Compartmentalized expression of kallikrein 4 (KLK4/hK4) isoforms in prostate cancer: nuclear, cytoplasmic and secreted forms

The prostate-specific antigen-related serine protease gene, kallikrein 4 (KLK4), is expressed in the prostate and, more importantly, overexpressed in prostate cancer. Several KLK4 mRNA splice variants have been reported, but it is still not clear which of these is most relevant to prostate cancer. Here we report that, in addition to the full-length KLK4 (KLK4-254) transcript, the exon 1 deleted KLK4 transcripts, in particular, the 5'-truncated KLK4-205 transcript, is expressed in prostate cancer. Using V5/His6 and green fluorescent protein (GFP) carboxy terminal tagged expression constructs and immunocytochemical approaches, we found that hK4-254 is cytoplasmically localized, while the N-terminal truncated hK4-205 is in the nucleus of transfected PC-3 prostate cancer cells. At the protein level, using anti-hK4 peptide antibodies specific to different regions of hK4-254 (N-terminal and C-terminal), we also demonstrated that endogenous hK4-254 (detected with the N-terminal antibody) is more intensely stained in malignant cells than in benign prostate cells, and is secreted into seminal fluid. In contrast, for the endogenous nuclear-localized N-terminal truncated hK4-205 form, there was less difference in staining intensity between benign and cancer glands. Thus, KLK4-254/hK4-254 may have utility as an immunohistochemical marker for prostate cancer. Our studies also indicate that the expression levels of the truncated KLK4 transcripts, but not KLK4-254, are regulated by androgens in LNCaP cells. Thus, these data demonstrate that there are two major isoforms of hK4 (KLK4-254/hK4-254 and KLK4-205/hK4-205) expressed in prostate cancer with different regulatory and expression profiles that imply both secreted and novel nuclear roles.

Molecular subtyping of feline immunodeficiency virus from domestic cats in Australia

Objective To determine the prevalent subtypes of feline immunodeficiency virus (FIV) present in the domestic cat population of Australia. Method Blood samples were collected from 41 FIV antibody positive cats from four cities across Australia. Following DNA extraction, polymerase chain reaction (PCR) was performed to amplify the variable V3-V5 region of the envelope (env) gene. Genotypes were assessed by direct sequencing of PCR products and comparison with previously reported FIV sequences. Phylogenetic analysis allowed classification of the Australian sequences into the appropriate subtype. Results Of the 41 FIV samples, 40 were found to cluster with previously reported subtype A isolates, whilst the remaining sample grouped within subtype B. Conclusions Subtype A was found to be the predominant FIV subtype present in Australia, although subtype B was also found. These results broaden our knowledge of the genetic diversity of FIV and the associated implications for preventative, diagnostic and therapeutic approaches.

Phylogenetic analysis to define feline immunodeficiency virus subtypes in 31 domestic cats in South Africa

Feline immunodeficiency virus (FIV), a lentivirus, is an important pathogen of domestic cats around the world and has many similarities to human immunodeficiency virus (HIV). A characteristic of these lentiviruses is their extensive genetic diversity which has been an obstacle in the development of successful vaccines. Of the FIV genes, the envelope gene is the most variable and sequence differences in a portion of this gene have been used to define 5 FIV subtypes (A, B, C, D and E). In this study, the proviral DNA sequence of the V3-V5 region of the envelope gene was determined in blood samples from 31 FIV positive cats from 4 different regions of South Africa. Phylogenetic analysis demonstrated the presence of both subtypes A and C, with subtype A predominating. These findings contribute to the understanding of the genetic diversity of FIV

Transcriptome analysis of a respiratory Saccharomyces cerevisiae strain suggests the expression of its phenotype is glucose insensitive and predominantly controlled by Hap4, Cat8 and Mig1

BACKGROUND: We previously described the first respiratory Saccharomyces cerevisiae strain, KOY.TM6*P, by integrating the gene encoding a chimeric hexose transporter, Tm6*, into the genome of an hxt null yeast. Subsequently we transferred this respiratory phenotype in the presence of up to 50 g/L glucose to a yeast strain, V5 hxt1-7Delta, in which only HXT1-7 had been deleted. In this study, we compared the transcriptome of the resultant strain, V5.TM6*P, with that of its wild-type parent, V5, at different glucose concentrations. RESULTS: cDNA array analyses revealed that alterations in gene expression that occur when transitioning from a respiro-fermentative (V5) to a respiratory (V5.TM6*P) strain, are very similar to those in cells undergoing a diauxic shift. We also undertook an analysis of transcription factor binding sites in our dataset by examining previously-published biological data for Hap4 (in complex with Hap2, 3, 5), Cat8 and Mig1, and used this in combination with verified binding consensus sequences to identify genes likely to be regulated by one or more of these. Of the induced genes in our dataset, 77% had binding sites for the Hap complex, with 72% having at least two. In addition, 13% were found to have a binding site for Cat8 and 21% had a binding site for Mig1. Unexpectedly, both the up- and down-regulation of many of the genes in our dataset had a clear glucose dependence in the parent V5 strain that was not present in V5.TM6*P. This indicates that the relief of glucose repression is already operable at much higher glucose concentrations than is widely accepted and suggests that glucose sensing might occur inside the cell. CONCLUSION: Our dataset gives a remarkably complete view of the involvement of genes in the TCA cycle, glyoxylate cycle and respiratory chain in the expression of the phenotype of V5.TM6*P. Furthermore, 88% of the transcriptional response of the induced genes in our dataset can be related to the potential activities of just three proteins: Hap4, Cat8 and Mig1. Overall, our data support genetic remodelling in V5.TM6*P consistent with a respiratory metabolism which is insensitive to external glucose concentrations.

Optic flow in human vision: MEG reveals a foveo-fugal bias in V1, specialization for spiral space in hMSTs, and global motion sensitivity in the IPS

Abstract We recorded MEG responses from 17 participants viewing random-dot patterns simulating global optic flow components (expansion, contraction, rotation, deformation, and translation) and a random motion control condition. Theta-band (3–7 Hz), MEG signal power was greater for expansion than the other optic flow components in a region concentrated along the calcarine sulcus, indicating an ecologically valid, foveo-fugal bias for unidirectional motion sensors in V1. When the responses to the optic flow components were combined, a decrease in MEG beta-band (17–23 Hz) power was found in regions extending beyond the calcarine sulcus to the posterior parietal lobe (inferior to IPS), indicating the importance of structured motion in this region. However, only one cortical area, within or near the V5/hMT+ complex, responded to all three spiral-space components (expansion, contraction, and rotation) and showed no selectivity for global translation or deformation: we term this area hMSTs. This is the first demonstration of an exclusive region for spiral space in the human brain and suggests a functional role better suited to preliminary analysis of ego-motion than surface pose, which would involve deformation. We also observed that the rotation condition activated the cerebellum, suggesting its involvement in visually mediated control of postural adjustment.

The investigation of oscillatory changes in the visual cortex using synthetic aperture magnetometry

This thesis is an exploration of the oscillatory changes occurring in the visual cortex as measured by a functional imaging technique known as Synthetic Aperture Magnetometry (SAM), and how these compare to the BOLD response, across a number of different experimental paradigms. In chapter one the anatomy and physiology of the visual pathways and cortex are outlined, introducing the reader to structures and terms used throughout the thesis whilst chapter two introduces both the technology and analysis techniques required to record MEG and fMRI and also outlines the theory behind SAM. In chapter three the temporal frequency tuning of both striate and extrastriate cortex is investigated, showing fundamental differences in both tuning characteristics and oscillatory power changes between the two areas. Chapter four introduces the concept of implied-motion and investigates the role of area V5 / MT in the perception of such stimuli and shows, for the first time, the temporal evolution of the response in this area. Similarly a close link is shown between the early evoked potential, produced by the stimulus, and previous BOLD responses. Chapter five investigates the modulation of cortical oscillations to both shifts in attention and varying stimulus contrast. It shows that there are both induced and evoked modulation changes with attention, consistent with areas previously known to show BOLD responses. Chapter six involves a direct comparison of cortical oscillatory changes with those of the BOLD response in relation to the parametric variation of a motion coherence stimulus. It is shown that various cortical areas show a linear BOLD response to motion coherence and, for the first time, that both induced oscillatory and evoked activity also vary linearly in areas coincidental with the BOLD response. The final chapter is a summary of the main conclusions and suggests further work.

Cortical activity during rotational and linear transformations

Neuroimaging studies of cortical activation during image transformation tasks have shown that mental rotation may rely on similar brain regions as those underlying visual perceptual mechanisms. The V5 complex, which is specialised for visual motion, is one region that has been implicated. We used functional magnetic resonance imaging (fMRI) to investigate rotational and linear transformation of stimuli. Areas of significant brain activation were identified for each of the primary mental transformation tasks in contrast to its own perceptual reference task which was cognitively matched in all respects except for the variable of interest. Analysis of group data for perception of rotational and linear motion showed activation in areas corresponding to V5 as defined in earlier studies. Both rotational and linear mental transformations activated Brodman Area (BA) 19 but did not activate V5. An area within the inferior temporal gyrus, representing an inferior satellite area of V5, was activated by both the rotational perception and rotational transformation tasks, but showed no activation in response to linear motion perception or transformation. The findings demonstrate the extent to which neural substrates for image transformation and perception overlap and are distinct as well as revealing functional specialisation within perception and transformation processing systems.

Engineering of a novel Saccharomyces cerevisiae wine strain with a respiratory phenotype at high external glucose concentrations

The recently described respiratory strain Saccharomyces cerevisiae KOY.TM6*P is, to our knowledge, the only reported strain of S. cerevisiae which completely redirects the flux of glucose from ethanol fermentation to respiration, even at high external glucose concentrations (27). In the KOY.TM6*P strain, portions of the genes encoding the predominant hexose transporter proteins, Hxt1 and Hxt7, were fused within the regions encoding transmembrane (TM) domain 6. The resulting chimeric gene, TM6*. encoded a chimera composed of the amino-terminal half of Hxt1 and the carboxy-terminal half of Hxt7. It was subsequently integrated into the genome of an hxt null strain. In this study, we have demonstrated the transferability of this respiratory phenotype to the V5 hxt1-7Δ strain, a derivative of a strain used in enology. We also show by using this mutant that it is not necessary to transform a complete hxt null strain with the TM6* construct to obtain a nonethanol-producing phenotype. The resulting V5.TM6*P strain, obtained by transformation of the V5 hxt1-7Δ strain with the TM6* chimeric gene, produced only minor amounts of ethanol when cultured on external glucose concentrations as high as 5%. Despite the fact that glucose flux was reduced to 30% in the V5.TM6*P strain compared with that of its parental strain, the V5.TM6*P strain produced biomass at a specific rate as high as 85% that of the V5 wild-type strain. Even more relevant for the potential use of such a strain for the production of heterologous proteins and also of low-alcohol beverages is the observation that the biomass yield increased 50% with the mutant compared to its parental strain. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Dyslexia research at the turn of the 21st century:behavioural, neuroimaging and genetic findings

Behavioural studies have shown that dyslexics are a heterogeneous population and between-group comparisons are thus inadequate. Some subjects do not develop dyslexia despite having a deficit implicated in this disorder, which points to protective factors. Dyslexia co-occurs with ADHD, DCD, SLI, and SSD, so that future behavioural studies will need to screen and/or statistically control for other disorders. Studies of multiple cases of DPs with other developmental disorders are necessary. Neuroimaging findings show structural and/or functional brain abnormalities in language areas, V5/MT and the cerebellum. Future neuroimaging studies need to investigate the whole reading network and multiple cases. Six dyslexia risk genes have been found, mostly involved in neural migration, which may suggest dyslexia is a deficit of neuronal migration. However, it is not clear how these genes can restrict migration to specific brain areas. As a complex and heterogeneous disorder, dyslexia is likely to be associated with several mutated genes. ADHD and SSD are characterised by genetic risk factors which are partially shared with dyslexia, resulting in comorbidity. Future genetic studies need to focus on identifying other risk genes and pleiotropic genes involved in comorbidities, and linking genotypes implicated in dyslexia with brain structure. Any theory of dyslexia needs to take into account a multitude of risk and protective factors across behavioural, neural and genetic domains.

Crisis and Welfare Society in Spain

Some arguments are briefly presented about the negative consequences of the deep global economic and financial crisis of 2008 on the economic activity and the social situation in Spain. Reformulation, sustainability and financial viability of social welfare in Spain require a new management through resource efficiency, increasing market presence and initiative of stakeholders as a whole. In this sense, the main credible argument of the welfare social in Spain depends on a new perspective on socialization and generosity of social protection system. Specifically, the solution to the crisis must come through economic growth, increased productivity, employment and competitiveness and not by the way of increasing levels of social protection.

Vanadate conformation variations in vanadium pentoxide nanostructures

We report the comparative structural-vibrational study of nanostructures of nanourchins, nanotubes, and nanorods of vanadium oxide. The tube walls comprise layers of vanadium oxide with the organic surfactant intercalated between atomic layers. Both Raman scattering and infrared spectroscopies showed that the structure of nanourchins, nanotubes, and nanorods of vanadium oxide nanocomposite are strongly dependent on the valency of the vanadium, its associated interactions with the organic surfactant template, and on the packing mechanism and arrangement of the surfactant between vanadate layers. Accurate assignment of the vibrational modes to the V-O coordinations has allowed their comparative classification and relation to atomic layer structure. Although all structures are formed from the same precursor, differences in vanadate conformations due to the hydrothermal treatment and surfactant type result in variable degrees of crystalline order in the final nanostructure. The nanotube-containing nanourchins contain vanadate layers in the nanotubes that are in a distorted γ- V5+ conformation, whereas the the nanorods, by comparison, show evidence for V5+ and V4+ species-containing ordered VOx lamina.

Comparative structural-vibrational study of nano-urchin and nanorods of vanadium oxide

We present a comparative structural–vibrational study of nanostructured systems of V2O5: nano-urchin (VONURs) which are spherical structures composed of a radially oriented array of VOx nanotubes (VOx-NTs) with a volumetric density of ∼40 sr–1, and vanadium oxide nanorods (VOx-NRDs) with an average length of ∼100 nm. The Raman scattering spectrum of the nano-urchin exhibits a band at 1014 cm–1 related to the distorted gamma conformation of the vanadium pentoxide (γ-V5+). The infrared vibrational spectra of the nanorods sample also exhibit a distorted laminar V2O5 structure with evidence observed for quadravalent V4+ species at 921 cm–1.

Development and Testing of An Automatic Lung IMRT Planning Algorithm

Knowledge-based radiation treatment is an emerging concept in radiotherapy. It

mainly refers to the technique that can guide or automate treatment planning in

clinic by learning from prior knowledge. Dierent models are developed to realize

it, one of which is proposed by Yuan et al. at Duke for lung IMRT planning. This

model can automatically determine both beam conguration and optimization ob-

jectives with non-coplanar beams based on patient-specic anatomical information.

Although plans automatically generated by this model demonstrate equivalent or

better dosimetric quality compared to clinical approved plans, its validity and gener-

ality are limited due to the empirical assignment to a coecient called angle spread

constraint dened in the beam eciency index used for beam ranking. To eliminate

these limitations, a systematic study on this coecient is needed to acquire evidences

for its optimal value.

To achieve this purpose, eleven lung cancer patients with complex tumor shape

with non-coplanar beams adopted in clinical approved plans were retrospectively

studied in the frame of the automatic lung IMRT treatment algorithm. The primary

and boost plans used in three patients were treated as dierent cases due to the

dierent target size and shape. A total of 14 lung cases, thus, were re-planned using

the knowledge-based automatic lung IMRT planning algorithm by varying angle

spread constraint from 0 to 1 with increment of 0.2. A modied beam angle eciency

index used for navigate the beam selection was adopted. Great eorts were made to assure the quality of plans associated to every angle spread constraint as good

as possible. Important dosimetric parameters for PTV and OARs, quantitatively

re

ecting the plan quality, were extracted from the DVHs and analyzed as a function

of angle spread constraint for each case. Comparisons of these parameters between

clinical plans and model-based plans were evaluated by two-sampled Students t-tests,

and regression analysis on a composite index built on the percentage errors between

dosimetric parameters in the model-based plans and those in the clinical plans as a

function of angle spread constraint was performed.

Results show that model-based plans generally have equivalent or better quality

than clinical approved plans, qualitatively and quantitatively. All dosimetric param-

eters except those for lungs in the automatically generated plans are statistically

better or comparable to those in the clinical plans. On average, more than 15% re-

duction on conformity index and homogeneity index for PTV and V40, V60 for heart

while an 8% and 3% increase on V5, V20 for lungs, respectively, are observed. The

intra-plan comparison among model-based plans demonstrates that plan quality does

not change much with angle spread constraint larger than 0.4. Further examination

on the variation curve of the composite index as a function of angle spread constraint

shows that 0.6 is the optimal value that can result in statistically the best achievable

plans.

Accuracy of computer-aided design/computer-assisted manufacture (CAD/CAM) fabricated dental restorations: a comparative study

Introduction: Computer-Aided-Design (CAD) and Computer-Aided-Manufacture (CAM) has been developed to fabricate fixed dental restorations accurately, faster and improve cost effectiveness of manufacture when compared to the conventional method. Two main methods exist in dental CAD/CAM technology: the subtractive and additive methods. While fitting accuracy of both methods has been explored, no study yet has compared the fabricated restoration (CAM output) to its CAD in terms of accuracy. The aim of this present study was to compare the output of various dental CAM routes to a sole initial CAD and establish the accuracy of fabrication. The internal fit of the various CAM routes were also investigated. The null hypotheses tested were: 1) no significant differences observed between the CAM output to the CAD and 2) no significant differences observed between the various CAM routes. Methods: An aluminium master model of a standard premolar preparation was scanned with a contact dental scanner (Incise, Renishaw, UK). A single CAD was created on the scanned master model (InciseCAD software, V2.5.0.140, UK). Twenty copings were then fabricated by sending the single CAD to a multitude of CAM routes. The copings were grouped (n=5) as: Laser sintered CoCrMo (LS), 5-axis milled CoCrMo (MCoCrMo), 3-axis milled zirconia (ZAx3) and 4-axis milled zirconia (ZAx4). All copings were micro-CT scanned (Phoenix X-Ray, Nanotom-S, Germany, power: 155kV, current: 60µA, 3600 projections) to produce 3-Dimensional (3D) models. A novel methodology was created to superimpose the micro-CT scans with the CAD (GOM Inspect software, V7.5SR2, Germany) to indicate inaccuracies in manufacturing. The accuracy in terms of coping volume was explored. The distances from the surfaces of the micro-CT 3D models to the surfaces of the CAD model (CAD Deviation) were investigated after creating surface colour deviation maps. Localised digital sections of the deviations (Occlusal, Axial and Cervical) and selected focussed areas were then quantitatively measured using software (GOM Inspect software, Germany). A novel methodology was also explored to digitally align (Rhino software, V5, USA) the micro-CT scans with the master model to investigate internal fit. Fifty digital cross sections of the aligned scans were created. Point-to-point distances were measured at 5 levels at each cross section. The five levels were: Vertical Marginal Fit (VF), Absolute Marginal Fit (AM), Axio-margin Fit (AMF), Axial Fit (AF) and Occlusal Fit (OF). Results: The results of the volume measurement were summarised as: VM-CoCrMo (62.8mm3 ) > VZax3 (59.4mm3 ) > VCAD (57mm3 ) > VZax4 (56.1mm3 ) > VLS (52.5mm3 ) and were all significantly different (p presented as areas with different colour. No significant differences were observed at the internal aspect of the cervical aspect between all groups of copings. Significant differences (p< M-CoCrMo Internal Occlusal, Internal Axial and External Axial 2 ZAx3 > ZAx4 External Occlusal, External Cervical 3 ZAx3 < ZAx4 Internal Occlusal 4 M-CoCrMo > ZAx4 Internal Occlusal and Internal Axial The mean values of AMF and AF were significantly (p M-CoCrMo and CAD > ZAx4. Only VF of M-CoCrMo was comparable with the CAD Internal Fit. All VF and AM values were within the clinically acceptable fit (120µm). Conclusion: The investigated CAM methods reproduced the CAD accurately at the internal cervical aspect of the copings. However, localised deviations at axial and occlusal aspects of the copings may suggest the need for modifications in these areas prior to fitting and veneering with porcelain. The CAM groups evaluated also showed different levels of Internal Fit thus rejecting the null hypotheses. The novel non-destructive methodologies for CAD/CAM accuracy and internal fit testing presented in this thesis may be a useful evaluation tool for similar applications.

Parametric CAD model based shape optimization using adjoint functions

Adjoint methods have proven to be an efficient way of calculating the gradient of an objective function with respect to a shape parameter for optimisation, with a computational cost nearly independent of the number of the design variables [1]. The approach in this paper links the adjoint surface sensitivities (gradient of objective function with respect to the surface movement) with the parametric design velocities (movement of the surface due to a CAD parameter perturbation) in order to compute the gradient of the objective function with respect to CAD variables.
For a successful implementation of shape optimization strategies in practical industrial cases, the choice of design variables or parameterisation scheme used for the model to be optimized plays a vital role. Where the goal is to base the optimization on a CAD model the choices are to use a NURBS geometry generated from CAD modelling software, where the position of the NURBS control points are the optimisation variables [2] or to use the feature based CAD model with all of the construction history to preserve the design intent [3]. The main advantage of using the feature based model is that the optimized model produced can be directly used for the downstream applications including manufacturing and process planning.
This paper presents an approach for optimization based on the feature based CAD model, which uses CAD parameters defining the features in the model geometry as the design variables. In order to capture the CAD surface movement with respect to the change in design variable, the “Parametric Design Velocity” is calculated, which is defined as the movement of the CAD model boundary in the normal direction due to a change in the parameter value.
The approach presented here for calculating the design velocities represents an advancement in terms of capability and robustness of that described by Robinson et al. [3]. The process can be easily integrated to most industrial optimisation workflows and is immune to the topology and labelling issues highlighted by other CAD based optimisation processes. It considers every continuous (“real value”) parameter type as an optimisation variable, and it can be adapted to work with any CAD modelling software, as long as it has an API which provides access to the values of the parameters which control the model shape and allows the model geometry to be exported. To calculate the movement of the boundary the methodology employs finite differences on the shape of the 3D CAD models before and after the parameter perturbation. The implementation procedure includes calculating the geometrical movement along a normal direction between two discrete representations of the original and perturbed geometry respectively. Parametric design velocities can then be directly linked with adjoint surface sensitivities to extract the gradients to use in a gradient-based optimization algorithm.
The optimisation of a flow optimisation problem is presented, in which the power dissipation of the flow in an automotive air duct is to be reduced by changing the parameters of the CAD geometry created in CATIA V5. The flow sensitivities are computed with the continuous adjoint method for a laminar and turbulent flow [4] and are combined with the parametric design velocities to compute the cost function gradients. A line-search algorithm is then used to update the design variables and proceed further with optimisation process.