Studies of subcellular localization and complex formation of the Agrobacterium tumefaciens transport ATPase VirB11

The VirB11 ATPase is an essential component of an Agrobacterium tumefaciens type IV bacterial secretion system that transfers oncogenic nucleoprotein complexes to susceptible plant cells. This dissertation investigates the subcellular localization and homo-oligomeric state of the VirB11 ATPase in order to provide insights about the assembly of the protein as a subunit of this membrane-associated transfer system. Subcellular fractionation studies and quantitative immunoblot analysis demonstrated that $\sim$30% of VirB11 partitioned as soluble protein and $\sim$70% was tightly associated with the bacterial cytoplasmic membrane. No differences were detected in VirB11 subcellular localization and membrane association in the presence or absence of other transport system components. Mutations in virB11 affecting protein function were mapped near the amino terminus, just upstream of a region encoding a Walker 'A' nucleotide-binding site, and within the Walker 'A' motif partitioned almost exclusively with the cytoplasmic membrane, suggesting that an activity associated with nucleotide binding could modulate the affinity of VirB11 for the cytoplasmic membrane. Merodiploid analysis of VirB11 mutant and truncation derivatives provided strong evidence that VirB11 functions as a homo- or heteromultimer and that the C-terminal half of VirB11 contains a protein interaction domain. A combination of biochemical and molecular genetic approaches suggested that VirB11 and the green fluorescence protein (GFP) formed a mixed multimer as demonstrated by immunoprecipitation experiments with anti-GFP antibodies. Second, a hybrid protein composed of VirB11 fused to the N-terminal DNA-binding domain of bacteriophage $\lambda$ cI repressor conferred immunity to $\lambda$ superinfection, demonstrating that VirB11 self-association promotes dimerization of the chimeric repressor. A conserved Walker 'A' motif, though required for VirB11 function in T-complex export, was not necessary for VirB11 self-association. Sequences in both the N- and the C-terminal halves of the protein were found to contribute to self-association of the full length protein. Chemical cross-linking experiments with His$\sb6$ tagged VirB11 suggested that VirB11 probably assembles into a higher order homo-oligomeric complex. ^

High resolution immunoelectron microscopic localization of functional domains of laminin, nidogen, and heparan sulfate proteoglycan in epithelial basement membrane of mouse cornea reveals different topological orientations

Thin and ultrathin cryosections of mouse cornea were labeled with affinity-purified antibodies directed against either laminin, its central segments (domain 1), the end of its long arm (domain 3), the end of one of its short arms (domain 4), nidogen, or low density heparan sulfate proteoglycan. All basement membrane proteins are detected by indirect immunofluorescence exclusively in the epithelial basement membrane, in Descemet's membrane, and in small amorphous plaques located in the stroma. Immunoelectron microscopy using the protein A-gold technique demonstrated laminin domain 1 and nidogen in a narrow segment of the lamina densa at the junction to the lamina lucida within the epithelial basement membrane. Domain 3 shows three preferred locations at both the cellular and stromal boundaries of the epithelial basement membrane and in its center. Domain 4 is located predominantly in the lamina lucida and the adjacent half of the lamina densa. The low density heparan sulfate proteoglycan is found all across the basement membrane showing a similar uniform distribution as with antibodies against the whole laminin molecule. In Descemet's membrane an even distribution was found with all these antibodies. It is concluded that within the epithelial basement membrane the center of the laminin molecule is located near the lamina densa/lamina lucida junction and that its long arm favors three major orientations. One is close to the cell surface indicating binding to a cell receptor, while the other two are directed to internal matrix structures. The apparent codistribution of laminin domain 1 and nidogen agrees with biochemical evidence that nidogen binds to this domain.

Softening the Lower Crust: Modes of Syn-Transport Transposition Around and Adjacent to a Deep Crustal Granulite Nappe, Parry Sound Domain, Grenville Province, Ontario, Canada

The Parry Sound domain is a granulite nappe-stack transported cratonward during reactivation of the ductile lower and middle crust in the late convergence of the Mesoproterozoic Grenville orogeny. Field observations suggest the following with respect to the ductile sheath: (1) Formation of a carapace of transposed amphibolite facies gneiss derived from and enveloping the western extremity of the Parry Sound domain and separating it from high-strain gneiss of adjacent allochthons. This ductile sheath formed dynamically around the moving granulite nappe through the development of systems of progressively linked shear zones. (2) Transposition initiated by hydration (amphibolization) of granulite facies gneiss by introduction of fluid along cracks accompanying pegmatite emplacement. Shear zones nucleated along pegmatite margins and subsequently linked and rotated. The source of the pegmatites was most likely subjacent migmatitic and pegmatite-rich units or units over which Parry Sound domain was transported. Comparison of gneisses of the ductile sheath with high-strain layered gneiss of adjacent allochthons show the mode of transposition of penetratively layered gneiss depended on whether or not the gneiss protoliths were amphibolite or granulite facies tectonites before initiation of transposition, resulting in, e.g., folding before shearing, no folding before shearing, respectively. Meter-scale truncation along high-strain gradients at the margins of both types of transposition-related shear zones observed within and marginal to Parry Sound domain mimic features at kilometer scales, implying that apparent truncation by transposition originating in a manner similar to the ductile sheath may be a common feature of deep crustal ductile reworking. Citation: Culshaw, N., C. Gerbi, and J. Marsh (2010), Softening the lower crust: Modes of syn-transport transposition around and adjacent to a deep crustal granulite nappe, Parry Sound domain, Grenville Province, Ontario, Canada, Tectonics, 29, TC5013, doi:10.1029/2009TC002537.

Metamorphism, Transient Mid-Crustal Rheology, Strain Localization and the Exhumation of High-Grade Metamorphic Rocks

We present a series of three-dimensional numerical models investigating the effects of metamorphic strengthening and weakening on the geodynamic evolution of convergent orogens that are constrained by observations from an exposed mid-crustal section in the New England Appalachians. The natural mid-crustal section records evidence for spatially and temporally variable mid-crustal strength as a function of metamorphic grade during prograde polymetamorphism. Our models address changes in strain rate partitioning and topographic uplift as a function of strengthening/weakening in the middle crust, as well as the resultant changes in deformation kinematics and potential exhumation patterns of high-grade metamorphic rock. Results suggest that strengthening leads to strain rate partitioning around the zone and suppressed topographic uplift rates whereas weakening leads to strain rate partitioning into the zone and enhanced topographic uplift rates. Deformation kinematics recorded in the orogen are also affected by strengthening/weakening, with complete reversals in shear sense occurring as a function of strengthening/weakening without changes in plate boundary kinematics.

SDR-based Passive Indoor Localization System for GSM

This study deals with indoor positioning using GSM radio, which has the distinct advantage of wide coverage over other wireless technologies. In particular, we focus on passive localization systems that are able to achieve high localization accuracy without any prior knowledge of the indoor environment or the tracking device radio settings. In order to overcome these challenges, newly proposed localization algorithms based on the exploitation of the received signal strength (RSS) are proposed. We explore the effects of non-line-of-sight communication links, opening and closing of doors, and human mobility on RSS measurements and localization accuracy. We have implemented the proposed algorithms on top of software defined radio systems and carried out detailed empirical indoor experiments. The performance results show that the proposed solutions are accurate with average localization errors between 2.4 and 3.2 meters.

Optimal target localization for subthalamic stimulation in patients with Parkinson disease

OBJECTIVE To further determine the causes of variable outcome from deep brain stimulation of the subthalamic nucleus (DBS-STN) in patients with Parkinson disease (PD). METHODS Data were obtained from our cohort of 309 patients with PD who underwent DBS-STN between 1996 and 2009. We examined the relationship between the 1-year motor, cognitive, and psychiatric outcomes and (1) preoperative PD clinical features, (2) MRI measures, (3) surgical procedure, and (4) locations of therapeutic contacts. RESULTS Pre- and postoperative results were obtained in 262 patients with PD. The best motor outcome was obtained when stimulating contacts were located within the STN as compared with the zona incerta (64% vs 49% improvement). Eighteen percent of the patients presented a postoperative cognitive decline, which was found to be principally related to the surgical procedure. Other factors predictive of poor cognitive outcome were perioperative confusion and psychosis. Nineteen patients showed a stimulation-induced hypomania, which was related to both the form of the disease (younger age, shorter disease duration, higher levodopa responsiveness) and the ventral contact location. Postoperative depression was more frequent in patients already showing preoperative depressive and/or residual axial motor symptoms. CONCLUSION In this homogeneous cohort of patients with PD, we showed that (1) the STN is the best target to improve motor symptoms, (2) postoperative cognitive deficit is mainly related to the surgery itself, and (3) stimulation-induced hypomania is related to a combination of both the disease characteristics and a more ventral STN location.

Active Testing for Face Detection and Localization

We provide a novel search technique which uses a hierarchical model and a mutual information gain heuristic to efficiently prune the search space when localizing faces in images. We show exponential gains in computation over traditional sliding window approaches, while keeping similar performance levels.

Improved Localization of Implanted Subdural Electrode Contacts on MRI Using an Elastic Image Fusion Algorithm in Invasive EEG Recording

BACKGROUND: Accurate projection of implanted subdural electrode contacts in presurgical evaluation of pharmacoresistant epilepsy cases by invasive EEG is highly relevant. Linear fusion of CT and MRI images may display the contacts in the wrong position due to brain shift effects. OBJECTIVE: A retrospective study in five patients with pharmacoresistant epilepsy was performed to evaluate whether an elastic image fusion algorithm can provide a more accurate projection of the electrode contacts on the pre-implantation MRI as compared to linear fusion. METHODS: An automated elastic image fusion algorithm (AEF), a guided elastic image fusion algorithm (GEF), and a standard linear fusion algorithm (LF) were used on preoperative MRI and post-implantation CT scans. Vertical correction of virtual contact positions, total virtual contact shift, corrections of midline shift and brain shifts due to pneumencephalus were measured. RESULTS: Both AEF and GEF worked well with all 5 cases. An average midline shift of 1.7mm (SD 1.25) was corrected to 0.4mm (SD 0.8) after AEF and to 0.0mm (SD 0) after GEF. Median virtual distances between contacts and cortical surface were corrected by a significant amount, from 2.3mm after LF to 0.0mm after AEF and GEF (p<.001). Mean total relative corrections of 3.1 mm (SD 1.85) after AEF and 3.0mm (SD 1.77) after GEF were achieved. The tested version of GEF did not achieve a satisfying virtual correction of pneumencephalus. CONCLUSION: The technique provided a clear improvement in fusion of pre- and post-implantation scans, although the accuracy is difficult to evaluate.

Localization of Epileptogenic Zone on Pre-surgical Intracranial EEG Recordings: Toward a Validation of Quantitative Signal Analysis Approaches

In patients diagnosed with pharmaco-resistant epilepsy, cerebral areas responsible for seizure generation can be defined by performing implantation of intracranial electrodes. The identification of the epileptogenic zone (EZ) is based on visual inspection of the intracranial electroencephalogram (IEEG) performed by highly qualified neurophysiologists. New computer-based quantitative EEG analyses have been developed in collaboration with the signal analysis community to expedite EZ detection. The aim of the present report is to compare different signal analysis approaches developed in four different European laboratories working in close collaboration with four European Epilepsy Centers. Computer-based signal analysis methods were retrospectively applied to IEEG recordings performed in four patients undergoing pre-surgical exploration of pharmaco-resistant epilepsy. The four methods elaborated by the different teams to identify the EZ are based either on frequency analysis, on nonlinear signal analysis, on connectivity measures or on statistical parametric mapping of epileptogenicity indices. All methods converge on the identification of EZ in patients that present with fast activity at seizure onset. When traditional visual inspection was not successful in detecting EZ on IEEG, the different signal analysis methods produced highly discordant results. Quantitative analysis of IEEG recordings complement clinical evaluation by contributing to the study of epileptogenic networks during seizures. We demonstrate that the degree of sensitivity of different computer-based methods to detect the EZ in respect to visual EEG inspection depends on the specific seizure pattern.

Accuracy of the withdrawal reflex for localization of the site of cervical disk herniation in dogs: 35 cases (2004-2007)

OBJECTIVE To evaluate the accuracy of neurologic examination versus magnetic resonance imaging (MRI) in localization of cervical disk herniation and evaluate the usefulness of withdrawal reflex testing in dogs. DESIGN Retrospective case series. ANIMALS 35 client-owned dogs with a single-level cervical disk herniation as determined via MRI. PROCEDURES 1 of 2 board-certified neurologists performed a complete neurologic examination in each dog. Clinical signs of a cervical lesion included evidence of neck pain and tetraparesis. The withdrawal reflex was used for neuroanatomic localization (C1-C5 or C6-T2). Agreement between results of neurologic and MRI examinations was determined. RESULTS Agreement between neurologic and MRI diagnoses was 65.8%. In 11 dogs in which the lesion was clinically localized to the C6-T2 segment on the basis of a decreased withdrawal reflex in the forelimbs, MRI revealed an isolated C1-C5 disk lesion. In 1 dog, in which the lesion was suspected to be at the C1-C5 level, MRI revealed a C6-T2 lesion. Cranial cervical lesions were significantly associated with an incorrect neurologic diagnosis regarding site of the lesion. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that the withdrawal reflex in dogs with cervical disk herniation is not reliable for determining the affected site and that a decreased withdrawal reflex does not always indicate a lesion from C6 to T2.

Microfabric memory of vein quartz for strain localization in detachment faults: A case study on the Simplon fault zone

This manuscript deals with the adaptation of quartz-microfabrics to changing physical deformation conditions, and discusses their preservation potential during subsequent retrograde deformation. Using microstructural analysis, a sequence of recrystallization processes in quartz, ranging from Grain-Boundary Migration Recrystallization (GBM) over Subgrain-Rotation Recrystallization (SGR) to Bulging Nucleation (BLG) is detected for the Simplon fault zone (SFZ) from the low strain rim towards the internal high strain part of the large-scale shear zone. Based on: (i) the retrograde cooling path; (ii) estimates of deformation temperatures; and (iii) spatial variation of dynamic recrystallization processes and different microstructural characteristics, continuous strain localization with decreasing temperature is inferred. In contrast to the recrystallization microstructures, crystallographic preferred orientations (CPO) have a longer memory. CPO patterns indicative of prism and rhomb glide systems in mylonitic quartz veins, overprinted at low temperatures (�400 �C), suggest inheritance of a high-temperature deformation. In this way, microstructural, textural and geochemical analyses provide information for several million years of the deformation history. The reasons for such incomplete resetting of the rock texture is that strain localization is caused by change in effective viscosity contrasts related to temporal large- and small-scale temperature changes during the evolution of such a long-lived shear zone. The spatially resolved, quantitative investigation of quartz microfabrics and associated recrystallization processes therefore provide great potential for an improved understanding of the geodynamics of large-scale shear zones.

Localization of impacted maxillary canines and root resorption of neighbouring teeth: a study assessing the diagnostic value of panoramic radiographs in two groups of observers.

OBJECTIVES To assess the diagnostic value of panoramic views (2D) of patients with impacted maxillary canines by a group of trained orthodontists and oral surgeons, and to quantify the subjective need and reasons for further three-dimensional (3D) imaging. MATERIALS AND METHODS The study comprises 60 patients with panoramic radiographs (2D) and cone beam computed tomography (CBCT) scans (3D), and a total of 72 impacted canines. Data from a standardized questionnaire were compared within (intragroup) and between (intergroup) a group of orthodontists and oral surgeons to assess possible correlations and differences. Furthermore, the questionnaire data were compared with the findings from the CBCT scans to estimate the correlation within and between the two specialties. Finally, the need and reasons for further 3D imaging was analysed for both groups. RESULTS When comparing questionnaire data with the analysis of the respective CBCT scans, orthodontists showed probability (Pr) values ranging from 0.443 to 0.943. Oral surgeons exhibited Pr values from 0.191 to 0.946. Statistically significant differences were found for the labiopalatal location of the impacted maxillary canine (P = 0.04), indicating a higher correlation in the orthodontist group. The most frequent reason mentioned for the further need of 3D analysis was the labiopalatal location of the impacted canines. Oral surgeons were more in favour of performing further 3D imaging (P = 0.04). CONCLUSIONS Orthodontists were more likely to diagnose the exact labiopalatal position of impacted maxillary canines when using panoramic views only. Generally, oral surgeons more often indicated the need for further 3D imaging.

Zazen meditation and no-task resting EEG compared with LORETA intracortical source localization

Meditation is a self-induced and willfully initiated practice that alters the state of consciousness. The meditation practice of Zazen, like many other meditation practices, aims at disregarding intrusive thoughts while controlling body posture. It is an open monitoring meditation characterized by detached moment-to-moment awareness and reduced conceptual thinking and self-reference. Which brain areas differ in electric activity during Zazen compared to task-free resting? Since scalp electroencephalography (EEG) waveforms are reference-dependent, conclusions about the localization of active brain areas are ambiguous. Computing intracerebral source models from the scalp EEG data solves this problem. In the present study, we applied source modeling using low resolution brain electromagnetic tomography (LORETA) to 58-channel scalp EEG data recorded from 15 experienced Zen meditators during Zazen and no-task resting. Zazen compared to no-task resting showed increased alpha-1 and alpha-2 frequency activity in an exclusively right-lateralized cluster extending from prefrontal areas including the insula to parts of the somatosensory and motor cortices and temporal areas. Zazen also showed decreased alpha and beta-2 activity in the left angular gyrus and decreased beta-1 and beta-2 activity in a large bilateral posterior cluster comprising the visual cortex, the posterior cingulate cortex and the parietal cortex. The results include parts of the default mode network and suggest enhanced automatic memory and emotion processing, reduced conceptual thinking and self-reference on a less judgmental, i.e., more detached moment-to-moment basis during Zazen compared to no-task resting.