Non-combinatorial library screening reveals subsite cooperativity and identifies new high-efficiency substrates for kallikrein-related peptidase 14

An array of substrates link the tryptic serine protease, kallikrein-related peptidase 14 (KLK14), to physiological functions including desquamation and activation of signaling molecules associated with inflammation and cancer. Recognition of protease cleavage sequences is driven by complementarity between exposed substrate motifs and the physicochemical signature of an enzyme's active site cleft. However, conventional substrate screening methods have generated conflicting subsite profiles for KLK14. This study utilizes a recently developed screening technique, the sparse matrix library, to identify five novel high-efficiency sequences for KLK14. The optimal sequence, YASR, was cleaved with higher efficiency (k(cat)/K(m)=3.81 ± 0.4 × 10(6) M(-1) s(-1)) than favored substrates from positional scanning and phage display by 2- and 10-fold, respectively. Binding site cooperativity was prominent among preferred sequences, which enabled optimal interaction at all subsites as indicated by predictive modeling of KLK14/substrate complexes. These simulations constitute the first molecular dynamics analysis of KLK14 and offer a structural rationale for the divergent subsite preferences evident between KLK14 and closely related KLKs, KLK4 and KLK5. Collectively, these findings highlight the importance of binding site cooperativity in protease substrate recognition, which has implications for discovery of optimal substrates and engineering highly effective protease inhibitors.

In the pursuit of effective affective computing : the relationship between features and registration

For facial expression recognition systems to be applicable in the real world, they need to be able to detect and track a previously unseen person's face and its facial movements accurately in realistic environments. A highly plausible solution involves performing a "dense" form of alignment, where 60-70 fiducial facial points are tracked with high accuracy. The problem is that, in practice, this type of dense alignment had so far been impossible to achieve in a generic sense, mainly due to poor reliability and robustness. Instead, many expression detection methods have opted for a "coarse" form of face alignment, followed by an application of a biologically inspired appearance descriptor such as the histogram of oriented gradients or Gabor magnitudes. Encouragingly, recent advances to a number of dense alignment algorithms have demonstrated both high reliability and accuracy for unseen subjects [e.g., constrained local models (CLMs)]. This begs the question: Aside from countering against illumination variation, what do these appearance descriptors do that standard pixel representations do not? In this paper, we show that, when close to perfect alignment is obtained, there is no real benefit in employing these different appearance-based representations (under consistent illumination conditions). In fact, when misalignment does occur, we show that these appearance descriptors do work well by encoding robustness to alignment error. For this work, we compared two popular methods for dense alignment-subject-dependent active appearance models versus subject-independent CLMs-on the task of action-unit detection. These comparisons were conducted through a battery of experiments across various publicly available data sets (i.e., CK+, Pain, M3, and GEMEP-FERA). We also report our performance in the recent 2011 Facial Expression Recognition and Analysis Challenge for the subject-independent task.

The relationship between cognitive ability, insight and self-regulatory behaviors: Findings from the older driver population

Self-regulation is often promoted as a coping strategy that may allow older drivers to drive safely for longer. Self-regulation depends upon drivers making an accurate assessment of their own ability and having a willingness to practice self-regulatory behaviors to compensate for changes in ability. The current study explored the relationship between older drivers’ cognitive ability, their driving confidence and their use of self-regulation. An additional study aim was to explore the relationship between these factors and older drivers’ interest in driving programs. Seventy Australian drivers aged 65 years and over completed a questionnaire about their driving and a brief screening measure of cognitive ability (an untimed Clock Drawing Test). While all participants reported high levels of confidence regarding their driving ability, and agreed that they would continue driving in the foreseeable future, a notable proportion performed poorly on the Clock Drawing Test. Compared to older drivers who successfully completed the Clock Drawing Test, those who failed the cognitive test were significantly less likely to report driving self-regulation, and showed significantly less interest in being involved in driving programs. Older drivers with declining cognitive abilities may not be self-regulating their driving. This group also appears to be unlikely to self-refer to driving programs.

SeqSLAM : visual route-based navigation for sunny summer days and stormy winter nights

Learning and then recognizing a route, whether travelled during the day or at night, in clear or inclement weather, and in summer or winter is a challenging task for state of the art algorithms in computer vision and robotics. In this paper, we present a new approach to visual navigation under changing conditions dubbed SeqSLAM. Instead of calculating the single location most likely given a current image, our approach calculates the best candidate matching location within every local navigation sequence. Localization is then achieved by recognizing coherent sequences of these “local best matches”. This approach removes the need for global matching performance by the vision front-end - instead it must only pick the best match within any short sequence of images. The approach is applicable over environment changes that render traditional feature-based techniques ineffective. Using two car-mounted camera datasets we demonstrate the effectiveness of the algorithm and compare it to one of the most successful feature-based SLAM algorithms, FAB-MAP. The perceptual change in the datasets is extreme; repeated traverses through environments during the day and then in the middle of the night, at times separated by months or years and in opposite seasons, and in clear weather and extremely heavy rain. While the feature-based method fails, the sequence-based algorithm is able to match trajectory segments at 100% precision with recall rates of up to 60%.

A Kallikrein 15 (KLK15) single nucleotide polymorphism located close to a novel exon shows evidence of association with poor ovarian cancer survival

KLK15 over-expression is reported to be a significant predictor of reduced progression-free survival and overall survival in ovarian cancer. Our aim was to analyse the KLK15 gene for putative functional single nucleotide polymorphisms (SNPs) and assess the association of these and KLK15 HapMap tag SNPs with ovarian cancer survival. Results In silico analysis was performed to identify KLK15 regulatory elements and to classify potentially functional SNPs in these regions. After SNP validation and identification by DNA sequencing of ovarian cancer cell lines and aggressive ovarian cancer patients, 9 SNPs were shortlisted and genotyped using the Sequenom iPLEX Mass Array platform in a cohort of Australian ovarian cancer patients (N = 319). In the Australian dataset we observed significantly worse survival for the KLK15 rs266851 SNP in a dominant model (Hazard Ratio (HR) 1.42, 95% CI 1.02-1.96). This association was observed in the same direction in two independent datasets, with a combined HR for the three studies of 1.16 (1.00-1.34). This SNP lies 15bp downstream of a novel exon and is predicted to be involved in mRNA splicing. The mutant allele is also predicted to abrogate an HSF-2 binding site. Conclusions We provide evidence of association for the SNP rs266851 with ovarian cancer survival. Our results provide the impetus for downstream functional assays and additional independent validation studies to assess the role of KLK15 regulatory SNPs and KLK15 isoforms with alternative intracellular functional roles in ovarian cancer survival.

Mastering the canonical loop of serine protease inhibitors : enhancing potency by optimising the internal hydrogen bond network

Background Canonical serine protease inhibitors commonly bind to their targets through a rigid loop stabilised by an internal hydrogen bond network and disulfide bond(s). The smallest of these is sunflower trypsin inhibitor (SFTI-1), a potent and broad-range protease inhibitor. Recently, we re-engineered the contact β-sheet of SFTI-1 to produce a selective inhibitor of kallikrein-related peptidase 4 (KLK4), a protease associated with prostate cancer progression. However, modifications in the binding loop to achieve specificity may compromise structural rigidity and prevent re-engineered inhibitors from reaching optimal binding affinity. Methodology/Principal Findings In this study, the effect of amino acid substitutions on the internal hydrogen bonding network of SFTI were investigated using an in silico screen of inhibitor variants in complex with KLK4 or trypsin. Substitutions favouring internal hydrogen bond formation directly correlated with increased potency of inhibition in vitro. This produced a second generation inhibitor (SFTI-FCQR Asn14) which displayed both a 125-fold increased capacity to inhibit KLK4 (Ki = 0.0386±0.0060 nM) and enhanced selectivity over off-target serine proteases. Further, SFTI-FCQR Asn14 was stable in cell culture and bioavailable in mice when administered by intraperitoneal perfusion. Conclusion/Significance These findings highlight the importance of conserving structural rigidity of the binding loop in addition to optimising protease/inhibitor contacts when re-engineering canonical serine protease inhibitors.

We just make the pictures…?’ How work is portrayed in children’s feature length films

This article explores how adult paid work is portrayed in 'family' feature length films. The study extends previous critical media literature which has overwhelmingly focused on depictions of gender and violence, exploring the visual content of films that is relevant to adult employment. Forty-two G/PG films were analyzed for relevant themes. Consistent with the exploratory nature of the research, themes emerged inductively from the films' content. Results reveal six major themes: males are more visible in adult work roles than women; the division of labour remains gendered; work and home are not mutually exclusive domains; organizational authority and power is wielded in punitive ways; there are avenues to better employment prospects; and status/money is paramount. The findings of the study reflect a range of subject matters related to occupational characteristics and work-related communication and interactions which are typically viewed by children in contemporary society.

Too sleepy to drive : self-perception and regulation of driving when sleepy

Background: Sleepiness is a direct contributor to a substantial proportion of fatal and severe road cashes. A number of technological solutions designed to detect sleepiness have been developed, but self-awareness of increasing sleepiness remains a critical component in on-road strategies for mitigating this risk. In order to take appropriate action when sleepy, drivers’ perceptions of their level of sleepiness must be accurate. Aims: This study aimed to assess capacity to accurately identify sleepiness and self-regulate driving cessation during a validated driving simulator task. Participants: Participants comprised 26 young adult drivers (20-28 years). The drivers had open licenses but no other exclusion criteria where used. Methods: Participants woke at 5am, and took part in a laboratory-based hazard perception driving simulation, either at mid-morning or mid-afternoon. Established physiological measures (including EEG) and subjective measures (sleepiness ratings) previously found sensitive to changes in sleepiness levels were utilised. Participants were instructed to ‘drive’ until they believed that sleepiness had impaired their ability to drive safely. They were then offered a nap opportunity. Results: The mean duration of the drive before cessation was 39 minutes (±18 minutes). Almost all (23/26) of the participants then achieved sleep during the nap opportunity. These data suggest that the participants’ perceptions of sleepiness were specific. However, EEG data from a number of participants suggested very high levels of sleepiness prior to driving cessation, suggesting poor sensitivity. Conclusions: Participants reported high levels of sleepiness while driving after very moderate sleep restriction. They were able to identify increasing sleepiness during the test period, could decide to cease driving and in most cases were sufficiently sleepy to achieve sleep during the daytime session. However, the levels of sleepiness achieved prior to driving cessation suggest poor accuracy in self-perception and regulation. This presents practical issues for the implementation of fatigue and sleep-related strategies to improve driver safety.

Barriers and facilitators to public bicycle scheme use : a qualitative approach

The purpose of this study was to explore barriers and facilitators to using CityCycle, a public bicycle share scheme in Brisbane, Australia. Focus groups were conducted with participants belonging to one of three categories. Group one consisted of infrequent and noncyclists (no bicycle riding over the past month), group two were regular bicycle riders (ridden a bicycle at least once in the past month) and group three was composed of CityCycle members. A thematic analytic method was used to analyse the data. Three main themes were found: Accessibility/spontaneity, safety and weather/topography. The lengthy sign-up process was thought to stifle the spontaneity typically thought to attract people to public bike share. Mandatory helmet legislation was thought to reduce spontaneous use. Safety was a major concern for all groups and this included a perceived lack of suitable bicycle infrastructure, as well as regular riders describing a negative attitude of some car drivers. Interestingly, CityCycle riders unanimously perceived car driver attitudes to improve when on CityCycle bicycles relative to riding on personal bicycles. Conclusions: In order to increase the popularity of the CityCycle scheme, the results of this study suggest that a more accessible, spontaneous sign-up process is required, 24/7 opening hours, and greater incentives to sign up new members and casual users, as seeing people using CityCycle appears critical to further take up.

Identifying familiarity to facilitate intuitive interaction for older adults

Almost every nation on the planet is experiencing increases in both the number and proportion of older adults. Research has shown that older adults use technology less intuitively than younger adults, and have more difficulty with using products effectively. With an ever-increasing population of older adults, it is necessary to understand why they often struggle to use technology, which is becoming more and more important in day to day living. Intuitive use of products is grounded in familiarity and prior experience. The aims of this research were twofold: (i) to examine the differences in familiarity between younger and older adults, to see if this could explain the difficulties faced by some older adults; (ii) to develop investigational methods to assist designers in identifying familiarity in prospective users. Two empirical studies were conducted. The first experiment was conducted in the field with 32 participants, divided across four age groups (18 – 44, 45 – 59, 60 – 74, and 75+). This experiment was conducted in the participants’ homes, with a product they were familiar with. Familiarity was measured through the analysis of data collected through interviews, observation and retrospective protocol. The results of this study show that the youngest group demonstrated significantly higher levels of familiarity with products they own than the 60 – 74 and the 75+ age groups. There were no significant differences between the 18 – 44 age group and the 45 – 59 age group and there were also no significant differences between the three oldest age groups. The second experiment was conducted with 32 participants, across the same four age groups. Four everyday products were used in this experiment. The results of Experiment 2 show that, with previously unused products, younger adults demonstrate significantly higher levels of familiarity than the three older age groups. The three oldest age groups had no significant differences between them. The results of these two studies show that younger adults are more familiar with contemporary products than older adults. They also demonstrate that in terms of familiarity, older adults do not differ significantly as they get older. The results also show that the 45 – 59 age group demonstrate higher levels of familiarity with products they have owned, in comparison with those they have not. The two older age groups did not demonstrate such differences. This suggests that interacting with products over time increases familiarity more for middle-aged adults than for older adults. As a result of this research, a method that can be used by designers to identify potential users’ product familiarity has been identified. This method is easy to use, quick, low cost, highly mobile, flexible, and allows for easy data collection and analysis. A tool has been designed that assists designers and researchers to use the method. Designers can use the knowledge gained from this tool, and integrate it into the design process, resulting in more intuitive products. Such products may lead to improvements in the quality of life of older adults, as a result of improved societal integration, better health management, and more widespread use of communications technology.