Search

The sequencing, assembly, and analysis of bacterial genomes is central to tracking and characterizing foodborne pathogens. The bulk of bacterial genome sequencing at the US Food and Drug Administration is performed using short-read Illumina MiSeq technology, resulting in highly accurate but fragmented genomic sequences. The MinION sequencer from Oxford Nanopore is an evolving technology that produces long-read sequencing data with low equipment cost. The goal of this study was to compare Campylobacter genome assemblies generated from MiSeq and MinION data independently, as well as hybrid genome assemblies combining both data types. Two reference strains and two field isolates of C. jejuni were sequenced using MiSeq and MinION, and the sequence data were assembled using the software programs SPAdes and Canu, respectively. Hybrid genome assembly was performed using the program Unicycler. Comparison of the C. jejuni 81-176 and RM1221 genome assemblies to the PacBio reference genomes revealed that the SPAdes assemblies had the most accurate nucleotide identity, while the hybrid assemblies were the most contiguous. Assemblies generated only from MinION data using Canu were the least accurate, containing many indels and substitutions that affected downstream analyses. The hybrid sequencing approach was the most useful for detecting plasmids, large genome rearrangements, and repetitive elements such as rRNA and tRNA genes. The full genomes of both C. jejuni field isolates were completed and circularized using hybrid sequencing, and a plasmid was detected in one isolate. Continued development of nanopore sequencing technologies will likely enhance the accuracy of hybrid genome assemblies and enable public health laboratories to routinely generate complete circularized bacterial genome sequences.

A collaborative validation study was performed to evaluate the performance of a new U.S. Food and Drug Administration method developed for detection of the protozoan parasite, Cyclospora cayetanensis, on cilantro and raspberries. The method includes a sample preparation step in which oocysts are recovered from produce using an enhanced produce washing solution containing 0.1% Alconox and a commercially available method to disrupt the C. cayetanensis oocysts and extract DNA. A real-time PCR assay targeting the C. cayetanensis 18S rDNA gene with an internal amplification control to monitor PCR inhibition provides species-specific identification. Five laboratories blindly analyzed a total of 319 samples consisting of 25 g of cilantro or 50 g of raspberries which were either uninoculated or artificially contaminated with C. cayetanensis oocysts. Detection rates for cilantro inoculated with 200, 10, and 5 oocysts, were 100%, 80%, and 31%, respectively. For raspberries, the detection rates for samples inoculated with 200, 10, and 5 oocysts were 100%, 90% and 50%, respectively. All uninoculated samples, DNA blank extracts, and no-template PCR controls were negative. Reproducibility between laboratories and analysts was high and the method was shown to be an effective analytical tool for detection of C. cayetanensis in produce.

Consumption of Campylobacter contaminated food or water is a leading cause of human acute gastroenteritis. Campylobacter jejuni, Campylobacter coli, and Campylobacter lari account for over 95% of total Campylobacter infections. A multiplex quantitative polymerase chain reaction (qPCR) for simultaneous identification of C. jejuni, C. coli, and C. lari was developed for use with the SmartCycler II system.We evaluated and combined previously described primers and probes for Campylobacter detection, designed a new internal amplification control, and optimized the multiplex qPCR for the detection of C. jejuni, C. coli, and C. lari.This method was 100% specific when tested against a panel of 32 target Campylobacter strains and 31 non-Campylobacter reference strains. Furthermore, there was no cross-reactivity with seven strains from four nontarget Campylobacter species. The amplification efficiency of each target in this multiplex qPCR was over 90%, and each coefficient of linearity was greater than 0.99. With artificially mixed genomic DNA, this method detected as few as two, three, and two genome copies of C. jejuni, C. coli, and C. lari, respectively. This method was also able to detect these three Campylobacter species in artificially contaminated milk with a sensitivity of five spiked cells of each target per reaction.The three Campylobacter targets were simultaneously identified using artificially mixed genomic DNA and spiked raw milk. This SmartCycler-based multiplex qPCR is a rapid, specific, and sensitive method to identify C. jejuni, C. coli, and C. lari.

Campylobacter jejuni causes more than 2 million cases of gastroenteritis annually in the United States, and is also linked to the autoimmune sequelae Guillan-Barre syndrome (GBS). GBS often results in flaccid paralysis, as the myelin sheaths of nerve cells are degraded by the adaptive immune response. Certain strains of C. jejuni modify their lipooligosaccharide (LOS) with the addition of neuraminic acid, resulting in LOS moieties that are structurally similar to gangliosides present on nerve cells. This can trigger GBS in a susceptible host, as antibodies generated against C. jejuni can cross-react with gangliosides, leading to demyelination of nerves and a loss of signal transduction. The goal of this study was to develop a quantitative PCR (qPCR) method and use whole genome sequencing data to detect the Campylobacter sialyltransferase (cst) genes responsible for the addition of neuraminic acid to LOS. The qPCR method was used to screen a library of 89 C. jejuni field samples collected by the Food and Drug Administration Pacific Northwest Lab (PNL) as well as clinical isolates transferred to PNL. In silico analysis was used to screen 827 C. jejuni genomes in the FDA GenomeTrakr SRA database. The results indicate that a majority of C. jejuni strains could produce LOS with ganglioside mimicry, as 43.8% of PNL isolates and 46.9% of the GenomeTrakr isolates lacked the cst genes. The methods described in this study can be used by public health laboratories to rapidly determine whether a C. jejuni isolate has the potential to induce GBS. Based on these results, a majority of C. jejuni in the PNL collection and submitted to GenomeTrakr have the potential to produce LOS that mimics human gangliosides.

Periventricular heterotopias is a malformation of cortical development, characterized by ectopic neuronal nodules around ventricle lining and caused by an initial migration defect during early brain development. Human mutations in the Filamin A (FLNA) and ADP-ribosylation factor guanine exchange factor 2 [ARFGEF2; encoding brefeldin-A-inhibited guanine exchange factor-2 (BIG2)] genes give rise to this disorder. Previously, we have reported that Big2 inhibition impairs neuronal migration and binds to FlnA, and its loss promotes FlnA phosphorylation. FlnA phosphorylation dictates FlnA–actin binding affinity and consequently alters focal adhesion size and number to effect neuronal migration. Here we show that FlnA loss similarly impairs migration, reciprocally enhances Big2 expression, but also alters Big2 subcellular localization in both null and conditional FlnA mice. FlnA phosphorylation promotes relocalization of Big2 from the Golgi toward the lipid ruffles, thereby activating Big2-dependent Arf1 at the cell membrane. Loss of FlnA phosphorylation or Big2 function impairs Arf1-dependent vesicle trafficking at the periphery, and Arf1 is required for maintenance of cell–cell junction connectivity and focal adhesion assembly. Loss of Arf1 activity disrupts neuronal migration and cell adhesion. Collectively, these studies demonstrate a potential mechanism whereby coordinated interactions between actin (through FlnA) and vesicle trafficking (through Big2-Arf) direct the assembly and disassembly of membrane protein complexes required for neuronal migration and neuroependymal integrity.

Objective Miller-Dieker syndrome (MDS) is a malformation of cortical development that results in lissencephaly (meaning smooth brain). This disorder is caused by heterozygous deletions on chromosome 17p13.3, including the lissencephaly 1 (LIS1) gene. Various mouse models have been used as an experimental paradigm in understanding human lissencephaly, but clear limitations exist in these studies, particularly because mice are naturally lissencephalic. Thus, the objective of this article was to establish human neural precursor cell lines from postmortem MDS tissue and to characterize the pathological cellular processes that contribute to the human lissencephalic phenotype. Methods Human neural precursors were isolated and expanded from the frontal cortices of a 33-week postmortem fetus with MDS and an age-matched control subject. Relative rates of proliferation and cell death were assessed in vitro, whereas the migration of precursors was examined after transplantation in vivo. Results Precursors showed haploinsufficiency of the LIS1 gene and a reduction in LIS1 protein. Precursors could also differentiate into both neurons and glia. MDS precursors demonstrated impairments in neuronal migration, diminished rates of cell proliferation, and increased cell death. Interpretation These results suggest that, in addition to migration, disruption in cell proliferation could play a more important role in the development of lissencephaly than previously suspected. Ann Neurol 2006

Periventricular heterotopia (PH) is a human malformation of cortical development associated with gene mutations in ADP-ribosylation factor guanine exchange factor 2 (ARFGEF2encodes for Big2 protein) and Filamin A (FLNA). PH is thought to derive from neuroependymal disruption, but the extent to which neuronal migration contributes to this phenotype is unknown. Here, we show thatArfgef2null mice develop PH and exhibit impaired neural migration with increased protein expression for both FlnA and phosphoFlnA at Ser2152. Big2 physically interacts with FlnA and overexpression of phosphomimetic Ser2512 FLNA impairs neuronal migration. FlnA phosphorylation directs FlnA localization toward the cell cytoplasm, diminishes its binding affinity to actin skeleton, and alters the number and size of paxillin focal adhesions. Collectively, our results demonstrate a molecular mechanism whereby Big2 inhibition promotes phosphoFlnA (Ser2152) expression, and increased phosphoFlnA impairs its actin binding affinity and the distribution of focal adhesions, thereby disrupting cell intrinsic neuronal migration.

Over the past century, sociocultural and technological developments have fostered the emergence of what Peterson and Kern (1996) call “omnivorous” music listeners, who listen to music from a variety of different genres. As well, non-hierarchical forms of categorization, such as tagging, have appeared in recent years. Despite such trends, genre remains the primary basis for categorizing music in systems with content, metadata, or both. Furthermore, techniques employed within many recommender systems, intended to aid listeners with finding music for recreational listening, indirectly continue to reflect genre-based categorization and taste. This paper provides an overview of the contexts in which such trends have emerged. It also considers prospects for incorporating actively nuanced dimensions of similarity into recommender systems, which could enable users to engage in cross-genre music discovery more easily than current systems allow. To provide further grounding for such possibilities, I am currently conducting a study to determine how “avid recreational music listeners” conceptualize musical similarity. This paper discusses the study’s methodology, which consists of semi-structured interviews and music-seeking exercises.

The impact of primary care practices adopting the patient-centered medical home (PCMH) model is analyzed by comparing per member per month (PMPM) costs and utilization among commercial HMO members with chronic illnesses in PCMH and non-PCHM practices in the Philadelphia area. Transforming primary care practices to conform to the PCMH model has shown early promise in reducing costs and improving outcomes, and chronically ill patients' frequent contact with the healthcare system and costly care make them ideal targets for such health system reforms.The impact of the PCMH model on PMPM costs was analyzed using a generalized linear regression model to adjust for age, gender, and baseline cost. The impact of the PCMH model on utilization per 1000 rates was analyzed with the Poisson regression model, adjusting for baseline differences in age, gender, and risk score.After accounting for differences at baseline, PCMH practices achieved lower total, inpatient, and specialist PMPM costs, as well as lower relative utilization of hospital admissions and specialist visits.These findings suggest that policy makers should maintain or expand incentives to adopt PCMH reforms and that targeting chronically ill patients may be the most effective way to leverage the benefits of PCMH adoption.

Agrobacterium tumefaciens and Agrobacterium rhizogenes are closely related plant pathogens that cause different diseases, crown gall and hairy root. Both diseases result from transfer, integration, and expression of plasmid-encoded bacterial genes located on the transferred DNA (T-DNA) in the plant genome. Bacterial virulence (Vir) proteins necessary for infection are also translocated into plant cells. Transfer of single-stranded DNA (ssDNA) and Vir proteins requires a type IV secretion system, a protein complex spanning the bacterial envelope. A. tumefaciens translocates the ssDNA-binding protein VirE2 into plant cells, where it binds single-stranded T-DNA and helps target it to the nucleus. Although some strains of A. rhizogenes lack VirE2, they are pathogenic and transfer T-DNA efficiently. Instead, these bacteria express the GALLS protein, which is essential for their virulence. The GALLS protein can complement an A. tumefaciens virE2 mutant for tumor formation, indicating that GALLS can substitute for VirE2. Unlike VirE2, GALLS contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. Both GALLS and VirE2 contain nuclear localization sequences and a C-terminal type IV secretion signal. Here we show that mutations in any of these domains abolished the ability of GALLS to substitute for VirE2.

Miller Dieker syndrome (MDS, type I lissencephaly) is a neuronal migration disorder, which is caused by deletions along the short arm of chromosome 17 (17p13.3). Recent studies would suggest that the cortical lamination in MDS is inverted, based on morphological criteria. The present neuropathological study examines the cerebral cortex from a 33-week old fetus with MDS using both neuronal and laminar-specific markers. These expression studies demonstrate a relatively preserved cortex and cortical lamination, overlying a layer of immature neurons in MDS brain. The findings are consistent with both a migratory and proliferative defect, giving rise to lissencephaly. Moreover, characterization of such rare human malformations of cortical development by immunohistochemical techniques will provide a greater understanding of the underlying mechanisms.

We report on the case of dizygotic twin boys, born prematurely to an asymptomatic mother. Bilateral periventricular heterotopias with enlarged ventricles were discovered at birth in both twins. One of the twins died prematurely of bronchopulmonary complications, and was shown to have several neuropathological anomalies (microgyria, thin corpus callosum, and reduced white matter). The surviving twin had mental retardation, without epilepsy. MRI of the mother showed asymptomatic periventricular heterotopias without ventricular enlargement. She had two affected daughters also with asymptomatic periventricular heterotopias. A point mutation in the last coding exon 48 of the Filamin A (FLNA) gene (7922c > t) was discovered on sequencing and segregated with the affected individuals. This family has a classical X-linked dominant BPNH pathology, with greater severity in males than females. The location of the FLNA mutation is discussed in light of the neuropathological anomalies and mental retardation in male patients.

Indexing and retrieval tools for music privilege genre-based categorization. Music recommender systems do not draw upon genre per se, but they utilize collaborative filtering algorithms that can give the appearance of doing so. This poster critiques genre’s pervasiveness, and suggests that recommender systems draw upon alternative notions of “similarity.”Les outils d’indexation et de repérage de la musique privilégient la catégorisation par genre. Les systèmes de recommandation de musique n’utilisent pas le genre en tant que tel, mais utilisent plutôt un algorithme de filtrage collaboratif qui en apparence fait la même chose. Cette affiche critique l’omniprésence du genre et suggère que les systèmes de recommandation devraient plutôt faire appel à la notion de « similarité ».

Threats are generally much easier to list than to describe, and much easier to describe than to measure. As a result, many organizations list threats. Fewer describe them in useful terms, and still fewer measure them in meaningful ways. This is particularly true in the dynamic and nebulous domain of cyber threats - a domain that tends to resist easy measurement and, in some cases, appears to defy any measurement. We believe the problem is tractable. In this report we describe threat metrics and models for characterizing threats consistently and unambiguously. The purpose of this report is to support the Operational Threat Assessment (OTA) phase of risk and vulnerability assessment. To this end, we focus on the task of characterizing cyber threats using consistent threat metrics and models. In particular, we address threat metrics and models for describing malicious cyber threats to US FCEB agencies and systems.

The developmental mechanisms underlying the formation of human cortical convolutions (gyri and sulci) remain largely unknown. Genetic causes of lissencephaly (literally ‘smooth brain’) would imply that disorders in neuronal migration cause the loss of cortical convolutions. However, prior studies have suggested that loss of sulci and gyri can also arise from impaired proliferation, disrupted lamination and loss of intracortical connections. To gain further insight into the mechanisms underlying the formation of cortical convolutions, we examined the progressive brain development of the gyrencephalic ferret. In this study, we used magnetic resonance imaging to follow the temporal and spatial pattern of neuronal migration, proliferation and differentiation in relation to the onset and development of cortical convolutions. In this manner, we demonstrate that the onset of gyrification begins largely after completion of neuronal proliferation and migration. Gyrification occurs in a lateral to medial gradient, during the period of most rapid cerebral cortical growth. Cortical folding is also largely complete prior to myelination of the underlying cortical axons. These observations are consistent with gyrification arising secondary to cortical processes involving neuronal differentiation.

Periventricular heterotopia (PH) is a malformation of cortical development characterized by the ectopic localization of neuronal nodules along the lateral ventricle. Mutations in X-linked filamin A gene are the most common cause of PH, although a rarer autosomal recessive form of PH with microcephaly due to ARFGEF2 mutations has been described [Sheen et al., 2001]. Affected individuals generally are of normal intelligence and most often present with adolescent onset seizures. The 1p36 deletion syndrome is associated with multiple congenital anomalies caused by haploinsufficiency of numerous contiguous genes. This deletion produces specific physical characteristics such as distinctive facial anomalies (pointed chin, flat nose, low set ears) and cardiovascular malformations (atrial septal defect, patent ductus arteriosus, tetralogy of Fallot). Central nervous system (CNS) defects include mental retardation, cranial nerve abnormalities (VI nerve palsies, optic disc anomalies, sensorineural hearing loss), neonatal hypotonia, cortical dysplasia, and seizures [Heilstedt et al., 2003; Kurosawa et al., 2005; Battaglia, 2005]. A reduction in the KCNAB2 potassium channel betasubunit has been hypothesized to be responsible for the seizures [Hirose et al., 2002]. Affected individuals can display outbursts, tendencies to strike people, and self-injurious behavior, as well as autistic-like behaviors [Slavotinek et al., 1999]. Previous neuroimaging and postmortem studies on individuals with 1p36 deletions have revealed mild CNS structural abnormalities. Deletions involving the most distal p-terminus (D1S508, 1p36.23-> 1pter) have only been associated with microcephaly, mild ventricular asymmetry, and ventricular enlargement [Kurosawa et al., 2005; Slavotinek et al., 1999]. An interstitial deletion (1p36.1->1p36.2) was suggested as causal for the development of neuroblastoma in a single case report [Keppler-Noreuil et al., 1995]. Finally, a series of three patients harboring 1p36.22->1pter deletions was reported to have hydrocephalus by cranial ultrasound but no mention of PH [Keppler-Noreuil et al., 1995]. To date, there have been no reported associations of PH with chromosome 1p36 deletions. Here, we describe the first case describing PH in an individual with a 1p36.22-> 1pter deletion. This study was approved by the IRB at the respective institutions in accordance with NIH. Informed consent was obtained from the participating subject’s parents. Genomic DNA was extracted from peripheral whole blood lymphocytes using standard blood DNA isolation techniques (Qiagen Inc., Valencia, CA). Metaphase chromosome analysis of lymphocytes was performed according to standard protocols. Initial routine karyotyping on the child was read as normal, although deletions on 1p36 are commonly missed due to the Giemsa-negative/poor staining in this region. Given the numerous clinical features seen in this subject, FISH analysis utilizing 41 subtelomere probes was performed (Genzyme laboratories (Hawthorne, NY), using Vysis probes (Downers Grove, IL). Results were confirmed with a chromosome 1p subtelomere probe (1pSUBTEL; Vysis) and the D1Z2 midi-satellite probe with repeats in band 1p36 (Oncor). The D1Z2 probe is used for confirmationof a commondeletion interval (1p36.3). A chromosome 1 centromere probe (D1Z5; Vysis)

Purpose: Familial periventricular heterotopia (PH) represents a disorder of neuronal migration resulting in multiple gray-matter nodules along the lateral ventricular walls. Prior studies have shown that mutations in the filamin A (FLNA) gene can cause PH through an X-linked dominant pattern. Heterozygotic female patients usually remain asymptomatic until the second or third decade of life, when they may have predominantly focal seizures, whereas hemizygotic male fetuses typically die in utero. Recent studies have also reported mutations in FLNA in male patients with PH who are cognitively normal. We describe PH in three male siblings with PH due to FLNA, severe developmental regression, and West syndrome.Methods: the study includes the three affected brothers and their parents. Video-EEG recordings and magnetic resonance image (MRI) scanning were performed on all individuals. Mutations for FLNA were detected by using polymerase chain reaction (PCR) on genomic DNA followed by single-stranded conformational polymorphism (SSCP) analysis or sequencing.Results: Two of the siblings are monozygotic twins, and all had West syndrome with hypsarrthymia on EEG. MRI of the brain revealed periventricular nodules of cerebral gray-matter intensity, typical for PH. Mutational analyses demonstrated a cytosine-to-thymidine missense mutation (c. C1286T), resulting in a threonine-to-methionine amino acid substitution in exon 9 of the FLNA gene.Conclusions: the association between PH and West syndrome, to our knowledge, has not been previously reported. Males with PH have been known to harbor FLNA mutations, although uniformly, they either show early lethality or survive and have a normal intellect. the current studies show that FLNA mutations can cause periventricular heterotopia, developmental regression, and West syndrome in male patients, suggesting that this type of FLNA mutation may contribute to severe neurologic deficits. Universidade Federal de São Paulo, Dept Neurol & Neurosurg, BR-04023900 São Paulo, Brazil Universidade Federal de São Paulo, Dept Radiol, BR-04023900 São Paulo, Brazil Univ Estadual Campinas, Dept Med Genet, São Paulo, Brazil Fed Univ Espirito Santo, Dept Pediat, Espirito Santo, Brazil Harvard Univ, Sch Med, Howard Hughes Med Inst, Beth Israel Deaconess Med Ctr,Dept Neurol, Boston, MA 02115 USA Universidade Federal de São Paulo, Dept Neurol & Neurosurg, BR-04023900 São Paulo, Brazil Universidade Federal de São Paulo, Dept Radiol, BR-04023900 São Paulo, Brazil Web of Science

Purpose The primary purpose of this study was to evaluate the impact of caspase-3 ablation on photoreceptor degeneration in the rd-1 mouse. Concurrently, the role of caspase-3 in postnatal retinal development was evaluated. Caspase-3 is an important effector caspase that mediates many of the terminal proteolytic events of apoptosis. Its activation has been demonstrated in rodent models of photoreceptor degeneration and its ablation results in exencephaly and neonatal death. Methods Retinal morphometry was performed at the light microscopic level in caspase-3 mutant mice from PN0 through PN23, and in rd-1/caspase-3 double mutant mice at PN14, -16, and -18. This was supplemented by terminal dUTP transferase nick end labeling (TUNEL) and immunohistochemical staining for activated caspase-3, rhodopsin, factor VII-related antigen and proliferating cell nuclear antigen (PCNA). Results Caspase-3-deficient animals display marginal microphthalmia, peripapillary retinal dysplasia, delayed regression of vitreal vasculature, and retarded apoptotic kinetics of the inner nuclear layer. Ablation of caspase-3 provided transient photoreceptor protection in rd-1, but TUNEL-positive rod death proceeded, despite the absence of caspase-3 activation. Conclusions In vivo, caspase-3 is not critical for rod photoreceptor development, nor does it play a significant role in mediating pathologic rod death. Peripapillary dysplastic lesions suggest that there is delayed fusion of the optic fissure, and inner nuclear layer abnormalities indicate a cell-specific dependency on the mitochondria-caspase axis during development. The temporal nature of apoptotic retardation in the absence of caspase-3 implies the presence of caspase-independent mechanisms of developmental and pathologic cell death.

Background Nonprogressive cerebellar ataxias are characterized by a persistent, nonprogressive ataxia associated with cognitive impairment. Cerebellar hypoplasia on imaging is variable but is not predictive of the degree of ataxia or cognitive impairment. Objective To describe a family with a nonprogressive cerebellar ataxia associated with cognitive and motor impairments that improve with age. Design Genetic study in a family with nonprogressive cerebellar ataxia. Clinical and imaging features are also described. Setting Community hospital. Patients Both parents and 3 children from an affected family. Main Outcome Measures Clinical features, magnetic resonance imaging findings, and genetic findings. Results A genome-wide single nucleotide polymorphism screen did not show clear linkage to known spinocerebellar ataxia loci, in particular spinocerebellar ataxia type 15. Repeat spinocerebellar ataxia loci expansions were excluded. Magnetic resonance images of all affected individuals demonstrated cerebellar vermian abnormalities. Conclusions These findings suggest that nonprogressive cerebellar ataxia is genetically heterogeneous and, when associated with gradual improvement in cognition and motor skills, likely represents a separate, distinct clinical entity.