This study was planned to collect data about causes, prevalence and responses to work-related musculoskeletal disorders reported by physiotherapists employed in Izmir, Turkey.A two-page survey with closed ended questions was used as the data collected method. This survey was distributed to 205 physiotherapists working in Izmir, Turkey, and 120 physiotherapists answered. Questions included occupational history of physiotherapists and musculoskeletal symptoms, special areas, tasks, job-related risk factors, injury prevention strategies, and responses to injury.Eighty-five percent of the physiotherapists have had a musculoskeletal injury once or more in their lifetime. Injuries have been occurred mostly in low back (26 %), hand-wrist (18 %), shoulders (14 %) and neck (12 %). The highest risk factor in causing the injury was transferring the patient at 15%. Sixty-nine percent of physiotherapists visited a physician for their injury and sixty-seven percent of the respondents indicated that they had not limited their patient contact time as a result to their injuryAccording to the results of this study, the rate of musculoskeletal disorders in physiotherapists in Izmir-Turkey has been found to be high due to their profession. Respondents felt that a change in work habits was required in order to decrease the risk of another injury. A work-related musculoskeletal disorder (WRMD) is defined as a musculoskeletal injury that results from a work-related event. This may result in lost work time, work restriction, or transfer to another job -4. TheseUnderstanding the issues related to musculoskeletal disorders in physical therapists requires some awareness of the context in which these professionals work. In Turkey, physiotherapists cannot treat patients directly. According to law, direct accesses to physiotherapists are not possible . FurtherThe purpose of this study was to investigate the prevalence and features of WRMDs in a group of Turkish physiotherapists working in various capacities in a large Turkish city, and to compare the findings to reports from other countries.The study was approved by the Ethics Committee of Dokuz Eylül University. The research population was selected physiotherapists who were employed in a broad spectrum of practice settings in İzmir, Turkey. All data were collected by a mailed questionnaire (Appendix 1 see ) that coThe questionnaire was based on another published survey ,6 and si2 were used to analyze influence personal characteristics (sex, age, number of years in physiotherapy practice, number of hours per week in direct patient care) to WRMDs.Data were analyzed using SPSS 10.0 for Windows. Results for the general information items were expressed as mean ± standard deviation, and results for items in the occupational portion were expressed as percentages. χThe 120 respondents included 92 females and 28 males of mean age 30.4 ± 6.9 years (range, 22–55 years). Total response rate was 59%, for women 59%, for men 58%. General information about the group is given in Table 2=.234, P=.629), age (χ2=.383, P=.536), number of years in physiotherapy practice (χ2=.067, P=.795), or number of hours per week in direct patient care (χ2=.151, P=.698) (Table Frequency of WRMDs was not correlated with sex (χOne hundred and two respondents (85%) reported that they had experienced work-related musculoskeletal pain or discomfort at some time in their occupational lives. Seventy-eight (65%) reported that they had sustained more than one WRMD.The lower back was the body part with the highest frequency of occupational injury (26%), and wrist-hand (18%), shoulder 14%), and neck (12%) were other sites frequently affected Table . The mai%, and neThe respondents who had experienced a WRMD indicated that lifting (18%), maintaining a position for prolonged period of time (17%), performing repetitive tasks 16%), and transferring patients (16%) were the activities that most often exacerbated their symptoms during clinical practice Table . Improve%, and trSixty-nine percent of the respondents with WRMDs had visited a physician for the problem, and 46% of whom stated that they had officially reported the injury to their employer. The respondents who had suffered WRMDs reported they used their own occupational knowledge, rest, medications, and exercise to treat the problem Table . Sixty-sMusculoskeletal system problems connected to occupational conditions are common among health care workers. The costs of these are substantial, both in terms of money and in terms of work time lost ,9,17,18.The results from studies on WRMDs in physiotherapists have generally been similar, though some have differed according to country. Such variations are linked to level of development, the status of the profession of physiotherapy in a given country, psychosocial, and epidemiological factors ,11,13,14In this study, we collected demographic and WRMD data from 120 physiotherapists in Izmir, Turkey, and analyzed rates of injury, risk factors, injury types and sites, and post-injury management. We asked to complete the self-administered questionnaire if they had more than 2 years of experience in practice. Thus, response rate of questionnaire was low 58.5 %) in our study. The survey answers revealed that 85% of the respondents had experienced WRMDs. Cromie .5 % in o investigConcerning sites of musculoskeletal injury during professional activities, the highest incidence is in the low-back region. Biomechanical studies have shown that physical loading factors, such as body flexion, rotation and weight loading, play a role in this . In a stStudies of WRMDs in health care of professionals have identified the lower back as the most commonly involved area of the body, followed by neck and upper extremities ,13,19-21According to the literature the work-related activities that most commonly lead to injury in health professionals are lifting heavy equipment and patients, transferring patient, maintaining the same posture for a long period, manual therapy practices, responding to patients' sudden movements, and repeated movements ,18,22-24Holder et al.  identifiIn Turkey, most physiotherapists work in general hospitals. In these facilities, the daily treated patient number is too high, and this number far exceeds the number of physiotherapists on staff. In addition, the majority of patients in these hospitals are seriously ill .etc.) is not ergonomic, and consequently the physiotherapist cannot follow ergonomic principles while doing his or her job.People who suffer injuries on the job may be treated with medication, rest and exercise. Physiotherapists have fundamental knowledge about ergonomics and biomechanics, and using this knowledge may vary depending on professional knowledge and skills. In Turkey, physiotherapists are trained in ergonomic working principles at the undergraduate level. However, in many working environments the equipment used (treatment table, chair, A study conducted by Cromie  in 2002 et al. [Previous research has shown that physiotherapists who have suffered a WRMD tend to change their professional attitudes to avoid additional injuries. Holder et al.  found thMierzejewski  reportedSome professions have very high WRMD prevalence, and this has led to more intensive research in recent years. These types of injury have major consequences for society, workers, employers, and the insurance sector due to loss of labor force, long-term disability and delay in returning to work, decreased productivity, and psychological effects on employees. Therefore, minimizing and preventing WRMDs has significant potential social and economic benefits. It is important to invest in studies aimed at reducing these types of injuries in physiotherapists.Our study has three main limitations. A physiotherapist's working time in a day is not long, but he or she treats a very large number of patients during this time. Our survey would have been more informative if we collected data for the number of daily treated patients. We also did not inquire about the physiotherapists' activity levels. This would have been valuable information, as sports and recreational activity may affect WRMD frequency. Another limitation of our study was comparing various areas of practice and techniques used by physiotherapists and investigating their relationship to WRMDs.Our survey reveals that the WRMDs in physiotherapists in Turkey are similar to rates reported in other countries. Physiotherapists in our country suffer similar work-related injuries as their counterparts elsewhere, despite different legal working conditions and cultural differences. This study provides data related to WRMDs in physiotherapists in Turkey. Further studies can be very useful if it research prevalence of WRMDs in physiotherapists who have employed different working conditions.None declared.YS participated in the design, managed the data collection and performed statistical analysis.AO participated in designed the study protocol, managed the coordination.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Table 1, Table 2. Questionnaire on occupational injuries in physical therapists. Turkish version of questionnaire on occupational injuries in physical therapistsClick here for file


Cholesterol ester transfer protein (CETP) plays a major role in regulating the levels of LDL- and HDL-cholesterol. We previously observed a fish-oil-induced elevation of low-density lipoprotein (LDL)-and very-low-density lipoprotein (VLDL)-cholesterol concentrations and a decrease in high-density lipoprotein (HDL)-cholesterol concentration in F1B hamsters. The molecular mechanism/s by which fish oil induces hyperlipidaemic effect was investigated in this study. We examined whether the effects of dietary fish oil on plasma lipoprotein concentrations are due to fish-oil-induced alterations in plasma CETP activity. MIX diet, a diet supplemented with a mixture of lard and safflower oil, was used as the control diet.We found that fish oil feeding in hamsters reduced CETP mass as well as CETP activity. Increasing the dietary fat level of fish-oil from 5% to 20% (w/w) led to a further decrease in CETP mass. Supplementation with dietary cholesterol increased both CETP mass and CETP activity in fish-oil and MIX-diet fed hamsters. However, there was no correlation between CETP mass as well as CETP activity and LDL-cholesterol concentrations.These findings suggest that cholesterol ester transfer between HDL and LDL is not likely to play a major role in determining fish-oil-induced changes in LDL- and HDL-cholesterol concentrations in F1B hamsters. A possible role of reduced clearance of LDL-particles as well as dietary fat level and dietary cholesterol dependent changes in LDL-lipid composition have been discussed. Fish oil, rich in n-3 polyunsaturated fatty acids (PUFA), is considered beneficial in lowering the risk of coronary heart disease ,2. The bOne of the factors that determine LDL concentrations is reverse cholesterol transport pathway, which removes cholesterol from peripheral tissues and returns to the liver. Cholesterol ester transfer protein (CETP) plays a major role in this pathway to transfer cholesterol esters from high-density lipoproteins (HDL) to VLDL and LDL, in exchange for triacylglycerols ,12. ThusCETP mediated exchange of cholesterol esters for triacylglycerols occurs mainly through an equimolar heteroexchange mechanism, which leads to alterations in cholesterol ester: triacylglycerol ratio. In addition to plasma CETP concentration, fatty acid composition of lipoproteins also modifies reverse cholesterol transfer. Cholesterol esters with n-3 polyunsaturated fatty acyl groups are more likely to transfer compared to cholesterol esters with saturated and monounsaturated fatty acyl groups . This efWe previously observed fish-oil-induced elevation of VLDL-and LDL-cholesterol concentrations and decrease in HDL-cholesterol concentration in F1B hamsters . In thisThe LDL-cholesterol, free cholesterol, cholesterol ester, phospholipid and triacylglycerol concentrations were measured in Bio F1B hamsters treated with fish-oil or MIX diet  to core lipids (cholesterol esters and triacylglycerols) ratio was higher in MIX diet fed hamsters compared to hamsters fed the fish oil diet, at both low and high fat levels Table . IncreasIt has been shown previously that there is a correlation between the level of CETP and that of LDL-cholesterol. To further investigate our observation of fish-oil-induced elevation of LDL-cholesterol concentrations and a decrease in HDL-cholesterol concentration in F1B hamsters, we measured CETP mass as well as CETP activity. The changes in CETP mass and CETP activity are shown in Figure CETP mass was significantly and positively correlated with CETP activity in low fat fish oil (r = 0.81, p < 0.03) and MIX diet (r = 0.96 p < 0.0003) fed hamsters Figure  and highThe correlations between CETP mass, CETP activity and LDL-cholesterol are shown in Figure CETP plays an important role in maintaining the levels of plasma LDL and HDL . IncreasCholesterol esters are more liable to transfer in fish-oil fed subjects since the core of the lipoprotein particles from these subjects have lower transition temperature  due to aIncreasing dietary fat level of fish oil decreased cholesterol ester transfer as reflected by decrease in CETP mass Figure . Thus, dHDL-total cholesterol and cholesterol ester concentrations were significantly lower in fish-oil diet fed hamsters compared to MIX-diet fed hamsters, while there was no difference in HDL-triacylglycerol concentrations Table . PreviouIncreasing the dietary fat level from low fat to high fat caused a decrease in HDL-total cholesterol and cholesterol ester levels Table , which mThe interactive effect of dietary cholesterol and n-3 PUFA on plasma cholesterol ester transfer is not yet known. Cholesterol supplementation caused an increase in plasma CETP activity in both fish-oil and MIX-diet fed hamsters at low fat levels  were obtained from Bio Breeders Inc. (Water Town, MA) and kept on chow diet for one week prior to feeding specific diets. After this equilibration period, hamsters were divided into 8 groups (n = 12) and each group was fed with one of the specified diets. The specified diets consisted of fat free semi-purified diet  that was supplemented with either fish oil  or a mixture of lard and safflower oil in 1.5:1 ratio (MIX diet) . The fatThe animals were maintained on specific diets for 2 weeks ad libitum. The food intake was measured daily during the study period, and the body weight was checked at the beginning of the study period, one week later and at the conclusion of the study. There was no difference in food intake and body weight gain between different diet groups. All animals were housed in individual cages in a single room with enriched environment. The Institutional Animal Care use Committee (IACC) approved all experimental procedures, which are in accordance with the principles and guidelines of the Canadian Council on Animal Care. After two weeks on specified diets the animals were sacrificed after 14 hrs of fasting. Blood was collected by cardiac puncture into tubes containing EDTA and centrifuged immediately to separate plasma. Plasma samples were stored at 4°C on ice until further use.g for 20 min at 12°C [Plasma was centrifuged at 15,500  at 12°C  to separ at 12°C . IsolateTotal cholesterol concentration was assayed in all lipoprotein fractions using cholesterol assay kit # 402 (Sigma Diagnostics Inc, St. Louis, MO). Total triacylglycerol concentration of lipoprotein fractions was assayed using triglyceride assay kit # 344 (Sigma Diagnostics Inc, St. Louis, MO). Free cholesterol in plasma and individual lipoprotein fractions was assayed using free cholesterol assay kit . Cholesterol ester concentration was determined by subtracting the free cholesterol concentration from total cholesterol concentration. Phospholipids were analyzed using the method of Bartlett [14C-cholesterol oleate [CETP in the plasma samples was assayed by a radioactive method  that wasl oleate . Plasma CETP mass in plasma samples was quantitatively assayed using CETP ELISA- DAIICHI kit  as previously published . ConcentP value was <0.05 [The effect of diet type, dietary fat level and dietary cholesterol was determined using 3-way analysis of variance, and a Tukey's post hoc test was used to test significant differences revealed by the ANOVA. Values are group means ± SD, n = 12; Differences were considered to be statistically significant if the associated as <0.05 .The author(s) declare that they have no competing interests.PPD conducted all the experiments in this study. PPD and AAM analyzed and interpreted the data, as well as drafted the manuscript. PJD is a collaborator on this project. SKC is the Principal investigator, has conceived the study, participated in its design and final approval of the version to be published. All Authors read and approved the final manuscript.


The speech signal contains both information about phonological features such as place of articulation and non-phonological features such as speaker identity. These are different aspects of the 'what'-processing stream (speaker vs. speech content), and here we show that they can be further segregated as they may occur in parallel but within different neural substrates. Subjects listened to two different vowels, each spoken by two different speakers. During one block, they were asked to identify a given vowel irrespectively of the speaker , while during the other block the speaker had to be identified irrespectively of the vowel (speaker categorization). Auditory evoked fields were recorded using 148-channel magnetoencephalography (MEG), and magnetic source imaging was obtained for 17 subjects.During phonological categorization, a vowel-dependent difference of N100m source location perpendicular to the main tonotopic gradient replicated previous findings. In speaker categorization, the relative mapping of vowels remained unchanged but sources were shifted towards more posterior and more superior locations.These results imply that the N100m reflects the extraction of abstract invariants from the speech signal. This part of the processing is accomplished in auditory areas anterior to AI, which are part of the auditory 'what' system. This network seems to include spatially separable modules for identifying the phonological information and for associating it with a particular speaker that are activated in synchrony but within different regions, suggesting that the 'what' processing can be more adequately modeled by a stream of parallel stages. The relative activation of the parallel processing stages can be modulated by attentional or task demands. This study explores attentional modulation within the 'what'-stream of the auditory modality during phoneme processing. Knowledge of speech sound representation in the auditory domain is still sparse. However, parallels to the extensively studied visual modality and also to the somatosensory domain are becoming evident. For example, columnar mapping of several stimulus properties  has been revealed in human and animal research: acoustic parameters like spectral bandwidth, periodicity, stimulus intensity ,2 or – f1-F2 distance) were located more posterior in auditory association cortex than responses to CORONAL vowels , and a topographical shift between these classes of vowels even when embedded in non-words has been reported [Given these parallels between sensory domains and the increasing preference for complex stimuli along the auditory central pathway, more complex topologies such as language-specific maps in auditory cortex are also plausible, and evidence for individually ordered mapping of speech sounds is growing -15  response to two vowels [o] and [ø], both produced by a male and a female speaker. Subject's attention was either on the vowel or on the speaker difference, in a counterbalanced order. How would a controlled shift of attention from specific phonological features of speech to features of speaker identity affect the speech sound mapping in timing and topography of the brain response? Two concurrent outcomes are conceivable here: First, from the numerous parallels between the auditory and other sensory domains, one might expect a blurring of differences of the phonological map in auditory cortex when features such as the speaker identity rather than phonological differences are attended over minutes. Second, phonological processing could be the default process needed in all speech-listening situations and should therefore activate phonological feature maps irrespectively of attentional demands. We would then expect that the separate mapping of DORSAL and CORONAL vowels described previously  is unaffIn 21 of 22 subjects, a clear waveform deflection around 100 ms post vowel onset was observed . However, the effect was significant in source space that is not influenced by varying head-to-sensor positions: The [ø] dipole source strength, an estimate for the amount of massed neuronal activity, was larger for the [ø] vowel than for the [o] by 25 % or 6 nAm (F1,16 = 9.36, p < .01). No hemispheric differences in signal power between vowel categories or tasks were apparent.Analysis of the N100m root mean square (RMS) peak latency revealed foremost a main effect of vowel (F1,16 = 7.73, p < .01), that is, the auditory processing in the DORSAL vowel [o] was reflected by a more posterior ECD location , a more vertical orientation (F1,16 = 5.81, p < .05) than the [ø] source, and from an angular difference between the two vowel categories in the sagittal plane (i.e. the [o] source was located more posterior and inferior, F1,16 = 10.91, p < .01) and in the axial plane . None of these effects showed an interaction with hemisphere, but data gained further validity as the right-hemispheric sources were all located more posterior (F1,16 = 8.88, p < .01), more inferior (F1,16 = 4.27, p < .06) and were tilted more vertically (F1,16 = 14.29, p < .01) than their left-hemispheric counterpart. Such a difference is to be expected from previously reported N100 asymmetries between cerebral hemispheres [In agreement with previous findings with a more comprehensive set of vowels , the vowispheres .1,16 = 4.72, p < .05) and marginally more posterior (F1,16 = 3.36, p < .10) ECD locations, which was also evident by an angular displacement in the sagittal plane (F1,16 = 4.6, p < .05). The effect seemed to be driven by changes in the left hemisphere but the task × hemisphere interaction never attained significance .The relative mapping of phonological features of the speech signal ,15 was n1,11 = 10.83, p < .01) and the axial plane (F1,11 = 7.16, p < .03). That is, subjects' attentional focus slightly affected the relative displacement of male and female voice-evoked brain responses: In both the sagittal plane and the axial plane, a significant 4° difference emerged in the phonological categorization task (both p < .05), which vanished in the speaker categorization task. In contrast, as reported above, no such task influence was evident in the relative position of vowel-evoked brain responses.When brain responses were analyzed separately for stimuli spoken by male and female speaker, which yielded satisfying dipole solutions only in 12 subjects, the most striking finding was a consistent speaker × task interaction of the dipole location in both the sagittal plane  and the speaker categorization task  did not differ significantly .This study was set up to explore potential influences of the attentional focus on the mapping of speech sounds within the auditory cortex. With subject's attention either on the phonological differences or on the speaker difference between vowel stimuli, we mapped the auditory evoked N100m and localized its sources that fitted well with a single dipole per hemisphere. All responses were located in the perisylvian region. Furthermore, the relative distribution of sources indicated an interesting pattern. As hypothesized and expected from previous studies, the fundamental location difference between the sources of the DORSAL vowel [o] source and the CORONAL vowel [ø] ,17 couldThat is, a shift of subjects' attention to a non-phonological acoustic feature, the speaker identity, did not blur the spatial segregation within the speech sound map. In contrast, the [ø] and [o] generators were slightly displaced towards more posterior and more superior locations when subjects focused on speaker identity.In most situations, a listener may automatically extract the phonological invariants from the speech signal in order to access lexical information, for example the meaning of the information inherent in speech. Speaker-dependent features such as pitch and periodicity should not play a crucial role in this phonological decoding process. This is what we mimicked by asking our subjects to detect a certain vowel in a stream of varying speech sounds. However, in cocktail-party-like situations there is the additional demand to attend acoustic properties of certain speech streams or speakers, and we implemented it by asking our subjects to detect a certain voice in a stream of varying speakers. Speaker identification comprises an important but not necessarily orthogonal process to phonological decoding in speech perception: areas in the upper bank of the superior temporal sulcus (STS) have been identified previously  to be voData presented here suggest that the systematic mapping of speech sounds within the auditory cortex is robust under changing attentional demands and not tied to phonological awareness. However, the general shift of activity when a non-phonological speaker categorization must be accomplished shows that speech sound representations are modulated in their locations in a context-dependent manner. Situational demands obviously influence the differential but time-synchronous involvement of specialized neuronal assemblies that contribute to speech sound decoding in a top-down fashion. Hence, the spectrally high-resolving analysis of the incoming speech stream is performed at the same time but in different locations, i.e. in a different mix of cell assemblies than the analysis of speaker-dependent features .Further spatially high-resolution brain imaging studies are needed to quantify as to which extent voice-selective areas in the upper bank of the STS  become i22 subjects  participated in the procedure. All subjects were monolingual native speakers of German. Only right-handers as ascertained by the Edinburgh Handedness Questionnaire  were incIn an auditory target detection task, subjects listened to randomized sequences of four German natural vowel exemplars: The DORSAL rounded vowel [o] in two exemplars, in one spoken by a male voice and in the other by a female voice, and the CORONAL rounded vowel [ø], also produced by both voices Fig. . 200 ms In a test sequence, subjects repeated vowels aloud and recognized all stimuli correctly, i.e. they distinguished between both vowel categories and voices without difficulty. Binaural loudness was slightly re-adjusted where necessary to ensure perception in the head midline.phonological categorization), subjects had to press a button with their right index finger whenever a given vowel  occurred, irrespective of the speaking voice. In a task B , subjects had to press a button whenever a given voice  uttered a vowel, irrespective of the uttered vowel category. Fig.In the actual measurement, vowel exemplars were presented in two randomized sequences with equal probability and a randomized stimulus onset asynchrony of 1.6 – 2 s. All subjects performed – in a counterbalanced order – two different tasks during these two sequences: In a task A  –,37 –15,3Data acquisition and analysis, including source modeling, closely followed the procedure described in : AuditorWe analyzed up to 150 artifact-free vowel responses that remained for both vowel categories [o] and [ø] after off-line noise correction, and averaged them separately for vowel category but across speaker voice. Splitting up vowel conditions into male and female speaker sub-conditions was not possible due to a resulting small number of averages. However, we also performed separate averages and analyses of male and female speaker across vowel categories. In any case, the resulting averages thus contained brain responses to two acoustically variant exemplars which makes results more comparable to our previous studies . A 20 HzThe N100m component was defined as the prominent waveform deflection in the time range between 90 and 160 ms Fig. . IsofielN100m peak latency was defined as the sampling point in this latency range by which the first derivative of the Root Mean Square (RMS) amplitude reached its minimum and second derivative was smaller than zero. RMS was calculated across 34 magnetometer channels selected to include the field extrema over the left and the right hemisphere, respectively.Prior to statistical analyses, all brain response latencies were corrected for a constant sound conductance delay of 19 ms in the delivery system. Using the same sets of channels, an equivalent current dipole (ECD) in a spherical volume conductor  was modeled at every sampling point separately for the left and the right hemisphere . The N10As source location displacements do not appear exactly and exclusively along the Cartesian axes of the source space (cf. ), we addJ.O., T.E. and C.E. conceived the experiment and drafted the manuscript. J.O. and C.E. prepared the exact experimental setup. J.O. supervised data acquisition, and performed all data and statistical analyses. All authors read and approved the final manuscript.


Gene expression technologies have the ability to generate vast amounts of data, yet there often resides only limited resources for subsequent validation studies. This necessitates the ability to perform sorting and prioritization of the output data. Previously described methodologies have used functional pathways or transcriptional regulatory grouping to sort genes for further study. In this paper we demonstrate a comparative genomics based method to leverage data from animal models to prioritize genes for validation. This approach allows one to develop a disease-based focus for the prioritization of gene data, a process that is essential for systems that lack significant functional pathway data yet have defined animal models. This method is made possible through the use of highly controlled spotted cDNA slide production and the use of comparative bioinformatics databases without the use of cross-species slide hybridizations.Using gene expression profiling we have demonstrated a similar whole transcriptome gene expression patterns in prostate cancer cells from human and rat prostate cancer cell lines both at baseline expression levels and after treatment with physiologic concentrations of the proposed chemopreventive agent Selenium. Using both the human PC3 and rat PAII prostate cancer cell lines have gone on to identify a subset of one hundred and fifty-four genes that demonstrate a similar level of differential expression to Selenium treatment in both species. Further analysis and data mining for two genes, the Insulin like Growth Factor Binding protein 3, and Retinoic X Receptor alpha, demonstrates an association with prostate cancer, functional pathway links, and protein-protein interactions that make these genes prime candidates for explaining the mechanism of Selenium's chemopreventive effect in prostate cancer. These genes are subsequently validated by western blots showing Selenium based induction and using tissue microarrays to demonstrate a significant association between downregulated protein expression and tumorigenesis, a process that is the reverse of what is seen in the presence of Selenium.Thus the outlined process demonstrates similar baseline and selenium induced gene expression profiles between rat and human prostate cancers, and provides a method for identifying testable functional pathways for the action of Selenium's chemopreventive properties in prostate cancer. Gene expression profiling, along with other methods to evaluate the global changes in genomes, provides the opportunity to understand whole scale changes present in human biology. Yet the sheer mass of data presented by these techniques often makes subsequent analysis difficult. Techniques such as gene expression profiling may result in the identification of hundreds if not thousands of differentially expressed genes that may be associated with the biological process, but may also represent noise related to the biological and technical variation. In an economic environment where limited resources are available for the follow-up and validation of potential target genes methods must be provided for the prioritization and sorting of data. Previous methods have relied heavily on the mapping of metabolic pathways or transcription factor binding sites -5. TheseSequence validated gene libraries for both the rat and human DNAs were obtained from Research Genetics (Huntsville, AL), and were supplemented with additional DNA samples obtained from the University of Iowa rat clone sequencing program . The majWe have sought to compare the rat and human prostate cancer transcriptomes in an effort to judge the degree of similarity between the two cell types. Because the use of differentially expressed genes would bias the comparison by eliminating the majority of genes that do not show any difference, we used the absolute level of expression for each gene and compared the rat and human genes for significant differences in absolute expression levels. In order to derive the absolute level of expression for individual genes in human or rat prostate cancer cells we used expression values derived from the associated self-self hybridizations performed for each cell line. The experiments were facilitated by the use of slides that have been quality controlled for the quantity of spotted target DNA through the use of a FITC label third dye . These sThe presence of a large quantity of genes whose expression may be related to general cellular functions, as opposed to prostate specific metabolism, could infuse a significant amount of homogeneity to the data. In the presence of such homogeneity it may be impossible to identify the true differences that are related to prostate cellular function, and thus the perceived similarities may be artifactual. To address this issue we sought to repeat the analysis using only prostate related genes. To generate a list of such genes we used cDNAs in eight normal human prostate cDNA libraries present in the NCI Cancer Genome Anatomy Project . Generatin-vitro studies of prostate cancer cells[While global gene expression profiles appear to be similar between rat and human prostate cancer cell lines one wonders whether the response to specific physiologic stimuli may elicit similar transcriptional changes. If so, one may be able to infer a degree of homology in their biological response to the stimuli. This has already been observed on a physiological level for the rat models of prostate cancer. For example, rat and human prostate cancers respond very similarly to chemotheraputic and environmental agents including hormonal agents (both respond), cyclophosphamide (neither respond), high fat diets (increased incidence), and soy isoflavones (decreased incidence) -22. In acer cells,29. ThesBecause some of the differences in the rat and human prostate cancer cell line transcriptomes may be related to confounding variables such as culture methods, cell passage number, or time in culture, an effort was made to focus on genes that are common, and as such may define the similar Selenium based cell proliferative changes. The subsets of 1123 and 1053 differentially expressed genes (6 hours and 5 days respectively) were analyzed for genes that demonstrate similar changes in expression with respect to Selenium across species. Of these differentially expressed genes, 291 and 309 demonstrated up-regulation in rat and human cells at 6 hours and 5 days respectively. Likewise, 261 (6 hours) and 216 (5 days) demonstrated down-regulation in the presence of Selenium. When these subsets were further analyzed to identify genes with similar levels of up or down-regulation (defined as ratio differences within 0.2 units of each other) 81 genes were identified at 6 hours and 73 at 5 days  the degree of diversity present within the samples may account for some of the residual differences still identified. In addition, the extended period of time that these cell lines have been used has allowed for the continued in-vitro evolution of the cells, and could possibly extend those genomic differences. Yet the common clustering of the rat and human cell lines together suggests there are still significant similarities in their biological potential. This is also demonstrated by the similar biological potential of the cell lines when treated with a given stimulus, in this example Selenium. This parallels the similar physiological properties observed in the rat models of human prostate cancer. Based on these features we demonstrate that it is possible to identify functionally significant genes related to Selenium response by using comparative genomics. These findings also support the use of animal models in the study of human prostate cancer by suggesting that there is enough inherent genomic similarity that valuable insights may be gained from animal systems.A true test of the profiling method is the identification of genes that have a functional significance to the experimental system. In this case we have identified a series of genes, which when examined with additional data mining techniques, identifies genes with associated roles related to apoptosis , antioxidant protection , cell cycle (CDC26-anaphase promoting complex, kinetochore associated protein), and protein balance (proteasome subunit beta-4, ubiquitin conjugating enzyme). In addition, the ability to sort the identified genes by their associated biomedical literature allowed the focus to shift to IGFBP3 and RXRalpha. Retinoids, through the retinoid X receptor, have been shown to induce the expression of IGFBP3 . In concUsing gene profiling on highly controlled spotted cDNA arrays we have demonstrated that similar baseline and selenium induced gene expression profiles can be identified between rat and human prostate cancer cells. This has allowed us to filter our gene expression data to identify genes whose transcriptional response to Selenium is similar across species, and by so doing focus our discovery process on specific common physiologic pathways. Two such proteins, RXR-alpha and IGFBP-3, which may be located in a common pathway, have been identified as dysregulated in human prostate cancers. This provides further support that the cross-species methods employed here can identify genes with roles in human prostate cancer.Cell lines were received from ATCC, Rockford, MD, (LNCap, DU-145, MatLyLu, AT3), from Drs. Paul Lindholm and Andre Kadjacsy-Balla (LN4, Pro4, PC3, PC3-NI(PC3US), PC3-I(PC3-S)), or Dr. Morris Pollard and Mark Suckow (PA-III). These cells were cultured in RPMI (DU-145) or DME medium supplemented with 10% fetal calf serum, 10 mM glutamine, and 10 mM sodium pyruvate, and passaged 1:8 or 1:10 when the cells reached 70–80% confluence with trypsin-EDTA. For the Selenium studies PC3 or PAIII cells from a single cell stock were seeded at 1 × 10EE4 cells per ml and grown to 50% confluence at which time the culture medium was changed to either standard growth medium (above) or medium supplemented with twenty-five micromolar Selenium (Seleno-DL-methionine, Sigma cat# S3875, St. Louis MO). The cells were then cultured for an additional 6 hours or 5 days. Cells that reached 80% confluence prior to the five day timepoint were split using trypsin-EDTA and replated in either control or selenium-containing medium for the duration of the experiment. Cells were monitored for viability and cell growth with parallel growth curves conducted in triplicate, this data demonstrated the previously described ,42 decreRNA was isolated from cells using Trizol (Invitrogen cat # 15596018, Carlsbad, CA) and subsequently examined for quality using agarose gel electrophoresis and Gelstar nucleic acid stain against known RNA standards and failed to demonstrate significant degradation based on the presence of high molecular weight RNA species, and intact 28s and 18s ribosomal RNA bands.2, 50 mM KCl, 0.2 mM each dNTP (Amersham, Piscataway, NJ), 1 M betaine, and 0.50 U Taq polymerase (Roche, Indianapolis IN). Reactions were amplified with a touchdown thermal profile consisting of 94°C for 5 min; 20 cycles of 94°C for 1 min, 60°C for 1 min (minus 0.5° per cycle), 72°C for 1 min; and 15 cycles of 94°C for 5 min; 20 cycles 94°C for 1 min, 55°C for 1 min, 72°C for 1 min; terminated with a 7 min hold at 72°. PCR reactions analyzed for single products by 1% agarose gel electrophoresis analysis. Products from replicate plates were pooled and then purified by size exclusion filtration using the Multiscreen 384 PCR filter plates (Millipore, Bedford, MA) to remove unincorporated primer and PCR reaction components. Forty wells of each 384-well probe plate were quantified by the PicoGreen assay (Molecular Probes, Eugene, OR) according to the manufacturers instructions. After quantification, all plates were dried down, and reconstituted at 150 ng/μl in 3% DMSO/1.5 M betaine.Sequence-verified rat and human libraries (Research Genetics, Huntsville, AL, and University of Iowa cDNA clone set, IA), consisting of 41,472 human clones and 36,000 rat clones were used as a source of probe DNA. A subset of 200 randomly selected clones were chosen from these libraries, resequenced locally, and demonstrated clone accuracy of 92%. We have opted to reformat libraries from 96 to 384-format for culture growth/archiving, PCR, purification, and printing. This has reduced the number of plates of our 41,472 human clone library from 432 to a more manageable 108, and the rat clone library from 375 to 94. The library was reformatted and subsequently manipulated using slot pin replicator tools (VP Scientific, San Diego, CA). Cultures were grown in 150 ul Terrific Broth (Sigma, St. Louis, MO) supplemented with 100 mg/ml ampicillin in 384 deep-well plates (Matrix Technologies, Hudson, NH) sealed with air pore tape sheets  and incubated with shaking for 14–16 hr. Clone inserts were amplified in duplicate in 384-well format from 0.5 μl bacterial culture diluted 1:8 in sterile distilled water or from 0.5 μl purified plasmid (controls only) using 0.26 μM of each vector primer {SK865 5'-fluorescein-GTC CGT ATG TTG TGT GGA A-3' and SK536: 5'-fluorescein-GCG AAA GGG GGA TGT GCT G-3'}  in a 20 μl reaction consisting of 10 mM Tris-HCl pH8.3, 3.0 mM MgCl2, deceleration: 100 cm/sec2. All slides were post-processed using the previously described nonaqueous protocol[A single printing array containing 19,200 elements (human) or 2 arrays of 9,600 (rat), were printed on poly-L-lysine coated slides prepared in-house (1–2 arrays/slide) as previously described [ protocol. Image f and parsed using local scripts and databases present in the Bioinformatics Program,[mdatta@mcw.edu.The experimental design utilized two biological replicates for each comparison with each replicate incorporating a Cy3/Cy5 dye flip. In addition, self-self hybridizations were performed for each sample to ensure experimental accuracy and evaluate expression bias. Comparisons were organized in a loop design for either human or rat prostate cancer cell lines, or were run as two-sample comparisons of baseline untreated control and Selenium treated cells. Array image TIFF files were analyzed with Gleams software (Nutec Sciences, Atlanta, GA). Additional TIFF file analysis, data normalization, clustering, and principle components analysis was performed using the Spotfinder, MIDAS and MultiExperiment Viewer Software from The Institute for Genomic Research (TIGR, Rockville, MD, , 12]) a) a12]) a Program,. AdditioProtein extracts were prepared and immunoprecipitations and/or western blots made from five day twenty-five micromolar Selenium treated or control PC3 or PAIII prostate cancer cell lines as described previously. In brieAfter expedited institutional review board approval normal prostate tissues and prostate cancer samples were obtained from de-identified discarded patient specimens. The formalin-fixed paraffin embedded specimens were prepared as 5 micron sections. Tissue microarrays were prepared from donor tissue blocks as 0.6 mm cores in 12 (4 × 4) or (5 × 5) grids with between 192 to 300 samples and used in the preparation of 5 micron sections. Immunohistochemistry was performed using primary rabbit polyclonal antibodies to the insulin-like growth factor binding protein 3 (IGFBP3, 1:300, Santa Cruz Biotechnology, Santa Cruz, CA), or retinoic-X-receptor alpha  using methods previously described ,49. In bNone declared.M.W.D. was responsible for the conception and implementation of this project in association with P.J.T., M.S., and M.P. H.L., X.W., and G.Z. were actively involved in the programming, database construction, and testing of the software. M.S. and M.H. were responsible for spotted cDNA construction, hybridization, and experimental analysis along with M.W.D. Cell culture, western blots, immunoprecipitations, and selenium treatments were performed by M.S. with assistance by B.M. Tissue microarray staining and analysis was performed by M.W.D., R.D., T.B., and B.M. All the authors reviewed and accepted the final version of the paper.Table 1, Word document, Table of the genes identified in the selenium gene expression studies.Click here for file


The multitude of motif detection algorithms developed to date have largely focused on the detection of patterns in primary sequence. Since sequence-dependent DNA structure and flexibility may also play a role in protein-DNA interactions, the simultaneous exploration of sequence- and structure-based hypotheses about the composition of binding sites and the ordering of features in a regulatory region should be considered as well. The consideration of structural features requires the development of new detection tools that can deal with data types other than primary sequence.) is a machine learning tool for the detection of conserved features in DNA. The software suite contains programs to extract different regions of genomic DNA from flat files and convert these sequences to indices that reflect sequence and structural composition or the presence of specific protein binding sites. The machine learning component allows the classification of different types of sequences based on subsamples of these indices, and can identify the best combinations of indices and machine learning architecture for sequence discrimination. Another key feature of GANN is the replicated splitting of data into training and test sets, and the implementation of negative controls. In validation experiments, GANN successfully merged important sequence and structural features to yield good predictive models for synthetic and real regulatory regions.GANN (available at GANN is a flexible tool that can search through large sets of sequence and structural feature combinations to identify those that best characterize a set of sequences. In bacteria, activator and repressor proteins bind to operator sequences that are typically found near the promoter, and promoter specificity is typically conferred through the sigma subunit of RNA polymerase, which binds the promoter directly [The minimal requirement for transcriptional activation is recruitment of an RNA polymerase complex to a promoter sequence of DNA upstream of an open reading frame (ORF). Most genes are also potentially under the control of DNA-binding regulatory proteins or directly . Eukaryodirectly . Regulatdirectly .linearly separable [The identity and spacing of these protein binding sites are key contributors to the responsiveness of a gene to changing cellular conditions, and sets of genes or operons that are expressed under similar conditions often have similar sets of regulatory elements in their 5' upstream regions. Recent programs such as ClusterBuster  and Promeparable , and woueparable ,8.additivity in DNA-protein interactions when position-specific scoring matrices (PSSMs) are used to model binding sites. In fact, binding affinity has been shown in some cases to depend on interactions between sites [Another issue in the detection and modelling of regulatory regions is the assumption of en sites -11, whicen sites ), but thcis-regulatory module detection, with the stochastic GA approach shown to yield similar outcomes to an exact search method in substantially less time. ClusterScan [The genetic algorithm (GA) is a powerful tool for combinatorial problems of model optimization and feature selection when the 'model space' is complex and has many local optima. Genetic algorithms carry out a number of simultaneous searches in model space, with one or more recombination operators to periodically combine the results of two or more searches, permitting large scale 'jumps' out of locally optimal regions. GAs have been applied to several tasks in computational biology, including sequence alignment (SAGA: ) and physterScan  is anothsterScan .Escherichia coli integration host factor (IHF) to its target sequence [Another important question in modelling regulatory regions involves the representation of binding sites. While position-specific scoring matrices (PSSMs) are a popular and effective way of representing conserved sites , other ssequence , the corsequence  and the sequence . DNA strsequence ). While sequence , and thesequence -27, careWe have developed GANN, a software suite that uses machine learning methods to identify combinations of the features listed above that best distinguish between a positive set (containing, for instance, a set of regulatory regions from co-expressed genes) and a negative set. GANN implements all of the binding site representations described above, allowing examination of models of different complexity as warranted by the type of binding site modelled and the amount of training data that is available.GANN contains a set of programs for sequence extraction, retrieval and grouping of requested patterns, neural network analysis of these patterns and collection of results. Each program in the suite can accept either the output of the previous program, or an appropriate set of data generated from an external method. The components of GANN and the flow of data through the system are shown in Figure The first program (GetSeq) reads in either a GenBank file of annotated genome sequence, or raw sequence and a list of open reading frames (ORFs) of interest generated with the NeuroGadgets Inc. Bioinformatics Web Service . GetSeq The Indices program takes as input a set of positive and negative set sequences (such as those generated by GetSeq), and can compute various properties of the sequences. The input sequences can be subdivided into overlapping windows of any size prior to the calculation of index values. The following indices can be calculated:k-mer within a window, then dividing by the length of that window. The program can determine the frequency of all k-mers of a specific length, or can assess any user-specified set of k-mers, which may include IUPAC notation to represent degenerate nucleotides.- Oligonucleotide frequencies are computed by counting the number of instances of a given - User-specified PSSMs can be counted for each sequence window. The user provides a set of scores for each type of nucleotide at each position within a PSSM, and a threshold score. The program will then count and record the number of sequence instances within each window that yield a PSSM score greater than the specified threshold.k-mer of a given length. The average score for a given sequence window is then computed by adding the scores for each overlapping k-mer within the sequence, and dividing by the total number of k-mers considered. Any numeric encoding of a complete set of k-mers can be specified: features sampled from publications such as [- Structure and flexibility rules are implemented via a text file, by assigning floating-point values to each  such as -31 are aAfter the extraction of indices, the Combine program merges the different index files into a single large file that is used as the input for the machine-learning software. Combine also allows the computation of Z-scores, thus representing each index value in terms of the number of standard deviations from the mean, and can identify peak values for a given index across a set of windows. Combine randomly subdivides the positive and negative index sets into training and test sets, and can also generate a negative control by randomly reassigning some positive and negative set members to the opposite category, yielding a disruption of patterns that were previously consistent within a single set. This type of control sets a 'baseline' for classification accuracy that can be compared to real experimental results.The core of GANN is the neural network classification system. The indices generated from 'Indices' and 'Combine' are presented as input to an artificial neural network, which is trained with either backpropagation or a genetic algorithm to maximize the discrimination between the positive and negative sets. Since the number of indices associated with each sequence is potentially very large, the Outer Genetic Algorithm (OGA) presents random subsets from the pool of indices to a series of neural networks. The unit of selection for the OGA is a 'Chromosome' that contains a predetermined number of indices sampled from the larger pool, and a set of parameters that define the architecture and connectivity of the ANN. The constitution of a population of OGA Chromosomes is determined randomly in the first generation, with random sampling of indices from the pool and ANN parameters sampled randomly from within a set of ranges specified by the user. Each OGA Chromosome is used to construct an ANN, which is then trained to yield optimal predictive accuracy on the training set defined by the Combine program above. At the end of training, performance on the test set is evaluated, and the fitness of the OGA Chromosome is equal to its predictive accuracy on the test set samples. The predictive accuracy is defined as follows:Pos|| and ||Neg|| the size of the positive and negative test sets, respectively. This formula assigns equal weight to the positive and negative sets regardless of their size, so the ANN cannot achieve an artificially high score by predicting every case as a member of the larger (training or test) set.Where TP and TN are the number of correctly classified positive and negative test set examples, and ||The OGA Chromosomes with highest fitness are then permitted to 'recombine', yielding new subsets of indices that are trained in the same manner, while less successful indices are gradually lost from the population. The classification potential of indices can be evaluated by examining the scores of neural networks that include these indices in their input set, and through a 'population genetics' approach that traces the frequency of indices through several rounds of OGA recombination and selection.training set. IGA Chromosomes with relatively high fitness are then subjected to stochastic recombination and mutation of parameters to yield a new population of Chromosomes that are used in the next round of training. While gradient-descent training methods for ANNs (such as backpropagation) can easily get trapped in local optima of the solution space, the recombination option of genetic algorithms permits a search to 'jump' through the solution space and escape local optima. The optimisation of network architecture and connection weights is similar to the 'structure evolution' method of [Training of the neural networks is performed using either backpropagation of errors  or with ethod of , which hethod of ,35, but There are many variables within GANN whose values can be specified by the user. While most default settings will be adequate in most situations, parameters such as the number of feature combinations generated by the OGA and the number of features in each combination should be chosen carefully. The 'DefineVars' program provides a set of menus that allow the user to set these parameters and write them to a configuration file that is input to GANN.In addition to reporting the scores of trained artificial neural networks, GANN will save information about the topology, connection weights and constituent indices of each neural network instance that achieves a generalization score above a specified minimum threshold. If GANN is invoked with any of these saved neural networks as input in addition to a table of indices, then it will use the input neural network to classify the new table. This process can yield functional predictions for sequences whose true classification is unknown.Escherichia coli K12 genome, are presented to illustrate the performance of GANN. Both of these experiments included a set of 250 nucleotide sequences, each 100 nucleotides (nt) in length, which were extracted from between convergently transcribed genes in the E. coli genome using the GetSeq program. These sequences were chosen because they are intergenic like upstream regulatory regions and not subject to the evolutionary constraints of protein-coding sequences, but are not expected to contain functional transcriptional regulatory features since they are exclusively 'downstream' of one gene in each direction. In the first experiment, we created an artificial positive set by inserting conserved sequences into a subset of the 250 sequences, with the remainder constituting the negative set. The entire set of 250 sequences was used as the negative set in the second experiment, while the positive set consisted of 212 upstream regulatory regions containing experimentally validated binding sites for the σ70 protein of E. coli.Two sets of detection experiments, both based on DNA sequences extracted from the Several run parameters were consistent across both experiments. Each OGA Chromosome contained a total of 8 indices, to allow the simultaneous representation of several sequence and structure properties. The population size (= number of OGA Chromosomes) was determined by multiplying the total number of indices by 10, then dividing by the number of indices (8) per OGA Chromosome. This formula ensured that indices would be represented 10 times each on average in the initial randomly generated population, and 99.9% of all indices should occur at least 3 times in the population according to the Poisson distribution. Thirty rounds of OGA Chromosome evaluation and selection were performed in each run. While GANN can evolve ANN architecture and learning parameters as well as combinations of indices, we chose reasonable ANN parameters (available at the GANN website) and fixed them for the entire run.k-mers) may yield multiple alternative solutions that are equally good. A final indicator of index performance is the composition of OGA Chromosomes that yield high predictive accuracy: if a set of features is important for characterization of a set of sequences, then each of those features should be represented by at least one index in the best OGA Chromosomes.The performance of different sets of indices was expressed in terms of the predictive accuracy (= score on the test set of sequences) described above. Differences in predictive accuracy are expected across replicates, because random partitioning of sequences into training and test sets is likely to yield variation in the frequency of some features that do not define the whole set. However, average predictive accuracy can be estimated by taking the mean across replicates. Indices that are retained in every replicate of an experimental run are more likely to reflect true characteristics of the sequences under consideration, though the redundancy of many indices (different window sizes, different percentiles for PSSM scores, and correlated frequencies of some In the first experiment, the positive set was constructed by adding conserved features to 76 of the 250 sequences ~30%) described above. Each member of the positive set was modified by adding a nucleotide decamer with high conformational mobility (CM). Five thousand unique decamers were generated randomly, and each of these was assigned a CM score based on the dinucleotide table in % describ, and eacThe construction of synthetic conserved regions is summarized in Figure Once the positive and negative set sequences were obtained and assembled, the Indices program was used to extract several different types of information from them, with varying window sizes depending on the features being examined. The overlap between adjacent windows was chosen to be 50% of the window length, so if a window of size 10 covered sites 1–10 in a sequence, the next window would cover sites 6–15.th, 95th, 90th, and 80th percentiles. The number of sequence matches above each PSSM threshold was computed for windows of size 20 and 40.- PSSMs for Lrp and CRP binding sites were constructed from the set of binding sites in RegulonDB. Frequency matrices for each site were constructed by dividing the number of occurrences of each residue at each site by the total number of sites (72 Lrp, 128 CRP). These frequencies were then divided by the 'background' frequency of each corresponding nucleotide in the set of intergenic sequences. Since the background frequency of each nucleotide was within the range 0.250 ± 0.005, background frequencies of 0.25 were assigned to each nucleotide. The PSSM was then obtained by taking the natural logarithm of each value in the corrected frequency matrix. Threshold values for PSSM predictions were determined by scoring 50 000 random sequences of the appropriate length against the PSSM, and identifying the scores that corresponded to the 99- The conformational mobility of sequence windows of size 10 and 20 was computed according to the dinucleotide values in .k-mers of size 1 (mononucleotides) and size 2 (dinucleotides) were computed for windows of size 10 and 20.- Counts of all k-mer counts, 64 describing the counts of PSSM 'hits' at different thresholds, and 36 describing the conformational mobility of different windows of sequence. These indices were then combined in four different ways to yield separate tests of different subsets. The k-mer frequencies were included in every set as a 'background' measure of predictive power with no explicit hypothesis. Set 1.1 included only the k-mer frequencies, while set 1.2 added the indices of conformational mobility, set 1.3 included the PSSM scores, and set 1.4 included all three types of index. We initially performed runs where index values were not standardized, but found that indices with values that were not close to zero, particularly the CM indices which ranged between 40 and 70, did not perform well and were consistently eliminated from the population of OGA Chromosomes. In response to this, we standardized all indices for the experiments described below and in Experiment 2.The computations described above yielded a total of 450 indices: 360 describing Generation of Sequence and Structure Indices above.Set 1.4, with the full set of 450 indices, was used to define the number of OGA Chromosomes. The formula at the beginning of this section yielded a recommendation of 562.5 OGA Chromosomes per generation, which was rounded up to 600 and applied to all four sets. 'Combine' was used to randomly subdivide the positive and negative set sequences into training and test sets with a ratio of 2:1. This random reassignment was repeated five times, and five corresponding negative control sets were generated as described in k-mer counts, yielded a mean generalization score of 0.880, a substantial improvement over set 1.1 with no overlap in the range of maximum scores between the two sets. Set 1.3, which considered k-mer counts and PSSM scores at several thresholds, yielded a mean best generalization score of 0.883, which was substantially better than set 1.1 and indistinguishable from set 1.2. Finally, set 1.4 yielded a small improvement over sets 1.2 and 1.3 in generalization score, with a mean of 0.900. These results suggest that the inclusion of PSSMs and flexibility indices yielded a substantial increase in predictive accuracy over the background of k-mer counts, with the combination of the two possibly producing a further slight increase.The mean of the best generalization scores achieved in each replicate over 30 rounds of OGA evaluation and selection is shown in Figure Figure th percentile of the CRP matrix between positions 60 and 99, another measuring the same quantity for Lrp sites between positions 20 and 59, an index describing the conformational mobility between sites 80 and 90, and two indices representing the count of GC dinucleotides and C mononucleotides between positions 29 and 39. The first three indices are easy to understand, as they correspond directly to features (binding sites and a high CM region) that were deliberately inserted into the positive sequence set. However, the third index is less clear until the Lrp sites from RegulonDB are examined in detail: these sites are G+C poor in general, and of the 72 × 11 = 792 dinucleotides contained in the full set of Lrp binding sites, only 20 of these are GC steps. If all 16 possible dinucleotides were present with equal frequencies, then the pair would be present 49 or 50 times, so GC is strongly underrepresented in this data set. Thus, it appears that the GC content in this region is included in many OGA Chromosomes because it is another indicator of the presence or absence of the Lrp binding site in this region.If sets of indices that yield the best predictive accuracy are preferentially selected for recombination by the OGA, then good indices should increase in frequency with successive rounds of testing and recombination. Only five indices were present in the final population of all five replicated experimental runs of set 1.4: one measuring the count of sites scoring in the 9970 subunit of RNA polymerase in E. coli. The 212 promoter-containing sequences in the positive set were extracted from a larger set defined in [The second experiment tested the ability of GANN to distinguish between the 250 unmodified intergenic sequences described earlier, and a set of sequences containing binding sites that are recognized by the 'housekeeping' σfined in . Where m70 were constructed from 250 promoter sequences in the data set, again with background frequencies of 25% for each nucleotide. As with CRP and Lrp above, the 99th, 95th, 90th and 80th percentile scores were generated for the -10 and -35 boxes from a set of random sequences, though only 1000 random sequences were generated for each case. Since the two halves of the σ70 consensus sequence are only six bases in length, indices based on PSSM matches for a window size of 10 nt as well as 20 nt and 40 nt were calculated. Indices of conformational mobility and k-mer frequency were calculated as in Experiment 1 above. A total of 618 standardized indices were generated in this experiment: 360 describing k-mer counts, 222 describing the counts of matches to the -10 and -35 PSSMs at different thresholds, and 36 describing the conformational mobility of different windows of sequence. Five sets of experiments were performed, with five experimental and five negative control runs in each. All sets (2.1 to 2.5) included the k-mer count indices, and these were the only indices considered in set 2.1. Sets 2.2 and 2.3 also included the -35 and -10 PSSMs respectively, while set 2.4 included both. All 618 indices were considered by set 2.5.Indices were constructed in a similar manner as in Experiment 1. Separate PSSMs for the -10 and -35 boxes recognized by σRuns were performed as in Experiment 1 above, with the exception of the OGA Chromosome population size. With 618 indices in total, the formula described at the beginning of this section yielded a recommendation of 772.5 OGA Chromosomes per generation, which was rounded up to 800 and applied to all five sets.k-mer counts alone, and that the inclusion of the σ70 PSSMs did not yield a more-precise model of the promoter sequence.The mean of the best generalization scores achieved in each replicate over 30 rounds of OGA evaluation and selection is shown in Figure th percentile) were favoured in Experiment 1, the PSSMs most commonly retained in the σ70 experiments favoured lower percentile thresholds, with a roughly even distribution of indices representing the 95th, 90th and 80th percentiles. This effect may be due to greater degeneracy of σ70 sites relative to the CRP and Lrp sites modelled earlier. Finally, four out of five replicate experimental runs of set 2.5 retained a CM index that covered positions 30–50 in the sequence. While no single index was retained in all five replicate runs of set 2.5, the OGA Chromosomes with the highest generalization scores all contained at least one instance of a PSSM for each of the two halves of the σ70 consensus and a flexibility feature. Thus, more-selective indices such as those based on PSSMs were included in the majority of OGA Chromosomes when they were present in the experimental set, even if the gain in predictive accuracy was marginal.While the predictive accuracy did not change across multiple sets of experiments, the type of indices that were selected by the OGA varied from set to set. The most successful index overall described the frequency of the dinucleotide 'TA' between positions 65 and 74, which corresponded to the position of the Pribnow box in the positive set sequences. This index was the only one present in all five replicates of sets 2.1, 2.3 and 2.4 after 30 rounds of OGA training, and was one of only three such indices for set 2.2. The other two indices that were retained by all five replicates of set 2.2 described the frequency of TA between positions 60 and 69, and the frequency of CA between positions 55 and 64. No single index was retained in all five replicate runs of set 2.5. In all sets that included PSSM representations, the appropriate PSSM component(s) were always retained in four out of five replicated runs. While PSSMs with a very high score threshold (9970 promoter set is similar to the sensitivity of 86% and specificity of 85% reported by [We have explored several features of GANN, most notably the ability to build classification rules for positive set members that form natural subsets, and the capacity to search through large sets of DNA sequence and structural indices to find combinations that yield optimal predictive accuracy. Our generalization accuracy of ~84% on the σorted by , though orted by , GANN coIn Experiment 1, we found that indices with mean values that are not close to zero should be standardized. A disadvantage of this approach is that standardization of a column of values is entirely sample dependent, since different finite samples from the same population of values will typically have different means and standard deviations, which may limit the accuracy of an ANN trained on one sample that is used to classify other subsamples from the same population. If standardization is to be applied, then there should be sufficient values in each index to yield a stable estimate of the population mean, as indicated with a low standard error. This is of particular concern since biological sequence samples often do not represent a random sample of all possible sequences, leading to biased estimates of sample mean and standard deviation.The two primary goals of GANN are to allow multiple alternative representations of DNA features, and to permit the discovery of important combinations of these features through the hybrid genetic algorithm / neural network approach. A long-term goal is to use GANN to identify important combinations of motifs predicted from programs such as PatSer , which w- Project name: GANN- Project home page: - Operating systems: Win32, UNIX- Programming language: C++, Perl- Other requirements: none- License: GNU GPL- Any restrictions to use by non-academics: noneCM – Conformational mobilityGANN – Genetic Algorithm Neural NetworksOGA – Outer Genetic AlgorithmPSSM – Position-specific scoring matrixRGB was responsible for software design and implementation, and manuscript writing. RLC contributed to the design, and to the implementation of the genetic algorithm classes in C++.


Schistosoma mansoni and malaria infections are often endemic in the same communities in sub-Saharan Africa, and both have pathological effects on the liver and the spleen. Hepatosplenomegaly associated with S. mansoni is exacerbated in children with relatively high exposure to malaria. Treatment with praziquantel reduces the degree of hepatosplenomegaly, but the condition does not completely resolve in some cases. The present analysis focused on the possibility that exposure to malaria infection may have limited the resolution of hepatosplenomegaly in a cohort of Kenyan schoolchildren.S. mansoni infection, a river, was molluscicided regularly over the following three years to limit S. mansoni re-infection, whereas malaria exposure was uninterrupted. Hepatic and splenic enlargement was assessed annually outside the malaria transmission season.Ninety-six children aged 6–16, from one community in Makueni district, Kenya, were treated with praziquantel. At baseline, all children had hepatomegaly and most had splenomegaly. The source of S. mansoni exposure but high exposure to malaria, and maximal in an area with relatively low exposure to both infections.Children living in an area of relatively high exposure to both infections presented with the largest spleens before treatment and at each follow-up. Spleens of firm consistency were associated with proximity to the river. The regression of hepatomegaly was also affected by location, being minimal in an area with relatively low The outcome of treating cases of hepatosplenomegaly with praziquantel in this cohort of Kenyan children depended strongly on their level of exposure to malaria infection. Furthermore, a residual burden of hepatosplenic morbidity was observed, which was possibly attributable to the level of exposure to malaria. The results suggest that exposure to malaria infection may be a significant factor affecting the outcome of praziquantel treatment to reduce the level of hepatosplenic morbidity. Schistosoma mansoni and malaria infections are endemic. Severely enlarged and hard organs may be an indicator of increased portal pressure, as well as pointing to an increased risk of portal hypertension and its sequelae, including oesophageal varices and haematemesis [S. mansoni infection, and on a global scale up to 8.5 million individuals may be affected [S. mansoni and malaria infections are endemic [P. falciparum schizont antigen (Pfs Ag) were higher in Kenyan children with S. mansoni infection and hepatosplenomegaly compared with children with infection but no hepatosplenomegaly [Hepatosplenomegaly is a widespread but neglected condition affecting many communities in sub-Saharan Africa where both atemesis . Historiaffected , with thaffected . Enlargeaffected ,5. One paffected . However endemic ,8, and i endemic ,10. Recenomegaly , furtherS. mansoni infection and seasonal malaria infection [S. mansoni egg count and the degree of both hepatomegaly and splenomegaly [S. mansoni infections [Given that malaria and schistosome infections are often endemic in the same communities, and are both targets of large-scale but independent, intervention programmes, it is important to understand how continued exposure to one parasite may affect the outcome of intervention against the other. The current longitudinal study evaluated praziquantel treatment on the regression of hepatosplenomegaly amongst children living in an area with endemic nfection ,13. Befonomegaly . Howeverfections . The preMbeetwani is situated in the district of Makueni, half way between Nairobi and Mombasa and approximately 10 km east of the main highway. The ethnic background of the population is predominantly Akamba. During the dry seasons, the river Kambu is the main source of water. Running from west to east, the river lies to the north of the community . Area-wide application and focal spraying using backpacks was undertaken twice yearly to coincide with periods when snail populations were reduced due to environmental conditions. Snail sampling activities at 9 sites on a monthly basis were undertaken to confirm that snail numbers were kept at very low levels (HC Kariuki, unpublished observations).Regular mollusciciding of the river Kambu was undertaken with Bayluscide ®, Shin Poon Pharmaceuticals, Seoul, Republic of Korea), at 40 mg/kg body weight. The children were again treated in 2001 and 2002 under the same regimen. No other source of treatment was available locally during this period. Tablets were administered together with a piece of bread and a soft drink in order to minimise gastrointestinal side effects. The study was approved by the Kenya Medical Research Institute Ethical Review Committee.The objectives of the study were carefully explained to the local community including the parents and teachers before the start of any activities. Informed assent was obtained from the parents or guardians of the children. After the clinical and ultrasound examination in October 1999, all children were treated with a single dose of praziquantel .Baseline data for each parameter were compared with 3 year follow up data amongst 71 children who attended the first and last surveys to estimate the number of children within each sector who presented with a lower degree of organomegaly at the end of the study. Repeated-measures ANOVA of each organ enlargement parameter was also undertaken for the 67 children who attended all surveys, with age, sex and sector of residence as fixed factors. Since ANOVA does not indicate the direction of any change, a S. mansoni egg counts and IgG3 responses within each sector are given elsewhere [S. mansoni only, and sector D was an area of low exposure to both infections.Figure lsewhere . BrieflySignificant variation in the prevalence of hard spleens over time was observed within each sector Table , attribuThe prevalence of firm livers in all sectors varied over time, except in sector B Table . In all post-hoc Wilcoxon Rank analysis . Figure MSL liver enlargement varied significantly over time (F = 3.78, p = 0.012), with the variation being affected by sector (F = 2.57, p = 0.008). There was no consistent decline in MSL hepatomegaly over time within any one sector Figure . AlthougThere was no evidence of periportal fibrosis in any of the children at any time point.S. mansoni and malaria varies over small areas [S. mansoni and malaria, but also for estimating the outcome of interventions.Many clinical surveys of sub-Saharan communities have reported that organomegaly of the spleen or liver is a very common condition, but attribution to a specific aetiology has always been problematic. Distinguishing the contribution of malaria from that of schistosome infections is particularly complex since they are often endemic in the same communities. However, it is well established that exposure to both ll areas -21, and S. mansoni and S. haematobium [S. mansoni egg counts at the western end of the study area. Such clustering is likely related to variation in the amount of surface water along the river, and hence variation in transmission potential due to the strong relationship between snail abundance and water level observed during long term studies of transmission in this area [The generation of these results was facilitated by several important features of the study design. The intermittent mollusciciding of the river Kambu was particularly important, since it reduced the potential for morbidity to rebound as a result of re-infection, as has been seen elsewhere in studies of both matobium . The timhis area . The cluhis area .Although we observed several significant changes following praziquantel treatment of the cohort, it is important to note that this was a retrospective analysis, which carries a few limitations. The observations made in this study were based on a small, case-only cohort, and therefore do not represent the outcome of an intervention programme involving mass treatment of a population. As this was a retrospective analysis, no control was possible concerning treatment for malaria during the follow-up period. The population had access to antimalarial drugs at local shops, but so far as we are aware there was no systematic intervention against malaria infection during the follow-up period. The results therefore encompass the effects of background treatment for, as well as exposure to, malaria infections.Reliability of the clinical measurements is an important factor, especially given the size of the cohort. We have assessed this procedure elsewhere and it has been found to be satisfactory (unpublished observations). The involvement of three or four clinicians at each examination also reduced the degree of imprecision in the organomegaly measurements, and previous analysis has demonstrated that interpretable changes in the measurements occurred within this cohort .S. mansoni re-infection, depended strongly on where members of the cohort were resident. Previously, we demonstrated that children within the same cohort that had relatively high-level exposure to both S. mansoni and malaria presented with significantly larger spleens before treatment than children highly exposed to either parasite alone [The major result from this analysis is that the outcome of treatment with praziquantel, combined with very limited te alone . This cote alone . Here, wS. mansoni infection [S. mansoni infection was the trigger for the improvement [Hardness of enlarged spleens at baseline was most commonly observed amongst children living close to the river, irrespective of their level of pre-treatment intensity of nfection . In the rovement . One posS. mansoni [S. mansoni infection and limiting re-infection, we may have abrogated any synergistic effects of co-infection on the spleen, and thereby uncovered the residual burden of chronic exposure to malaria. Importantly, the rate of regression of MAL splenomegaly was not affected significantly by location, which suggests that the effects of praziquantel treatment on splenomegaly attributable to S. mansoni infection are not dependent on the level of exposure to either malaria or S. mansoni.Three years after treatment with praziquantel, and with very limited re-infection by  mansoni , the preS. mansoni infection was a critical factor for improvement. However, upon analysing hepatomegaly data across sectors, it emerged that children from the sector with relatively high anti-Pfs IgG3 responses, but relatively low S. mansoni egg counts, had the lowest rate of regression. A possible explanation of this observation is that an aetiological agent other than S. mansoni was responsible for the observed hepatomegaly. A likely candidate is malaria infection. Enlargement of the liver in acute malaria infection is temporary and recedes rapidly after treatment [The effects of praziquantel treatment on hepatomegaly in this cohort were more subtle. There was gradual decline in the prevalence of hepatomegaly after praziquantel treatment  indicatireatment ; howeverreatment ,26. Our S. mansoni nor malaria infection. Alternatively, because they were relatively lightly infected with S. mansoni, and experienced relatively low exposure to malaria infection, it is possible that they were more likely to regress in terms of hepatomegaly within the follow-up period.Children from the sector with relatively low exposure to both infections experienced a significant decrease in liver enlargement along the mid sternal line when baseline data were compared with data from the third year follow-up. It is possible that hepatomegaly in these children was attributable to neither S. mansoni and malaria infections. Specifically, although the degree of hepatomegaly or splenomegaly may be correlated with S. mansoni infection, there is likely to be further exacerbation of the condition if a child is concurrently exposed to malaria. We have now observed that the apparent benefits of treating a case of hepatosplenomegaly with praziquantel may be reduced if a child lives in an area of relatively high exposure. We have therefore confirmed the long-standing, but untested, hypothesis that co-infections of S. mansoni and malaria may obscure the clinical evaluation associated with infection by either species. Our results also introduce the necessity of considering the level of exposure to malaria when evaluating the clinical outcome of praziquantel treatment for S. mansoni infection.In conclusion, our observations lend further support to the hypothesis that severity of hepatosplenomegaly in Kenyan school-aged children is related to their degree of exposure to both The author(s) declare that they have no competing interests.MB conceived of, and conducted, the analysis, and drafted the manuscript. BJV and AEB performed clinical examinations and participated in the design of the study. CA and AO conducted clinical examinations. CHK, GK, JM, JHO and EM participated in the planning and execution of field activities. DWD participated in the design of the study and in fieldwork.The pre-publication history for this paper can be accessed here:


Despite heavy recent emphasis on blood pressure (BP) control, many patients fail to meet widely accepted goals. While access and adherence to therapy certainly play a role, another potential explanation is poor quality of essential care processes (QC). Yet little is known about the relationship between QC and BP control.We assessed QC in 12 U.S. communities by reviewing the medical records of a randomly selected group of patients for the two years preceding our study. We included patients with either a diagnosis of hypertension or two visits with BPs of ≥140/90 in their medical records. We used 28 process indicators based on explicit evidence to assess QC. The indicators covered a broad spectrum of care and were developed through a modified Delphi method. We considered patients who received all indicated care to have optimal QC. We defined control of hypertension as BP < 140/90 in the most recent reading.Of 1,953 hypertensive patients, only 57% received optimal care and 42% had controlled hypertension. Patients who had received optimal care were more likely to have their BP under control at the end of the study (45% vs. 35%, p = .0006). Patients were more likely to receive optimal care if they were over age 50 (76% vs. 63%, p < .0001), had diabetes (77% vs. 71%, p = .0038), coronary artery disease (87% vs. 69%, p < .0001), or hyperlipidemia (80% vs. 68%, p < .0001), and did not smoke (73% vs. 66%, p = .0005).Higher QC for hypertensive patients is associated with better BP control. Younger patients without cardiac risk factors are at greatest risk for poor care. Quality measurement systems like the one presented in this study can guide future quality improvement efforts. Hypertension affects approximately 58 million Americans . LowerinOne potential explanation for uncontrolled hypertension is suboptimal quality of care. Although the U.S. Joint National Committee on Prevention, Detection, and Treatment of High Blood Pressure (JNC-VII)  has codiIf deficits in process quality are indeed related to BP control, then which patients are failing to receive optimal care? The literature suggests that ethnic minorities and older patients are less likely to have controlled hypertension , but we We hypothesized that overall process quality for hypertensive care is associated with better BP control. We developed indicators of hypertensive care and determined whether patients had received the indicated care by reviewing medical records for a national sample of patients receiving care in a variety of settings. We also investigated whether patients with other cardiac risk factors received better hypertensive care and had better BP control.Our examination of hypertensive care was part of a larger study called the Community Quality Index Study (CQI), a cross-sectional study that assessed effectiveness of care for 32 different clinical conditions by examining medical records for patients in 12 randomly selected communities with populations greater than 200,000 (Boston, Cleveland, Greenville, Indianapolis, Lansing, Little Rock, Miami, Newark, Orange County, Phoenix, Seattle, and Syracuse) . The metWe developed process indicators for hypertensive quality of care by reviewing the scientific literature and clinical practice guidelines  pertainiAfter a two-week training course, twenty nurse abstractors used a computer-based abstraction tool with branching logic to abstract from the medical records we collected the following data for the two-year study period: a diagnosis of hypertension; a diagnosis of co-morbid disease, including CAD, diabetes, and hyperlipidemia; BP readings; laboratory results, including serum creatinine, cholesterol, trigylcerides, sodium, potassium, and urinalyses; prescriptions for anti-hypertensive agents; and counseling for lifestyle modification. The Kappa statistic for inter-rater reliability for indicator eligibility was .75 and, given agreement on eligibility, the kappa statistic for the indicated care was .91.86% of patients with a visit in the last 13 months of the study period had a blood pressure measured. Patients were included in the study if their provider noted a diagnosis of hypertension in their medical record or if they had BP readings greater than 140/90 on two occasions at least two weeks apart. However, patients were only classified as having a new diagnosis of hypertension if this was specifically noted in their medical records. Per JNC-VI, we defined Stage 1 hypertension as BP 140/90–159/99 and Stage 2 hypertension as BP > 160/100; we determined stage by averaging a patient's BP over the study period, regardless of notation of diagnosis or treatment regimen. We defined uncontrolled hypertension as a last systolic BP ≥ 140 or a last diastolic BP ≥ 90.We determined both individual and overall quality scores for each patient. We calculated individual scores by determining a patient's eligibility for an indicator and whether s/he received the indicated care. We calculated overall quality scores by adding the number of instances in which a patient was eligible for and received the indicated care and dividing this number by the total number of instances for which s/he was eligible for the indicated care; this score was expressed as a percentage. We defined optimal quality as patients having received all of the indicated care for which they were eligible. We adjusted the scores for non-response using multivariate models that weighted respondents to be representative of the population from which they were drawn. We used the bootstrap method to directly estimate standard errors for all of the individual indicator scores . StatistOf the 6,712 adult patients for whom we analyzed medical records1,953 (29%) had a diagnosis of hypertension noted in their medical record or repeated BP readings indicative of hypertension. Of these, 241 (12%) were newly diagnosed during the study period; 1,102 (57%) had hypertension noted in the medical record, but the diagnosis was not new; 610 (31%) had repeated BPs ≥ 140 systolic and/or 90 diastolic evidencing hypertension, but no diagnosis in the chart. The sample included 1,070 women (55%), 1,326 patients over age 50 (68%), and 918 (47%) patients with CAD, hyperlipidemia, or diabetes noted in their medical records. The average last systolic BP was 139.2 and the average last diastolic BP was 82.0. Of the 1,368 patients (70%) who were receiving pharmacological treatment for hypertension, 25% were prescribed beta-blockers, 37% diuretics, 30% calcium channel blockers, and 35% angiotensin converting enzyme inhibitors or angiotensin II receptor antagonists. Patients with a diagnosis of hypertension noted in their records were more likely to have received pharmacotherapy during the study period than those with elevated BP readings but no notation of a diagnosis (88% vs. 31%, p < .0001).On average, patients were eligible for 3.8 indicators (range: 1–21 and visited their providers 13 times during the study period. Table The last BP reading for 42% (825) of patients indicated good control (<140/90). Table A logistic regression (not shown) revealed that the relationship between higher quality scores and BP control persisted when controlling for number of physician visits, baseline blood pressure control, gender, age, smoking status, and presence of CAD, diabetes, or hyperlipidemia. Other than quality, only baseline BP control was significantly associated with BP control at the end of the study. The relationship between quality and BP control also persisted when we restricted the study sample to those with at least two BP readings 12 months apart (n = 1,321). Likewise, there were no changes in direction or significance when we controlled for BP at 160/100 or used the continuous measures of last systolic or diastolic BP reading as the outcome.Figure Our data suggest that quality of care for hypertensive patients falls short of the ideal. Overall, patients received about 72% of recommended essential care processes, and 77% of patients with persistently elevated blood pressure had some change in therapy noted in the medical record over the course of two years. Though still concerning, these rates are higher than in some previous studies in more limited populations ,19. SomePatients with additional risk factors for cardiovascular events were more likely to receive recommended care, suggesting that providers may be targeting patients at highest risk for hypertensive complications. Alternatively, these patients may have had better BP control because they were already engaged in treatment for other conditions. Smoking was the exception to this pattern. Smokers received lower quality of care even though they are at higher risk for cardiac complications from hypertension. This is particularly surprising because cigarette smoking is the only measured risk factor that directly and immediately affects BP, though its effect on long-term hypertension is unclear . FurtherAs in previous studies of hypertensives who have visited their physician, just under half of hypertensive patients had controlled BP. However, patients who received optimal care  were more likely to have controlled BP at the end of the study, supporting the hypothesis that a broad range of hypertensive process quality is associated with better intermediate outcomes. The study was not powered to analyze which specific subcomponents of hypertensive care were most associated with BP control. However, because controlled hypertension is correlated with fewer myocardial infarctions and cerebrovascular accidents, it also seems likely that better overall care processes would eventually be associated with fewer complications from hypertensive care, although we could not directly test for this.This process-outcome relationship between hypertensive care and BP control was observed in almost all subgroups, but differences in quality were statistically significant primarily in patients without other cardiac risk factors . UnfortuIn addition to poor process quality, there are other possible explanations for the high rates of uncontrolled BP observed in this study. Even among patients with at least one medical visit in the last two years, impaired access to care might be a factor, and this problem was likely worse in the 863 patients with no visits during the study period who were not included in the analysis . Among tOur study is the first to examine the relationship between the quality of hypertensive care and outcomes among a national population; however, some study limitations exist beyond those inherent in retrospective medical record reviews. First, in constructing the indicators, our expert panel was instructed to rate indicators highly only if documentation of the process in the medical record was common and required for good quality care. Nonetheless, it is possible that documentation differences rather than true quality differences explain some of the observed variations in process quality or outcomes. A previous study found that differences in process scores were 10% lower for medical record abstraction compared to standardized patients with audiotapes of encounters ; thus, oQuality assessment in hypertension is only useful if it is linked with efforts to improve care. There is some evidence that quality improvement (QI) programs can lead to BP control among hypertensive patients. Godley has recently showed that a QI program in a group-model managed care organization increased BP control from 37% to 49% . Our resWe found that the average hypertensive patient in the United States did not receive one in four essential care processes, and those with sub-optimal care had worse BP control. Our data indicate that improvements in processes of care may lead to better outcomes. While it appears that providers appropriately focus attention on patients with additional cardiovascular risk factors, they are under-treating other hypertensive patients who (as previous research would suggest) are nonetheless at increased risk for the same adverse outcomes. Future research should test if the application of routine measurement of hypertensive process quality in provider groups or plans leads to improved processes and BP control.Drs. Asch, McGlynn, Adams, and Kerr declare they have no competing interests. Ms. Hiatt, Hicks, and DeCristofaro declare they have no competing interests. Drs. Chen and LaPuerta are employees and own shares of Bristol-Myers Squibb Company.SA and BM served as principal investigators of this study and were involved in all aspects, as were EK and LH. PL and RC helped arrange partial funding and were involved in the analysis and writing of the manuscript. JA led the analysis, AD and JH were involved in supervising the data collection and participated in the analysis.The pre-publication history for this paper can be accessed here: