to control for SCM in this way substantially exaggerated the number of voxels, apparently demonstrating a between‐group difference in task response. Hum. Brain Mapping 7:38–48, 1999.

Common and Distinct Neural Substrates for Pragmatic, Semantic, and Syntactic Processing of Spoken Sentences: An fMRI Study

Extracting meaning from speech requires the use of pragmatic, semantic, and syntactic information. A central question is: Does the processing of these different types of linguistic information have common or distinct neuroanatomical substrates? We addressed this issue using functional magnetic resonance imaging (fMRI) to measure neural activity when subjects listened to spoken normal sentences contrasted with sentences that had either (A) pragmatical, (B) semantic (selection restriction), or (C) syntactic (subcategorical) violations sentences. All three contrasts revealed robust activation of the left-inferior-temporal/fusiform gyrus. Activity in this area was also observed in a combined analysis of all three experiments, suggesting that it was modulated by all three types of linguistic violation. Planned statistical comparisons between the three experiments revealed (1) a greater difference between conditions in activation of the left-superior-temporal gyrus for the pragmatic experiment than the semantic/syntactic experiments; (2) a greater difference between conditions in activation of the right-superior and middletemporal gyrus in the semantic experiment than in the syntactic experiment; and (3) no regions activated to a greater degree in the syntactic experiment than in the semantic experiment. These data show that, while left- and right-superior-temporal regions may be differentially involved in processing pragmatic and lexico-semantic information within sentences, the left-inferior-temporal/fusiform gyrus is involved in processing all three types of linguistic information. We suggest that this region may play a key role in using pragmatic, semantic (selection restriction), and subcategorical information to construct a higher representation of meaning of sentences.

Frontotemporal white matter changes in amyotrophic lateral sclerosis

AbstractCognitive dysfunction can occur in some patients with amyotrophic lateral sclerosis (ALS) who are not suffering from dementia. The most striking and consistent cognitive deficit has been found using tests of verbal fluency. ALS patients with verbal fluency deficits have shown functional imaging abnormalities predominantly in frontotemporal regions using positron emission tomography (PET). This study used automated volumetric voxel-based analysis of grey and white matter densities of structural magnetic resonance imaging (MRI) scans to explore the underlying pattern of structural cerebral change in nondemented ALS patients with verbal fluency deficits. Two groups of ALS patients, defined by the presence or absence of cognitive impairment on the basis of the Written Verbal Fluency Test (ALSi, cognitively impaired, n = 11; ALSu, cognitively unimpaired n = 12) were compared with healthy age matched controls (n = 12). A comparison of the ALSi group with controls revealed significantly (p < 0.002) reduced white matter volume in extensive motor and non–motor regions, including regions corresponding to frontotemporal association fibres. These patients demonstrated a corresponding cognitive profile of executive and memory dysfunction. Less extensive white matter reductions were revealed in the comparison of the ALSu and control groups in regions corresponding to frontal association fibres. White matter volumes were also found to correlate with performance on memory tests. There were no significant reductions in grey matter volume in the comparison of either patient group with controls. The structural white matter abnormalities in frontal and temporal regions revealed here may underlie the cognitive and functional imaging abnormalities previously reported in non–demented ALS patients. The results also suggest that extra–motor structural abnormalities may be present in ALS patients with no evidence of cognitive change. The findings support the hypothesis of a continuum of extra–motor cerebral and cognitive change in this disorder.

Motor response suppression and the prepotent tendency to respond : a parametric fMRI study

In the present study we utilised functional magnetic resonance imaging (fMRI) to examine cerebral activation during performance of a classic motor task in which response suppression load was parametrically varied. Linear increases in activity were observed in a distributed network of regions across both cerebral hemispheres, although with more extensive involvement of the right prefrontal cortex. Activated regions included prefrontal, parietal and occipitotemporal cortices. Decreasing activation was similarly observed in a distributed network of regions. These response forms are discussed in terms of an increasing requirement for visual cue discrimination and suppression/selection of motor responses, and a decreasing probability of the occurrence of non-target stimuli and attenuation of a prepotent tendency to respond. The results support recent proposals for a dominant role for the right-hemisphere in performance of motor response suppression tasks that emphasise the importance of the right prefrontal cortex.

Measuring brain stem and cerebellar damage in parkinsonian syndromes using diffusion tensor MRI

Objective: To use diffusion tensor MRI to quantify and compare degeneration of the pons and cerebellar peduncles in multiple system atrophy (MSA), progressive supranuclear palsy (PSP), and Parkinson disease (PD) and to relate changes in diffusion measures to clinical features and localized atrophy. Methods: We used a region-of-interest approach to measure changes in fractional anisotropy and mean diffusivity in the middle cerebellar peduncles, decussation of the superior cerebellar peduncles, and pons in 17 patients with MSA, 17 with PSP, 12 with PD, and 12 healthy volunteers. We also evaluated atrophy of the cerebellar peduncles and pons on T2-weighted magnetic resonance images in patients with MSA and PSP. Results: In MSA, fractional anisotropy was markedly reduced in the middle cerebellar peduncles, and mean diffusivity increased both here and in the pons compared with other groups, whereas in PSP, mean diffusivity was strikingly increased in the decussation of superior cerebellar peduncles. Cerebellar ataxia was related to mean diffusivity in the middle cerebellar peduncles (r = 0.71, p = 0.001) and pons (r = 0.60, p = 0.01) in MSA. Diffusion measures were related to localized atrophy in both MSA and PSP. Conclusions: Diffusion tensor MRI can be used to quantify neurodegenerative processes in different brain stem and cerebellar structures in multiple system atrophy and progressive supranuclear palsy during life, and may have diagnostic value. Larger studies of early, undifferentiated parkinsonian syndromes are indicated to provide estimates of the relative diagnostic value of diffusion measures, atrophy measures, and visual assessment of scans.

Cloning and comparison of repeated DNA sequences from the human filarial parasite Brugia malayi and the animal parasite Brugia pahangi

A 320-base-pair repeated sequence was observed when DNA samples from the filarial parasites Brugia malayi and Brugia pahangi were digested with the restriction endonuclease Hha I. A 640-base-pair dimer of the repeated sequence from B. malayi was inserted into the plasmid pBR322. When dot hybridization was used, the copy number of the repeat in B. malayi was found to be about 30,000. The 320-base-pair Hha I repeated sequences are arranged in direct tandem arrays and comprise about 12% of the genome. B. pahangi has a related repeated sequence that cross-hybridizes with the cloned B. malayi Hha I repeat. Dot hybridization with the cloned repeat shows that the sequence is present in B. malayi and in B. pahangi but not in four other species of filarial parasites. The cloned repeated DNA sequence is an extremely sensitive probe for detection of Brugia in blood samples. Hybridization with the cloned repeat permits the detection of DNA isolated from a single parasite in an aliquot of blood from animals infected with B. malayi. There are differences in the restriction sites present in the repeated sequences that can be used to differentiate between the two Brugia species. The B. malayi repeated DNA sequence is cleaved by Alu I and Rsa I but the B. pahangi sequence is not. A comparison of repeated sequences between the two species by DNA sequence analysis indicates that some regions of individual repeats are over 95% homologous, while other short regions are only 60-65% homologous. These differences in DNA sequence will allow the construction of species-specific hybridization probes.

A proton magnetic resonance spectroscopic study in ALS: Correlation with clinical findings

Objective: To evaluate neuronal dysfunction in the motor region subcortical white matter in ALS using volumetric localized proton magnetic resonance spectroscopy (1H-MRS). Methods: Sixteen patients with El Escorial definite, probable, or possible ALS and eight healthy age-matched control subjects were studied. The ALS patients were divided into those with limb onset (n = 8) and those with bulbar onset (n = 8). Measurements of the metabolic ratios N-acetylaspartate (NAA)/creatine and phosphocreatine (Cr+PCr), NAA/choline (Cho), and Cho/(Cr+PCr) were correlated with clinical assessments. Results: We found no differences in the metabolic peak area ratios in the motor region when comparing the total ALS group and the control subjects. However, correlations were found between the NAA/(Cr+PCr) ratio and the El Escorial category (p = 0.03), the ALS severity scale (p = 0.01), and the Medical Research Council score (p = 0.06). No correlations were found between the NAA/(Cr+PCr) ratio and the Ashworth Spasticity Scale, reflex score, or disease duration (p > 0.16). Bulbar-onset patients had a lower NAA/(Cr+PCr) ratio in the motor region compared with limb-onset patients (p = 0.03). Conclusion: In vivo 1H-MRS of the subcortical white matter in the motor region is unlikely to be sensitive enough to detect early disease changes in ALS because there is considerable overlap between the metabolic peak area ratios from patients with ALS and normal control subjects. However, changes in the NAA/(Cr+PCr) metabolic peak area ratios correlate with clinical measures of disease severity, and this measure may be useful in monitoring disease progression.

Identification of Potential Vaccine and Drug Target Candidates by Expressed Sequence Tag Analysis and Immunoscreening of Onchocerca volvulus Larval cDNA Libraries

ABSTRACT The search for appropriate vaccine candidates and drug targets against onchocerciasis has so far been confronted with several limitations due to the unavailability of biological material, appropriate molecular resources, and knowledge of the parasite biology. To identify targets for vaccine or chemotherapy development we have undertaken two approaches. First, cDNA expression libraries were constructed from life cycle stages that are critical for establishment of Onchocerca volvulus infection, the third-stage larvae (L3) and the molting L3. A gene discovery effort was then initiated by random expressed sequence tag analysis of 5,506 cDNA clones. Cluster analyses showed that many of the transcripts were up-regulated and/or stage specific in either one or both of the cDNA libraries when compared to the microfilariae, L2, and both adult stages of the parasite. Homology searches against the GenBank database facilitated the identification of several genes of interest, such as proteinases, proteinase inhibitors, antioxidant or detoxification enzymes, and neurotransmitter receptors, as well as structural and housekeeping genes. Other O. volvulus genes showed homology only to predicted genes from the free-living nematode Caenorhabditis elegans or were entirely novel. Some of the novel proteins contain potential secretory leaders. Secondly, by immunoscreening the molting L3 cDNA library with a pool of human sera from putatively immune individuals, we identified six novel immunogenic proteins that otherwise would not have been identified as potential vaccinogens using the gene discovery effort. This study lays a solid foundation for a better understanding of the biology of O. volvulus as well as for the identification of novel targets for filaricidal agents and/or vaccines against onchocerciasis based on immunological and rational hypothesis-driven research.