Dear Editor,I have read the nice paper published in your estimable journal of Tobacco Induced Diseases Vol. 2, No. 3: 145-150 (2004) about "Oral White Lesions Associated with Chewing Khat". This was a clinical descriptive study done by Gorsky et al. on 47 Yemeni Israeli who had chewed khat more than 3 years and 55 Yemeni non-chewers as group control.I kindly wanted to express my thoughts about some points mentioned in the paper including:1. The phrase [chewing khat] mentioned in the paper is a misnomer and should be replaced by "takhzeen al-qat" as chewing does not infer the exact meaning of what Yemeni people used to do. They used to do "takhzeen" which means in Arabic chewing and storing of qat for several hours. Therefore, the Arabic word takhzeen is used to properly describe this habit1. And the word qat with letter "q" is more commonly used than khat, particularly in the recent studies .2. The authors addressed people who chewed qat for more than 3 years (neglecting the frequency) as chronic chewers, in fact chronocity is dependent on two main factors; the duration (time in years), and frequency of chewing per week. For instance, many persons may use qat for more than 10 years in low frequency (once per month), yet, not considered as chronic users. On the other hand, others may practise it daily for only 2 years and considered as chronic. A recent study strongly correlated the effect of this habit with the frequency3. The authors mentioned in their study a very exaggerated percentages of white lesions in both control (16%) and chewer (83%) groups. Whereas the highest international reported incidence4 of oral white lesions among the normal population is not more than 11.6% and among qat chewers1 (n = 1528) is 22.3%. Furthermore, the authors did not mention any description for the white lesions they found in the control group. What types of white lesions were found?4. The authors addressed 14.6% of the diagnosed white lesions as non-homogenous, at the same time they said "No white lesion was felt to be clinically suspicious for malignant or premalignant changes", on the other hand, non-homogenous white lesions are considered in the international reports as aggressive lesions -7. Moreo5. Finally, all recent references cited in this paper are not related to qat habit but to other habits in other regions of the world. While authors did not quote several studies done in the last 10 years on this habit in the Middle East, which are available through the Medline,11,12.Looking forward to hearing from youYours sincerelyAli A. A.The authors declare that they have no competing interests.


It is uncertain whether the range and frequency of Diagnostic Thinking Processes (DTP) and pieces of information (concepts) involved in dental restorative treatment planning are different between students and expert clinicians.2, and Fisher's. Scott's π was used to determine inter-coder reliability.We video-recorded dental visits with one standardized patient. Clinicians were subsequently interviewed and their cognitive strategies explored using guide questions; interviews were also recorded. Both visit and interview were content-analyzed, following the Gale and Marsden model for clinical decision-making. Limited tests used to contrast data were t, χFifteen dentists and 17 senior dental students participated in visits lasting 32.0 minutes (± 12.9) among experts, and 29.9 ± 7.1 among students; contact time with patient was 26.4 ± 13.9 minutes (experts), and 22.2 ± 7.5 (students). The time elapsed between the first and the last instances of the clinician looking in the mouth was similar between experts and students. Ninety eight types of pieces of information were used in combinations with 12 DTPs. The main differences found in DTP utilization had dentists conducting diagnostic interpretations of findings with sufficient certainty to be considered definitive twice as often as students. Students resorted more often to more general or clarifying enquiry in their search for information than dentists.Differences in diagnostic strategies and concepts existed within clearly delimited types of cognitive processes; such processes were largely compatible with the analytic and (in particular) non-analytic approaches to clinical decision-making identified in the medical field. Because we were focused on a clinical presentation primarily made up of non-emergency treatment needs, use of other DTPs and concepts might occur when clinicians evaluate emergency treatment needs, complex rehabilitative cases, and/or medically compromised patients. Since seminal work was carried out decades ago in Medicine -3, few iClinical decision-making is a complex phenomenon which presents the task of solving an "unknown problem" , a problIsomorphic models have gained acceptance over time. Early work by Elstein et al.  hinged uOur own past research has attempted to characterize diagnostic thinking in restorative dentistry ,10. We pIn the present manuscript, we build on our prior research and expand the sophistication of our interpretation of the clinical decision-making processes in restorative dentistry  by observing both expert clinicians and competent senior dental students. Although Crespo et al.  found diThe study design followed ethical guidelines at Indiana University (IRB 0606-57) to protect all participants' individual rights (standardized patient and clinicians).Gale and Marsden  describeThe goal was to find an "everyday" type of dental patient, who could be of either sex ranging in age from 25 to 60 years old and:• who had at least 20 teeth, with some carious lesions but no major prosthetic or endodontic needs,• with existing restorative needs limited to class I, II, or III amalgam and composite fillings,• with no more than 3-6 units of crown and bridge work,• with a minor periodontal pocket profile in the 2-4 mm range and with gingival recession generally not exceeding 2 mm,• with no urgent dental treatment needs,• not medically compromised or treated for head or neck cancer,• not wearing or needing removable prostheses, or having more than one dental implant,• not having diagnosable TMD problems, exhibiting significant dental phobias, or needing pre-medication before dental appointments, and• who did not have severe fluorosis/erosion/wear; severe malocclusion, cervical abrasions, soft tissue disorders, or obvious signs of dental/craniofacial trauma.Initial screening involved subjects invited from a pool of simulated patients. An oral soft tissue examination was conducted. Their teeth were thoroughly examined using the International Caries Detection & Assessment System (ICDAS) criteria . : 1280 × 960 pixel pictures in TIFF format. Teeth were dried prior to imaging. After being captured, each picture was reviewed on computer monitor, and once it was deemed to be of an appropriate quality and resolution, it was saved to a data file and subsequently serialized in a MS Office PowerPointCompetent fourth year dental students and dentists were compensated $100 U.S. and 3 hours continuing education credits for participating in this study, respectively. Using the standardized patient in a regular dental setting, each clinician was supplied with a description of how the patient contact would unfold, the components available in the dental visit, and the sequence of the (i) dental visit and (ii) interview exercises. Conventional video and audio-recording equipment was used to record the dental visit (TAPE 1) until terminated by the clinician. A researcher kept track of the interaction between clinician and patient, noting potential decision-making events. The clinician and researcher moved to a separate office after the clinician indicated that s/he was satisfied with the information, and a treatment plan was sketched. TAPE 1 of the dental visit was then replayed for the researcher and the clinician. The researcher interviewed the clinician while s/he had available a full series of radiographs, complete medical and dental history, and intra-oral images of teeth and mouth. A second video/audio recording was completed (TAPE 2) as the clinician recalled his/her thoughts with the researcher, to gather an account of the strategies pursued and the relevant pieces of information (concepts) used in the dental consultation.Content analysis ,45 was c® version 15. Data were analyzed using Student's t test for differences between means, and either χ2 test for associations or Fisher's exact test for percentages. As the research was a non-experimental design, we did not attempt to adjust the α level. Our approach at data analysis was not 'mapping' or time-sequencing of actions or perceived strategies [This is an inherently qualitative study. We followed standard recommendations to systematically quantify findings. The coded DTP and concept data were entered into SPSSrategies  but rathContent-analysis reliability was evaluated by establishing the stability (intra-coder variability) of DTP coding  in four A total of 32 clinicians participated in the dental visit and the research interview phases of the study, with 15 dentists  and 17 senior dental students  (p = 0.42). The overall visit intervals lasted a mean of 32.0 minutes (standard deviation 12.9) among experts, and 29.9 (± 7.1) among students (p = 0.57). The length of time between when clinicians first began reviewing patient information and when they requested having the patient brought into the operatory was on average 5.6 minutes (± 4.9) among experts, and 7.6 (± 3.7) among students (p = 0.20). The average face-to-face dental visit time spent with the standardized patient was 26.4 minutes (± 13.9) for experts, and 22.2 (± 7.5) for students (p = 0.25). On average 10.8 minutes (± 6.4) elapsed for experts between the first instance of looking into the mouth of the standardized patient and the last instance; these intervals were not statistically significantly different between experts (10.8 min ± 6.4) and students (13.2 ± 5.1), however, substantial variations in time did exist across all clinicians.Fifteen dentists evaluated the radiographs before seeing the patient .'Failures to make specific or general inquiries'), which were not used by students or dentists. The differences in overall utilization across DTPs were considerable, with DTP3  being used 227 times versus DTP13 being used only 2 times ('Active elimination of an interpretation')  had a lower mean use of 0.9 instances among students versus 2.1 among dentists (p = 0.0004), and DTP3  had a higher average of 8.3 instances among students versus 5.7 among dentists (p = 0.0001). Although statistically not significantly different, DTP9 ('Routine enquiry') was found to be evident on average 2.9 times for students versus 3.7 for dentists. Please note that the use of DTPs described above is outlined for the entire length of the dental visit.Conversely, differences for each one of the DTPs used by students or by dentists were relatively small. Only two DTPs were found to be statistically differently between dentists and students: DTP2 (''Enquiry responsive to elicited information') and 12 ('Routine enquiry') students were seemingly  more prone to use these diagnostic strategies later in the visit, compared with dentists.We wanted to ascertain the sequencing of DTP and concept uses: we made these distinctions explicit by breaking down the overall visit time for each clinician into four segments of equal length, thereby suggesting a first contact and initial gathering of information in the first 25% of the visit time, and closure and presentation of treatment plan in the last 25% of the visit time. Table I think there is some incipient decay on this occlusal surface" would be DTP1 used jointly with concept 'undefined lesion (possible presence in situ)'. Note that the latter is different from concepts 'undefined lesion ', 'residual lesion (possible presence in situ)', or 'recurrent lesion (possible presence in situ)'. A total of 18 concepts were in the category 112 called "Dental Caries", which constituted one of the arrays of concepts used.We have described above the strategies employed by clinicians; we will now describe their use of pieces of information. There were 15 categories of 98 concepts Table  used by 'Health Status, Overall' when the entire visit was examined ; 'Attendance To Dental Care' ; 'Oral Hygiene (Appearance)' ; 'Oral Status, Overall' ; 'Radiographic Information' (different in the first quarter); 'Restorative Item Status, Specific' ; and 'Caries' (different for the first and second quarters).Overall use of concepts across both types of clinicians increased as the visit progressed from the initial quarter to the third , and then decreased in the final quarter (27.5%). There was a very similar frequency of utilization of concepts dealing with Dentists and students (as groups) took about as long to complete their visits, to be ready to see the patient for the first time in the operatory area, spent about the same time with the patient, and invested similar time evaluating the patient's mouth. Furthermore, both groups of clinicians used about the same number of DTPs, had a similar range of DTPs in conducting their visits, and they each omitted the use of the same two DTPs (DTP10 and DTP11). All in all, experts and students had broad similarities in their diagnostic strategies - a finding that has been previously reported for medical clinicians ,48,49. AIn the present study we found differences in DTP use between experts and students. We will first discuss what those differences were, and separately contrast their significance against the common perception that experts are better able to discriminate earlier and faster the significance of information ,56.'Diagnostic interpretation of clinical information'). Students, meanwhile, resorted to searching for clarifying pieces of information through DTP3  more often to make sense of clinical information. By the third quarter, or halfway through the visit, students had continued to judge the need for further general or clarifying enquiry (DTP3) more often than dentists, suggesting that well into the second half of the visit students still had horizons of interpretation that demanded searches for clarifying information about the patient's signs or symptoms.The differences we found were telling in how the groups approach the processing of information. Dentists identified key pieces of clinical information through their more frequent use of DTP2 ('Routine enquiry') did not reach statistical significance when the four quarters of the visit were jointly examined, dentists were eliciting information on routine information (DTP9) in the final quarter. Students, meanwhile, had the bulk of the clinical history preferentially covered in earlier quarters of the visit  that are important to determine the route of the clinical encounter . Even thit Table . StudentDo differences in DTP and concept utilization constitute the hallmark of expertise?  The prevailing interpretation among scholars is that clinicians go about solving diagnostic problems using approaches that are in part mandated by the complexity posed by the case ,59 but aanalytic and non-analytic processing of information. In the first one, a careful evaluation of signs and symptoms and the possible associated diagnoses was conducted, and these linkages were explicitly scrutinized  or a sign elicited . According to the second interpretation , clinicians may identify salient aspects of the signs and symptoms posed by a clinically meaningful amount of information. For example, in this study clinicians had to decide to restore or extract third molars: they looked decayed, were difficult to keep free of plaque, and while in reasonable occlusion positions, their value as teeth were lower than other teeth. If the path to attain a sufficient degree of certainty about the diagnosis is short and direct, a pattern is recognized and we can label it as 'non-analytical.' It is to be expected that the two approaches coexist in differing degrees and at diverse stages of the restorative dentistry decision-making effort [Clinical training has traditionally implied that a careful analysis of the relation between signs, symptoms and diagnoses is the hallmark of clinical expertise . But theg effort  or they g effort  rather tSecondly, an important question is how the pattern is acquired in a non-analytical approach. Regardless of whether such recognition occurs instantly or progressively, a decision leading to clinical action is made. A decision to intervene seemingly depends on one clinical decision-making feature being significant enough to justify the restoration on its own , or an accumulation of features giving rise to the same effect. It has been established that a series of negative factors influences a clinician's decision making process such as when seeing a tooth with an old and 'ugly' filling, or when it presents marginal defects. These negative patient attributes can exert a cumulative influence on teeth being assigned for restoration more often than teeth with fewer negative factors ,64.Finally, case complexity also likely plays a role for different levels of clinician expertise in defining a case as either routine or emergency, or simple or difficult ,37,38. WI forgot to ask about Z symptom..."), we assume that any clinician who suddenly realizes he/she has forgotten to follow a line of enquiry would correct this immediately by asking about the matter. Such amendment(s) could not be categorized as DTP10 nor DTP11. Participants in the Gale and Marsden's study considered that cases were of similar complexity and typical of their usual performance [The absence of DTP10 and DTP11 was not unexpected, as we had reported the same feature in an unrelated group of dental students . Althougformance : a situaformance  dismissaThe use of concepts throughout the visit followed to a large extent the trends identified within the DTPs. An examination of Table The present body of information is derived from a fairly typical number of participants in qualitative research. The attention to detail to dissect the recorded visit and interview materials afforded a mosaic of cognitive strategies during the evaluation of clinical needs. We adhered to the theoretical approach used in Gale and Marsden's model by contrasting levels of expertise: seasoned dentists involved in private dental practice, compared with senior dental students in the last seven months of their clinical training. Students were able to provide comprehensive care and engage in relevant clinical decision-making efforts. A separate methodological consideration pertains to the fidelity enhancement of a real-life situation by using a 'live' clinical encounter: by removing the nuances that a live patient scenario can introduce into standardized observations we aimed to characterize human thinking. Because we used a secluded environment with a single patient who was trained to provide identical answers to similar questions, and to interact in a homogeneous way with every clinician, we feel that we procured as uninfluenced a setting as possible. However, a realistic interpretation of what the research setup offered was not an entirely unobtrusive clinical encounter. The remote possibility that interviewees are affected by their 'observed' status is acknowledged; our findings may be tainted by clinicians trying to 'excel' under test conditions, or trying to provide the information that the interviewer wants to hear. The only complete assurance against such potential biases is to undertake recordings of clinical encounters without informed consent - clearly an unethical design. Finally, the statistical analysis must be taken with reservations as qualitative research does not aim to render representative results. Therefore, the unadjusted alpha level may be a limitation of the study. This is always the case with non-experimental designs, and the lack of comparable studies made it unrealistic to undertake detailed power calculations.In the present manuscript we have made no attempt to ascertain whether individual clinicians or groups of clinicians reached more appropriate diagnoses or treatment plans. Instead we subjectively ascertained that the management measures proposed fell within the limits of what was customary and reasonable for a clinical case. Interestingly, the considerable variation across individual clinicians in terms of how much time they took to evaluate the clinical case, did not have an influence on the time differences between the groups of students and experts. In terms of DTPs and concepts, differences did exist between students and dentists. Differences were not major, probably because we were very specifically focused on restorative treatment needs: associated decisions may resort more (or more frequently) to contrasts between idealized images of what appropriate restorations should look like -11 insteTo our knowledge, this is one of the few reports that examine in detail the developmental cognitive processes involved in dental decision-making using a 'real' live standardized patient. We recognized similarities and differences directly ascribable to two levels of decision-making experience/expertise by using a cognitive psychology model. We then identified concepts clinically relevant to restorative dentistry. The underlying benefit derived from using a single patient case is that stability and fidelity were enhanced as much as possible. Differences across the two groups were thus more likely to be developmental, which connote expert versus student heuristics, rather than case-specific differences derived from multiple clinical cases. Future work should address the strategies and concepts used to diagnose and ascertain treatment needs for presenting complaints, as opposed to 'routine' evaluation of restorative needs, as a likely example of how we may further tease out the use of analytic (deductive reasoning) and non-analytic (pattern recognition) approaches in dental clinical decision-making. By creating more clinical scenarios and testing them as cases to be solved by student and expert clinicians  we should be able to expand the knowledge base in this area. Specifically, to ascertain the tolerance to risk and the perception of risk; the minimim sufficient profile of information that allows a clinical decision to be made; and the qualitative and quantitative differences in information processing that are specific to acute clinical care  or to diagnostically complex scenarios , in contrast to the more 'routine' restorative care for patients of record. At a more general level, appraising the costs ascribable to various decision outcomes will help defend improved pedagogical and clinical guidelines.DTP: diagnostic thinking processes. ICDAS: International Caries Detection & Assessment System criteria The authors declare that they have no competing interests.GM led the design and analysis phases of the study, as well as steering the manuscript and associated presentations to completion/delivery. SS supported study design and acted as the main advisor in qualitative methods and stability assessment throughout the research; he had secondary responsibility for manuscript development. SM was in charge of data management, quantitative contrasts, and supported the various stages of manuscript development. LG provided major input to study design and undertaking, as well as assembling the contacts with expert clinicians; he had secondary responsibility for manuscript development. HE conducted most clinical arrangements, supported standardized patient recruitment and selection, conducted the screening of pool of patients, and assembled components of the clinical case; he had secondary responsibility for manuscript development. All authors read and approved the final manuscript.Not applicableThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6831/10/11/prepubAppendix 1. Identification of DTP and examples in transcripts.Click here for fileAppendix 2. List of concepts identified and allocated to 98 categories in 15 groups of concepts.Click here for file


Despite the use of anti-platelet agents such as acetylsalicylic acid (ASA) and clopidogrel in coronary heart disease, some patients continue to suffer from atherothrombosis. This has stimulated development of platelet function assays to monitor treatment effects. However, it is still not recommended to change treatment based on results from platelet function assays. This study aimed to evaluate the capacity of a static platelet adhesion assay to detect platelet inhibiting effects of ASA and clopidogrel. The adhesion assay measures several aspects of platelet adhesion simultaneously, which increases the probability of finding conditions sensitive for anti-platelet treatment.2 (TXB2)-levels and by static platelet adhesion to different protein surfaces. The results were subjected to Principal Component Analysis followed by ANOVA, t-tests and linear regression analysis.With a randomised cross-over design we evaluated the anti-platelet effects of ASA combined with clopidogrel as well as monotherapy with either drug alone in 29 patients with a recent acute coronary syndrome. Also, 29 matched healthy controls were included to evaluate intra-individual variability over time. Platelet function was measured by flow cytometry, serum thromboxane B2 = 0.49). In opposite, TXB2-levels decreased with ASA compared to clopidogrel. Serum TXB2 and ADP-induced platelet activation could both be regarded as direct measures of the pharmacodynamic effects of ASA and clopidogrel respectively. Indirect pharmacodynamic measures such as adhesion to albumin induced by various soluble activators as well as SFLLRN-induced activation measured by flow cytometry were lower for clopidogrel compared to ASA. Furthermore, adhesion to collagen was lower for ASA and clopidogrel combined compared with either drug alone.The majority of platelet adhesion measures were reproducible in controls over time denoting that the assay can monitor platelet activity. Adenosine 5'-diphosphate (ADP)-induced platelet adhesion decreased significantly upon treatment with clopidogrel compared to ASA. Flow cytometric measurements showed the same pattern (r2 for evaluation of anti-platelet treatment. This should be further evaluated in future clinical studies where screening opportunities with the adhesion assay will be optimised towards increased sensitivity to anti-platelet treatment.The indirect pharmacodynamic measures of the effects of ASA and clopidogrel might be used together with ADP-induced activation and serum TXB Also, the PFA-100™, Multiplate™ and the VerifyNow™ are examples of instruments commercially developed for evaluation of anti-platelet therapy. However, no studies have investigated the usefulness of altering treatment based on laboratory findings of ASA resistance [Anti-platelet drugs such as acetylsalicylic acid (ASA) and clopidogrel are routinely used to prevent thrombosis in cardiovascular disease. The benefits of ASA have been clearly demonstrated by the Anti-platelet Trialists' Collaboration . They fosistance . Regardisistance ,21.2-levels and by measuring static platelet adhesion to proteins in microplates. The aim was to evaluate the usefulness of the static platelet adhesion assay for measuring the effects of ASA and clopidogrel. Static adhesion is an aspect of platelet function that has not been investigated in earlier studies of the effects of platelet inhibiting drugs. Consequently, static platelet adhesion is not measured by any of the current candidate assays for clinical evaluation of platelet function. The static platelet adhesion assay offers an opportunity for simultaneous measurements of the combined effects of several different platelet activators on platelet function. In this study, platelet adhesion to albumin, collagen and fibrinogen was investigated in the presence of soluble platelet activators including adenosine 5'-diphosphate (ADP), adrenaline, lysophosphatidic acid (LPA) and ristocetin. Collagen, fibrinogen, ADP and adrenaline are physiological agents that are well-known for their interactions with platelets. Ristocetin is a compound derived from bacteria that facilitates the interaction between von Willebrand factor (vWf) and glycoprotein (GP)-Ib-IX-V on platelets, which otherwise occurs only at flow conditions [The current study used a randomised cross-over design in order to investigate the effects on platelets of dual therapy with ASA and clopidogrel as well as the effects of either drug alone in patients with a recent acute coronary syndrome. Platelet function was assessed by means of flow cytometry, serum TXBnditions . The standitions . LPA is nditions . It was nditions . Thus, bnditions . This isThe study was approved by the Research Ethics Committee of Linköping University, Linköping, Sweden and the Medical Product Agency, Sweden (EudraCT Number 2005-003927-38). A total of 33 patients recently diagnosed with acute coronary syndrome were included on a consecutive basis from the Department of Cardiology at the University Hospital in Linköping, Sweden Figure . ExclusiBlood was drawn from patients at three different occasions Figure . The fir2 and (3) tubes containing sodium citrate  for flow cytometric analysis (patients only). To obtain platelet rich plasma (PRP) for platelet adhesion analysis, 8 mL blood was transferred from sodium heparin tubes to a single plastic centrifuge tube. This single tube was then centrifuged for 20 min at 205 × g resulting in the production of a PRP supernatant. Blood obtained in serum tubes were allowed to clot at room temperature followed by centrifugation for 10 min at 1000 × g. The serum was transferred to eppendorf-tubes and stored at -70°C until analysis of TXB2. For patients, blood samples were also drawn into lithium heparin-tubes and K2EDTA-tubes for biochemical analysis at the accredited Department of Clinical Chemistry at the University Hospital in Linköping, Sweden. The lithium heparin-tubes were used for analysis of plasma concentrations of C-reactive protein (CRP), cholesterol, triglycerides, LDL-cholesterol, HDL-cholesterol, apolipoprotein-A1 (Apo-A1) and apolipoprotein-B (Apo-B), utilising the clinical chemistry analyzer Advia 1650 from Roche. Concentrations of platelets and leukocytes were determined from the K2EDTA-samples.Whole blood was drawn from antecubital veins and collected in (1) tubes containing sodium heparin  for platelet adhesion analysis, (2) tubes with no additives for measurements of serum TXB2  and 25 μL of platelet activators. The soluble platelet activators were ADP and LPA from Sigma-Aldrich , adrenaline from Merck NM AB  and ristocetin from Diagnostica Stago   was added. Background absorbance was measured at 405 nm using a Spectramax microplate reader  and the microplates were then incubated for 40 min at room temperature during shaking. In parallel, 50 μL PRP as well as 50 μL 0.9% NaCl were added to wells on a separate microplate. Both PRP and NaCl wells were treated with 140 μL of the sodium citrate/citric acid buffer described above followed by background absorbance measurements and consequently served as controls for 100% and 0% adhesion respectively. During the 40 min incubation, an enzymatic reaction occurred between added phosphatase substrate and platelet acid phosphatase. Adding 100 μL 2 mol/L NaOH to all wells (including 100% and 0%) stopped the reaction and resulted in a colour change of the developed product. Absorbance was measured at 405 nm with automatic reduction of background absorbance and percentage platelet adhesion was calculated.Static platelet adhesion was measured as previously described . Ninety-is study ,28. The Platelet expression of P-selectin and binding of fibrinogen were measured by flow cytometry as indicators of platelet activation -32. To t2 were measured with a commercial enzyme immuno assay (EIA) kit according to the manufacturers' instructions . Amount of TXB2 present in serum was calculated with the use of a data analysis tool developed by Cayman Chemical [Serum levels of TXBChemical .2-levels) consisted of data from three measurements on patients and two on controls. All other variables were only analysed on patients, which resulted in three measurements that were included in the PCA. A variable was considered to be part of a factor when its loading was ≥ 0.4. After finding distinct factors, the composite variables included in the PCA were standardised according to Z-scores. This procedure transforms all variables to the same scale having a mean value of 0 and a standard deviation of 1. For each individual, a mean was calculated from the Z-scores of the variables that were found to belong to the same factor. From the Z-mean of the individuals, a Z-mean of the whole factor was calculated and further used for statistical comparisons of means. The factors, as well as some representative variables, were then analysed for treatment effects and for intra-individual variations within controls by Repeated Measures ANOVA. Differences between controls and patients were analysed by One-sample t-test. Correlations between factors were investigated with linear regression.The variables measured were subjected to Principal Component Analysis (PCA) with direct obliminal rotation using SPSS 14.0 software . This technique analyses to what extent different variables are measuring the same concept and allows correlating variables to be ordered into separate factors . The PCA2 formed a separate factor. Visual inspection of the data of the healthy controls for the initial factor solution revealed possibilities for making the factors corresponding to platelet adhesion even simpler. Attention was paid at (1) different concentrations of the same soluble agonist on a specified surface, (2) the effects of weak agonists compared to basal adhesion and (3) the effect of an agonist compared to its combination with another agonist.In total the PCA grouped the initial 69 variables of platelet activation and routine clinical chemistry analyses into 15 different factors that we renamed according to the aspects they measured .The second scenario regarding the effect of weak agonists can be exemplified by Factor 5. It is possible that weak agonists do not increase platelet adhesion significantly compared to adhesion to the surface alone. As was the case for different doses of ADP, the weak agonist will then not contribute any relevant information regarding adhesion and could therefore be excluded. For Factor 5, adrenaline at 1 μmol/L was the only agonist that induced significantly increased adhesion compared to the surface alone and all others were consequently excluded from this factor. As for Factor 1, other reasons motivated the exclusion of adrenaline at 1 μmol/L as well from Factor 5 (see next section).2+ showed that both adrenaline and LPA induced a weak albeit significant decrease in platelet adhesion. Since both LPA and adrenaline are platelet agonists, the decreased adhesion observed was considered irrelevant in this case and the variables were excluded.A special case was observed for Factor 8. Pairwise analysis of the data regarding adhesion to collagen in the presence of MgFactor 4, 6 and 7 belongs to the third scenario in which comparisons were made between single agonist addition and addition with the same agonist in the same concentration combined with a second agonist. The combined addition was excluded unless it resulted in significantly increased adhesion compared to single agonist addition.Finally, Factor 2 contained only variables that can be regarded as negative controls resulting in no platelet adhesion, as exemplified by albumin without any soluble activator. Such conditions can never detect inhibiting effects of drugs, which prompted us to exclude the whole factor.2-levels were performed on healthy controls on two separate occasions  in order to investigate the presence of intraindividual variation in platelet reactivity and clotting-induced TXB2-production. The standardised Z-scores from the simplified factors were used for analysis by Repeated Measures ANOVA of the data from the healthy controls. We found significantly decreased platelet adhesion at the second compared to the first visit for ADP-induced adhesion  and for adhesion to fibrinogen . This intra-individual variability over time makes it difficult to draw any conclusions regarding effects of anti-platelet treatment. We therefore further analysed the individual variables constituting Factors 1 and 5 with Repeated Measures ANOVA in order to distinguish the variables that varied significantly over time. Variables being significantly different between visit 1 and visit 2 were then excluded and a new Repeated Measures ANOVA was performed on the new factors. After this modification, none of the factors corresponding to adhesion showed variation over time and these factors were then used for analysis on patients. Serum levels of TXB2, which constituted a separate factor, varied significantly in healthy controls at two separate occasions , ristocetin-induced adhesion Factor 4) and adhesion to fibrinogen (Factor 5). Regarding adhesion factors detecting treatment effects, ADP-induced adhesion  and ristocetin-induced adhesion to albumin (Factor 6) were significantly decreased for patients treated with clopidogrel alone or in combination with ASA compared to healthy controls  consistently decreased for patients compared to the reference values , correlated with an r2-value of 0.28. Regarding TXB2, regression analyses were only performed on samples with clopidogrel monotherapy since levels of TXB2 were totally suppressed when platelets were treated with ASA. However, serum TXB2-levels did not correlate with any of the other measurements.Linear regression analyses were primarily focused on investigating possible correlations between any of the factors and (1) ADP-induced platelet adhesion and (2) serum TXB2 formed a distinct group not correlated to any of the other measurements.With the aim of finding variables sensitive to clopidogrel and ASA-treatment, this study used a screening approach and measured several different variables simultaneously. To reduce the complexity of the material we performed PCA in order to find correlating variables that measured the same property. In this way the 54 measurements of platelet adhesion were reduced to 8 factors. Visual inspection revealed that each factor represented a separate entity of platelet adhesion and the factors could therefore be renamed according to the aspect they measured. We thus conclude that future studies must not involve all 54 adhesion variables, but instead, one variable from each factor should be enough to cover 8 different aspects of platelet adhesion. In addition to the adhesion data, the remaining 15 variables also formed distinct factors that were possible to rename according to measured property. It is notable that serum TXBin vivo platelet adhesion occurring during flow conditions. However, platelet adhesion to collagen and fibrinogen is dependent on α2β1- and αIIbβ3-receptors respectively in the current assay [It is important that laboratory assays used for clinical purposes are reproducible and that they measure parameters that are not confounded by other variables. Some of the measurements performed in this study  are used for clinical analysis at accredited laboratories at the University hospital in Linköping. However, the reproducibility of the platelet adhesion assay was mostly unknown before this study . Our inint assay . This sunt assay .We found that platelet adhesion to albumin, collagen and fibrinogen induced by ADP was suppressed during treatment with clopidogrel alone or in combination with ASA compared to treatment with ASA alone. The same pattern was also seen for the flow cytometric measures of ADP-induced activation. Furthermore, clopidogrel treatment suppressed both ADP-induced adhesion and ADP-induced activity measured by flow cytometry below the levels observed for healthy controls. These results are clear indications that both assays are able to detect the direct effect of inhibition of ADP-signalling by clopidogrel. It is also interesting to note that, upon ADP-challenge, the different assays correlated relatively well to each other. The variation between those factors not explained by the other can probably be ascribed to the different aspects of platelet function that the two assays measure. Flow cytometry measures the expression of activation-dependent receptors when platelets are in solution, while the adhesion assay measures the ability of platelets to adhere to a surface .2-levels in healthy controls revealed significant intra-individual differences. This effect may be attributed to varying stress levels for the controls at the separate occasions since urinary levels of the TXA2 metabolite 2,3-dinor-TXB2 has been shown to correlate with urinary cortisol [2 follows a seasonal variation pattern [2-levels were completely suppressed after ASA treatment compared to both clopidogrel treatment and controls. Consequently, despite the intra-individual variations, this shows that serum TXB2 is a good indicator of the ability of ASA to inhibit the cyclooxygenase pathway.Measurements of serum TXBcortisol . Also, u pattern . AccordiSome adhesion measures showed decreased adhesion by clopidogrel alone compared with clopidogrel plus ASA. Since dual therapy was always the initiating treatment, this difference may reflect the presence of more active platelets in the time frame closest to the index event and coronary revascularization. It has been proposed that reendothelialization after insertion of a bare metal stent is complete after approximately 3 months  and that2 levels measure the effects of ASA. However, these measures focus on the primary interaction between the drugs and the platelets, which could be problematic when trying to evaluate the complex in vivo treatment effect. It has previously been found that only 12 of 682 ASA-treated patients (≈ 2%) had residual TXB2 serum levels higher than 2 standard deviations from the population mean [2 [2-formation with platelet aggregation [2-effects (in our case ADP-induced activation measured by adhesion or flow cytometry and serum TXB2-levels) must be combined with measures that are only partly dependent on ADP and TXA2 respectively. For instance, an adhesion variable partly dependent on TXA2 might be able to detect ASA resistance caused by increased signalling through other activating pathways. Such a scenario would be characterized by serum TXB2 values showing normal COX-inhibition while platelet adhesion is increased. This study employed a screening procedure in order to find such indirect measures of the effects of ASA and clopidogrel. Our results show inhibiting effects of clopidogrel compared to ASA on adhesion to albumin in the presence of LPA or ristocetin. This was also observed for our flow cytometric measurements with SFLLRN as activator, which confirms that SFLLRN is able to induce release of granule contents in platelets [in vitro [in vivo setting of myocardial infarction. The collagen surface is different from the other stimuli since dual therapy results in significantly depressed platelet adhesion compared to both monotherapies. This indicates that adhesion to collagen is dependent on both ADP and TXA2 and this measure was the only adhesion-related factor that showed potential for being partly dependent on TXA2. However, the significant effects observed between treatments were rather small. Nevertheless, it has earlier been shown that platelet activation induced by collagen is reduced by intake of ASA [From this discussion it is evident that the adhesion assay as well as flow cytometry can measure effects of clopidogrel when using ADP as activating stimuli. It is also evident that serum-TXBion mean . Measureion mean . Thus, oion mean . Several mean [2 -49. In lregation . We furtlatelets ,52. SFLLin vitro . This stin vitro  makes ite of ASA ,53. Rega2. One such measure is adhesion to a collagen surface, which should be more thoroughly investigated for its ability to detect effects of clopidogrel and ASA. Likewise, due to its connection to atherosclerosis and myocardial infarction, the LPA-induced effect should be further evaluated for its ability to detect effects of clopidogrel. In conclusion, the screening procedure undertaken in this study has revealed suggestions on which measures of platelet activity to combine in order to evaluate platelet function. Speculatively, the ADP-mediated effects in the present adhesion assay in combination with serum TXB2, may be combined with LPA and collagen-induced adhesion for an optimal monitoring of clopidogrel and ASA therapy.In this study we employed different assays in order to evaluate platelet function in patients treated with different anti-platelet regimens. Among these assays, the platelet adhesion assay had a certain role since it had not been used before for this clinical purpose. Actually, there are no assays of static platelet adhesion that have been used in previous studies aimed at investigating treatment effects of platelet inhibiting drugs. Importantly, this study shows that the static platelet adhesion assay is reproducible over time. We also showed that the static platelet adhesion assay as well as flow cytometry detected the ability of clopidogrel to inhibit platelet activation induced by ADP. Our results further suggest that other measures of platelet adhesion and platelet activation measured by flow cytometry are indirectly dependent on secreted ADP or TXAThe authors declare that they have no competing interests.2-levels and performed the statistical analyses. All authors participated in the design of the study, co-operated in the drafting of the manuscript and read and approved the final version of the manuscript.ACE carried out the analysis of static platelet adhesion and serum TXBAll variables measured in the study. A table showing all the variables that were measured in the study.Click here for fileThe final factors used for ANOVA analyses. A table showing the factors used for ANOVA analyses.Click here for file


The purpose of this study was to measure the impact of an educational intervention on parents of children taking methotrexate (MTX) for juvenile idiopathic arthritis (JIA).t-test  was performed on individuals who answered both questionnaires.This study was conducted using a pre- and postsurvey design. The parents of 100 children with JIA taking MTX for at least 2 months were surveyed during a routine office visit. The parents completed an initial questionnaire regarding the safe use, adverse effects, and guidelines for monitoring the toxicity of MTX. An educational intervention was then administered, and an identical follow-up questionnaire was given during the next office visit. Statistical analysis using a paired P < 0.0001). Individuals scored the lowest (49%) on the question that addressed MTX's impact on pregnancy and fertility.There were 100 responses to the initial questionnaire and 67 responses to the follow-up questionnaire. The mean length of time between surveys was 2.9 ± 0.9 months. In those who completed both questionnaires, the overall correct score increased significantly from 75.8% to 93.4%, respectively (MTX knowledge may be less than expected in the parents of children with JIA. Brief educational interventions in the pediatric subspecialty practice can significantly affect a family's understanding of their child's medications. Methotrexate (MTX) is the most commonly used disease-modifying antirheumatic drug (DMARD) for the treatment of juvenile idiopathic arthritis (JIA) . It is aThe most commonly reported adverse effects are gastrointestinal (nausea and vomiting) although folic acid can be used to reduce these symptoms. Ulcerative stomatitis and alopecia also occur but are much less common. Prescribers must be cautious in using MTX for women of childbearing age because of fetal death and/or congenital anomalies in pregnancy. Bone marrow suppression may occur and result in anemia, leukopenia, neutropenia, and/or thrombocytopenia. Immune suppression may lead to opportunistic infections. MTX has been associated with elevated transaminases and potential hepatotoxicity. Renal damage, pulmonary disease and dermatologic reactions can occur as well ,4-6.Patients are advised to obtain routine laboratory tests to monitor for potential toxicities. These include a complete blood count, liver function tests, blood urea nitrogen, creatinine and urinalysis. Periodic liver biopsy is not recommended for toxicity monitoring as it was in the past. For some patients with pulmonary disease, a chest x-ray and pulmonary function tests are also obtained periodically; however, MTX-associated interstitial lung disease is exceedingly rare in children .In pediatric rheumatology practice, every parent and age-appropriate patient receives instructions and information on MTX use at the start of therapy. These instructions can vary across practitioners and may not be repeated on follow-up visits. A previous study on adults with RA taking MTX showed that knowledge of the toxicity and safe use of MTX were significantly improved by a patient education program . There iThe primary aim was to determine whether educational interventions, including review of a MTX questionnaire and dispersal of a MTX brochure, were effective in parental education regarding MTX use in their son or daughter with JIA. It was also determined which question was answered incorrectly most often, and therefore which topics are important to emphasize with parents regarding MTX therapy.This quality improvement study was conducted as a pre- and post-survey design over a six month period at the Children's Hospital of Pittsburgh Rheumatology Clinic. Patients taking MTX were identified through chart review the day of their visit. Parents of children with JIA were asked to answer a five item questionnaire during a routine outpatient visit:1. How often should blood work be checked on a child taking methotrexate?(a) Every 4 weeks(b) Every 6-8 weeks(c) Every 3 months(d) Every 6 months2. What are the most common side effects of methotrexate?(a) Upset stomach and nausea(b) Skin rash and hives(c) Dizziness and headaches(d) Blurry vision and forgetfulness3. Why should blood work be checked regularly in a child taking methotrexate?(a) Monitor for worsening of the arthritis(b) Monitor for potential side effects(c) For research studies(d) Compare results to other patients with similar disease4. Why do we prescribe folic acid?(a) It helps with the arthritis symptoms(b) It helps to prevent side effects of methotrexate(c) It helps prevent the eye disease associated with arthritis(d) It helps with morning stiffness5. How will methotrexate affect a child's ability to have children in the future?(a) It has no affect but will cause birth defects if taken by women at the time of conception or pregnancy(b) It will cause problems for women who want to get pregnant many years after the drug is stopped(c) It will cause lower sperm counts in men many years after the drug is stopped(d) It has no consequence on fertility or pregnancy*Correct Responses: 1(b); 2(a); 3(b); 4(b); 5(a).The questionnaire was developed by consensus of the physicians in the division and was based on information that was thought to be essential for parents to understand regarding MTX therapy. The questions were mainly derived from a brochure provided by the Arthritis Foundation which is given to patients that are initiated on MTX therapy for rheumatic conditions. There was no pilot testing of the questions; however, some content was similar to that from a previous study conducted on adults with RA , providit-test  was used to compare mean scores (pre- and post-) for those individuals who answered both questionnaires. It was also determined whether the percentage correct was different between male versus female parents, length of MTX use, and subtype of disease using the Mann-Whitney U test . In the few cases in which both parents came to the initial visit, only one parent answered the questionnaire.A single physician (AP) reviewed the results of the questionnaire with the parents in a standardized fashion and provided literature on MTX. The literature provided was a brochure from the Arthritis Foundation from which the questions were derived. During the next routine clinic visit, a follow-up questionnaire identical to the first was given to the parents. Some patients did not have a scheduled follow-up visit in the allotted time, were taken off MTX, or had a different caregiver present at the second visit; therefore, fewer participants answered the follow-up questionnaire. The patient demographics were not significantly different between those who answered the follow-up questionnaire and those who did not. Statistical analysis with a paired n = 67), the overall score on the initial questionnaire was 75.8% ± 20.8 and the overall score on the follow-up questionnaire was 93.4% ± 10.7. The difference between these scores using a paired t-test was statistically significant (P < 0.0001). The mean length of time between questionnaires was 2.9 ± 0.9 months.The first 100 parents who were approached answered the initial questionnaire. Sixty-seven of the initial 100 answered the follow-up questionnaire. The patient female-to-male ratio was 3:1 with an age range of 1-21 years old (mean of 9.9 ± 4.7 and median of 10). The most common subtype of JIA was rheumatoid factor (RF) (-) polyarticular disease (39%). The range of MTX dose was 5-25 mg (mean of 17.6 ± 5.1 and median of 18), with 56% taking oral tablets and 44% receiving subcutaneous injections. The duration of MTX therapy when initially surveyed ranged from 2-96 months (mean of 24.3 ± 21.8 months and median of 18). In those who completed both questionnaires . The average age of the patient during this study was 9.9 years old, reflecting that many were prepubertal when MTX was initiated. Healthcare workers may not have discussed pregnancy-related issues during the initiation of MTX due to the young age of the population. Another possibility is that the parents may not have been concerned about these issues because of the age of their child. Regardless of the cause, healthcare providers should consider discussing MTX and pregnancy more often and at an earlier age to establish awareness. There is limited data on children with JIA and pregnancy; however, adolescent pregnancy is always a concern, regardless of chronic disease.A study of quality indicators showed that there was significant variability in providing adult rheumatoid arthritis patients with discussions on MTX and in obtaining appropriate monitoring studies . ProvidiIt has been recommended that educating families about goals of treatment and how to minimize treatment side effects may enhance adherence . A direcIn summary, there can be a lack of knowledge regarding the use and common adverse effects of MTX in JIA. The parent understands the use and toxicity of MTX can be improved with education. This can be done with a combination of verbal information by a healthcare provider and written literature on MTX. Pediatric rheumatologists should consider repetitive education when teaching families about DMARDs, which can be achieved in a short amount of time and enhance parental knowledge of MTX use and toxicity.The authors declare that they have no competing interests.AP distributed the questionnaires and performed the educational interventions of the study. AP and KT performed the statistical analysis. All the authors participated in the design of the study and helped draft the manuscript. All authors read and approved the final manuscript.


Secondary metabolism is an example of a complex network of biochemical pathways, which, although well mapped from a biochemical point of view, is not well understood with regards to its physiological roles and genetic and biochemical regulation. Many of the metabolites produced by this network such as higher alcohols and esters are significant aroma impact compounds in fermentation products, and different yeast strains are known to produce highly divergent aroma profiles. Here, we investigated whether we can predict the impact of specific genes of known or unknown function on this metabolic network by combining whole transcriptome and partial exo-metabolome analysis.YMR210W, BAT1, AAD10, AAD14 and ACS1 were selected for overexpression in commercial wine yeast, VIN13. Analysis of the data show a statistically significant correlation between the changes in the exo-metabome of the overexpressing strains and the changes that were predicted based on the unbiased alignment of transcriptomic and exo-metabolomic data.For this purpose, the gene expression levels of five different industrial wine yeast strains that produce divergent aroma profiles were established at three different time points of alcoholic fermentation in synthetic wine must. A matrix of gene expression data was generated and integrated with the concentrations of volatile aroma compounds measured at the same time points. This relatively unbiased approach to the study of volatile aroma compounds enabled us to identify candidate genes for aroma profile modification. Five of these genes, namely The data suggest that a comparative transcriptomics and metabolomics approach can be used to identify the metabolic impacts of the expression of individual genes in complex systems, and the amenability of transcriptomic data to direct applications of biotechnological relevance. Saccharomyces cerevisiae are currently produced and sold in the global industry. Many research and development programs have focused on improving specific aspects of wine yeast strains [Commercial wine yeast strains have been selected to meet specific requirements of wine producers with regard to phenotypical traits such as fermentation performance, general stress resistance, the profile of aromatic compounds produced, the ability to release enzymes or mannoproteins of oenological relevance and many more . As a re strains . However strains . In thisSaccharomyces cerevisiae strains convert sugars to ethanol, but also produce a large number of volatile aroma compounds, including fatty acids, higher alcohols and esters  for the five yeast strains in our study. At the time points considered, the variation in gene expression between the different strains was in the range of about 50–400 transcripts. Strains that appear to be most similar to one another on a gene expression level were the EC1118 and DV10 strains, as well as the BM45 and 285 strains. The VIN13 strain was least similar to any of the other four strains. This pattern is in line with the differences observed in aroma production for all of these strains.Numerous and substantial changes in the expression of genes involved in pathways that lead to the production of volatile aroma compounds were evident both between strains at comparable stages of fermentation and for individual strains at different fermentative stages. To identify relevant transcriptional variation in the context of aroma compound production, PCA analysis and PLS1 and PLS2 models were constructed for the compounds in tables The general intrastrain trend revealed a decrease in the transcript levels of enzymes involved in the synthesis of aromatic and branched-chain amino acids, while transcript levels encoding aldehyde and alcohol dehydrogenases, as well as certain acetyltransferases were generally increased. Fold changes for differentially expressed transcripts, both between different strains at either day 2 or day 5 of fermentation and between day 2 and day 5 in individual strains, can be viewed as additional material [see Additional data file Figure The X-Y scores and loading plots figure  are cleaOf the 22 volatile aroma compound measured in this study, 13 were amenable to PLS1 modeling (using transcriptome data) based on our selected criteria for model validation . The details of these models are summarized in a table that can be viewed as additional material [see Additional data file BAT1, AAD10, AAD14, ACS1 and YMR210W. AAD10 and AAD14 encode aryl alcohol dehydrogenases which are believed to be responsible for the putative role of degrading the complex aromatic compounds in grape must into their corresponding higher alcohols [BAT1 encodes a mitochondrial branched-chain amino acid aminotransferase that is involved in catalyzing the first transamination step of the catabolic formation of fusel alcohols via the Ehrlich pathway [YMR210 gene codes for a putative acyltransferase enzyme (similar to EEB1 and EHT1) and is believed to play a role in medium-chain fatty acid ethyl ester biosynthesis. Lastly, the ACS1 gene (encoding an acetyl-coA synthetase isoform) codes for the enzyme responsible for the conversion of acetate to acetyl-coA, which is an intermediate or reactant in several of the aroma compound producing pathways [Of the genes listed in the tables presented in the supplementary material, five were chosen for in-depth analysis due to their significant contributions to the respective prediction models for several of the important higher alcohols and esters, as well as their amenability to easy cloning and vector construction. These genes were alcohols . BAT1 en pathway . The YMRpathways .BAT1 overexpressing strain was already available for use [An in-house  for use . For theFermentations were carried out as before with the 5 transformed cell lines and a VIN13 control. Samples for HPLC and GC-FID analysis were taken at the same time points, namely days 2, 5 and 14 of fermentation. No significant differences were observed regarding the glucose and fructose utilization of the overexpression strains during fermentation (Data not shown). Slight differences were found for ethanol production, while some changes in glycerol production were evident for the different strains Figure .Figure YMR210W overexpressing strain did not show any changes, and is therefore not included in the figures below. We did not further investigate whether this absence of changes in aroma production is due to problems with the expression construct or reflects the absence of aroma–related activity of the gene product.Four of the five overexpressing strains showed significant changes in the aroma profiles produced at the end of fermentation. Only the Significant differences were evident in the aroma profiles of the four transformed yeast strains under consideration. We investigated whether the observed changes in aroma compound concentrations at the end of fermentation can be reconciled with the anticipated changes based on multivariate prediction models. Figure To clarify, the predicted influence of a given gene on a particular compound is represented on a scale from -1 to +1, based on statistical projections related to PLS loading weights. On this scale a value of -1 suggests a strong probability of significant concentration decreases of a given compound (for overexpression of the gene), while a value of +1 is indicative of a strong positive correlation between the expression levels of the gene of interest and the compound in question. A value of zero indicates no expected influence of gene expression on the relevant aroma compound.Likewise, log-normalization was carried out on the actual metabolite concentrations measured in the overexpression strains to represent these values on a scale from -1 to 1, relative to the corresponding concentrations of the control fermentations. Figure The aim of this study was to determine whether the transcription profiles of the various strains during fermentation could be reconciled with the volatile aroma compound production of these strains, and whether this comparative analysis could be used to predict the impact of individual gene expression levels on aroma compounds and profiles.AAD10, AAD14 and BAT1, were very significantly aligned with the changes that were predicted from the alignment of transcriptome and metabolome data alone. The predictions, as can be seen from the alignment of predicted vs. observed changes in metabolite levels in a qualitative manner, indeed proved fairly reliable. The model was able to assign positive and negative influences on a particular compound with relative accuracy. Although the extent/magnitude of the increase/decrease is not always well aligned with model values, the absolute direction of the change holds true in most cases. An absolute alignment would not be expected, since the level of expression in a plasmid-based system can not be adjusted to the differences of expression observed between the different strains. In the case of AAD10, only the influence of the overexpression on decanoic acid was not in line with the projection. Predictions for AAD14 and BAT1 were well matched with the observed changes in metabolite profiles. Predicted and real changes did not match satisfactorily in only one case, ACS1. Nevertheless, even in this case, eight out of the thirteen compounds evolved in the predicted direction. It should also be noted that the expression of this gene had generally a less severe impact on changes in the aroma profile than those of the other three genes.The data generated by the overexpression of four of the genes whose expression was statistically most significantly linked to the production of aroma profiles suggest that this approach has been successful. Indeed, overexpression of the selected genes had a far reaching impact on the aroma profiles produced by the fermenting yeast, and this impact was generally well aligned with the impact predicted from the comparative omics analysis. Indeed, the data aligned better than we, considering the significant challenges when approaching complex systems, had expected. Our data show that the metabolic changes observed upon overexpression of three of the four genes, Considering the complexity of the system, the rate of success achieved in this study can be considered as highly significant. To our knowledge, this is the first report to exploit such an intra- and interstrain comparative approach to identify genes that play a significant role in a complex metabolic network.While we were clearly able to identify genes with significant impact on aroma compound production in a specific industrial environment, and which in some cases had not been previously directly linked to these pathways, the data do not allow a firm conclusion on the exact metabolic role of these genes. Indeed, the vast number of significant changes to metabolite levels makes it difficult to identify the specific 'point of influence' of any overexpressed gene in a given pathway.The increases/decreases in specific volatile compounds seen for the VIN13(pBAT1-s) strain is in keeping with the results reported in colombar fermentations . The twoACS1 gene did not lead to such numerous and substantial increases/decreases in volatile production as was the case for the other three genes. Interestingly, valeric and isovaleric acid were below detection levels in these fermentations. Concentrations of isoamyl acetate, ethyl acetate, butanol and butyric acid were significantly higher, and ethyl caprate lower relative to control fermentations.Overexpression of the On the whole though, our analysis shows that the cross-comparison of gene expression data with metabolite levels has the potential to identify points of interest on a genomic scale. This also opens new possibilities to design improved yeast enhancement strategies for optimized aroma production and fermentation performance.Many other genes showed significant variation in expression between different strains and/or time points, as well as high loadings on PLS models and strong negative or positive correlations with specific aroma compounds. These genes encode enzymes that either are known to participate in aroma compound production, or have activities  that could suggest such roles. Here we discuss some of the most relevant of these enzymes, which fall into several categories, either according to their place in a specific metabolic pathway such as the metabolisms of branched chain amino acids or of aromatic amino acids, or based on their specific activity such as dehydrogenases  and acetyl transferases.BAT1 has been discussed above. Other genes that encode enzymes in this pathway and that were identified in our study for their strong statistical link between expression levels and the production of specific compounds include LEU2, encoding a beta-isopropylmalate dehydrogenase that catalyzes the third step in the leucine biosynthesis pathway, and, to a lesser degree, LEU1, which encodes an isopropylmalate isomerase [ILV5, which encodes an acetohydroxyacid reductoisomerase involved in branched-chain amino acid biosynthesis [Of the enzymes involved in branched chain amino acid metabolism, somerase ,23. Bothynthesis , showed BAT1 expression showed a significant positive correlation with a large number of the volatile compounds measured in our study, the cytosolic isoform (BAT2) of this enzyme showed no significant correlations with any of these aroma compounds. Although this isoform is supposedly highly expressed during stationary phase and repressed during the logarithmic phase, BAT2 expression levels in our study were found to stay constant, if not to decrease slightly upon entry into stationary phase in comparison to the exponential phase at day 2. In addition, BAT2 expression levels were generally considerably lower throughout fermentation when compared to BAT1.While ARO1, which encodes a pentafunctional arom protein, ARO7, which encodes a chorismate mutase responsible for the conversion of chorismate to prephenate and ARO8, which codes for an aromatic aminotransferase showed statistically significant correlations between expression levels and metabolite production [2 = 0.7) with 2-phenyl ethanol and mild negative correlations with all the other compounds. Only octanoic acid showed a very strong (r2 = 0.82) negative correlation with ARO8 expression at day 2 of fermentation. Despite its seemingly crucial role, ARO10, which encodes a phenylpyruvate decarboxylase corresponding to the first specific step in the Ehrlich pathway did not show any noteworthy correlations between its expression and any of the volatile compounds in our study [Of the genes involved in aromatic amino acid metabolism, three, oduction ,26. All ur study . Of courALD5, ALD6, ADH6 and ADH7 showed a substantial decline in expression levels between days 2 and 5 of fermentation, while others  increased during this time. The distinct expression patterns during fermentation reflects the different regulatory mechanisms governing the expression of these genes (i.e. expression of ALD3 is glucose-repressed and stress-induced) and suggests that the different ALD gene products have specific roles during different stages of fermentation [Several specific enzyme activities were also overrepresented in our list. Such enzymes include many dehydrogensases. Aldehyde and alcohol dehydrogenases such as those encoded by entation .ALD4 and ALD5 , and ALD3 and ALD6 (cytoplasmic) encode aldehyde dehydrogenases involved in the conversion of acetaldehyde to acetate [ acetate .ALD4 encodes a mitochondrial aldehyde dehydrogenase (utilizing NADP+ or NAD+) that is required for growth on ethanol and conversion of acetaldehyde to acetate [ALD4 is also glucose repressed, and increases 2–4 -fold from day 2 to 5 of fermentation. ALD4 expression shows a very strong correlation to the amount of hexyl acetate (R2 = 0.82) produced by the fermenting yeast, as well as to ethyl acetate (0.77), isoamyl alcohol (0.91) and isoamyl acetate (0.85). acetate . ExpressALD6 encodes a constitutively expressed cytosolic aldehyde dehydrogenase (utilizes NADP+ as the preferred coenzyme) and is required for conversion of acetaldehyde to acetate [ALD6 expression showed a very strong positive correlation to the levels of acetic acid produced by the fermenting cells (0.92). Also, expression was very strongly inversely correlated to ethanol production (R2 = 0.81). Interestingly, fairly strong positive correlations were also evident for 2-phenyl ethanol (R2 = 0.79) and 2-phenyl ethyl acetate (R2 = 0.67). acetate . Not surADH6 encodes an NADPH-dependent cinnamyl alcohol dehydrogenase family member with broad substrate specificity [ADH4, ADH5 and ADH7 on the other hand showed only modest correlations with the above-mentioned, or any other aroma compounds for that matter.cificity . ExpressAAD3, AAD10 and AAD14 showed the greatest variation in expression, both on an intra- and interstrain level. Expression of AAD10 and AAD14, for example, was increased more than twofold in most of the strains at day 5 relative to day 2 of fermentation. No distinct physiological role has been established for the products of these genes [With respect to the aryl alcohol dehydrogenase family of genes, the transcripts for se genes , but it AAD10 showed weak yet significant positive correlations with a number of the aroma compounds. Expression of AAD14 between different strains and time points was also highly variable. Highest expression levels were noted for the DV10 strain, and significant positive correlations with ethyl acetate (0.67) and ethyl caprate (0.74) were observed for this gene.This hypothesis is supported by the data generated through the overexpression of these genes. Indeed, overexpression yielded changes to the aroma profile that were very similar to those predicted from the alignment of transcriptome and metabolome data sets. The expression of ATF1 nor ATF2, the two most prominent alcohol acetyl transferases, showed statistically strong correlations between expression levels and metabolite production. EEB1, on the other hand, which encodes an acyl-coenzymeA:ethanol O-acyltransferase and is responsible for the major part of medium-chain fatty acid ethyl ester biosynthesis during fermentation [Acetyl transferases are another family of enzymes of relevance to aroma compound metabolism . Howeverentation , showed EHT1 encodes an acyl-coenzymeA:ethanol O-acyltransferase that plays a role in medium-chain fatty acid ethyl ester biosynthesis, but also contains a known esterase activity [EHT1 expression increased somewhat as fermentation progressed and inter-strain expression at both day 2 and 5 of fermentation varied significantly. Interestingly, EHT1 expression showed a fairly strong inverse correlated with 2-phenylethyl acetate (R2 = 0.74) and octanoic acid (R2 = 0.75), as well as a weaker yet significant inverse correlation with decanoic acid (R2 = 0.59). This could indicate that the esterase activity of Eht1p could predominate under certain conditions.activity . EHT1 exYMR210W encodes a putative acyltransferase with similarity to both Eeb1p and Eht1p, and may have a minor role in medium-chain fatty acid ethyl ester biosynthesis [ynthesis . ExpressAYT1 gene, encoding a transferase of unknown substrate specificity, also showed considerable variation at different fermentative stages [In addition to these relatively well studied acetyltransferases, the mRNA levels of the e stages .BAT1, AAD10, AAD14 and ACS1, the match between predicted and real changes is highly significant. This is the first study linking metabolic networks to transcriptome analysis through the comparative analysis of different wine yeast strains.The impact of these individual genes on aroma compound metabolism has to be assessed individually. However, from the data presented here, it is clear that an analysis based on the comparison of transcriptome and metabolome data derived from different commercial yeast strains can help to identify genes that most significantly impact a metabolic network in specific environmental and industrial conditions. Our over-expression analysis of five genes that were randomly selected from the list of ORFs identified for their statistically significant impact on aroma production also clearly suggests that the method has significant predictive power regarding the reorientation of metabolic flux through the network in response to changes in gene expression levels. Indeed, for four out of five selected genes, Saccharomyces cerevisiae strains used in industrial wine fermentations. Yeast cells were cultivated at 30°C in YPD synthetic media 1% yeast extract , 2% peptone , 2% glucose . Solid medium was supplemented with 2% agar .The yeast strains used in this study are listed in table Fermentation experiments were carried out with synthetic must MS300 which approximates to a natural grape must as previously described . The med2 outlet. The fermentation temperature was approximately 22°C and no continuous stirring was performed during the course of the fermentation. Fermentation bottles were inoculated with YPD cultures in the logarithmic growth phase (around OD600 = 1) to an OD600 of 0.1 . The cells from the YPD pre-cultures were briefly centrifuged and resuspended in MS300 to avoid carryover of YPD to the fermentation media. The fermentations followed a time course of 14 days and the bottles were weighed daily to assess CO2 release and the progress of fermentation. Samples of the fermentation media and cells were taken at days 2, 5 and 14 as representative of the exponential, early stationary and late stationary growth phases respectively. It should be stressed that early stationary phase in these conditions is metabolically active, since growth arrest is due to ethanol toxicity. Sugar levels and fermentative activity are still high at this stage.All fermentations were carried out under microaerophilic conditions in 100 ml glass bottles (containing 80 ml of the medium) sealed with rubber stoppers with a COX, Bio-Tek Instruments) by measuring the optical density (at 600 nm) of 200 μl samples of the suspensions over the 14 day experimental period.Cell proliferation (i.e. growth) was determined spectrophotometrically  on an AMINEX HPX-87H ion exchange column using 5 mM H-1. To each of these samples 1 ml of solvent (diethyl ether) was added and the tubes sonicated for 5 minutes. The top layer in each tube was separated by centrifugation at 3000 rpm for 5 minutes and the extract analyzed. After mixing, 3 μl of each sample was injected into the gas chromatograph (GC). All extractions were done in triplicate.Each 5 ml sample of synthetic must taken during fermentation was spiked with an internal standard of 4-methyl-2-pentanol to a final concentration of 10 mg.l-1, with hydrogen used as the carrier gas for a flame ionisation detector held at 250°C. The oven temperature was increased from 35°C to 230°C at a ramp of 3°C min-1.The analysis of volatile compounds was carried out on a Hewlett Packard 5890 Series II GC coupled to an HP 7673 auto-sampler and injector and an HP 3396A integrator. The column used was a Lab Alliance organic-coated, fused silica capillary with dimensions of 60 m × 0.32 mm internal diameter with a 0.5 μm coating thickness. The injector temperature was set to 200°C, the split ratio to 20:1 and the flow rate to 15 ml.minInternal standards  were used to calibrate the machine for each of the compounds measured.T-tests and anova analyses were conducted using Statistica (version 7). HCL and KMC clustering were carried out using TIGR MeV v2.2 .® microarrays were performed according to Affymetrix instructions, starting with 6 μg of total RNA. Results for each strain and time point were derived from three independent culture replicates. The quality of total RNA, cDNA, cRNA and fragmented cRNA were confirmed using the Agilent Bioanalyzer 2100.Sampling of cells from fermentations and total RNA extraction was performed as described . Probe p® Operating Software (GCOS) version 1.4. All arrays were scaled to a target value of 500 using the average signal from all gene features using GCOS. Genes with expression values below 12 were set to 12 + the expression value as previously described in order to eliminate insignificant variations [Acquisition and quantification of array images and data filtering were performed using Affymetrix GeneChipriations .Variable (gene) selection is important for the successful analysis of gene expression data since most of the genes are unchanged and irrelevant to the prediction and analysis of phenotypic measurements. These non-informative genes should be removed before further analysis. One approach is by significance analysis of microarrays . DetermiIn terms of design, the samples represent the different fermentations (three independent replicates for each of the five strains) at different time points. The variables considered are the expression levels of the pre-selected genes  as well as aroma compound concentrations in the synthetic must. The patterns within the different sets of data were investigated by principal-component analysis (PCA), while the correlations between different sets of data were determined by using partial least-squares (PLS) regression . PCA is a bilinear modeling method which gives a visually interpretable overview of the main information in large, multidimensional datasets. By plotting the principal components it is possible to view statistical relationships between different variables in complex datasets and detect and interpret sample groupings, similarities or differences, as well as the relationships between the different variables .PLS regression is a bilinear modeling method for identifying the variations in a data matrix for explanatory or predictive purposes . By plotThe data were analyzed by using test-set validation with centered data and the variables were weighted according to their standard deviations. One strain was used as the test segment at each of the time points. Day 2 and 5 data were considered together as representative of the full scope of fermentation variability as the period from the start of fermentation until day 5 represents the period of maximum aroma compound production.The Y variables were the respective aroma compounds measured and the X variables were the gene expression levels of the gene set that was pre-selected for analysis . Genes wAll plasmids used in this study are listed in table Eshericia coli DH5α (GIBCO-BRL/Life Technologies) was used as the host for the construction and propagation of the plasmids listed in table The primers listed in table DR carried out the experimental work and contributed to the experimental design and data analysis. She also drafted the manuscript. TN contributed to the statistical analysis of the data. FFB conceived of the study and participated in its design and coordination. All authors read and approved of the final manuscript.Identified ORFs and functional description. The table lists all ORFs whose expression pattern correlated significantly with aroma compound production. A short functional annotation is also provided.Click here for fileInter- and intra-strain differences in expression levels of all identified ORFs. The three tables show the differences in expression levels of identified ORFs between different strains at day 2 (Table  2 Table  and at d 2 Table .Click here for fileSummaries of PLS1 models. The table summarises PLS1 models used for interpretation and selection of genes for overexpression.Click here for fileComparison between metabolites produced in the strains overexpressing individual ORFs. The table shows a comparison between the volatile aroma metabolites that are produced in the strains overexpressing individual ORFs at day 2 and day 5 of fermentation.Click here for file


Observations were made over a 4-year period on squamous cell carcinomas on the ear and other areas poorly covered by hair or wool, sheep pastured in the hot, dry environment of north-western Queensland. Overall incidence in the flock was higher than in flocks kept at greater latitudes. Increased incidence with advancing age was demonstrated, and ewes appeared to be more susceptible than wethers. Metastases were observed in 4 of 33 affected ewes (12%) submitted to detailed necropsy examination. Measurements of tumour growth in 4 ewes revealed an increase in size of about 3-5 mm per month. Ovine aural squamous cell carcinoma was considered to be a good model for studies on skin cancer in man.


Plasmodium falciparum Merozoite Surface Protein 1 (MSP1) is synthesized during schizogony as a 195-kDa precursor that is processed into four fragments on the parasite surface. Following a second proteolytic cleavage during merozoite invasion of the red blood cell, most of the protein is shed from the surface except for the C-terminal 19-kDa fragment (MSP119), which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP119 during the parasite's subsequent intracellular development using immunochemical analysis of metabolically labeled MSP119, fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP119 remains intact and persists to the end of the intracellular cycle. This protein is the first marker for the biogenesis of the food vacuole; it is rapidly endocytosed into small vacuoles in the ring stage, which coalesce to form the single food vacuole containing hemozoin, and persists into the discarded residual body. The food vacuole is marked by the presence of both MSP119 and the chloroquine resistance transporter (CRT) as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP119 does not simply follow a classical lysosome-like clearance pathway, instead, it may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase. However, it is known that a fragment of MSP1 -linked precursor, which is directed to the parasite's surface . The latter comprises two epidermal growth factor (EGF)-like domains and is carried into the interior of the infected-RBC on the merozoite surface 19 has been detected on the surface of the early ring-stage parasite by both IFA 19 that are present in the culture medium at the time of invasion, can be internalized when bound to MSP119 on the parasite surface 19 after invasion has not been studied in any detail.MSP1 is synthesized by intracellular 19 by IFA in the food vacuole of late rings/trophozoites, suggesting that this organelle is able to receive molecules endocytosed from the parasite surface. Although there is no clear morphological evidence for the existence of a classical eukaryotic endosome-lysosome system in Plasmodium, the food vacuole may act as a lysosome-like compartment as it contains proteases , are internalized to form one or more food vacuoles Early after invasion, numerous small food vacuoles form within the ring stage parasite 19 using a combination of metabolic labeling, IFA and IEM techniques applied to highly synchronized parasite cultures, together with antibodies directed against well-defined regions of MSP1. We find that after endocytosis into small food vacuoles in the ring stage, MSP119 persists through the cycle as a coherent component of the food vacuole wall without further processing or addition of newly synthesised MSP1. This behavior indicates that the molecule does not simply follow a classical lysosome-like clearance pathway, but may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase.In the present study we have explored in detail the post-invasion fate of MSP1P. falciparum lines 3D7 in vitro as described lbuMAX® I in RPMI 1640 medium. Parasites were synchronized using combinations of a magnet 19; rabbit polyclonal antibodies raised against either affinity purified MSP1 (Wellcome type) 1919 (i.e. not reacting with MSP119) 19 is position 1526. Rabbit antibodies raised against peptides of CRT P. falciparum antigens, for IEM.A number of antibodies against different regions of MSP1 were used: monoclonal antibody (mAb) 1E1 35S Met/Cys; GE Healthcare, Little Chalfont, UK) essentially as described previously Mature stage parasites (3D7) were metabolically labeled with ProMix  in PBS to identify the nuclei. The films were subsequently mounted in glycerol/PBS solution containing anti-quenching agent  and viewed under oil immersion. The parasites were viewed using a Zeiss Axioplan2 microscope equipped with a Plan-APOCHROMAT 100×/oil immersion lens and appropriate filters. Images were analysed and processed using Adobe Photoshop  and Microsoft PowerPoint software.Thin blood films were made on microscope slides at intervals from time 0 (when newly invaded erythrocytes had been synchronized and returned to culture), air-dried, frozen without fixation and stored desiccated at −20°C until required. The time-course slides were assayed as follows: samples were thawed and dried quickly, fixed in fresh 1% formaldehyde in PBS for 30 minutes, rinsed in phosphate buffered saline (PBS), permeabilized with 0.1% Triton X-100/PBS for 10 minutes, rinsed in PBS and blocked overnight at 4°C in 3% BSA/PBS. Cells were probed consecutively with primary antibodies in 3% BSA/PBS, and with Alexa Fluor 488 (green) or Alexa Fluor 594 (red)-conjugated affinity purified goat anti-mouse IgG or anti-rabbit IgG secondary antibody (Molecular Probes) as appropriate at 37°C for 30 minutes each, followed by three washes in PBS. Dual labeling experiments were performed on thin films and probed successively with different primary antibodies derived from different species (mouse and rabbit). This was followed by staining with appropriate secondary antibody (anti-IgG) conjugated to Alexa Fluor 488 or Alexa Fluor 594. Control incubations were carried out in the absence of primary or secondary antibodies in addition to a range of antibody controls described above. Under no condition was a false fluorescence signal from the hemozoin observed. Finally, the films were stained with 0.5 µg ml19 with antibody (i.e. prior to invasion) synchronous mature parasites were cultured and allowed to invade in the presence of mAb 1E1: mature schizonts were purified using a combination of gel flotation and Percoll purification. Fresh erythrocytes were added to give a parasitemia of 5% (at 17% hematocrit) and samples were split into two fractions; one-half was placed in normal medium; the other in medium containing 500 µg ml−1 mAb 1E1. Following further culture with gentle shaking for 1 hour, remaining schizonts were removed using a Percoll cushion followed by sorbitol treatment of the pellet. A sample was taken (t = 0) and smeared for Giemsa staining and for IFA, then the remaining parasites were cultured and smears were prepared every 3 hours over a 48-hour time period. The overall window for invasion was 2 hours.To pre-label MSP1P. falciparum clones 3D7 and C10, and line IT04) were prepared for IEM as described in Schizonts  onwards indicates that the protein is associated with food vacuoles, and that the small food vacuoles had increased in size but decreased in number, suggesting their fusion. The subcellular location of mAb 1E1 bound to MSP119 was identical to that of an MSP119-specific polyclonal antibody that was applied to the parasites post-fixation   , panel B vacuole , panel A19 was detectable in fixed parasites in two independent experiments with rabbit polyclonal anti-MSP119 and mAb 1E1 antibodies at least until 36 and 30 hours post-invasion, respectively, after which time the presence of newly synthesized MSP1 on the parasite surface masked the labeling of internalized MSP119  was used, no labeling was found on any ring structure (data not shown).IEM with the polyclonal anti-MSP1membrane  and occamembrane . Howevermembrane , and, inmembrane , none ofMorphological EM of trophozoites and schizonts, in which a single vacuole is present in each parasite, showed that food vacuole size varied with post-invasion age. In trophozoites and early schizonts the vacuole was rounded or elliptical in section and larger than in rings  19. The persistence of this fragment may therefore also reflect an equal persistence of the associated lipid raft components in the food vacuole wall.The IEM results show that after endocytosis, MSP119 are clustered into one or more patches of high concentration on the vacuole wall, rather than being spread evenly around its perimeter. This suggests that MSP119 molecules and the membrane lipids with which they associate form a coherent unit integrated into the vacuole wall membrane, preserving a group identity separate from other membrane components of the wall. This behavior implies some form of lateral interaction between MSP119 molecules either directly with each other or indirectly through clustering of their linked membrane lipids. Interestingly, inspection of published IEM reports of CRT localization to the food vacuole wall 19 and CRT does not show a total colocalization, suggesting that they cover overlapping or different areas of the food vacuole wall.The IEM results additionally show that in the single vacuole of trophozoites and early schizonts, the molecules of MSP119-attached membrane is only a part. Such an arrangement may not be too surprising, considering what must be the highly dynamic nature of this boundary, which constantly receives new membrane from the cytostomes and probably by fusion of Golgi-derived vesicles carrying proteolytic enzymes These findings indicate that the membrane lining the food vacuole may be a mosaic of different molecular compositions, of which MSP119 increases until at the end it covers most or all of the inner surface of the food vacuole   see . As show19 the results reported here throw new light on the process of endocytosis into the food vacuole at different stages of the cycle. It is plain that food vacuoles formed in rings are in a number of respects different from those of later stages. The avid uptake of MSP119 from the ring surface contrasts strongly with the absence of uptake of full length MSP1 into the schizont food vacuole, as shown by the failure of N-terminal-directed antibodies to detect vacuolar MSP1. It is likely that both GPI-anchored forms of this molecule are associated with cholesterol-rich lipid microdomains 19 is carried into the vacuole by association with such specific lipids. In later stages when it would be wasteful for the parasite to allow vacuolar degradation of newly synthesized GPI-anchored surface proteins destined for the next brood of merozoites, some mechanism must exist at the mature cytostome for ensuring MSP1 is excluded from endocytosis. The molecular basis for this selective process is not at present apparent.Besides the potential functional importance of MSP119 in food vacuoles is merely an aspect of the food vacuole's ability to store undegradable biomolecules, already evident in hemozoin sequestration. However, as MSP119 is located at the inner surface of these organelles, it is likely that any function relates to that boundary. There is experimental evidence that some anti-MSP119 antibodies (though not the anti-MSP119 mAb 1E1 used here) carried into the red cell on the surface of the merozoite can interfere with intracellular development 19 along the food vacuole wall rather than in isolated clumps or vesicles within its lumen (as typical of degraded structures in secondary lysosomes of many eukaryotes: (see 19 is not merely discarded as a functionless end-product.It is possible that the presence of MSP119 suggests that it may serve as a protective protease-resistant protein coat on the inside of the food vacuole, guarding other membrane proteins against the powerful proteases within this organelle. As it covers only part of the wall, it is unlikely to be the only protective agent, and other molecules with similar resistance may also participate, including MSP8, which has a similar EGF domain structure. However, gene disruption experiments have shown that MSP8 is not essential for parasite progress The protease-resistant structure of MSP119 might be involved in nucleating hemozoin crystallization, a role posited for a number of parasite molecules including HRPII, which is either transported directly to the food vacuole or first trafficked to the red cell cytoplasm and then taken back into the food vacuole during feeding 19 has the capacity to nucleate or accelerate hemozoin crystallization.Another possibility is that MSP119 is important in mechanically stabilizing the vacuole membrane especially in late schizonts when the vacuole is packed with sharp-ended hemozoin crystals. Further experiments are needed to test these possibilities.A third possibility is that by virtue of its association with membrane lipids including those forming cholesterol-rich lipid microdomains, MSP1Whatever the true function, the findings reported here point to an important but as yet undefined role for merozoite proteins in the intracellular phase of the parasite's erythrocytic cycle, and they also emphasize our lack of understanding of the degradative pathways within the parasite. Both of these aspects of the parasite's biology deserve attention as potential targets for the development of antimalarial strategies.