Intervention studies have shown that lycopene-containing foods may reduce cholesterol levels and lipid peroxidation, factors implicated in the initiation of cardiovascular disease. The objective of this study was to determine whether consumption of lycopene rich foods conferred cardiovascular protection to middle-aged adults as indicated by plasma lipid concentrations and measures of Ten healthy men and women consumed a low lycopene diet with no added lycopene (control treatment) or supplemented with watermelon or tomato juice each containing 20 mg lycopene. Subjects consumed each treatment for three weeks in a crossover design. Plasma, collected weekly was analyzed for total cholesterol, high density lipoprotein cholesterol (HDL-C) and triglyceride concentrations and for the antioxidant biomarkers of malondialdehyde formation products (MDA), plasma glutathione peroxidase (GPX) and ferric reducing ability of plasma (FRAP). Data were analyzed using Proc Mixed Procedure and associations between antioxidant and lipid measures were identified by Pearson's product moment correlation analysis.Compared to the control diet, the lycopene-containing foods did not affect plasma lipid concentrations or antioxidant biomarkers. Women had higher total cholesterol, HDL-C and triglyceride concentrations than did the men. Total cholesterol was positively correlated to MDA and FRAP while HDL-C was positively correlated to MDA and GPX. GPX was negatively correlated to triglyceride concentration.The inclusion of watermelon or tomato juice containing 20 mg lycopene did not affect plasma lipid concentrations or antioxidant status of healthy subjects. However, plasma cholesterol levels impacted the results of MDA and FRAP antioxidant tests. Howevoperties -10.Recent epidemiological studies have linked reductions in risks of cardiovascular disease with diets rich in lycopene containing foods. These reductions in risk have been primarily attributed to the antioxidant properties of lycopene ,12. Imprvivo as antioxidants or alter cholesterol levels.Other studies have assessed response of plasma lipids to lycopene-rich diets. In one study, six healthy men were supplemented with 60 mg/day for three months with tomato lycopene (LycoRed) with a 14% reduction in LDL-C and no change in HDL-C . Researcvivo biomarkers.The objectives of this study were to compare the ability of two lycopene containing foods, tomato and watermelon to provide cardiovascular protection to middle-aged adults by measuring changes in cholesterol levels and antioxidant ex Samples for this study came from a larger study, which has been reported in detail . This sttrans lycopene and 6% cis isomers, primarily 5-cis and 13-cis with minimal amounts of other cis isomers [trans lycopene and 10.8% cis isomers, primarily identified as 5-cis, 9-cis, 13-cis, and 15-cis, and minimal amounts of other cis isomers [Watermelon juice for the study was prepared at a pilot plant at the USDA Citrus and Subtropical Products Laboratory, Winter Haven, FL without heat treatment as previously described . Canned  isomers . For tom isomers .Plasma samples were thawed on ice for four hours then mixed by vortexing, prior to preparing for assays. Serum total cholesterol and triglyceride concentrations were determined enzymatically using kits from Roche Diagnostics . Serum HDL-cholesterol was determined by a direct method  that utilizes the combined action of polymers, polyanions, and detergent to solubilize cholesterol from HDL but not from VLDL, LDL, and chylomicrons as previously described . AnalysiPlasma from subjects was analyzed for plasma glutathione peroxidase using an ELISA kit . Two replicates per sample of 20 μl of plasma were diluted 1:25 with TRIS-HCl buffer then pippetted into pre-coated polyclonal antibodies microplate wells specific for human plasma glutathione peroxidase (GPX). The amount of enzyme present was determined by reaction with para-nitrophenyl-phosphate and was read using a microplate reader at 405 nm . The concentration of plasma GPX was determined from a standard curve for each plate using five dilutions of GPX standard.Malondialdehyde compounds were determined colorimetrically using a commercial kit specific for measuring free and total malondialdehyde compounds . Two replicates per sample of 210 μl of plasma were added to each test tube with 11 μl of 500 mM butylated hydroxytoluene and 5.3 μl of concentrated hydrochloric acid. Tubes were capped, mixed then incubated at 60°C for 80 minutes, cooled to room temperature and 680 μl of N-methyl-2-phenylindole in acetonitrile was added. Then tubes were mixed, and centrifuged at 13,000 g for 5 minutes. New tubes were prepared and 660 μl of clear supernatant was added with 115 μl of concentrated HCl. Tubes were capped, mixed and incubated at 45°C for 60 minutes. Samples were centrifuged at 13,000 g for 5 min and the supernatant was read on a spectrophometer at 575 nm. Concentration of samples was determined using a five point standard curve.This assay was conducted according to previously published methods . In brieExperimental procedures for the clinical trial were approved by the Institutional Review Board at the Johns Hopkins University Bloomberg School of Hygiene and Public Health; subjects gave their written informed consent to participate. The plasma cholesterol and antioxidant studies were approved by the Institutional Review Board at Oklahoma State University, Stillwater, OK. Data were analyzed using Proc Mixed Procedure and mean separation was performed using LSMEANS, correlation analysis was performed using Spearman's Correlation Coefficient Analysis .Because there were significant four way interactions with gender × intervention period × treatment × weeks with MDA, FRAP, GPX and cholesterol analysis, trends by treatment, intervention period or week of treatment were not seen. Supplementing the diet with 20 mg/day of lycopene of either food did not change the plasma antioxidant status of the subjects and values ranged from 0.66–2.20, 540–1094, and 1296–2596 :mol/L for MDA, FRAP and GPX respectively. These levels are similar to levels reported for healthy subjects in other studie ,23.Intervention with 20 mg of lycopene to the diet of subjects did not alter their total cholesterol, HDL-C or triglyceride  status. However, there were gender differences and the women had higher average levels of plasma triglycerides, total cholesterol and HDL-C than men Figure . The higThere was a significant positive correlation between each pair of total cholesterol and MDA and MDA and FRAP and between HDL-C and MDA and HDL-C and GPX. A significant negative correlation was found between triglycerides and GPX Table .Because of the correlation between cholesterol concentrations and antioxidant analysis, a preliminary analysis of data was conducted to determine if cholesterol levels impacted antioxidant results. Subjects were separated into two groups based upon baseline concentrations of plasma triglycerides, total cholesterol and LDL-C above or below 200, 180 and 160, respectively. Five subjects, two men and three women, fit the criteria of moderately hypercholesterolemic Table .Analyses showed an interaction of cholesterol level × treatment period × treatment factor for MDA and FRAP analysis. Higher MDA and FRAP levels were found in the group having higher cholesterol levels compared to the other group Table . No trenWe found no improvement in the antioxidant status of healthy middle-aged adults supplemented with two lycopene-containing foods. In previous antioxidant studies, reduced lipid peroxidation was reported in subjects supplemented from one to four weeks with 5 to 45 mg lycopene containing tomato products ,16,23,26in vitro in the FRAP assay [The reports from lycopene intervention studies that measured FRAP activity are not in agreement. One study reported improvement in FRAP levels of plasma in subjects supplemented with tomato juice and olive oil , while tAP assay ,29. In tUnlike a previous report by Fuhrman et al., neither lycopene intervention with watermelon nor tomato affected cholesterol levels . DiffereFruits and vegetables are excellent sources of antioxidant compounds and the average American consumes only 1.5 and 3.1 servings per day . In manyThe positive correlation between total cholesterol and MDA antioxidant analysis has been reported in studies with hypercholesterolemic subjects compared to normocholesterolemic subjects ,36. The The trend correlating higher FRAP with higher cholesterol levels has not been previously reported. The significance of this trend is speculative, since the FRAP assay measures the oxidation and reduction potential of compounds based on the reduction of the ferric to ferrous iron , lipid pin vitro [Long-term supplementation studies where diet is controlled will probably be necessary to identify the benefits provided by lycopene. There may be real health benefits associated with lycopene especially since it is stored in various tissues and exhibits strong antioxidant activity in vitro ,10,39,40in vitro ,12,41,42in vitro ,44. The ex vivo antioxidant studies may be due to the cholesterol levels of subjects thus warranting further research.The interaction between cholesterol levels and antioxidant values needs more research. Contradictory findings of this study with other None declared.Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap. The article cited was prepared by a USDA employee as part of his/her official duties. Copyright protection under U.S. copyright law is not available for such works. Accordingly, there is no copyright to transfer. The fact that the private publication in which the article appears is itself copyrighted does not affect the material of the U.S. Government, which can be freely reproduced by the public.JKC: conception and design of the study, drafted the manuscript, BHA: design of study, editing, PLC:design of the study, statistical analysis, PPV: conception of study, editing, RAB: editing, technical assistance, BAC:conception of study, editing. All authors read and approved the final manuscript.


Complex biological database systems have become key computational tools used daily by scientists and researchers. Many of these systems must be capable of executing on multiple different hardware and software configurations and are also often made available to users via the Internet. We have used the Java Data Object (JDO) persistence technology to develop the database layer of such a system known as the SigPath information management system. SigPath is an example of a complex biological database that needs to store various types of information connected by many relationships.Using this system as an example, we perform a critical evaluation of current JDO technology; discuss the suitability of the JDO standard to achieve portability, scalability and performance. We show that JDO supports portability of the SigPath system from a relational database backend to an object database backend and achieves acceptable scalability. To answer the performance question, we have created the SigPath JDO application benchmark that we distribute under the Gnu General Public License. This benchmark can be used as an example of using JDO technology to create a complex biological database and makes it possible for vendors and users of the technology to evaluate the performance of other JDO implementations for similar applications.The SigPath JDO benchmark and our discussion of JDO technology in the context of biological databases will be useful to bioinformaticians who design new complex biological databases and aim to create systems that can be ported easily to a variety of database backends. Biological databases are key computational tools used daily by biologists. Such a large number of biological databases have been developed for biology that the Nucleic Acids Research Journal has published an annual database issue since 1996. From the point of view of the user, these resources are most useful when they are regularly updated and when they provide user-friendly ways to browse, search and view information. These user needs are generally recognized as important requirements by the designers and developers of biological databases. To cope with these requirements, bioinformaticians who develop the biological databases have typically responded by developing increasingly customized software to manage the data and the information . In doiIn this article, we report on our experience with the Java Data Objects persistence technology and take a critical view at the advantages and drawbacks of this emerging Java persistence standard for the development of advanced biological databases. We have ported the SigPath information management system (see below) to the JDO API and have defined an application-specific benchmark. We used this benchmark to evaluate the performance of two JDO implementations that target either a relational or an object database backend. This article summarizes the performance results that we obtained, announces the availability of the SigPath JDO benchmark (available under the GPL license), and identifies areas where the JDO API could be refined to facilitate portability and scalability of applications.Biological databases are built with software that executes on computers. Most biological databases are of a size that could fit entirely in the central memory of modern computers. However, because computers may need to be shutdown for maintenance – or may crash inadvertently – data for a given database cannot be kept in computer memory for the life of a biological database. This problem is not specific to biological databases so that a variety of data persistence approaches and technologies are available. The key role of these technologies is to guarantee that data persists safely between the invocations of the programs that may modify the data.text files with limited structure and important information can be stored in unstructured text files expressed in English. Unstructured flat-files do not help perform large-scale analyses, structured queries or integrate data across multiple sources, all of which are important requirements for biological databases. Therefore, unstructured files are now widely recognized throughout the field as inadequate for the management of biological information.Data can be stored in Highly structured data formats, such as ASN.1 . T. T23]. TMost traditional biological databases focus on one type of database entry  and store information in database entries. This approach has been very useful to create detailed catalogs of biological parts and is a critical and essential element of the bioinformatics resources that support modern biological research. However, certain integrative studies, such as systems biology and modeling and simulation of biochemical pathways call for databases that integrate several types of information.The SigPath IMS is an example of an advanced biological database that encodes information through a number of information types and a set of relationships among them. Figure A fragment of the SigPath ontology is given on Figure The SigPath system supports several types of biological information, ranging from information to represent small molecules and proteins to the interactions between these molecules. The main information types supported by SigPath are listed on Table This multiplicity of SigPath information types and the variety of relationships among them makes it important to clearly define what type of information can be represented by the system . This information is formalized in the SigPath ontology. This ontology is implemented in a JDO database schema. . The JDO schema consists of the set of SigPath Java classes that are persistent and of meta-data about these classes. Meta-data is expressed in JDO files and provides information about the classes that cannot be expressed directly in the Java language, for instance, type of the elements for the collection field of the persistent classes. Figure The SigPath code base has specific characteristic that make it a useful resource for evaluating JDO technology:• SigPath is an open-source project released under the GPL, so that the benchmark code is freely available for others to study, reproduce our results, or extend the benchmark to other JDO implementations or database backends.• SigPath is both a web-based application and a batch-oriented application.• The SigPath code-base includes unit tests  that hel• The SigPath system provides varied use cases that exercise different behaviors of the database backend and JDO implementation (see use cases below).In the next section, we present the methods that we used to evaluate JDO technology for the creation of advanced biological databases.This section describes the results of the SigPath JDO benchmark and addresses the portability and performance questions described in the introduction.We modified the FastObjects JDO version of SigPath to compile indifferently with the FastObjects and Kodo implementations of JDO. The modifications that we had to make to the project were (i) modifications to the JDO file, (ii) modifications of the code base and (iii) modification of the code base and application data.The Kodo enhancer tool performs stricter semantic validations on the JDO files than the FastObjects enhancer. Modifications needed to pass the validation tests were:1. Added persistence-capable-superclass attribute to classes that have a persistence capable superclass. This attribute is optional for FastObjects, which uses Java reflection by default, but is strictly required by the Kodo implementation (in agreement with the JDO specification).2. Removed all interfaces from the JDO file. Enhancing with Kodo failed when interfaces were listed in this file. Since FastObjects requires interfaces to be listed as persistent classes, the SigPath build script conditionally includes such statements in the JDO file when FastObjects is configured. The JDO specification does not mention interfaces, so that the behaviour of JDO implementation is left undefined.3. (As a result of 2.) Replaced references to interfaces with references to implementation  throughout the JDO file.4. Added collection element types to all persistent collections. FastObjects requires the type to be specified when the collection is used in a query. Kodo requires the type to be defined for each collection, otherwise Kodo will try to serialize the collection and store it as a binary object. If the persistent class is not serializable, this mechanism will fail. Therefore, for this benchmark, we explicitly defined the collection types for each collection.5. Removed field definitions from sub-classes when they refer to fields of a super-class. . Removing these duplicate declarations is consistent with the JDO specification.Furthermore, the Kodo enhancer expects classes to be listed in the JDO file in a specific order. The enhancer fails if a class appears in the JDO file before another class that the first class references. Therefore, we reordered the class definitions in the JDO file. .Finally, we added Kodo extensions to the JDO file to create indexes on the tables that were used extensively in queries. All changes to the JDO file were consistent with the JDO specification. Index tuning was performed by running the boot and test part of the benchmark and the small molecule import with various indexes choices.We modified the code base to work-around a limitation of the Kodo implementation. With Kodo, instances of classes that contain java.lang.Object fields are made persistent with the object field stored as a BLOB in the database. Storing objects as BLOBs puts strong limitations on their use. For instance, it is impractical to query for these objects by their fields . Storing such fields as BLOBS was therefore not acceptable for certain types of persistence objects, and we implemented the work-around shown on Figure Another code modification was required to work-around a problem with the database backend that did not handle appropriately empty strings (“”). The database backend used for this benchmark stored empty strings as null. Reading these strings back from the database resulted in null being obtained from Kodo instead of empty strings. This resulted in several unexpected NullPointerException being thrown during the JUnit tests. Figure Finally, we had to modify the code base to put a limit on the length of long strings. Using a relational database backends imposes to define the maximum length of each string attribute defined in the persistent classes of the application. For instance, a limit must be set on the name attribute of the SigPathEntityImpl shown on Figure A brief summary of the performance measurements obtained with the SigPath benchmark is given in Table The raw data used for the calculation of these performance measures is provided in the supplementary material and on the SigPath JDO benchmark pages. These pages also provide the logs from which the raw data has been collected.Our port of SigPath confirms that JDO greatly facilitates the porting of a bioinformatics application from one database backend to another. However, we report here several modifications that we had to make to the SigPath system to achieve this level of portability. This suggests that there is a need to develop JDO compliance tests that could be used to test that a specific implementation of a JDO-aware database is really compliant with the standard. This test suite would validate that JDO enhancers accept correct JDO files and correctly reject JDO files that break the specification. The differences in the interpretation of JDO files that we noticed between FastObjects and Kodo (see Results section) practically limit the portability of JDO applications. This article has presented techniques that can be used to work around these limitations until a JDO compliance test is developed and used. We note that the work arounds that we described may be specific to the two JDO implementations that we tested, and that other work arounds may be needed to achieve portability with other JDO compliant backends.Surprisingly, we found that an outstanding portability problem is in the way the different JDO back-ends store long strings of characters. While the FastObjects backend put no limitation of the length of long strings, the relational back-end used with Kodo limited the length of long strings to 4,000 characters. This limit had to be chosen and set for each persistent string field used in the application . Although 4,000 characters may appear a large limit, it is likely to be reached in bioinformatics application either with textual or with sequence data. When this happens, the application will have to be re-engineered to work around the fixed limit. A work-around could be to use a data type that does not have a length limitation, but these data types also have other limitations . Whichever solution is chosen, this issue must be considered early during the design of the application. It would be useful if the JDO standard offered a mechanism for the application developers to specify which string length their application requires to function properly with JDO backends. Each enhancer could then check that the application is requesting a maximum string length that is compatible with the database backend and fail early if it does not. The SigPath benchmark provides precise measurements of the performance of one biological database application against two JDO compliant database backends. The measurements were performed on use cases that are typical of the activities needed to develop the code of SigPath and to deploy a production SigPath system.  the complexity of the database schema (75 persistent classes) and (ii) the number of connections that exist among instances of these various classes.Finally, these results and our distribution of the SigPath benchmark source code can help vendors diagnose performance problems with their implementation of the JDO implementation, and provide users with an objective measure of the performance a given JDO implementation, for similar types of applications.During our evaluation of the JDO technology, we have noted that two common requirements of advanced biological databases are currently not being addressed by JDO.When designing a biological database schema, it is often useful to express that one class shares the properties of two or more classes. In a programming language such as C++ this can be represented as multiple inheritance (one class inherits from two parents) while in a programming language such as Java, this concept is represented with interfaces (one class implements two interfaces). In the context of JDO, consider the class diagram shown on Figure Biological databases often need to manage large number of objects . For instance, SigPath stores information about several hundred of thousands of proteins. We found that JDO 1.0.1 lacks some features that would facilitate writing scalable applications.An example is that the JDO standard does not provide a scalable way to determine the number of persistent instances of a given class. The JDO compliant way to accomplish this operation is to obtain a reference to a collection of instances of this class (using a JDO Extent), and to call the size method on this collection. Since the collection must first be obtained from the database server before the size method can be invoked, this procedure takes a time proportional to the number of instances of this class. Most database backends store the number of instances of a certain class in the database and can determine this information in a constant time, so a standard way to obtain this information from a JDO implementation would be very helpful .A second example is that JDO 1.0.1 does not provide support for queries that return large result sets. Under standard JDO 1.0.1 behavior, traversing a persistent collection (by accessing each element of the collection in turn) brings the entire contents of that collection into memory. This behavior is appropriate for small result sets. However, there are cases where the complete set of instances returned by a query cannot be processed within a single transaction. This occurs for instance when all the results returned by a query do not fit in the fixed memory limit allocated to the Java Virtual Machine. In such cases, it may be necessary to obtain the result of a query in chunks of a certain number of records/instances , and process them in independent transactions. Upon transaction commit, memory associated with a chunk is released and can be used to process the next chunk. Implementing this type of scalable processing in an efficient manner usually requires making modifications both in the persistent class of elements in the result set and in the query filter. The class of the elements in the result set can be modified to add an instance identifier that can be used both to sort the instances and to select only those within the current processing chunk. The query filter can be modified to add a clause that selects only instances of the next chunk, based on the identifier introduced in each element. An alternative is to provide an API call to notify the JDO implementation that instances which have been processed can be evicted from memory. Since several vendors already have their own extensions to provide scalability feature, it would be useful for JDO to support such features through a standard API.Here, we have shown that it is possible to develop a bioinformatics database that can be reconfigured automatically and recompiled to run either against a relational database backend or against an object database backend. The key advantage of this added flexibility is that the bioinformatics database becomes portable with respect to the database backend. This has important implications for the development of open-source bioinformatics databases. In such projects, usually more than one laboratory contributes to developing the software of a specific biological database. Therefore, it is useful if each laboratory can choose a database backend for development and deployment, yet contribute to the project in a shared code base.The Java Data Objects standard offers the productivity gains of transparent object persistence, and a fine-grained object persistence model useful to represent many biological concepts. We discussed why JDO can appear as an attractive option for the development of advanced biological databases and the type of problems that we encountered when implementing and deploying a biological database against two different JDO implementations. The future JDO standard (JDO 2.0) should address some of the issues that we discussed in this article . When JDO 2 implementations become available, we expect that JDO technology will have a significant impact on the design of high-performance biological databases that need to represent and manage complex biological information types.To address the performance question, we have developed benchmark use cases. The benchmark use cases were designed to be representative of performance that one would observe when either (i) developing the software of the SigPath system or (ii) preparing a new release of the SigPath IMS (includes loading the database with information from other databases). Our benchmark thus considers both the development and the production stages of the life-cycle of the application.The use cases, or benchmark steps and a summary of their purpose in the context of the SigPath project are listed on Table The boot and test steps make it possible for the SigPath developers to create a sample database and test that important functionalities of the application are working satisfactorily.boot – The boot step compiles the sources of the project, enhances the JDO persistent classes , creates an empty database and imports information into the database. Importing this information involves parsing an XML file that contains the information, validating this file against the SigPath information exchange XML Schema, validating against additional semantic rules that cannot be expressed with XML Schemas , and saving new persistent instances to the database. The boot sample data is designed to contain at least one instance of each type of information that can be stored in the SigPath IMS.test – The test step runs JUnit tests against the data that was imported during the boot step. The JUnit tests assert that information stored in the database corresponds to the information in the boot XML file. For instance, the tests check that the number of persistent instances matches the number found in the boot import file, but also that specific elements of information have been saved accurately. Furthermore, the tests assert various semantic properties of the application and database access code, running queries against the database, navigating through objects, creating new persistent instances or deleting them, etc. The complete set of operations performed in the test is fully described in the source code for the JUnit tests .small molecule import – This step creates SmallMoleculeImpl persistent instances . For this benchmark, XML import instantiates 14 SmallMoleculeImpl, 8 ProteinImpl, 121 ComplexImpl, 77 modified chemicals , 92 ConcentrationMeasurement, 165 ReactionImpl, 75 EnzymaticReactionImpl, 23 Model, 3 Pathway and 27 PendingReviews.The benchmarks were run as described on the SigPath Project web site ([??] see the "JDO benchmark" tab). Each benchmark  was run on a two Xeon 3GHz processor machine with hyper-threading on and 6 Gb of memory. The machine was running Red Hat Advanced Server Linux 2.4.21-4.0.1.ELsmp, and was used both as database server and database client (to minimize the impact of the network on performances). No significant other processes were running on the benchmark machine while the tests were executed. We benchmarked FastObjects t7 server version 9.0.7.185 and Kodo JDO version 2.5.3. Each benchmark was run four times to average the effect of variability in the computational environment that may not have been controlled by our benchmark procedure. The results report the coefficient of variations (mean divided by the standard deviation) of the total running time for each use case and this helps point out cases when the computational environment had an effect on measured times. We believe that these variations are common in a production environment and report the average total running time as well as the minimum total running time for each use case.Marko Srdanovic and US implemented significant components of the FastObjects and Kodo JDO ports. Marko Srdanovic collected benchmark data at WMC and US collected similar data at FastObjects. Michael Schwieger and FC designed the study and contributed to the JDO ports. FC drafted the manuscript. All authors read and approved the final manuscript.


This paper provides an overview of initial research conducted in the development of pressure-sensitive foam and its application in wearable sensing. The foam sensor is composed of polypyrrole-coated polyurethane foam, which exhibits a piezo-resistive reaction when exposed to electrical current. The use of this polymer-coated foam is attractive for wearable sensing due to the sensor's retention of desirable mechanical properties similar to those exhibited by textile structures.The development of the foam sensor is described, as well as the development of a prototype sensing garment with sensors in several areas on the torso to measure breathing, shoulder movement, neck movement, and scapula pressure. Sensor properties were characterized, and data from pilot tests was examined visually.The foam exhibits a positive linear conductance response to increased pressure. Torso tests show that it responds in a predictable and measurable manner to breathing, shoulder movement, neck movement, and scapula pressure.The polypyrrole foam shows considerable promise as a sensor for medical, wearable, and ubiquitous computing applications. Further investigation of the foam's consistency of response, durability over time, and specificity of response is necessary. Today we are faced with a significant information overload problem as users struggle to locate the right information in the right way at the right time. In response, a number of researchers have suggested that adaptive information technologies may hold the key to the next generation of ubiquitous information systems, systems that automatically adapt to changes in their environment and usage in order to deliver users a more intelligent, proactive and personalized information service. In this paper we provide an overview of initial research conducted as part of the Adaptive Information Cluster Sensing in the wearable environment is crucial for many applications, but existing sensor technologies pose significant wearability problems when integrated into the user's peri-personal space. One of the most compelling needs for wearable technology is in the continuous monitoring of the human body, be that for medical monitoring or to inform the operation of a context-aware computerized application. While many technologies that are often made wearable (such as music players or telephones) function nearly as well (or sometimes better) as portable devices, almost all continuous body-sensing technologies must be worn to be effective. However, because of their ubiquitous, constant-wear nature, such technologies must prioritise the effects of the technology on the user's physical comfort as well as social comfort. Traditional sensing technologies are rarely designed for continuous, on-body use: those that require skin contact are generally designed to be used in a hospital or doctor's office, and those that do not are generally designed for use in stationary devices. Consequently, the achievement of certain design goals for existing sensors (such as durability) is ultimately detrimental to the user's comfort when applied to the wearable environment. For example, durability often equals stiffness, which results in a solid device that can cause discomfort by localizing pressure.Textile-based sensors offer a compromise solution to this problem, by retaining the characteristics associated with comfort and wearability . Many textile-based sensors are actually sensing materials used to coat a textile  or sensiIn previous work , a novelCharacterisation for the PPy-coated PU foam was carried out using a number of methods as described in . It was Once a predictable reaction was observed from the foam, it was applied to the wearable environment to explore its utility in garment systems. It was integrated into a torso garment in several ways to investigate the ability of the foam sensor to monitor specific body changes and physiological signals. The test garment contained foam sensors in 6 locations: the top outer edge of each shoulder, the back of the neck, the superior protrusion of each scapula, and the right side rib cage under the bust Figure . Sensor The test garment was a sleeveless, collared shirt, closely fitted and nonextensile. The outer garment layer was a 100% polyester satin weave, and the inner layer was a 100% acrylic satin weave. The collar was 80% nylon, 20% elastine jersey knit. The structure of the garment was crucial to the quality of data obtained, as its textile composition, design, and fit moderated the amount of force present between the body and the sensors. In this study, the prototype garment was fitted to one test subject, to eliminate inter-subject anthropometric variation.Sensors were sewn between the two garment layers, allowing them to be easily removed and interchanged. In each test two wire leads were attached to the foam sensors and to a constant current digital multi-meter, HP, Leixlip, Ireland. Data was collected at a rate of 3 points per second. The finished prototype garment is shown in Figure The breathing sensor was attached on the subject's left-side rib cage, under the bust. The sensor measured 2.75 × 1.5 × 0.5 cm. Data was gathered with the subject standing, and the subject was instructed to breathe deeply for a period of approximately one minute.Two shoulder movement sensors were attached at the outer edge of the garment at the apex of each shoulder (above the subject's axilla). The sensors measured 1.5 × 2.0 × 0.5 cm. Data was gathered with the subject seated, and the subject was instructed to raise one shoulder repeatedly to its maximum height.nd vertebra) to 2.5 cm below the neckline of the garment (approximately 4th vertebra). The sensor measured 1.5 × 5.5 × 0.5 cm. Data was gathered with the subject seated, and the subject was instructed to perform four full neck extensions (backwards movement) and three full neck flexions (forward movement).The neck motion sensor was attached vertically along the subject's spine, at the back of the neck extending from 4 cm below the top of the collar . The conductivity of the modified foam depends on the weight of conducting polymer deposited, which in turn depends on the number of coating layers deposited on to the foam substrate. It has been shown previously [1 mS/cm. Integrating the foam sensors into the torso garment caused little alteration in the visual or tactile properties of the garment. The largest sensors, the scapula pressure pads, caused the only visible change to the appearance of the garment, as these were the only sensors that possessed enough volume to change the surface topology of the garment. Although comfort was not a measured variable, there appeared to be no change in the tactile comfort of the garment when the sensors were added. In demonstration, both the test subject and other viewers had difficulty locating the sensors within the garment without direction.t, Rt, by the initial baseline resistance, R0. It can be seen in Figure As seen in Figure The response of the foam to shoulder movements was an approximate 100% decrease in relative resistance as seen in Figure The foam responded to full neck extensions, section A in Figure 2 versus 2–12 cm2 for the other sensors and so the response time for the shoulder-blade foam sensor would be slightly slower than that for the other sensor positions, e.g. 4 seconds for shoulder lift foam sensor.The foam responded with a 60% increase in the relative resistance when the subject moved from supine (applying pressure to the scapula area) to a seated position (no pressure), as seen in Figure As demonstrated, pressure sensing in the wearable environment can provide useful descriptive information about the physical state of the user. Conducting electroactive polymers are attractive for sensing in a garment-integrated context because of their ability to retain the tactile and mechanical properties of a textile-based structure. In the garment integration, the foam sensors had little effect on the comfort or wearability of a standard garment. However, more investigation is necessary to determine the accuracy of the foam sensor responses, particularly the repeatability of response.As seen in the torso sensor evaluation, the age of the sensor had a significant impact on the absolute resistance of the sensors. It has been shown previously that if PPy is left to open to atmosphere then there is a gradual increase in the electrical resistance due to oxidation of the polymeric backbone . HoweverIn the torso integration, the raw pilot test data indicates that foam sensors can provide detectable responses to all of the body signals investigated, although careful sensor placement is important to the quality of data gathered. In this study, inter-subject anthropometric variation was controlled by limiting the number of subjects to one, and by custom-engineering the garment to fit that subject precisely. However, in a real-world scenario such control would not be possible, and sensor locations across a broad variety of body shapes and sizes would be hard to predict. Similar issues would arise with sizing, fit, and sensor locations on the foot. Because of the increased number of sensors and precision of locations, this variable would become even more difficult to control, however were the number and locations of sensors increased still more to create a uniform grid of pressure sensors, the fit issue could be avoided.An additional problem of hysteresis caused by the PU foam substrate results in the gradual and positive increase in the resistance of the foam sensor. Since the position and the relative resistance of the PPy-coated PU sensors are crucial to their sensitivity, calibration of the sensors would be required on a regular basis. This calibration would involve setting the baseline resistance and range of the measured resistance of the sensors as determined through a series of standard repeatable exercises by the subject. Once these parameters are set subject monitoring could be commenced.There are many applications of wearable sensing for which this type of sensor is particularly well suited. For example, in the monitoring of high-pressure body areas for individuals with reduced tactile sensation (such as diabetics suffering from neuropathy) the foam sensor would allow pressure points to be monitored without introducing a solid sensor element into a pressurized area close to the skin that could create more irritation. Rigid sensors in such an area could easily create more irritation and exacerbate the problem, but a foam sensor not only would not create irritation, it could actually protect the body from irritants by providing an additional layer of cushioning on key pressure points.Outside of medical applications, knowledge of the state of the body is essential in many wearable, mobile, and ubiquitous computing applications. It is common in wearable and ubiquitous computing applications for a system to make decisions based on its perception of the needs and wants of the user. A subtle, comfortable sensor that demands no attention or adaptation from the user can allow the application to function invisibly, reducing the cognitive load on the user.Based on these preliminary data, polypyrrole-coated conductive foam shows considerable promise as a basic sensing technology, and for use in detecting body movements, physiological functions, and body state from body-garment interactions. Importantly, the sensor maintains the attractive structural properties of foam, consistent with the objectives of wearability and comfort in a smart garment.Further study is necessary to fully understand the ability of the foam to serve as a reliable sensor over time and under the hostile conditions that garments must usually face. For instance, further work is required to understand and determine the effects of oxidation on baseline drift, the influence of variable conductance responses, calibrations of these responses and the optimal locations for sensors. In addition, processing algorithms for extraction of patterns from gathered data are required, as well as wearable and wireless hardware to allow the data to be used in real-time.Future work includes in-depth analysis of foam responses in controlled environments, and evaluation of optimal sensor location for monitoring of specific activities and conditions.The author(s) declare that they have no competing interests.LED created the garment prototypes, participated in the prototype pilot evaluations, and drafted the manuscript. SB created the foam sensors, participated in the prototype pilot evaluations, and drafted the manuscript. BS participated in the project organization and supervised the research. DD participated in the project organization and supervised the research. All authors read and approved the final manuscript.


The objective of this study is to investigate the perceptions of first and third year medical students on self-study and reporting processes of Problem-based Learning (PBL) sessions and their usage of learning resources.The questionnaire applied to the students consisted of; questions about students' perceptions on searching and preparing phases of the self-study process, the breadth and depth of discussion during reporting phase and the usage of learning resources.First-year students spent more time for self-study and more highly rated the depth of discussion compared to third-year students. The searching and preparing phases of the self-study process were considered as statistically important factors strongly influencing the breadth and depth of discussion during the reporting phase. The effect of extensiveness of searching on the depth of discussion was negative among the first-year students, and positive among third-year students.The relative shortness of third-year students' self-study periods can be related to their mental weariness, decreased motivation or first-year students' slowness in accessing appropriate resources. The third-year students' more frequent use of textbooks may be due to the improvement of their abilities in reaching relevant learning resources. The findings implied that the increase in students' PBL experience paralleled the development of their discussion skills using different learning resources. The institution of the present study, Dokuz Eylul University School of Medicine (DEUSM), has been implementing Problem-based Learning (PBL) in its undergraduate curriculum since the 1997–1998 academic year. The duration of undergraduate medical education is six years. PBL is the principal educational strategy in the first three years. Task-based learning strategy was adopted as an educational strategy for clerkships in the 2000–2001 academic year. During the first three years of undergraduate education, PBL sessions are the main focus of a modular structure.The three objectives of PBL are; acquisition of essential knowledge, use of knowledge in clinical contexts and self-directed learning . In a PBPBL curriculum emphasises the development of self-regulating skills. Rather than being passive recipients of information, students are expected to be actively involved . The divIn PBL, students are encouraged to take substantial responsibility for their learning. Small group discussions stimulate independent and active learning. They guide the students during their independent and self-directed learning. During a PBL group session, limited only by the boundaries of their prior knowledge, students try to clarify the issues being discussed. The issues that cannot be explained thoroughly are formulated as student-generated learning objectives which will guide the students during their independent and self-directed learning.During the searching phase of individual study, students are expected to refer to different learning resources and search for literature relevant to their learning issues. The findings are evaluated and prepared for the group discussion. The extensiveness of different learning resources used is an indicator of students' self-directed learning skill. The consultation of diverse information sources influences the breadth and depth of discussion of the tutorial group during the reporting phase .The iteration of self-directed learning periods leads the students to organise and review the learning resources critically. Through this critical approach, a major educational objective of PBL, the learning resources can be efficiently and effectively accessed and relevant information can be elaborated to form the theoretical basis leading to the solution of the problem . StudentDuring the first week of their medical education, first-year students attend a PBL orientation course. This course consists of basic principles of PBL, student and tutor roles and presentation of available learning resources in DEUSM. All tutors are initially required to take a PBL course and regularly attend weekly tutor meetings . The tutThe research questions of this study are;√ What were the differences between first and third-year students' perceptions with respect to self-study and reporting processes of PBL?√ What were the length of students' self-study times and their usage of learning resources?√ What was the overall and year-specific impact of self-study process on the breadth and depth of discussion during the reporting phase?The objective of the present study is to investigate and compare the perceptions of first and third-year PBL students on self-study and reporting processes and their usage of learning resources.The first-year students of DEUSM who were recently introduced to PBL programme and completed their first semester and the third-year students who had a two and a half year experience in the PBL programme were included in this study.The questionnaire consisted of questions about students' perceptions on searching and preparing phases of the self-study process, the breadth and depth of discussion during reporting phase and the usage of learning resources. The questionnaire implemented and tested for validity and reliability in Maastricht University  was tranThe participants reflected their perceptions of 22 questionnaire items, grouped under the headings of searching and preparing phases of self-study process, breadth and depth of discussion during the reporting phase, on a five-point scale ranging from 1 = totally disagree to 5 = totally agree. The points attributed to the items of the scale were evaluated between 1 to 5 points .The pilot study of the questionnaire was applied to 10 medical students and favourable results were obtained. At the beginning of a PBL session in February 2002, the questionnaire was distributed to first and third-year students and collected 15 minutes later. Before the application of the questionnaire, the purpose of the study was briefly explained to the students and their oral consents were obtained.The response rate was 78.8% (115/146) for first-year students and 85.5% (142/166) for third-year students.SPSS 10.0 for Windows was used and the reliability coefficient was calculated . Chi-Square Test was used to investigate students' frequency of use of learning resources. Independent Samples T Test was used to compare the scores of self-study and reporting processes of both classes. The effects of independent variables on the breadth and depth of discussion were analysed with Multiple Regression Analysis Test.The weekly self-study times of the first and third-year students during a two-week module were 15.00 ± 8.83 and 11.57 ± 7.04 hours respectively (p = 0.001).2 = 11.1, p = 0.01), educational CDs 14.8% and 6.3% , lecture handouts 95.7% and 95.8%  and medical journals 8.7% and 9.9% . Lecture handouts were used very frequently. It was found that third-year students referred more frequently to textbooks than first-year students.The first and third-year students' percentages of learning resources usage and statistical difference between them were respectively as follows; textbooks 24.3% and 44.4%  , but a positive effect among third-year students .A probable explanation for the relative shortness of third-year students' self-study times compared with those of first-year students' was third-year students' mental weariness due to continuous and intensive effort in reaching learning objectives. Another probable explanation was first-year students' slowness in accessing appropriate resources due to their lack of familiarity with self-directed learning.It was found that first and third-year students frequently used lecture handouts. The reasons for the high frequency of use of handouts were considered as their wide availability due to their provision at the end of lectures that are limited to one hour a day. In order not to hinder the curiosity of students and their motivation for self-directed learning, handouts are designed as brief outlines prepared by lecturers and include topic titles, schemata, algorithms and tables.Since the Learning Resources Centre was inaugurated in 2001, third year students could only use it during their second and third years, whereas first-year students started using it since the beginning of their medical education. More frequent use of Learning Resources Centre CDs by first-year students may be related to their early encounter with these learning facilities.Third-year students' more frequent use of textbooks may reflect the development of their ability in reaching essential resources.Third-year students' higher ratings for learning issue driven searching and extensiveness of searching are consistent with the general expectation stating that experienced students can search more learning resources The first-year students attributed higher scores to statements regarding explanation oriented preparing Table . This fiIn the Maastricht study, first-year students' average scores for learning issue driven searching, extensiveness of searching and explanation oriented searching were 3.1 ± 0.3, 2.8 ± 0.4 and 3.2 ± 0.3 respectively .The scores of the entire study group attributed to breadth and depth of discussion during the reporting phase varied between 3.2 ± 0.9 and 3.7 ± 0.7. Although third-year students were expected to become more competent in PBL discussions, third-year students' scores, especially the ones attributed to the depth of discussion, were lower than those of first-year students' , though not statistically significant, negatively affected the depth of discussion. During a PBL session, in the presence of different resources, in-depth understanding of newly acquired information requires a well-structured discussion. This may be difficult for first-year students due to their lack of familiarity with PBL and self-directed learning. In the group session, first-year students may encounter difficulties while tutoring a discussion based on several resources . When reThe design of the present study based on students' perceptions was its main limitation. The assumptions regarding the causes of differences between first and third-year students' perceptions were based on the PBL experience of authors.First-year students' longer self-study times, higher explanation oriented preparing scores, and higher depth of discussion scores compared with the scores of third-year students were considered as research questions which need to be investigated in future studies.None declared.BM carried out research design, statistical data analysis, data evaluation, research report, YG carried out research design, data evaluation, research report, HCT carried out data evaluation and research report, SO carried out data evaluation and contributed to the research report, AT contributed to research design, data collection and data evaluation. All authors read and approved the final manuscript.DEUSM: Dokuz Eylul School of MedicinePBL: Problem-based LearningSelf-study and Reporting Processes QuestionnaireYear: 1 □ 3 □weekly average study time in a two-week module?What is your • (..............) hours.What kind of resources do you use during your self-study process?•  Textbook  Lecture handouts  Others ..................... Educational CDs  Medical journalsPlease indicate your level of agreement with the following statements about self-study process, by rating them between 1-to-5.• 1 2 3 4 5Search Phase Learning Issue Driven Searching1. I use the learning issues as a starting point for my search and search the resources accordingly.During studying2. I always check the learning issues to decide whether I study deep enough.3. I remain attached to the learning issues.4. I check the learning issues to decide whether the resources I study fully cover the learning issues.5. I use the learning issues as a guide while studying the resources step by step.Extensiveness of searching6. When searching the resources, I try to evaluate the relevancy of different books with the subject to be studied.7. When searching the resources, I try to compare different resources about the same subject.8. I spent a lot of time and effort on searching the resources before I start studyingPreparing phase Explanation Oriented9. I study the subjects such that I can explain them without looking at the resources.10. I study the subjects such that I can comment about the theories being discussed11. I study such that I can explain the content of the resource in my own words.12. I study such that I know what needs to be discussed in each learning issue.13. I study by making summaries of the selected resources.14. I study by making notes and algorithms.Reporting Phase Breadth of Discussion15. Many different issues/findings are discussed.16. When a student from the group reaches an information not included in the learning issues explains it to others.17. The members of the group question different aspects of the resources.18. Different/contradicting resources are compared.Depth of Discussion19. During discussions new concepts are discussed and explained in detail.20. The issues are discussed in depth.21. The problems of the scenario are questioned and clarified using the newly learned knowledge.22. The discussion in the reporting makes very useful contributions to newly learned knowledge in the self-study process.The pre-publication history for this paper can be accessed here:


Saccharomyces cerevisiae. This simple on/off switch contrasts with a more complex circuitry in higher eukaryotes. Here we show that a heretofore unrecognized pathway operates in yeast to regulate the transcription of HAC1. The resulting increase in Hac1p production, combined with the production or activation of a putative UPR modulatory factor, is necessary to qualitatively modify the cellular response in order to survive the inducing conditions. This parallel endoplasmic reticulum–to–nucleus signaling pathway thereby serves to modify the UPR-driven transcriptional program. The results suggest a surprising conservation among all eukaryotes of the ways by which the elements of the UPR signaling circuit are connected. We show that by adding an additional signaling element to the basic UPR circuit, a simple switch is transformed into a complex response.Nonconventional splicing of the gene encoding the Hac1p transcription activator regulates the unfolded protein response (UPR) in  The unfolded protein response in yeast was thought to be a simple on/off switch. Here, a second signaling element is revealed, transforming this simple switch into a complex response In eukaryotes, the endoplasmic reticulum (ER) serves as the first station of the secretory pathway, through which all secreted and membrane proteins must pass. Within the ER, proteins are folded into their native structure and multisubunit protein complexes are assembled. The ER is a dynamic organelle, capable of sensing and adjusting its folding capacity in response to increased demand: when misfolded proteins accumulate in the ER, a signaling pathway, termed the unfolded protein response (UPR), is activated  or tunicamycin (both agents that cause protein misfolding selectively in the ER) and monitored HAC1 mRNA by Northern blot analysis  to spliced iHAC1 mRNA (i for UPR-induced). Quantitation of the results shows that the relative abundance of HAC1 mRNA (the sum of uHAC1 and iHAC1 mRNAs) remained unchanged over at least 12 h   or only a mild effect  on HAC1 mRNA abundance.In light of these observations, we were surprised to find that blocking the secretory pathway distal to the ER resulted in a pronounced increase in s 13–16) . SplicinHAC1 mRNA levels in response to altered conditions in the secretory pathway. In the experiments described above, we observed HAC1 mRNA induction only in sec mutants that block transport distal to the ER, not in those that block protein entry into the ER. One common consequence of blocking the secretory pathway at later stages is that proteins in transit will eventually back up into the ER  cells can be induced to upregulate HAC1 mRNA: (1) ER protein misfolding combined with a temperature shift from 23 °C to 37 °C . Thus, HAC1 mRNA induction requires a bipartite signal, consisting of one input provided by unfolded proteins in the ER , and the other input provided by inositol starvation or temperature shift .To determine the nature of this second signal, we sought conditions that induce to 37 °C A and (2)to 37 °C B. IntrigSSA1, a known target of the heat shock response  flanked by ACT1 untranslated regions (HAC1pro-GFP). The resulting heterologous GFP mRNA therefore contained no HAC1 mRNA sequences. Under conditions providing both the UP and I/T signals, the change in abundance of the GFP mRNA .The results presented so far define a novel regulatory mechanism whereby cells adjust the amount of HAC1 mRNA levels under otherwise identical growth conditions. To this end, we engineered a yeast strain unable to transcriptionally upregulate HAC1. In these cells, HAC1 mRNA expression was removed from the control of the HAC1 promoter and was instead driven by the heterologous ADH1 promoter (ADH1pro-HAC1), at levels closely approximating the uninduced HAC1 state . Expression from the ADH1 promoter was constitutive, and the levels of HAC1 mRNA did not change significantly under the various inducing conditions described above. As expected, induction of the UPR in these strains led to efficient HAC1 mRNA splicing and Hac1p production. This strain therefore allowed us to fix the cellular Hac1p concentration to a level closely approximating the basal HAC1 expression state observed during the UPR.To assess the physiological role of the S-UPR, we sought conditions that would allow us to directly monitor the consequences of changes in HAC1 mRNA levels were required for cell growth. Therefore, we subjected WT cells and the engineered strain described above to the combinations of stresses described in HAC1 from the endogenous or from the ADH1 promoter grew equally well on plates lacking inositol  and redox proteins . (2) Class 2 targets  Class 3 targets . It could also be due to a combination of these two scenarios. To distinguish among these possibilities, we determined the target gene induction profile in cells in which the HAC1 mRNA concentration was artificially elevated to a similar level as that found after S-UPR induction. We took advantage of a specific 15-bp deletion in the HAC1 promoter HI),, splicing of HAC1 mRNA was somewhat reduced upon UPR induction ; however, even with this reduction, HIHAC1pro cells produced approximately 2.5-fold more spliced iHAC1 mRNA than WT cells . Otherwise, however, high levels of Hac1p did not fully reconstitute the induction seen during the S-UPR. For example, while the Class 3 gene INO1 was induced 7.5-fold more in the S-UPR than in the UPR, it was induced only 3-fold more by high levels of Hac1p, compared to normal levels. We conclude that elevated Hac1p levels are sufficient to selectively increase the induction of a few UPR targets, but that the full transcriptional program of the S-UPR predicts the production or activation of an additional transcriptional activator, which we term UPR modulatory factor (UMF).The ability to set ADH1pro-HAC1 cells, which are prevented from achieving high level Hac1p expression, and compared the mRNA expression profile against the UPR in WT cells. In this analysis, Hac1p levels were approximately equivalent in the two conditions, so variations from the normal UPR transcriptional program reflect the activity of UMF. The results are shown in ADH1pro-HAC1 cells, compared to induction during the UPR in WT cells, and the y-axis shows the number of genes at any given ratio. Not surprisingly, the induction of Class 1 targets (YOR289W and YHR087W (both of unknown function) reach near WT S-UPR induction levels, without elevated levels of Hac1p; for these targets, UMF likely plays a leading role in their induction, with Hac1p having less influence. Most Class 2 and Class 3 targets  produced or activated by the S-UPR (UMF), allows the cell to mount a modified transcriptional response to cope with the inducing stress conditions.In this paper, we describe a novel ER surveillance pathway in yeast that modulates the UPR, resulting in a new physiological state that we term the S-UPR. In response to a bipartite signal transmitted from the ER by an HAC1 produces uHAC1 mRNA, which is translationally inactive due to the presence of the inhibitory intron. In response to unfolded proteins, Ire1p performs an on/off operation, excising the intron from uHAC1 mRNA to generate spliced iHAC1 mRNA, which is translated to produce the Hac1p transcription activator. The S-UPR provides another layer of regulation superimposed on the UPR (in blue). If ER folding stress is combined with either a shift to elevated temperature or inositol starvation, an AND gate integrates this bipartite signal and boosts HAC1 mRNA levels. In turn, this regulation causes increased Hac1p production. Together with UMF, Hac1p induces UPR target genes, with particular genes responding differentially to differences in Hac1p and UMF concentration. Thus the S-UPR can be seen as an adaptation of the classical  UPR, fine-tuning the activation of select targets to produce a response suited to the challenge faced by the cell.HAC1 proceeds normally in Δgcn4 cells (unpublished data). A recent report from Ogawa and coworkers , elevated Hac1p concentrations are sufficient to increase transcriptional induction, while for others, S-UPR-derived UMF is also required. We find evidence for both kinds of regulation. The promoters of target genes, therefore, display differential responsiveness to Hac1p concentration and UMF activity.HAC1 mRNA abundance can be used as a gene-specific gain control for target activation. This control is similar to that observed in regulation of phosphate metabolism, where the differential affinity of certain Pho4p phosphoforms for target promoters allows for the selective activation of a subset of phosphate-responsive genes . Promoters with lesser affinity for Hac1p would increase in occupancy, and hence transcriptional activation, as Hac1p levels rose during the S-UPR, and would possibly achieve full transcriptional activity only with the additional binding of UMF . Such a mechanism of promoter-encoded differential responsiveness to transcription factor concentration would explain the selective regulation of subsets of UPR target genes.The HAC1 in yeast  and are otherwise genotypically identical to W303. The cHSF1 strain was a kind gift of Hillary Nelson  and contains the R222A allele of HSF1  or with pPW599 . Strains used in experiments described in HAC1pro-HAC1 and Δhac1 (described above) and Δhac1 transformed with pPW600  was made by subjecting HAC1pro-HAC1 to QuikChange mutagenesis  following the manufacturer's protocol, using oligonucleotides to remove the 15 bp at coordinates −338 to −323 (+1 representing the start site of transcription).The WT strain W303–1A, the Δeviously . All sec of HSF1  replacinged HAC1  under itcription  and exten vector  and tran600≈ 0.5). For temperature shift experiments, cultures were transferred to a preheated 37 °C water bath shaking incubator. DTT  was added to a final concentration of 6 mM, and tunicamycin  was added to a final concentration of 1 μg/ml. For experiments involving inositol deprivation in liquid medium, yeast cells were grown in liquid complete synthetic medium described by Yeast cultures were grown in YPD medium (unless otherwise specified) at the indicated temperatures to midlog phase (OD600 ≈ 0.5), transferred to a 96-well microtiter plate, and serially 5-fold diluted in fresh YPD. Using a liquid transfer prong (“frogging”) tool , approximately 3 μl of all serial dilutions of all strains was simultaneously transferred to complete synthetic plates lacking inositol (described above), either in the absence or presence of 0.2 μg/ml tunicamycin. After approximately 2 d of incubation at 30 °C, plates were photographed using the Epi Chemi II Darkroom GelDoc system .For the experiment described in HAC1. The mRNA abundance was quantitated using a PhosporImager . The membranes were then stripped with two serial washes using 0.1% SDS at 65 °C for 60 min each and incubated with a probe directed against the 3′ exon of ACT1, and mRNA abundance was again quantitated. To control for the variable strength of Northern blot probes across multiple experiments, the relative HAC1/ACT1 mRNA abundance ratio is always normalized to the untreated (t = 0) sample. For the detection of other mRNAs, membranes were incubated with the additional relevant probes  concurrent with the HAC1 probe. All data shown are an average of at least two independent experiments.Isolation of total RNA from yeast cells was carried out with the modified hot-phenol extraction method described in http://derisilab.ucsf.edu/core/, was performed as in http://microarrays.org/. All array data are the average of two independent experiments. For this study, we were obliged to evaluate UPR targets differently than in HAC1-independent responses, whereas the former study specifically isolated genes induced by unfolded proteins via Hac1p (z-score ≥ 3.6 σ). Here, UPR targets were defined as those genes that met the following three criteria in a parallel set of microarray experiments using WT (W303) and Δhac1 (JC408) strains. First, induction (log2 of the fold change in gene expression) in WT cells treated with DTT must be at least one standard deviation greater than the mean . Second, induction in WT cells treated with tunicamycin must be at least one standard deviation greater than the mean . Third, induction in Δhac1 cells treated with DTT must be at least one standard deviation less than the induction in WT cells treated with DTT /σWT,DTT] − ) − μhac1,DTTWT,DTT − Δ]/σhac1,DTTWT,DTT − Δ ≥ 1).PolyA+ mRNA was isolated from total RNA using the PolyATract system  according to the manufacturer's instructions. Microarray analysis, using yeast ORF arrays printed at the University of California, San Francisco, Core Center for Genomics and Proteomics , and Western blots were visualized using SuperSignal West Dura Extended Duration ECL Substrate  according to the instructions of the manufacturer. Hac1p was detected using a polyclonal antibody raised against the carboxy terminus  was grown in YPD at 23 °C to OD600 ≈ 0.5 and then shifted to a 37 °C water bath, shaking at 250 RPM. To induce the UPR, DTT was added to a final concentration of 6 mM. Cells were harvested and total RNA isolated, at 20 min intervals, as described above.The yeast strain JC218 ; rbp1–1 The GenBank accession numbers of the sequences discussed in this paper are Hac1p (NP_ 116622), Ire1p (NP_011946), and tRNA ligase (NP_012448).Microarray data can be accessed at the Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) database as platform number GPL999 and sample numbers GSM16978–GSM1984.


Tyrosine kinases regulate signaling pathways that control cell growth, proliferation, motility, and other critical cellular processes. Mutations in tyrosine kinase genes can lead to abnormal kinase activity, and some tumors become dependent upon this activity for growth and survival. Thus, kinases are attractive targets for anti-cancer drugs. Examples of new kinase inhibitors include gefitinib and erlotinib, which have recently shown promise in treating non-small-cell lung cancer. Unfortunately, gefitinib and erlotinib work only in a subset of patients, and they can have severe side effects, albeit infrequently. So researchers have been trying to find ways to predict who will benefit from therapy with these drugs and who won't.Following the work of Lynch et al. (N Engl J Med 350: 2129–2139) and Paez et al. (Science 304: 1497–1500), William Pao and colleagues have previously shown that the epidermal growth factor receptor (EGFR), a tyrosine kinase, is often mutated in non-small-cell lung cancers, and that tumors that harbor such mutations are sensitive to gefitinib and erlotinib.In this new study, they focused on a signaling protein called KRAS, which functions downstream of many tyrosine kinases, including EGFR. The KRAS gene is also often mutated in lung cancers, but very few cancers have mutations in both EGFR and the KRAS gene. To find out whether KRAS mutations could help to predict which patients would respond to gefitinib or erlotinib, the researchers looked for mutations in EGFR and KRAS genes in 60 tumors for which sensitivity to either drug was known.They extended their earlier findings that EGFR mutations (which were found in 17 of the tumors) were associated with sensitivity to the kinase inhibitors, and found that tumors that had mutations in KRAS  were refractory  to either drug.These results need to be validated in larger and prospective trials that use standardized mutation detection techniques. If they are confirmed, knowing the mutation status of EGFR and KRAS in tumors could help physicians decide which patients should receive gefitinib and/or erlotinib. As Inoue and Nukiwa state in a Perspective that accompanies the article, “By combining all the factors that relate to response or resistance, patients who will benefit from treatment can hopefully be identified. Undoubtedly we have taken a great step forward in molecular therapy for lung cancer treatment.”


Biological systems are so complex that we must ask: "What purpose does all this complexity serve?" Lander argues that computational biology may help provide answers Why is the sky blue? Any scientist will answer this question with a statement of mechanism: Atmospheric gas scatters some wavelengths of light more than others. To answer with a statement of purpose—e.g., to say the sky is blue in order to make people happy—would not cross the scientific mind. Yet in biology we often pose “why” questions in which it is purpose, not mechanism, that interests us. The question “Why does the eye have a lens?” most often calls for the answer that the lens is there to focus light rays, and only rarely for the answer that the lens is there because lens cells are induced by the retina from overlying ectoderm.It is a legacy of evolution that teleology—the tendency to explain natural phenomena in terms of purposes—is deeply ingrained in biology, and not in other fields . NaturalAs a group, molecular biologists shy away from teleological matters, perhaps because early attitudes in molecular biology were shaped by physicists and chemists. Even geneticists rigorously define function not in terms of the useful things a gene does, but by what happens when the gene is altered. Molecular biology and molecular genetics might continue to dodge teleological issues were it not for their fields' remarkable recent successes. Mechanistic information about how a multitude of genes and gene products act and interact is now being gathered so rapidly that our inability to synthesize such information into a coherent whole is becoming more and more frustrating. Gene regulation, intracellular signaling pathways, metabolic networks, developmental programs—the current information deluge is revealing these systems to be so complex that molecular biologists are forced to wrestle with an overtly teleological question: What purpose does all this complexity serve?In response to this situation, two strains have emerged in molecular biology, both of which are sometimes lumped under the heading “systems biology.” One strain, bioinformatics, champions the gathering of even larger amounts of new data, both descriptive and mechanistic, followed by computerbased data “mining” to identify correlations from which insightful hypotheses are likely to emerge. The other strain, computational biology, begins with the complex interactions we already know about, and uses computer-aided mathematics to explore the consequences of those interactions. Of course, bioinformatics and computational biology are not entirely separable entities; they represent ends of a spectrum, differing in the degree of emphasis placed on large versus small data sets, and statistical versus deterministic analyses.Computational biology, in the sense used above, arouses some skepticism among scientists. To some, it recalls the “mathematical biology” that, starting from its heyday in the 1960s, provided some interesting insights, but also succeeded in elevating the term “modeling” to near-pejorative status among many biologists. For the most part, mathematical biologists sought to fit biological data to relatively simple mathematical models, with the hope that fundamental laws might be recognized . This stNowadays, many computational biologists avoid modeling-as-data-fitting, opting instead to create models in which networks are specified in terms of elements and interactions (the network “topology”), but the numerical values that quantify those interactions (the parameters) are deliberately varied over wide ranges. As a result, the study of such networks focuses not on the exact values of outputs, but rather on qualitative behavior, e.g., whether the network acts as a “switch,” “filter,” “oscillator,” “dynamic range adjuster,” “producer of stripes,” etc. By investigating how such behaviors change for different parameter sets— an exercise referred to as “exploring the parameter space”—one starts to assemble a comprehensive picture of all the kinds of behaviors a network can produce. If one such behavior seems useful (to the organism), it becomes a candidate for explaining why the network itself was selected, i.e., it is seen as a potential purpose for the network. If experiments subsequently support assignments of actual parameter values to the range of parameter space that produces such behavior, then the potential purpose becomes a likely one.For very simple networks , possible global behaviors are usually limited, and computation rarely reveals more than one could have gleaned through intuition alone. In contrast, when networks become even slightly complex, intuition often fails, sometimes spectacularly so, and computation becomes essential.For example, intuitive thinking about MAP kinase pathways led to the long-held view that the obligatory cascade of three sequential kinases serves to provide signal amplification. In contrast, computational studies have suggested that the purpose of such a network is to achieve extreme positive cooperativity, so that the pathway behaves in a switch-like, rather than a graded, fashion . AnotherAs the power of computation to identify possible functions of complex biological networks is increasingly recognized, purely (or largely) computational studies are becoming more common in biological journals. This raises an interesting question for the biology community: In a field in which scientific contributions have long been judged in terms of the amount of new experimental data they contain, how does one judge work that is primarily focused on interpreting  the experimental data of others? At the simplest level, this question poses a conundrum for journal editors. At a deeper level, it calls attention to the biology community's difficulty in defining what, exactly, constitutes “insight” .In yesterday's mathematical biology, a model's utility could always be equated with its ability to generate testable predictions about new experimental outcomes. This approach works fine when one's ambition is to build models that faithfully mimic particular biological phenomena. But when the goal is to identify all possible classes of biological phenomena that could arise from a given network topology, the connection to experimental verification becomes blurred. This does not mean that computational studies of biological networks are disconnected from experimental reality, but rather that they tend, nowadays, to address questions of a higher level than simply whether a particular model fits particular data.PLoS Biology  are laid down. In the early fruit fly embryo, it is known that a network involving cell-to-cell signaling via the Wingless (Wg) and Hedgehog (Hh) pathways specifies the formation and maintenance of alternating stripes of gene expression and cell identity. This network is clearly complex, in that Wg and Hh signals affect not only downstream genes, but also the expression and/or activity of the components of each other's signaling machinery.When they used a network topology based only upon molecular and generegulatory interactions that were firmly known to take place in the embryo, they were unable to produce the necessary output (stable stripes), but upon inclusion of two molecular events that were strongly suspected of taking place in the embryo, they produced the desired pattern easily. In fact, they produced it much more easily than expected. It appeared that a remarkably large fraction of random parameter values produced the very same stable stripes. This implied that the output of the network is extraordinarily robust, where robustness is meant in the engineering sense of the word, namely, a relative insensitivity of output to variations in parameter values.Because real organisms face changing parameter values constantly—whether as a result of unstable environmental conditions, or mutations leading to the inactivation of a single allele of a gene—robustness is an extremely valuable feature of biological networks, so much so that some have elevated it to a sort of sine qua non . Indeed,There is little doubt that von Dassow's computational study extracted an extremely valuable insight from what might otherwise seem like a messy and ill-specified system. But Ingolia now argues that something further is needed. He proposes that it is not enough to show that a network performs in a certain way; one should also find out why it does so.Ingolia throws down the gauntlet with a simple hypothesis about why the von Dassow network is so robust. He argues that it can be ascribed entirely to the ability of two positive feedback loops within the system to make the network bistable. Bistability is the tendency for a system's output to be drawn toward either one or the other of two stable states. For example, in excitable cells such as neurons, depolarization elicits sodium entry, which in turn elicits depolarization—a positive feedback loop. As a result, large depolarizations drive neurons to fully discharge their membrane potential, whereas small depolarizations decay back to a resting state. Thus, the neuron tends strongly toward one or the other of these two states. The stability of each state brings with it a sort of intrinsic robustness— i.e., once a cell is in one state, it takes a fairly large disturbance to move it into the other. This is the same principle that makes electronic equipment based on digital  signals so much more resistant to noise than equipment based on analog circuitry.Ingolia not only argues that robustness in the von Dassow model arises because positive feedback leads to network bistability, he further claims that such network bistability is a consequence of bistability at the single cell level. He strongly supports these claims through computational explorations of parameter space that are similar to those done by von Dassow et al., but which also use strippeddown network topologies , test specifically for bistability, correlate results with the patterns formed, and ultimately generate a set of mathematical rules that strongly predict those cases that succeed or fail at producing an appropriate pattern.sloppy-paired, in Hh signaling, removes some of the more tenuous connections, and promptly recovers a biologically essential behavior that the original von Dassow network lacked: the ability to maintain a fixed pattern of gene expression even in the face of cell division and growth.At first glance, such a contribution might seem no more than a footnote to von Dassow's paper, but a closer look shows that this is not the case. Without mechanistic information about why the von Dassow network does what it does, it is difficult to relate it to other work, or to modify it to accommodate new information or new demands. Ingolia demonstrates this by deftly improving on the network topology. He inserts some new data from the literature about the product of an additional gene, Taken as a pair, the von Dassow and Ingolia papers illustrate the value of complementary approaches in the analysis of complex biological systems. Whereas one emphasizes simulation , the other emphasizes analysis . Whereas one emphasizes exploration (exploring a parameter space), the other emphasizes the testing of hypotheses (about the origins of robustness). The same themes can be seen in sets of papers on other topics. For example, in their analysis of bacterial chemotaxis, Leibler and colleagues  found a From these and many other examples in the literature, one can begin to discern several of the elements that, when present together, elevate investigations in computational biology to a level at which ordinary biologists take serious notice. Such elements include network topologies anchored in experimental data, fine-grained explorations of large parameter spaces, identification of “useful” network behaviors, and hypothesisdriven analyses of the mathematical or statistical bases for such behaviors. These elements can be seen as the foundations of a new calculus of purpose, enabling biologists to take on the much-neglected teleological side of molecular biology. “What purpose does all this complexity serve?” may soon go from a question few biologists dare to pose, to one on everyone's lips.


The low-grade inflammation seen with aging is noted particularly in subjects with the metabolic syndrome of aging. Insulin resistance, obesity/abdominal obesity, and risks for many age-related diseases characterize this common syndrome. It is becoming clear  that this increased adipose tissue is not simply a  reservoir for excess nutrients, but rather an active  and dynamic organ capable of expressing several  cytokines and other fat-derived peptides (FDP). Some, but not all, FDP may have a role in development of the metabolic syndrome but there is no evidence that these FDP are causing inflammation directly. We suggest that high levels of inflammatory peptides are markers for obesity/abdominal obesity seen with aging, but some may not necessarily have a causative role in the development of inflammation. Because bone marrow and adipocytes are derived from the same stem cells, it is not surprising to find so many inflammatory peptides expressed in fat tissue. Many of these are classical inflammatory peptides derived from components of the adipose tissue and all are known also as fat derived peptides (FDP). With aging, there is a linear accumulation of adipose cells and percent of body fat increases. This increased body fat is characterized by increased visceral adiposity  and occuThe metabolic syndrome is a common disorder consisting of a cluster of abnormalities including insulin resistance, dyslipidemia, and hypercoagulability and is associated with increased risk for cancer, Alzheimer's disease, type-II diabetes and atherosclerosis . It is aα, and other (non-cytokine) FDP, such as PAI-1, adiponectin and resistin, which may play a role in the pathogenesis, and/or serve as markers of risk in the metabolic syndrome. The fact that many of these peptides are derived from adipose tissue leads us to the question of whether adipose tissue itself is the underlying pathophysiological link between obesity and the poor clinical outcomes associated with the metabolic syndrome. We will provide a brief overview of some of the peptides associated with the metabolic syndrome.Increased inflammatory peptides are being studied as possible modifiable markers of the increased risk predictors of disease and possibly the underlying link between obesity and the poor clinical outcomes seen with the metabolic syndrome. More specifically, C-reactive protein (CRP) is the most well established inflammatory cytokine in the clinical setting but there are other inflammatory cytokines including IL-6, leptin, TNF-TNFα, leptin, and IL-6 are examples for cytokines that may have a role in the metabolic syndrome. TNFα, previously known as lymphotoxin and cachetin, is believed to be involved in the wasting that occurs during acute and chronic illness and malignancy. In the basal state TNFα is directly proportional to fat mass and has been shown to be involved in the development of insulin resistance [α decreases the insulin receptor tyrosine phosphorylation, and down regulates several steps in the insulin signaling pathway [α have been shown to improve insulin resistance. [TNFα is not only a classical cytokine but may be causal in the insulin resistance of the metabolic syndrome of aging.sistance . In-vitr pathway  while neistance.  Thus, TNLeptin is a peptide derived from adipose tissue and like other cytokines acts through a cytokine receptor. It is expressed and secreted in direct proportion to fat mass. Leptin exerts is effect predominantly through receptors in the hypothalamus but it may also have peripheral actions [Leptin appears to have an important role in energy regulation but no apparent role in development of inflammation. actions . Leptin  actions  With obeIL-6 is another cytokine derived from adipose tissue. Its expression and circulating levels correlate directly with obesity, and weight loss will lower circulating levels. Elevation of circulating IL6 is a predictor of the development of cardiovascular disease and diabetes [IL-6 plays a role in the development of insulin resistance and may directly cause induction of CRP.diabetes . Infusiodiabetes  Additiondiabetes IL-6 playIL-1, IL-8, IL18, Serum Amyloid A, have been shown to be increased with obesity and may have a yet undetermined role in the syndrome. These cytokines are other examples of inflammatory markers which do not have a clear role in the causation of systemic inflammation.Other inflammatory cytokines such as Adiponectin is highly expressed in adipose tissue, and is the one non-cytokine FDP that is protective from inflammation. Unlike most FDP, circulating levels are inversely proportional to obesity and therefore tend to be low in obesity. Adiponectin levels increase with weight loss and with use of insulin sensitizing drugs. [g drugs.  Adiponecg drugs.  Low leveg drugs.  Animal mg drugs.  The pro-Plasminogen activator inhibitor type-1 (PAI-1) is the primary inhibitor of fibrinolysis and is highly expressed in adipose tissue. Levels of PAI-1 are elevated in acute conditions such as deep venous thrombosis, and chronic conditions such as obesity, the metabolic syndrome of aging and diabetes. PAI-1 levels are correlated with adiposity and significantly overexpressed in the adipose tissue of obese compared to lean animals. [animals.  Levels aanimals. . The relAngiotesinogen (AGT) is a peptide that is produced in the liver and in adipose tissue. The strong correlation between obesity and hypertension implies that adipose tissue may play a role in blood pressure regulation and in fact there is a correlation between circulating AGT levels and obesity/hypertension [rtension . Animal rtension .Resistin is a peptide which is elevated in obesity and appears to play a role in glucose homeostasis in rodents. In experimental models, resistin induces hepatic insulin resistance while anti-resistin antibodies have the opposite effect [e effect . In humavivo metabolism, we suggest that some levels of cytokines are increased because of the hyperplastic characteristic of adipose tissue, and their levels are better serve as marker of adipose tissue hypertrophy, rather than having a causal role in aging. Thus, whether aging is inflammatory state or whether it is a state associated with increased inflammatory marker is subject for further studies.Low grade inflammation is a predominant feature in the metabolic syndrome of aging and seems to be linked to the development of diabetes and poor vascular outcomes. We have briefly named several cytokines and other FDP that are generally increased with fat mass. Although many of these FDP have a role in metabolic homeostasis, many seem to lack distinct role in inflammatory pathogenesis. While many FDP have roles in


Escherichia coli lacking the ability to digest lactose, called −lac  mutants, regain that ability when presented with lactose as their only food source.For a human, the normal response to stress is to reduce it through some purposeful action, be it indulging in chocolate or calling in sick, at a rate which we can vary to fit the circumstances. For a strain of bacteria faced with stress, the choice is often more stark: it must mutate or die. Among evolutionary theorists, an important question has been whether the rate of mutation is fixed, or instead can adaptively increase in response to stress, thereby increasing the likelihood of a favorable mutation. Something like this latter possibility was envisioned by Darwin, but fell out of favor among some neo-Darwinists, for whom a steady rate of mutation was more in keeping with their overall model of evolutionary gradualism. This debate is taken up in a new study in this issue by P. J. Hastings and colleagues, who examined the mechanism by which −lac  mutants to a +lac state can be achieved by either of two genetic events: amplification, which creates numerous copies of the nonfunctional lac gene, and point mutations, which give rise to functional versions of the gene . According to the gradualist view, amplification precedes mutation, and the rapid appearance of +lac cells is explained by a normal mutation rate acting on multiple copies of the gene. In contrast, according to the hypermutation view, amplification and mutation are independent events, and +lac cells arise quickly because the mutation rate has increased.It has been known for some time that the reversion of While some results from previous studies have supported the gradualist interpretation, the experiments of Hastings et al. show that hypermutation is the most plausible explanation. A variety of procedural improvements allowed them to analyze more individual cells at an earlier stage of colony development. For instance, they analyzed colonies composed of as few as one hundred cells, rather than the ten thousand cells in prior experiments, and even nascent colonies at the two-cell stage.+lac colonies, before amplification can account for the number of +lac revertants observed. Amplification is not only independent from mutation, but occurs relatively late under starvation. The researchers found that amplification, but not point mutation, requires the presence of a particular DNA polymerase, further strengthening the case that amplification need not precede mutation. They also showed that amplification by itself does not induce a so-called SOS response. The SOS system includes a group of genes that cause an increase in mutation in response to stress, and one hypothesis arising from the gradualist model was that amplification turned on the SOS response.The study produced clear evidence that point mutations arise very early in the development of Based on their data, the authors reject the strict gradualist model for the adaptive mutation mechanism in the Lac system. They propose that amplification and hypermutation are independent responses to stress, each of which increases the likelihood of adaptive change. They also suggest that a stress-induced increase in the rate of point mutations may have implications for a variety of mutation-related phenomena, from tumor formation to development of resistance to antibiotics and chemotherapeutic drugs.