Evaluation of silk sericin as a biomaterial: In vitro growth of human corneal limbal epithelial cells on Bombyx mori sericin membranes

Sericin and fibroin are the two major proteins in the silk fibre produced by the domesticated silkworm, Bombyx mori. Fibroin has been extensively investigated as a biomaterial. We have previously shown that fibroin can function successfully as a substratum for growing cells of the eye. Sericin has been so far neglected as a biomaterial because of suspected allergenic activity. However, this misconception has now been dispelled, and sericin’s biocompatibility is currently indisputable. Aiming at promoting sericin as a possible substratum for the growth of corneal cells in order to make tissue-engineered constructs for the restoration of the ocular surface, in this study we investigated the attachment and growth in vitro of human corneal limbal epithelial cells (HLECs) on sericin-based membranes. Sericin was isolated and regenerated from the silkworm cocoons by an aqueous procedure, manufactured into membranes, and characterized (mechanical properties, structural analysis, contact angles). Primary cell cultures from two donors were established in serum-supplemented media in the presence of murine feeder cells. Membranes made of sericin and fibroin-sericin blends were assessed in vitro as substrata for HLECs in a serum-free medium, in a cell attachment assay and in a 3-day cell growth experiment. While the mechanical characteristics of sericin were found to be inferior to those of fibroin, its ability to enhance the attachment of HLECs was significantly superior to fibroin, as revealed by the PicoGreen® assay. Evidence was also obtained that cells can grow and differentiate on these substrata.

A conceptual model to link deep customer insights to both growth opportunities and organisational strategy in SME’s as part of a design led transformation journey

Although Design Led Innovation activities aim to raise the value of design within the business, knowledge about which tools are available to support companies and how to apply them to make the connection between design for new product development and design as a strategic driver of growth is needed. This paper presents a conceptual method to supplement existing process and tools to assist companies to grow through design. The model extends the authors’ previous work to explore how through storytelling, customer observation can be captured and translated into new meaning, then creating new design propositions shaped into product needs, which can drive internal business activities, brand and the strategic vision. The paper contributes to a gap in the theoretical frameworks and literature by highlighting the need to align and scale design processes which match the needs of SME’s as they transition along a trajectory to become design led businesses.

Evaluation of novel Streptococcus pyogenes vaccine candidates incorporating multiple conserved sequences from the C-repeat region of the M-protein

A major challenge for Streptococcus pyogenes vaccine development is the identification of epitopes that confer protection from infection by multiple S. pyogenes M-types. Here we have identified and characterised the distribution of common variant sequences from individual repeat units of the C-repeat region (CRR) of M-proteins representing 77 different M-types. Three polyvalent fusion vaccine candidates (SV1, SV2 and SV3) incorporating the most common variants were subsequently expressed and purified, and demonstrated to be alpha-helical by Circular Dichroism (CD), a secondary conformational characteristic of the CRR in the M-protein. Antibodies raised against each of these constructs recognise M-proteins that vary in their CRR, and bind to the surface of multiple S. pyogenes isolates. Antibodies raised against SV1, containing five variant sequences, also kill heterologous S. pyogenes isolates in in vitro bactericidal assays. Further structural characterisation of this construct demonstrated the conformation of SV1 was stable at different pHs, and thermal unfolding of SV1 a reversible process. Our findings demonstrate that linkage of multiple variant sequences into a single recombinant construct overcomes the need to embed the variant sequences in foreign helix promoting flanking sequences for conformational stability, and demonstrates the viability of the polyvalent candidates as global S. pyogenes vaccine candidates.

Representational change and strategy use in children's number line estimation during the first years of primary school

Background: The objective of this study was to scrutinize number line estimation behaviors displayed by children in mathematics classrooms during the first three years of schooling. We extend existing research by not only mapping potential logarithmic-linear shifts but also provide a new perspective by studying in detail the estimation strategies of individual target digits within a number range familiar to children. Methods: Typically developing children (n = 67) from Years 1 – 3 completed a number-to-position numerical estimation task (0-20 number line). Estimation behaviors were first analyzed via logarithmic and linear regression modeling. Subsequently, using an analysis of variance we compared the estimation accuracy of each digit, thus identifying target digits that were estimated with the assistance of arithmetic strategy. Results: Our results further confirm a developmental logarithmic-linear shift when utilizing regression modeling; however, uniquely we have identified that children employ variable strategies when completing numerical estimation, with levels of strategy advancing with development. Conclusion: In terms of the existing cognitive research, this strategy factor highlights the limitations of any regression modeling approach, or alternatively, it could underpin the developmental time course of the logarithmic-linear shift. Future studies need to systematically investigate this relationship and also consider the implications for educational practice.

Pre-clinical evaluation of amphiphilic silicone oligomers for scar remediation

The formation of hypertrophic scars is a frequent outcome of wound repair and often requires further therapy with treatments such as silicone gel sheets (SGS; Perkins et al., 1983). Although widely used, knowledge regarding SGS and their mechanism of action on hypertrophic scars is limited. Furthermore, SGS require consistent application for at least twelve hours a day for up to twelve consecutive months, beginning as soon as wound reepithelialisation has occurred. Preliminary research at QUT has shown that some species of silicone present in SGS have the ability to permeate into collagen gel skin mimetics upon exposure. An analogue of these species, GP226, was found to decrease both collagen synthesis and the total amount of collagen present following exposure to cultures of cells derived from hypertrophic scars. This silicone of interest was a crude mixture of silicone species, which resolved into five fractions of different molecular weight. These five fractions were found to have differing effects on collagen synthesis and cell viability following exposure to fibroblasts derived from hypertrophic scars (HSF), keloid scars (KF) and normal skin (nHSF and nKF). The research performed herein continues to further assess the potential of GP226 and its fractions for scar remediation by determining in more detail its effects on HSF, KF, nHSF, nKF and human keratinocytes (HK) in terms of cell viability and proliferation at various time points. Through these studies it was revealed that Fraction IV was the most active fraction as it induced a reduction in cell viability and proliferation most similar to that observed with GP226. Cells undergoing apoptosis were also detected in HSF cultures exposed to GP226 and Fraction IV using the Tunel assay (Roche). These investigations were difficult to pursue further as the fractionation process used for GP226 was labour-intensive and time inefficient. Therefore a number of silicones with similar structure to Fraction IV were synthesised and screened for their effect following application to HSF and nHSF. PDMS7-g-PEG7, a silicone-PEG copolymer of low molecular weight and low hydrophilic-lipophilic balance factor, was found to be the most effective at reducing cell proliferation and inducing apoptosis in cultures of HSF, nHSF and HK. Further studies investigated gene expression through microarray and superarray techniques and demonstrated that many genes are differentially expressed in HSF following treatment with GP226, Fraction IV and PDMS7-g-PEG7. In brief, it was demonstrated that genes for TGFβ1 and TNF are not differentially regulated while genes for AIFM2, IL8, NSMAF, SMAD7, TRAF3 and IGF2R show increased expression (>1.8 fold change) following treatment with PDMS7-g-PEG7. In addition, genes for αSMA, TRAF2, COL1A1 and COL3A1 have decreased expression (>-1.8 fold change) following treatment with GP226, Fraction IV and PDMS7-g-PEG7. The data obtained suggest that many different pathways related to apoptosis and collagen synthesis are affected in HSF following exposure to PDMS7-g-PEG7. The significance is that silicone-PEG copolymers, such as GP226, Fraction IV and PDMS7-g-PEG7, could potentially be a non-invasive substitute to apoptosis-inducing chemical agents that are currently used as scar treatments. It is anticipated that these findings will ultimately contribute to the development of a novel scar therapy with faster action and improved outcomes for patients suffering from hypertrophic scars.

Evaluation cortical bone elasticity in response to pulse power excitation using ultrasonic technique

This paper presents the ultrasonic velocity measurement method which investigates the possible effects of high voltage high frequency pulsed power on cortical bone material elasticity. Before applying a pulsed power signal on a live bone, it is essential to determine the safe parameters of pulsed power applied on bone non-destructively. Therefore, the possible changes in cortical bone material elasticity due to a specified pulsed power excitation have been investigated. A controllable positive buck-boost converter with adjustable output voltage and frequency has been used to generate high voltage pulses (500V magnitude at 10 KHz frequency). To determine bone elasticity, an ultrasonic velocity measurement has been conducted on two groups of control (unexposed to pulse power but in the same environmental condition) and cortical bone samples exposed to pulsed power. Young’s modulus of cortical bone samples have been determined and compared before and after applying the pulsed power signal. After applying the high voltage pulses, no significant variation in elastic property of cortical bone specimens was found compared to the control. The result shows that pulsed power with nominated parameters can be applied on cortical bone tissue without any considerable negative effect on elasticity of bone material.

From the margin to the centre : towards an art strategy for the ethnic enclaves in the city

Sunnybank represents a distinctly Australian take on the classic ‘Chinatown’ – or indeed other ethic community enclaves such as ‘little Italy’, ‘little Bombay’, ‘little Athens’ and so on. In the Northern Hemisphere these tended to grow up in the dense working class neighbourhoods of industrial cities, especially in port cities like Liverpool, London, New York and San Francisco. The existing Chinatowns of Sydney and Melbourne, and to some extent Brisbane’s Fortitude Valley, are of this variety. In the late 1970s, with the growth of suburbanisation and the de-industrialisation and consequent dereliction of the ‘inner city’, these ethnic communities were one of the few signs of life in the city. Apart from the daily commute into the CBD, business with the city council or a trip to the big shopping streets these areas were one of the few reasons for visiting city centres stigmatised by urban decay and petty crime.

Rational design of serine protease inhibitors

Proteases regulate a spectrum of diverse physiological processes, and dysregulation of proteolytic activity drives a plethora of pathological conditions. Understanding protease function is essential to appreciating many aspects of normal physiology and progression of disease. Consequently, development of potent and specific inhibitors of proteolytic enzymes is vital to provide tools for the dissection of protease function in biological systems and for the treatment of diseases linked to aberrant proteolytic activity. The studies in this thesis describe the rational design of potent inhibitors of three proteases that are implicated in disease development. Additionally, key features of the interaction of proteases and their cognate inhibitors or substrates are analysed and a series of rational inhibitor design principles are expounded and tested. Rational design of protease inhibitors relies on a comprehensive understanding of protease structure and biochemistry. Analysis of known protease cleavage sites in proteins and peptides is a commonly used source of such information. However, model peptide substrate and protein sequences have widely differing levels of backbone constraint and hence can adopt highly divergent structures when binding to a protease’s active site. This may result in identical sequences in peptides and proteins having different conformations and diverse spatial distribution of amino acid functionalities. Regardless of this, protein and peptide cleavage sites are often regarded as being equivalent. One of the key findings in the following studies is a definitive demonstration of the lack of equivalence between these two classes of substrate and invalidation of the common practice of using the sequences of model peptide substrates to predict cleavage of proteins in vivo. Another important feature for protease substrate recognition is subsite cooperativity. This type of cooperativity is commonly referred to as protease or substrate binding subsite cooperativity and is distinct from allosteric cooperativity, where binding of a molecule distant from the protease active site affects the binding affinity of a substrate. Subsite cooperativity may be intramolecular where neighbouring residues in substrates are interacting, affecting the scissile bond’s susceptibility to protease cleavage. Subsite cooperativity can also be intermolecular where a particular residue’s contribution to binding affinity changes depending on the identity of neighbouring amino acids. Although numerous studies have identified subsite cooperativity effects, these findings are frequently ignored in investigations probing subsite selectivity by screening against diverse combinatorial libraries of peptides (positional scanning synthetic combinatorial library; PS-SCL). This strategy for determining cleavage specificity relies on the averaged rates of hydrolysis for an uncharacterised ensemble of peptide sequences, as opposed to the defined rate of hydrolysis of a known specific substrate. Further, since PS-SCL screens probe the preference of the various protease subsites independently, this method is inherently unable to detect subsite cooperativity. However, mean hydrolysis rates from PS-SCL screens are often interpreted as being comparable to those produced by single peptide cleavages. Before this study no large systematic evaluation had been made to determine the level of correlation between protease selectivity as predicted by screening against a library of combinatorial peptides and cleavage of individual peptides. This subject is specifically explored in the studies described here. In order to establish whether PS-SCL screens could accurately determine the substrate preferences of proteases, a systematic comparison of data from PS-SCLs with libraries containing individually synthesised peptides (sparse matrix library; SML) was carried out. These SML libraries were designed to include all possible sequence combinations of the residues that were suggested to be preferred by a protease using the PS-SCL method. SML screening against the three serine proteases kallikrein 4 (KLK4), kallikrein 14 (KLK14) and plasmin revealed highly preferred peptide substrates that could not have been deduced by PS-SCL screening alone. Comparing protease subsite preference profiles from screens of the two types of peptide libraries showed that the most preferred substrates were not detected by PS SCL screening as a consequence of intermolecular cooperativity being negated by the very nature of PS SCL screening. Sequences that are highly favoured as result of intermolecular cooperativity achieve optimal protease subsite occupancy, and thereby interact with very specific determinants of the protease. Identifying these substrate sequences is important since they may be used to produce potent and selective inhibitors of protolytic enzymes. This study found that highly favoured substrate sequences that relied on intermolecular cooperativity allowed for the production of potent inhibitors of KLK4, KLK14 and plasmin. Peptide aldehydes based on preferred plasmin sequences produced high affinity transition state analogue inhibitors for this protease. The most potent of these maintained specificity over plasma kallikrein (known to have a very similar substrate preference to plasmin). Furthermore, the efficiency of this inhibitor in blocking fibrinolysis in vitro was comparable to aprotinin, which previously saw clinical use to reduce perioperative bleeding. One substrate sequence particularly favoured by KLK4 was substituted into the 14 amino acid, circular sunflower trypsin inhibitor (SFTI). This resulted in a highly potent and selective inhibitor (SFTI-FCQR) which attenuated protease activated receptor signalling by KLK4 in vitro. Moreover, SFTI-FCQR and paclitaxel synergistically reduced growth of ovarian cancer cells in vitro, making this inhibitor a lead compound for further therapeutic development. Similar incorporation of a preferred KLK14 amino acid sequence into the SFTI scaffold produced a potent inhibitor for this protease. However, the conformationally constrained SFTI backbone enforced a different intramolecular cooperativity, which masked a KLK14 specific determinant. As a consequence, the level of selectivity achievable was lower than that found for the KLK4 inhibitor. Standard mechanism inhibitors such as SFTI rely on a stable acyl-enzyme intermediate for high affinity binding. This is achieved by a conformationally constrained canonical binding loop that allows for reformation of the scissile peptide bond after cleavage. Amino acid substitutions within the inhibitor to target a particular protease may compromise structural determinants that support the rigidity of the binding loop and thereby prevent the engineered inhibitor reaching its full potential. An in silico analysis was carried out to examine the potential for further improvements to the potency and selectivity of the SFTI-based KLK4 and KLK14 inhibitors. Molecular dynamics simulations suggested that the substitutions within SFTI required to target KLK4 and KLK14 had compromised the intramolecular hydrogen bond network of the inhibitor and caused a concomitant loss of binding loop stability. Furthermore in silico amino acid substitution revealed a consistent correlation between a higher frequency of formation and the number of internal hydrogen bonds of SFTI-variants and lower inhibition constants. These predictions allowed for the production of second generation inhibitors with enhanced binding affinity toward both targets and highlight the importance of considering intramolecular cooperativity effects when engineering proteins or circular peptides to target proteases. The findings from this study show that although PS-SCLs are a useful tool for high throughput screening of approximate protease preference, later refinement by SML screening is needed to reveal optimal subsite occupancy due to cooperativity in substrate recognition. This investigation has also demonstrated the importance of maintaining structural determinants of backbone constraint and conformation when engineering standard mechanism inhibitors for new targets. Combined these results show that backbone conformation and amino acid cooperativity have more prominent roles than previously appreciated in determining substrate/inhibitor specificity and binding affinity. The three key inhibitors designed during this investigation are now being developed as lead compounds for cancer chemotherapy, control of fibrinolysis and cosmeceutical applications. These compounds form the basis of a portfolio of intellectual property which will be further developed in the coming years.

Rethinking comprehension and strategy use in second language listening instruction

In second language classrooms, listening is gaining recognition as an active element in the processes of learning and using a second language. Currently, however, much of the teaching of listening prioritises comprehension without sufficient emphasis on the skills and strategies that enhance learners’ understanding of spoken language. This paper presents an argument for rethinking the emphasis on comprehension and advocates augmenting current teaching with an explicit focus on strategies. Drawing on the literature, the paper provides three models of strategy instruction for the teaching and development of listening skills. The models include steps for implementation that accord with their respective approaches to explicit instruction. The final section of the paper synthesises key points from the models as a guide for application in the second language classroom. The premise underpinning the paper is that the teaching of strategies can provide learners with active and explicit measures for managing and expanding their listening capacities, both in the learning and ‘real world’ use of a second language.

Strategic project evaluation for open pit mining ventures using real options and allied econometric techniques

Open pit mine operations are complex businesses that demand a constant assessment of risk. This is because the value of a mine project is typically influenced by many underlying economic and physical uncertainties, such as metal prices, metal grades, costs, schedules, quantities, and environmental issues, among others, which are not known with much certainty at the beginning of the project. Hence, mining projects present a considerable challenge to those involved in associated investment decisions, such as the owners of the mine and other stakeholders. In general terms, when an option exists to acquire a new or operating mining project, , the owners and stock holders of the mine project need to know the value of the mining project, which is the fundamental criterion for making final decisions about going ahead with the venture capital. However, obtaining the mine project’s value is not an easy task. The reason for this is that sophisticated valuation and mine optimisation techniques, which combine advanced theories in geostatistics, statistics, engineering, economics and finance, among others, need to be used by the mine analyst or mine planner in order to assess and quantify the existing uncertainty and, consequently, the risk involved in the project investment. Furthermore, current valuation and mine optimisation techniques do not complement each other. That is valuation techniques based on real options (RO) analysis assume an expected (constant) metal grade and ore tonnage during a specified period, while mine optimisation (MO) techniques assume expected (constant) metal prices and mining costs. These assumptions are not totally correct since both sources of uncertainty—that of the orebody (metal grade and reserves of mineral), and that about the future behaviour of metal prices and mining costs—are the ones that have great impact on the value of any mining project. Consequently, the key objective of this thesis is twofold. The first objective consists of analysing and understanding the main sources of uncertainty in an open pit mining project, such as the orebody (in situ metal grade), mining costs and metal price uncertainties, and their effect on the final project value. The second objective consists of breaking down the wall of isolation between economic valuation and mine optimisation techniques in order to generate a novel open pit mine evaluation framework called the ―Integrated Valuation / Optimisation Framework (IVOF)‖. One important characteristic of this new framework is that it incorporates the RO and MO valuation techniques into a single integrated process that quantifies and describes uncertainty and risk in a mine project evaluation process, giving a more realistic estimate of the project’s value. To achieve this, novel and advanced engineering and econometric methods are used to integrate financial and geological uncertainty into dynamic risk forecasting measures. The proposed mine valuation/optimisation technique is then applied to a real gold disseminated open pit mine deposit to estimate its value in the face of orebody, mining costs and metal price uncertainties.

Personalised search – a hybrid approach for web information retrieval and its evaluation

Nowadays, everyone can effortlessly access a range of information on the World Wide Web (WWW). As information resources on the web continue to grow tremendously, it becomes progressively more difficult to meet high expectations of users and find relevant information. Although existing search engine technologies can find valuable information, however, they suffer from the problems of information overload and information mismatch. This paper presents a hybrid Web Information Retrieval approach allowing personalised search using ontology, user profile and collaborative filtering. This approach finds the context of user query with least user’s involvement, using ontology. Simultaneously, this approach uses time-based automatic user profile updating with user’s changing behaviour. Subsequently, this approach uses recommendations from similar users using collaborative filtering technique. The proposed method is evaluated with the FIRE 2010 dataset and manually generated dataset. Empirical analysis reveals that Precision, Recall and F-Score of most of the queries for many users are improved with proposed method.

Understanding evaluation of learning support in mathematics and statistics

With rapid and continuing growth of learning support initiatives in mathematics and statistics found in many parts of the world, and with the likelihood that this trend will continue, there is a need to ensure that robust and coherent measures are in place to evaluate the effectiveness of these initiatives. The nature of learning support brings challenges for measurement and analysis of its effects. After briefly reviewing the purpose, rationale for, and extent of current provision, this article provides a framework for those working in learning support to think about how their efforts can be evaluated. It provides references and specific examples of how workers in this field are collecting, analysing and reporting their findings. The framework is used to structure evaluation in terms of usage of facilities, resources and services provided, and also in terms of improvements in performance of the students and staff who engage with them. Very recent developments have started to address the effects of learning support on the development of deeper approaches to learning, the affective domain and the development of communities of practice of both learners and teachers. This article intends to be a stimulus to those who work in mathematics and statistics support to gather even richer, more valuable, forms of data. It provides a 'toolkit' for those interested in evaluation of learning support and closes by referring to an on-line resource being developed to archive the growing body of evidence. © 2011 Taylor & Francis.

Practical decision support system evaluation for consultant selection

The widespread development of Decision Support System (DSS) in construction indicate that the evaluation of software become more important than before. However, it is identified that most research in construction discipline did not attempt to assess its usability. Therefore, little is known about the approach on how to properly evaluate a DSS for specific problem. In this paper, we present a practical framework that can be guidance for DSS evaluation. It focuses on how to evaluate software that is dedicatedly designed for consultant selection problem. The framework features two main components i.e. Sub-system Validation and Face Validation. Two case studies of consultant selection at Malaysian Department of Irrigation and Drainage were integrated in this framework. Some inter-disciplinary area such as Software Engineering, Human Computer Interaction (HCI) and Construction Project Management underpinned the discussion of the paper. It is anticipated that this work can foster better DSS development and quality decision making that accurately meet the client’s expectation and needs

Technology transfer evaluation in the high technology industry : an interdisciplinary perspective

The underlying objective of this study was to develop a novel approach to evaluate the potential for commercialisation of a new technology. More specifically, this study examined the 'ex-ante'. evaluation of the technology transfer process. For this purpose, a technology originating from the high technology sector was used. The technology relates to the application of software for the detection of weak signals from space, which is an established method of signal processing in the field of radio astronomy. This technology has the potential to be used in commercial and industrial areas other than astronomy, such as detecting water leakages in pipes. Its applicability to detecting water leakage was chosen owing to several problems with detection in the industry as well as the impact it can have on saving water in the environment. This study, therefore, will demonstrate the importance of interdisciplinary technology transfer. The study employed both technical and business evaluation methods including laboratory experiments and the Delphi technique to address the research questions. There are several findings from this study. Firstly, scientific experiments were conducted and these resulted in a proof of concept stage of the chosen technology. Secondly, validation as well as refinement of criteria from literature that can be used for „ex-ante. evaluation of technology transfer has been undertaken. Additionally, after testing the chosen technology.s overall transfer potential using the modified set of criteria, it was found that the technology is still in its early stages and will require further development for it to be commercialised. Furthermore, a final evaluation framework was developed encompassing all the criteria found to be important. This framework can help in assessing the overall readiness of the technology for transfer as well as in recommending a viable mechanism for commercialisation. On the whole, the commercial potential of the chosen technology was tested through expert opinion, thereby focusing on the impact of a new technology and the feasibility of alternate applications and potential future applications.

A strategy for delayed research method selection : deciding between grounded theory and phenomenology

This paper presents a strategy for delayed research method selection in a qualitative interpretivist research. An exemplary case details how explorative interviews were designed and conducted in accordance with a paradigm prior to deciding whether to adopt grounded theory or phenomenology for data analysis. The focus here is to determine the most appropriate research strategy in this case the methodological framing to conduct research and represent findings, both of which are detailed. Research addressing current management issues requires both a flexible framework and the capability to consider the research problem from various angles, to derive tangible results for academia with immediate application to business demands. Researchers, and in particular novices, often struggle to decide on an appropriate research method suitable to address their research problem. This often applies to interpretative qualitative research where it is not always immediately clear which is the most appropriate method to use, as the research objectives shift and crystallize over time. This paper uses an exemplary case to reveal how the strategy for delayed research method selection contributes to deciding whether to adopt grounded theory or phenomenology in the initial phase of a PhD research project. In this case, semi-structured interviews were used for data generation framed in an interpretivist approach, situated in a business context. Research questions for this study were thoroughly defined and carefully framed in accordance with the research paradigm‟s principles, while at the same time ensuring that the requirements of both potential research methods were met. The grounded theory and phenomenology methods were compared and contrasted to determine their suitability and whether they meet the research objectives based on a pilot study. The strategy proposed in this paper is an alternative to the more „traditional‟ approach, which initially selects the methodological formulation, followed by data generation. In conclusion, the suggested strategy for delayed research method selection intends to help researchers identify and apply the most appropriate method to their research. This strategy is based on explorations of data generation and analysis in order to derive faithful results from the data generated.