Current guidelines recommend thrombus aspiration in patients with ST-elevation myocardial infarction (STEMI); however, there are insufficient data to unequivocally support thrombectomy in patients with non-STEMI (NSTEMI).The TATORT-NSTEMI  trial is a prospective, controlled, multicenter, randomized, open-label trial enrolling 460 patients. The hypothesis is that, against a background of early revascularization, adjunctive thrombectomy leads to less microvascular obstruction (MO) compared with conventional percutaneous coronary intervention (PCI) alone, as assessed by cardiac magnetic resonance imaging (CMR) in patients with NSTEMI. Patients will be randomized in a 1:1 fashion to one of the two treatment arms. The primary endpoint is the extent of late MO assessed by CMR. Secondary endpoints include early MO, infarct size, and myocardial salvage assessed by CMR as well as enzymatic infarct size and angiographic parameters, such as thrombolysis in myocardial infarction flow post-PCI and myocardial blush grade. Furthermore, clinical endpoints including death, myocardial re-infarction, target vessel revascularization, and new congestive heart failure will be recorded at 6 and 12 months. Safety will be assessed by the incidence of bleeding and stroke.The TATORT-NSTEMI trial has been designed to test the hypothesis that thrombectomy will improve myocardial perfusion in patients with NSTEMI and relevant thrombus burden in the culprit vessel reperfused by early PCI.http://www.clinicaltrials.gov: NCT01612312.The trial is registered under  Use of manual thrombectomy devices during percutaneous coronary intervention (PCI) in patients with acute ST-elevation myocardial infarction (STEMI) has been shown to decrease the incidence of cardiac death and repeat myocardial infarction -3. ConseThis emphasizes the need for an adequately powered randomized controlled trial (RCT) to address the current role of thrombus aspiration in patients with NSTEMI and a relevant thrombus burden in the culprit vessel. The TATORT-NSTEMI  trial is designed to test the hypothesis that against a background of early revascularization, adjunctive thrombectomy leads to less microvascular obstruction (MO) compared with conventional PCI alone, as assessed by cardiac magnetic resonance imaging (CMR).The TATORT-NSTEMI trial is a prospective, multicenter, randomized, controlled, open-label study to compare PCI plus routine thrombus aspiration with PCI without thrombus aspiration in patients with NSTEMI and relevant thrombus burden who are undergoing early invasive reperfusion therapy. The study will determine if adjunctive thrombectomy is superior than PCI alone with respect to the extent of no-reflow/MO assessed by CMR in patients with NSTEMI.The primary study endpoint of the TATORT-NSTEMI trial is the extent of late MO assessed by CMR on days 1 to 4 after randomization. Using CMR, zones of MO within the infarcted myocardium can be visualized and quantified, representing the CMR equivalent of the no-reflow phenomenon ,14. MO ahttp://www.euroqol.org) questionnaire during the 6-month and 12-month follow-ups.Secondary endpoints include other CMR parameters associated with patient prognosis, such as early MO, infarct size, myocardial salvage, and left ventricular ejection fraction (LVEF), as well as angiographic markers of reperfusion success such as the thrombolysis in myocardial infarction (TIMI) flow post-PCI and myocardial blush grade ,21. In aSafety assessment will include stroke and bleeding until hospital discharge. Stroke will be defined as any new neurological symptoms in association with signs of ischemia or hemorrhage in cranial computed tomography or magnetic resonance imaging. Bleeding will be defined in accordance with the GUSTO  criteria as 1) severe or life-threatening, 2) moderate, or 3) mild bleeding . HoweverThe study population will consist of 460 patients with NSTEMI enrolled at eight centers in Germany. Informed consent will be required prior to randomization.Patients will be eligible for the study if they have: 1) ischemic symptoms such as angina pectoris for more than 20 minutes; 2) occurrence of previous symptoms less than 72 hours before randomization; 3) cardiac troponin levels above the 99th percentile; and 4) culprit lesion containing thrombus (TIMI thrombus grade 2 to 5 within the lesion) and intended early PCI.Exclusion criteria will be presence of cardiogenic shock, STEMI, no identifiable culprit lesion, coronary morphology ineligible for thrombectomy , indication for acute bypass surgery, age less than 18 or more than 90 years, pregnancy, current participation in another clinical study, co-morbidity with limited life expectancy of less than 6 months or contraindications to CMR at study entry , and contraindications for treatment with heparin, aspirin, or thienopyridines.®; Terumo Europe, Leuven, Belgium). In the standard PCI group, patients will be treated by conventional PCI in accordance with local practice without thrombectomy. Cross-over is not planned; however, if this occurs, the reasons will be recorded. Manual thrombectomy will be the only difference between the groups, and all other therapeutic procedures will be similar in both treatment arms.Patient randomization will be performed centrally, with a randomization ratio of 1:1  and secondary endpoints such as early MO, infarct size, and myocardial salvage will be assessed by CMR performed on a 1.5 or 3.0 Tesla scanner. Imaging will be performed on days 1 to 4 after randomization, using a standard scanning protocol Figure . In brieBoth before and after PCI, the culprit vessel should be visualized in several projections in order to obtain particularly precise display of the distal segments. Evaluation of thrombus burden will be performed before and after PCI, in accordance with the TIMI thrombus grade scale.The flow within the infarct-related artery will be assessed using the TIMI criteria and the corresponding myocardial blush grades ,29. For g for 10 minutes, and aliquots are stored at -80°C until used. Multiple biomarkers such as markers of CHF, inflammation, or myocardial-derived microRNAs (miRNAs) will be analyzed. In addition, assessment of platelet function will be performed.In a single-center sub-study  blood will be collected from the femoral sheath  and from the infarct-related coronary artery for biomarker sub-studies for approximately 200 patients. In this technique, the plasma is separated by centrifugation at 3500 In a two-center study  that will include approximately 100 patients, the composition and key determinants of aspirated thrombi will be analyzed. This will include histological and immunohistological analysis, and analysis of specific biomarkers such as thrombus miRNAs. The probes will be stored at -80°C and transferred to the core facility at the University of Tübingen.The aim of the study is to demonstrate superiority of manual thrombus aspiration versus standard PCI without thrombus aspiration, based on the primary endpoint of late MO in the study patients.At the time of planning the study, the only available data on MO in NSTEMI were from a study on a small number (n = 25) of patients . Thus, wIn the intervening time, two other studies on MO and NSTEMI were published, which reported a lower prevalence and extent of MO than expected ,32. ThusPre-specified subgroup analyses by gender, presence of diabetes, TIMI thrombus burden (2 to 4 versus 5), TIMI flow pre-PCI (0 to 1 versus 2 to 3), and time from most recent symptoms to occurrence of PCI (<6 versus ≥6 hours), and additional administration of glycoprotein IIb/IIIa inhibitors will be performed.t-test. In addition, as a secondary analysis, the differences between the treatment groups in the main outcome will be assessed using an unpaired t-test adjusted by baseline values (ANCOVA). Categorical variables will be analyzed by Fisher’s exact test. The 95% confidence interval (CI) for differences in quantitative data will be displayed. For clinical events, relative risks and 95% CI will be reported. To identify factors associated with the observed results, univariate and multivariate regression analysis will be performed. To assess clinical outcome, Kaplan-Meier curves with log-rank comparison and Cox regression analyses will be performed. A two-tailed P-value of <0.05 will be considered significant.All patients will be analyzed on an intention-to-treat basis. The differences in primary and secondary endpoints between the treatment groups will be assessed using an unpaired The TATORT-NSTEMI Steering Committee is chaired by Prof. Dr. Holger Thiele , co-chaired by Prof. Dr. Uwe Zeymer, , and Prof. Dr. Bruno Scheller . The Steering Committee is responsible for the scientific content of the protocol and oversees the trial operations, and will perform the preparation of the primary manuscript and other publications arising from the TATORT-NSTEMI trial. Each participating center has a coordinator with extensive clinical-trial experience. All trial-related processes will follow the standard operating procedures of the IHF, which is an independent clinical-research organization providing biometry, study coordination, monitoring, and data management. Central monitoring will include a timely query-management process based on consistency and plausibility checks automatically generated from the database, combined with a dunning process for missing documentation and a reminder system in advance for upcoming visits. On-site monitoring will include initiation, regular visits, and close-out. During recruitment and follow-up, all centers will be visited regularly to check adherence to good clinical practice and trial protocol. The focus of the visits will be on the verification of informed-consent documents, eligibility criteria, key primary and secondary endpoints, and safety aspects.The trial will be monitored by an independent DSMB. The CEC will adjudicate all clinical endpoints. The trial follows a standard study organization with a Steering Committee, DSMB, and CEC.The study has been approved by the central ethical committee at the University of Leipzig and at all local ethics committees at the participating sites.Despite early invasive strategies and optimal medical treatment, morbidity and mortality rates for NSTEMI and STEMI continue to be high. Thus, further improvement of the initial therapy is necessary to improve patient outcome. The pivotal TAPAS  trial of patients with STEMI, which compared a strategy with thrombectomy with a strategy without thrombectomy, showed a reduction in cardiac mortality at 1 year ,34. HoweNotably, in the majority of both STEMI and NSTEMI cases, the key pathophysiological substrate is rupture of a vulnerable plaque with subsequent coronary thrombosis. Whereas in STEMI the thrombus is mostly fibrin-rich leading to total vessel occlusion, the thrombus in many patients with NSTEMI is predominantly platelet-rich and unstable. leading to partial or intermittent occlusion of the coronary vessel . The majHowever, RCTs are necessary to investigate whether thrombus aspiration in NSTEMI results in improved angiographic, functional, and clinical outcomes compared with conventional PCI. Currently, one additional randomized single-center study comparing thrombectomy with PCI alone is ongoing (TAPAS-II study) . The priPrevious data for patients with STEMI indicate that in approximately 10% of all patients the thrombus aspiration catheter fails to cross the lesion . BecauseIn clinical trials using CMR parameters as surrogate endpoints, the time period for CMR image acquisition after the index event or randomization is of importance for the final quality and reproducibility of the results. However, previous studies analyzing the influence of timing of CMR image acquisition on CMR parameters led to inconsistent results. In contrast to previously published animal data with stable infarct size and MO measurements between day 2 and 7 after the index event ,there haRecruitment began in March 2011 and of December 2012, almost 360 patients had been enrolled. Given these inclusion rates, completion of enrollment is expected by July 2013. It is expected that complete data will be available in September 2013.AHA/ACC: American Heart Association/American College of Cardiology; CEC: Clinical Endpoints Committee; CHF: congestive heart failure; CI: Confidence interval; CMR: Cardiac magnetic resonance; DSMB: Data Safety Monitoring Board; ESC: European Society of Cardiology; IHF: Institut für Herzinfarktforschung; LV: Left ventricle; LVEF: Left ventricular ejection fraction; MO: Microvascular obstruction; NSTEMI: Non-ST-elevation myocardial infarction; PCI: Percutaneous coronary intervention; RCT: Randomized controlled trial (RCT); STEMI: ST-elevation myocardial infarction; TATORT-NSTEMI: Thrombus Aspiration in ThrOmbus containing culpRiT lesions in Non-ST-Elevation Myocardial Infarction; TIMI: Thrombolysis in myocardial infarction.The authors do not have competing interests concerning the TATORT-NSTEMI trial.All authors have made substantial contributions to conception and design of the current trial and have been involved in drafting the manuscript or revising it critically for important intellectual content. All authors have given final approval of the manuscript.


Patients diagnosed with Systemic Lupus Erythematosus have similar levels of neuropsychological dysfunction  as those with Neuropsychiatric Systemic Lupus Erythematosus (NPSLE). We hypothesized a gradient between cognition and white matter integrity, such that strongest brain-behavior relationships would emerge in NPSLE, intermediate in non-NPSLE, and minimal in controls. We studied thirty-one patients , ranging in age from 18 to 59 years old , and eighteen age and gender matched healthy controls. DTI examinations were performed on a 1.5T scanner. A broad neuropsychological battery was administered, tapping attention, memory, processing speed, and executive functioning. The Total z-score consisted of the combined sum of all neuropsychological measures. In control subjects, we found no significant FA-Total z-score correlations. NPSLE, non-NPSLE, and control subjects differed significantly in terms of Total z-score . In non-NPSLE subjects, FA within the right external capsule was significantly correlated with Total z-score. In NPSLE subjects, the largest FA-Total z-score clusters were observed within the left anterior thalamic radiation and right superior longitudinal fasciculus. In subsequent analyses the largest number of significant voxels linked FA with the Processing Speed z-score in NPSLE. The current results reflect objective white matter correlates of neuropsychological dysfunction in both NPSLE and (to a lesser degree) in non-NPSLE. non-NPSLE and NPSLE subjects did not differ significantly in terms of depression, as measured by the GDI; thus, previous hypotheses suggesting moderating effects of depression upon neuropsychological performance do not impact the current FA results. Cognitive dysfunction is a significant  comorbid characteristic of systemic lupus erythematosus post hoc.” Based on previous research Few prospective studies have linked cognitive decline to white matter changes across NPSLE and SLE patient cohorts. In SLE subjects (here forward “non NPSLE”), subtle correlations between neuropsychological measures and white matter integrity, measured by diffusion tensor imaging (DTI), have been observed Written informed consent was obtained from all subjects prior to participation in the experimental protocol. The study was approved by the institutional review board of the University of New Mexico, and consistent with the Declaration of Helsinki.Thirty-one female patients were recruited from the Rheumatology Clinics of the University of New Mexico, and ranged in age from 18 to 59 years old. All subjects were diagnosed with lupus based on the 1997 American College of Rheumatology Revised Criteria. The SLE Disease Activity Index (SLEDAI) was used to assess current disease activity post hoc. The current sample differs from our previous report in that we focus exclusively on female subjects . We encourage readers to review that open access paper for additional information regarding clinical characteristics of patients, lesion load, and statistical comparisons referred to within the current study .Sixteen patients met the American College of Rheumatology case definitions for Neuropsychiatric (NP) events in SLE, defined as acute stroke or transient ischemic attack (TIA), acute confusional state, moderate cognitive dysfunction, seizures, or psychosis MR examinations were conducted on a 1.5T scanner using an 8-channel phased array head coil. DTI was acquired as follows: . DTI was performed twice and averaged to increase signal to noise ratio.The neuropsychological battery is in general compliance with the recommendations of the American College of Rheumatology Brain atrophy and lesions were quantified by one of the authors (WLS), blind to group status of individual scan . Methodology by which lesions loads are quantified in NPSLE cohorts have been described previously by our group post hoc analysis, the fractional anisotropy (FA) image of each subject was normalized to a 1×1×1 mm3 FA template in the Montreal Neurological Institute (MNI) space using the non-linear registration algorithm FNIRT/FSL (www.fmrib.ox.ac.uk/fsl/). We first correlated the Total z score with FA across each subject group  using FSL's General Linear Model (GLM) tool (www.fmrib.ox.ac.uk/fsl/fsl/list). Age was entered into the model as a nuisance variable. All results are corrected at p-values<.05 after controlling for family wise error rate. If significant, then subdomain z-scores  were then correlated with FA for each subject group. The correlation inferences were tested using permutation methods with FSL's Randomise (www.fmrib.ox.ac.uk/fsl/randomise). We ran 5000 two-tailed Monte Carlo permutation tests,  for each of the correlations between neuropsychological composite measures and FA. Tract-Based Spatial Statistics (TBSS) allows measurement of water movement along white matter tracts on a voxel-by-voxel basis across the entire brain NPSLE, non-NPSLE, and control subjects did not differ significantly in terms of age , and premorbid intellectual functioning . Control subjects differed from both non-NPSLE  and NPSLE  on the GDI, a measure of depressive symptomatology ; however, NPSLE and non-NPSLE scores on the GDI were not significantly different .post hoc analysis indicating significant NPSLE-non-NPSLE differences on Total z-score . The Total z-score represents roughly 1 standard deviation decline (from premorbid estimates) in overall cognitive functioning for the non-NPSLE group, and a 2 standard deviation decline in functioning for the NPSLE group. Only one subdomain z-score (Processing Speed) showed significant NPSLE-non-NPSLE differences .One way ANOVA revealed that NPSLE, non-NPSLE, and control subjects differed significantly in terms of Total z-score , with In control subjects, we found no significant regions in which FA was correlated with Total z-score. In non-NPSLE subjects, FA within the right external capsule was significantly correlated with Total z-score (p = .029). No subcThis is the first study to evaluate neuropsychological and white matter functioning in a well-matched cohort of female NPSLE, non-NPSLE, and control subjects. Importantly, non-NPSLE and NPSLE subjects did not differ significantly in terms of depression, as measured by the GDI; thus, previous hypotheses suggesting moderating effects of depression upon neuropsychological performance These finding represent preliminary evidence that the neurocognitive effects of NPSLE differ significantly from non-NPSLE even when tightly matched by age, sex, medication use, mood symptoms, and non-neurological disease activity and accumulated disease burden. Moreover, these neurocognitive effects are well correlated with FA changes within white matter regions outside of overt lesions Neuroimaging of complex cognition has evolved beyond strict localization of function There are limitations to this approach, including the fact that five of the NPSLE patients had overt lesions due to stroke, which in turn could have led to Wallerian degeneration at least partially responsible for diffuse white matter changes


Development of tuberculosis (TB) is determined by various risk factors and the interactions of temporal and spatial distributions. The aim of this study was to identify the most salient risk factors for TB disease as well as multidrug resistant TB (MDR-TB) at the oblast  level in Kazakhstan.Correlational and descriptive analyses were conducted at the oblast and national level using data provided by the country’s National Institute of Geography (NIG) and the National Tuberculosis Program (NTP). Reported incident case notification rates (CNRs) and prevalence vary by oblast, thus the study investigated which determinants contributed to this regional variation and compared burdens among oblasts.The results showed that while tuberculosis CNRs decreased over the study period, MDR-TB conversely increased. Two oblasts -Atyrauskaya and Mangystauskaya - presented especially significant anomalies with large decreases in TB incident CNRs coupled with comparatively large increases in MDR-TB incident CNRs.Understanding the distribution of TB and MDR-TB cases and associated risk factors, especially the “unknown risk factor” categorization points to the need for future research. Despite World Health Organization’s (WHO) recent report describing a decline in tuberculosis (TB) morbidity and mortality, the disease remains an acute threat to global public health . The incIn several FSU republics including Kazakhstan, multidrug resistant forms of the disease increasingly threaten TB control efforts ,6. The NTB incidence peaked in Kazakhstan in 2003 and 2004, since then the NTP has attained some success in restraining further increases; however, MDR-TB presents a growing challenge to controlling TB. The WHO reports that 14% range 11-18%) of all newly diagnosed TB cases in Kazakhstan are multidrug resistant. Among retreatment cases, 45% test positive for MDR-TB ,10. The -18% of aInterest in the social, economic, and environmental determinants of TB has grown as CNRs have risen during the past two decades. The contextual milieu in which individuals live and work is critical to understanding and stemming the disease . A literThis study presents the first comprehensive description of the spatial and temporal burden of TB disease constructed from data obtained from the Kazakhstani national TB surveillance system. Study findings identify salient risk factors for TB disease as well as MDR-TB at the oblast  level in Kazakhstan. Reported incident CNRs and prevalence vary by oblast, thus the study investigated which determinants contribute to this regional variation. By analyzing aggregate and individual case records provided by the country’s National Institute of Geography and the National Tuberculosis Program, we compared burdens among oblasts and identified significant risk determinants.Surveillance data from the National Tuberculosis Program (NTP) and the National Institute of Geography (NIG) were obtained for the years 2006 through 2010. NTP surveillance data included all new tuberculosis cases diagnosed and reported to the NTP from January 1, 2006 through December 31, 2010 and tuberculosis and MDR-TB national incidence and prevalence (2006 – 2010) estimates. The NIG provided surveillance data on oblast population estimates (2006 – 2010). De-identified data was obtained in Microsoft Access 2010, Excel 2010, and Word 1997–2003. Standard data security and management procedures were followed to ensure the highest level of data safety. The study was conducted under a protocol approved by the Institutional Review Boards of Columbia University and Nazarbayev University.As described above, the NTP provides free diagnosis and treatment of TB and MDR-TB. Kazakhstan’s laboratory network holds a Class B rating by the WHO . RoutineIndividual risk factors recorded by the NTP and included in this analysis are alcohol use, child or youth from a vulnerable group, known diabetes diagnosis, history of illicit drug use, incarceration history within the past two years, migrant status, non-regular uptake of anti-tuberculosis medication (less relevant for new cases), prison system staff member, recent mother (having given birth within one year of diagnosis), registered contact of a TB or MDR-TB case, TB health care staff member, and a category listed as “unknown individual risk factors.” Additionally, categories for employment status included currently incarcerated, detainee, farmer, officer, pensioner, child or youth, prison medical staff, self-employed, student, TB clinic medical staff, worker, unemployed, or other. Individual level data was not available for geocoding beyond the rayon (community) level; therefore, analysis on residential related variables could not be conducted.Descriptive statistical analyses on the oblast and national levels were conducted in Microsoft Excel 2010 and SAS 9.2. National and oblast level incident CNRs and prevalence data presented here were reported directly from the NTP and are assumed to have an underlying Poisson distribution ,22. PoisTable National MDR-TB incident CNR has conversely increased over the same time period, from 5.8 cases per 100,000 in 2007 to 10.5 cases per 100,000 in 2010 (p = 0.12). Four out of fourteen oblasts observed a statistically significant change in MDR-TB incident CNR . Almatynskaya oblast was one of the four oblasts that did not experience a statistically significant decrease in TB CNR over the same time period. National MDR-TB prevalence increased from 54.4 cases per 100,000 in 2007 to 61.6 cases per 100,000 in 2010 (p = 0.25).Nationally aggregated TB and MDR-TB CNRs and prevalence data was available for 2006. Its inclusion in the analysis supports the observed decrease of TB CNR coupled with the increase in MDR-TB CNR. Figure Tuberculosis and MDR-TB have different spatial distributions within Kazakhstan , being incarcerated within the past two years (p = 0.03), having unknown risk factors (p = 0.03), self-identifying as an officer (p = 0.05), self-identifying as a worker (p = 0.05), self-identifying as unemployed (p = 0.04), and self-identifying as a detainee (p = 0.03).Kazakhstan has a high rate of tuberculosis overall, and one of the highest rates of MDR-TB in the world. This study provides a comprehensive description of the epidemiology of tuberculosis and MDR-TB in this rapidly developing Central Asian nation. The findings demonstrate that although the overall CNR of tuberculosis has declined significantly over the past five years, the CNR of MDR-TB has risen. It is probable that there is both under- and over-diagnosis of TB cases in Kazakhstan because a substantial proportion of cases are identified clinically and unconfirmed by culture. Some of these cases may represent prior, healed TB with exacerbations of bronchiectasis, for example. In addition, it is likely that the lack of routine access to culture and drug susceptibility testing may result in considerable under-diagnosis of tuberculosis cases overall, and perhaps differential under-diagnosis of drug-resistance. It is quite probable that a low CNR for MDR-TB represents under-diagnosis, and this under-diagnosis is contributing to the spread of MDR-TB in several oblasts. Rapid diagnosis of TB and drug susceptibility testing using the WHO-recommended Cepheid GenExpert is expected in Kazakhstan in late 2012. Cepheid GenExpert should improve detection and treatment of MDR-TB cases.The national data on CNRs and MDR-TB are not uniformly distributed; Atyrauskaya and Mangystauskaya oblasts present anomalies with large decreases in TB CNRs coupled with comparatively large increases in MDR-TB CNRs. The lack of uniformity in distribution may result from differential socio-environmental factors and a lack of access to treatment. After the FSU collapse, access to anti-tuberculosis chemotherapy became limited and sporadic, and supervision and surveillance of patients with tuberculosis was inadequate. These factors undoubtedly contributed to the proliferation of MDR-TB cases in Russia and FSU republics that is apparent today. Understanding the reasons for the differential socio-environmental factors and lack of access to treatment will be critical to developing effective strategies for controlling TB in Kazakhstan and limiting the proliferation of MDR strains of tuberculosis.Kazakhstan’s national TB control program classified a high percentage of patients (83% – 87% throughout the study period) as being without identified risk factors for tuberculosis. Such risk factors may be related to undetected TB transmission in the community or in institutional settings. Many instances of nosocomial transmission of tuberculosis, especially of MDR-TB, have been reported around the world, including diverse locations such as New York and Johannesburg, and it is possible that a similar situation has occurred in Kazakhstan. It is also possible that some MDR strains currently present in Kazakhstan are particularly virulent and capable of generating a large number of secondary cases before a new index case is diagnosed. Risks may also be related to selection of the single drug resistant strains induced by previous treatments.An influx of migrant workers has been attracted to the country because of its rapid development. Migrants typically are at increased risk of tuberculosis as they have limited access to health care services, including anti-tuberculosis chemotherapy, and reside in crowded conditions that favor the rapid transmission of tuberculosis. It is also possible that immigrants are entering Kazakhstan knowing that they have MDR-TB because they perceive that services and treatment are available that are not attainable in their home countries. This could lead to a rise in MDR-TB cases that outpaces the rise in drug-susceptible cases. This explanation is supported by the large increases in MDR-TB prevalence reported in the two oblasts (Atyrauskaya and Mangystauskaya) with the highest immigration rates. Most migrants come to these areas from other high TB incidence Central Asian countries. Previous studies have reported higher risks for MDR-TB in foreign-born individuals . Future Another possibility for increasing MDR-TB CNRs is that although DOTS is available for patients reported to the NTP, delayed detection of drug resistance among clinically and microbiologically confirmed cases of tuberculosis limits DOTS effectiveness. Patients may not be receiving ideal therapy in a timely fashion. Use of the Cepheid GenExpert system could ameliorate this situation. GenExpert will be introduced in four regions of Kazakhstan in 2012 with the help of a USAID-funded pilot project. The sustainability of the GenExpert system long-term is yet to be determined. Furthermore, if cases of MDR-TB are not promptly diagnosed, nosocomial transmission of MDR-TB could be playing a substantial role in the rising MDR-TB rates, particularly as the practice in Kazakhstan is to treat all TB patients with prolonged periods of hospitalization.Positive HIV/AIDS status is an important risk factor related to increasing MDR-TB transmission rates. Kazakhstan, along with other eastern European and Central Asian countries, belongs to the region with the fastest growing HIV epidemic in the world. Several studies noted that HIV-infected patients with TB develop drug-resistance, particularly to rifampicin, in response to erratic administration of chemotherapy ,27. ThisOther aspects of the current study deserve particular comment. Compared with previous research, recent incarceration or employment in a prison was not highly represented as a reported risk factor for new TB cases. This could represent a true improvement in control and treatment programs within the prison system or an artifact of the reporting process.The study findings represent an analysis of all the data reported to the NTP. However, through a detailed review and analysis of the data provided for this study, various inconsistencies in reporting, variable definitions, and collection procedures were uncovered when comparing NTP data with other published documents. The data might not accurately reflect the diverse collection and reporting methods currently functioning across the country. Divergent MDR-TB CNRs could be reflective of the quality of the NTP or an artifact of diverse testing practices. Atyrauskaya and Mangystauskaya could report higher incidence of MDR-TB because the oblasts may have more financial resources available and impetus to test suspected TB cases than areas such as South-Kazakhstan. These inconsistencies are being investigated through continued collaboration with our study partners at the NTP and NIG. Accurate surveillance and reporting is critical to controlling tuberculosis in any locale.In conclusion, this study expands current knowledge of the epidemiology of tuberculosis in Kazakhstan. Notwithstanding the limitations detailed above, understanding the distribution of TB and MDR-TB cases and associated risk factors provides context for future studies. The continuing rise of MDR cases in Kazakhstan and other countries in the FSU should be considered a global health emergency. The findings presented here provide a roadmap to further investigations that can lead to the underlying causes of the problem. Understanding the ‘unknown risk factor’ categorization and reporting practices across the country are two ideal starting points for further research that will yield benefits to the entire public health system in Kazakhstan.WHO: World Health Organization; TB: Tuberculosis; FSU: Former Soviet Union; MoH: Kazakhstan’s Ministry of Health; DOTS: Directly Observed Therapy Short Course; NTP: National Tuberculosis Program; MDR-TB: Multidrug Resistant Tuberculosis; NIG: National Institute of Geography; CNR: Case notification rates.The author(s) declare that they have no competing interests.AT had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: AT, NE, SG, NS. Acquisition of data, critical review of intellectual content and final approval of the version to be published: TA, TM, FA, AS, ZZ. Analysis and interpretation of data: SH, SG, NS, NE. Drafting of the manuscript: SH and LB with feedback from SG, NE, NS, AT. Critical revision of the manuscript for important intellectual content: SG, NS, NE, AT. Statistical analysis: SH, SG. Obtained funding: AT, NE, SH. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/262/prepub


We report a case of childhood onset, generalized dystonia due to slowly progressive bilateral striatal necrosis associated with anti-N-methyl-D-aspartate receptor (NMDAR) antibodies. This clinical phenotype has not been previously associated with NMDA receptor autoimmunity.An eighteen year old man presented with a history of childhood-onset, progressive generalized dystonia. Clinical examination revealed a pure generalized dystonia with no cognitive or other neurological findings. Magnetic resonance imaging showed bilateral high T2 signal striatal lesions, which were slowly progressive over a period of nine years. New parts of the lesion showed restricted water diffusion suggesting cytotoxic oedema. Positron emission tomography of the brain showed frontal hypermetabolism and cerebellar hypometabolism. Antibodies against the NR1 subunit of the NMDA receptor were detected in the patient’s serum and cerebrospinal fluid. There was no neoplasia or preceding infection or vaccination.This is the first report of chronic progressive bilateral striatal necrosis associated with anti-NMDAR antibodies. Our findings expand the clinical spectrum of disease associated with anti-NMDAR antibodies and suggest that these should be included in the work-up of dystonia with striatal necrosis. Generalized dystonia due to bilateral striatal necrosis (BSN) has a heterogeneous etiology including hereditary, toxic, infectious and immune mediated disorders. Immune mediated BSN is a rare clinical entity  which haBasal ganglia involvement occurs commonly in the encephalitis associated with anti-N-methyl-D-aspartate receptor (NMDAR) antibodies. This acute or subacute illness can manifest psychiatric and neurological symptoms including epileptic seizures, autonomic instability, hypoventilation and decreased levels of consciousness. Patients often develop hyperkinetic movement disorders including choreoathetosis, myoclonus and dystonia -7 duringWe report a case of childhood onset, pure generalized dystonia due to slowly progressive BSN, associated with anti-NMDAR antibodies. Our patient’s presentation, course and clinical features have not been previously associated with anti-NMDAR autoimmunity.The patient, now an eighteen year old man, was born after normal pregnancy and delivery and had normal early psychomotor development. From the age of three years he experienced episodic migraine-like headache, which resolved spontaneously at the age of sixteen. From the age of nine he noticed slowly progressive difficulties with handwriting due to focal stiffness and abnormal postures of his right hand. There was no infection or vaccination prior to the onset of symptoms. Subsequently, he developed abnormal postures and involuntary movements in his whole right upper limb described as slow, strained abduction and elevation of the shoulder and flexion of the elbow, wrist and fingers. The motor symptoms gradually progressed and, over a period of approximately one year, involved both upper limbs, the neck and face, truncal muscles and the lower limbs. From the age of twelve years he lost unsupported locomotion. He is now eighteen years old and wheel-chair dependent with severe generalized dystonia. He has completed his primary and secondary education and shows no evidence of cognitive or psychiatric dysfunction.Physical examination by the authors at the age of fifteen revealed severe generalized dystonia with predominantly tonic features, axial and appendicular rigidity and abnormal posturing involving the neck, trunk and all four extremities  imaging with fluor-18-deoxy-glucose (FDG) was performed at the age of eighteen on a Siemens Biograph 40 PET-CT. FDG-PET of the brain showed glucose hypermetabolism in the frontal cortex and hypometabolism in the cerebellum. There was no tracer uptake in the putamen and reduced uptake in the left caudate Figure . PET-CT CSF analysis on several occasions showed normal cell count and mildly elevated protein (0.52-0.53 g/L), but no oligoclonal bands. Antibodies against the NR1 subunit of the NMDA receptor were detected using transfected cells produced by Euroimmun Medizinische Labordiagnostika AG (D-2356 Lübeck) in the patient’s serum and CSF. Screening for other autoantibodies to nervous system antigens was negative including anti-Hu, Ri, Yo, Tr, MAG, myelin, Ma/Ta, GAD, amphiphysin, AMPA, GABA-b receptor, LGI1, CASPR2 and glycine receptors.SURF1 mutations were excluded. He was thoroughly evaluated for mitochondrial disease including a muscle biopsy and qualitative/quantitative mitochondrial DNA analysis with normal findings. Analysis of DYT1, RRM2B, SUCLA2 and OPA1 genes was normal.He had normal metabolic screening in blood, CSF and urine. Wilson’s and Leigh disease due to mitochondrial DNA or The patient received five intravenous immunoglobulin (IVIg) infusions (Kiovig 0.4 g/day) followed by two more infusions a month later and a new screening for anti-NMDAR antibodies one month later (2 months after treatment start) was negative in serum and CSF. Three months after the first IVIg infusion his clinical status and MRI findings, including lesional ADC, are stable but there have not been signs of clinical improvement. He receives regular intramuscular injections of Incobotulinum toxin A in the upper limbs resulting in moderate improvement in motor function.This work has been deemed quality control by our local ethical committee .We report a case of generalized dystonia due to BSN associated with anti-NMDAR antibodies. Unlike the cases of anti-NMDAR encephalitis, our patient has a pure, predominantly tonic dystonia with an insidious onset and slowly progressive course. The selectivity of the brain lesions is striking. There is involvement of the dorsal striatum (putamen and caudate nucleus), which is predominantly involved in motor function and sparing of the ventral striatal structures , which are mostly connected to the limbic system and involved in cognitive and emotional functions. This localization correlates well with the clinical features of our patient who had a pure movement disorder with no signs of psychiatric or cognitive dysfunction or impaired consciousness.One patient with isolated hemidystonia and anti-NMDAR antibodies has been reported . Unlike The finding of restricted diffusion suggests cytotoxic edema affecting neurons in the newest parts of the lesions. The etiology of this damage is unclear, but may reflect direct antibody-mediated damage and/or excitotoxicity. Neuronal hyperexcitability is reported with anti-NMDAR antibodies  and the Treatment with IVIg decreased the patient’s antibody titer below detection levels, but has thus far not produced apparent clinical effects. This is not surprising given the extensive damage in the patient’s striatum and the short observation time. On the positive side, his disease has not shown further progression, but longer follow-up is required before the treatment response can be confidently assessed.While our study is based on only one case, it is highly unlikely that the anti-NMDAR antibodies in our patient are an incidental finding. Anti-NMDAR antibodies are rare and false positive occurrence in both serum and CSF has not been reported. A pathogenic role for the antibodies in our patient is also supported by the FDG-PET findings of frontal hypermetabolism and cerebellar hypometabolism, which although not specific, are consistent with findings reported in patients with encephalitis . As the Our findings raise the possibility anti-NMDAR autoimmunity may be an unrecognized cause of dystonia with BSN. While further cases are needed to elaborate the mechanisms involved, we feel that our study highlights the need to thoroughly investigate cases with BSN as early identification may provide the opportunity for better and more effective treatment. We suggest, therefore, that anti-NMDAR antibodies should be included in the work up of chronic movement disorders with MRI evidence of striatal necrosis.Written informed consent was obtained from the patient for publication of this case report and any accompanying images or videos. A copy of the written consent is available for review by the Editor of this journal.This work has been deemed quality controlled by our local ethics committee (REK vest).ADC: Apparent diffusion coefficient; BSN: Bilateral striatal necrosis; CSF: Cerebrospinal fluid; FDG: Fluor-18-deoxy-glucose; IVIg: Intravenous immunoglobulin; MRI: Magnetic resonance imaging; NMDA: N-methyl-D-aspartate; NMDAR: N-methyl-D-aspartate receptor; PET: Positron emission tomography.The authors declare that they have no competing interests.CT, LB: clinical evaluation, acquisition, analysis and interpretation of data, drafting of the manuscript. MH, CV, GT, MB, TS: analysis and interpretation of data, critical revision of the manuscript. LAB, AS, IOG, CV: critical revision of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/13/55/prepubVideo of the patient at the age of eighteen.Click here for file


Transat detects all conserved helices, including those expected in a final structure, but also transient, alternative and pseudo-knotted helices. RNA-Decoder uses unique evolutionary models to detect conserved RNA secondary structure in alignments which may be partly protein-coding. SimulFold simultaneously co-estimates the potentially pseudo-knotted conserved structure, alignment and phylogenetic tree for a set of homologous input sequences. CoFold predicts the minimum-free energy structure for an input sequence while taking the effects of co-transcriptional folding into account, thereby greatly improving the prediction accuracy for long sequences. R-chie is a program to visualise RNA secondary structures as arc diagrams, allowing for easy comparison and analysis of conserved base-pairs and quantitative features. The web site server dispatches user jobs to a cluster, where up to 100 jobs can be processed in parallel. Upon job completion, users can retrieve their results via a bookmarked or emailed link. e-RNA is located at http://www.e-rna.org.e-RNA offers a free and open-access collection of five published RNA sequence analysis tools, each solving specific problems not readily addressed by other available tools. Given multiple sequence alignments,  Recent studies have shown the expanding number of functional RNAs within the cell . In manyTransat (RNA-Decoder (SimulFold (CoFold (R-chie (We here focus on the prediction of RNA secondary structure (henceforth referred to simply as structure), i.e. the set of Watson-Crick and wobble base-pairs. We have developed a web server hosting five tools previously published by our group, giving users a user-friendly interface for predicting specific sequence features currently only possible with our tools. Transat  is able -Decoder  was desiimulFold  outputs   with corresponding prior probabilities for the predicted annotation state of each alignment column . In scanning mode, RNA-Decoder returns base-pairing probabilities for each individual alignment column rather than a structure with specific base-pairs , the multiple sequence alignment and the phylogenetic tree.All programs that detect conserved RNA structure based on a fixed multiple sequence input alignment require a high-quality input alignment to reliably detect the conserved structure. Often, this implies that a alignment of high quality cannot be compiled unless the conserved structure is known upfront, amounting to a serious chicken-egg problem. A few existing tools tackle this problem by simultaneously aligning and folding a given multiple sequence alignment, such as oldalign , carnac   RNA structure that minimises the overall free energy of the molecule. This MFE structure, however, does not account for the effects of co-transcriptional folding that may influence structure formation in vivo . By crea . For the full description of all input and other options, please refer to the original manuscript (R-chie returns figures in either PNG or PDF format, and also specifies the corresponding command line that users could use to reproduce the same figure with their own copy of R-chie.The most basic usage of nuscript . R-chie e-RNA provides a collection of unique programs for RNA secondary structure prediction and visualisation, with an emphasis on the detection of conserved RNA structure features. Using a multi-core computer cluster and a job scheduling system, our servers can process up to 100 jobs in parallel. Upon job completion, users can either retrieve their results via a job-specific bookmarked link, or optionally receive the link via email, if the email address was supplied during job submission. For most of our tools, a tar-ball file containing the full job input, intermediate files, output and figures is provided for download for further analysis by the user. We provide this server in the hope that it will be useful, but it is provided as is without any warranty of any kind, expressed or implied. Finally, as research continues in our lab, we will continue to make additions and updates to e-RNA.


The mechanisms of several neurological disorders, such as epilepsy, have been linked to NTs. Because the number of diagnosed cases is increasing, the diagnosis and treatment of such diseases are important. To detect biomolecules including NTs, microtechnology, micro and nanoelectronics have become popular in the form of the miniaturization of medical and clinical devices. They offer high-performance features in terms of sensitivity, as well as low-background noise. In this paper, we review various devices and circuit techniques used for monitoring NTs  It has been shown that the mechanisms of several brain-level diseases are linked to NTs, such as Parkinson's disease and epilepsy. Regarding these two disorders alone, statistics show that over 300,000 Canadians and 50 million people worldwide have epilepsy , while ain vivo. Additionally, it has been shown that neurotransmission occurs on the millisecond to minute time scale, which makes its real-time analysis easy to achieve ..et al. det al. used a molecularly-imprinted polymer (MIP) electropolymer of OPPy for the in vivo detection of DA [In another study, Dengler on of DA . The MIPDifferent polymers, such as PPy, poly, polyphenol and polythiophenol, are electroactive functional monomers that have been used widely for the development of chemosensors based on MIP . Figure et al. presented the first FSCV microelectrode arrays (MEA) implemented in vivo with the ability to sample from various regions in close spatial proximity (equidistant within 1 mm), providing information about neurotransmission across multiple brain compartments [The functional monomer of pyrrole plays an important role in the MIP in terms of recognizing the desired DA target molecules. Zachek artments . The men2.1.et al. described a biocompatible implantable voltammetric microsensor that can be applied to the targets of midbrain DA systems, detecting sub-second DA dynamics with sub-second temporal resolution over months in vivo in rats and mice [et al. developed an integrated flexible implantable probe on a polyimide-film substrate for sensing NTs. The flexibility of the probe helps to prevent scar formation in tissues during long-term in vivo monitoring. The assembled sensors were calibrated and tested at various concentrations of L-GLU with and without the presence of interfering molecules. The results demonstrated good sensitivity and selectivity  at a carbon fiber microelectrode (CFM). It has been shown that the WINCS supports FSCV at a fiber CFM to provide superior temporal and spatial resolution, including chemical specificity [in vitro and in vivo. These results demonstrate that WINCS is well-suited to the in vivo monitoring of AD, and the clinical application of AD measurements may prove important in achieving a better understanding of neurochemical mechanisms. In another study, Kang et al. used ultra-microelectrode arrays (UMEAs) (The wireless instantaneous NT concentration system (WINCS) also provides real-time co-monitoring of NTs. Shon cificity . The WIN (UMEAs) , a squaret al. found that electroactive species, such as DA, can be adsorbed to an electrode and then rapidly released by varying the potential of that electrode [UMEAs offer several advantages over macroelectrodes, because of having micron-sized electrodes, which enhance the rates of mass transport of the analyte to the electrode surfaces. The compatibility of microfabricated nanodiamond UMEA with the FSCV technique was evaluated, which was found to offer high temporal resolution for NT detection. Because DA is an electrochemically active compound and forms DA-ortho-quinone (DOQ), via oxidation, transferring to electrons and protons, the behavioral and environmental events of DA release occur on a short timescale. Dengler lectrode . The des2.2.et al. used a composite-modified platinum (Pt) electrode for the simultaneous determination of catecholamines, UA and AA. Atta et al. also constructed an electrochemical sensor based on gold NPs and graphite for the selective detection of DA [et al. showed that an electrode modified by a poly film of a negatively-charged compound could work as an electrocatalyst for DA and UA oxidation and as a discriminating layer for DA against AA and UA; and that it could be used for the simultaneous determination of these compounds. In addition, using this modified electrode, the oxidation current of DA and UA increased significantly, as did the reproducibility and stability. The proposed methods can be applied to the detection of DA in human urine samples [The simultaneous measurement of NTs is especially important in plasma and urine samples. Normally, AA, DA and UA coexist in body fluids. Because the oxidation potentials of AA, DA and UA are very close to one another, their selective detection using bare electrodes is a challenging task. Additionally, due to the surface fouling of bare electrodes caused by the adsorption of oxidized products of AA on the electrode surface, stability and reproducibility cannot be achieved. At bare electrodes, the selective detection of some NTs, such as DA, is impossible, because of interference from other compounds, such as AA and UA, which have oxidation potentials that are very close to that of DA . To monion of DA . The gol samples .et al. developed a method to study the electrochemical behaviour of AA, DA and NA using differential pulse stripping voltammetry (DPSV) [et al. compared three key coating materials to show their selectivity and fouling resistance to electrodes: Nafion, base hydrolyzed cellulose acetate (BCA) and fibronectin [et al. used Sulfonazo III-modified film on a glassy carbon (GC) electrode. Sulfonazo III, a 2,7-bis(2-sulfophenylazo) chromotropic acid tetra sodium salt, is a pH indicator [et al. also presented the preliminary results of the simultaneous determination of DA and UA in the presence of AA by using a poly-modified glassy carbon electrode (3-TPA/GC) [To monitor DA in the presence of AA and UA, Ni y (DPSV) . This meronectin . They fondicator . Thus, t-TPA/GC) . They shin vitro and in vivo applications. In the following section, we will further discuss carbon nanotubes, one of the most used modified electrodes in NT measurement.Overall, one of the most important aspects of voltammetry is the low noise and high sensitivity in biosensor applications, enabling the simultaneous detection of multiple analytes. Although voltammetry is one of the sensitive electrochemical analyses, expensive equipment and complicated procedures are required. More work needs to be done in this area to design simple and affordable devices for 3.Carbon nanotubes (CNTs) were first discovered by Sumio Iijima in 1991 . Since tin vivo, as well as in vitro [Recently, several groups have applied CNT-based electrochemical biosensors for the monitoring of diverse biological structures, such as DNA, viruses, antigens and disease markers. These molecules can promote electron transfer in electrochemical reactions. The carbon nanotube coating on the electrodes is biocompatible and can be used in vitro . Carbon-in vitro .To expand the array of analytes beyond electroactive molecules, enzymes are often incorporated into biosensors to selectively detect an analyte and create an electroactive product after an enzymatic reaction. Biomolecules, such as proteins, enzymes and DNA are easily adsorbed on to the surface of CNTs and can be attached directly to functional groups on the CNTs . Adding et al. introduced a new method for coating CNTs on the electrodes [in vivo. Gholizadeh et al. also reported a sensitive GLU biosensor based on GLU dehydrogenase/vertically-aligned CNTs . The results showed that the GLDH/VACNT electrode was a suitable basic electrode for the detection of GLU, even without an electron mediator. They also reported the fabrication of a vertically-aligned carbon nanotube nanoelectrode array (VACNT-NEA) via the photolithography method [Dip coating CNTs is not reproducible, and sometimes, CNTs lay flat on the surface of the electrode, resulting in limited access to the electroactive sites. Xiao ectrodes . They aly method . The fabet al. modified carbon-fiber microelectrodes (CFMEs) with three differently functionalized single-wall carbon nanotubes and measured their responses to 5-HT, DA and AA using FSCV. Both carboxylic-acid-functionalized and amide-functionalized CNTs increased the oxidative current of CFMEs by approximately 2–6-fold for the cationic NTs, 5-HT and DA, but octadecylamine-functionalized CNTs resulted in no significant signal change. PPy, as a conducting polymer, can also be used in the design of bioanalytical sensors [et al. developed a biosensor by using a simple electrocodeposition of MWCNT, PPy and Laccase (Lac) on a platinum (Pt) electrode surface [et al. has also developed a new biosensor by a simple electrocodeposition of multi-walled carbon nanotubes (MWCNT), PPy and Lac on the platinum (Pt) electrode surface. The NT biosensor was applied to the detection of DA in urine samples using DPV [et al. proposed using oPPY-CNT electrodes instead of Nafion-CNT electrodes for repeated in vivo use, because of their reproducible fabrication, selectivity over anions and larger currents in vivo [in vivo and preserved selectivity over anions.In another study, Jacobs  sensors . Cesarin surface . The NT sing DPV . Peairs  in vivo . Immobil3.1.et al. developed implantable carbon microelectrodes that were modified with CNTs dispersed in nafion to quantify pharmacological alterations in 5-HT release via DPV [et al. also reported the simultaneous determination of catecholamines EP and NE using square wave voltammetry (SWV) and a multi-walled carbon-nanotube-modified edge plane pyrolytic graphite electrode (MWNT/EPPGE) [et al. similarly reported the electrochemical behavior of AA, DA and UA on the surface of the iron (II) complex-MWNT-modified GC electrode [The simultaneous determination of NTs could be facilitated by using CNT. Njagi  via DPV . Goyal eT/EPPGE) . SWV hasT/EPPGE) . Generallectrode . The modet al. used a novel captopril/thiophenol (Capt/TP) mixed SAM as a substrate for the selective adsorption of 5-HT in a solution matrix containing AA and the other commonly encountered NTs. As a result, the charge-transfer impedances underwent large changes caused by very small changes in the electrode surface due to adsorption, and these changes were detectable [et al. demonstrated that the simultaneous determination of adrenalin (AD) and paracetamol (PAR) can be performed on a single-walled carbon nanotube/chitosan/ionic-liquid-modified glassy carbon electrode (SWCNT-CHIT-IL/GCE). They showed that a biocompatible polymeric matrix, chitosan (CHIT), displays excellent film-forming abilities, high-water permeability and susceptibility to chemical modifications, and chitosan chains can associate with CNTs to form a biocompatible nanotube aqueous solution [A self-assembled monolayer (SAM) can also be used for the modification of electrode surfaces, providing fast responses, high sensitivity, antifouling effects and chemical specificity. Pătraşcu tectable . Babaei solution .3.2.in vivo. Koehne et al. integrated a carbon nanofiber (CNF)   electrodin vitro and in vivo. Overall, CNTs could facilitate higher sensitivity and develop electron transfer kinetics for NT detection.CNFs have been shown to be useful as neurochemical recording electrodes, because of their high sensitivity, fast electron transfer kinetics, widespread potential window, biocompatibility and manufacturing versatility. Additionally, FSCV is a particularly powerful technique that can be used to monitor very rapid events, such as neurochemical release. Due to their small size, both micro- and nano-electrodes exhibit a double-layer capacitance, which aids in the rapid change of electrode potentials and eliminates the distortions in the applied voltage waveform that are observed with larger electrodes. WINCS , which win vitro to cells and in vivo to mice; however, further systematic investigations are required. Optimization of the surface functionalization to minimize toxic side effects is important. In spite of challenges on the way towards the clinic, carbon nanotubes offer great potential for biomedicine and can bring opportunities for different applications in biomedicine [Although numerous encouraging results using CNTs in biomedicine have been published in the past several years, more work is yet needed before CNTs can enter the clinic. One of the important issues for CNT application is the concern of long-term toxicity. It has been shown that well-functionalized carbon nanotubes are not toxic medicine –85. Tabl4./Pt/Au being stacks deposited and structured via lift-off techniques and the plasma-enhanced vapor deposition of silicon oxides and nitrides, followed by platinum (Pt) sputtering to create electrodes [et al. have presented an integrated electrochemical measurement system based on CMOS technology that allows the detection of several analytes in parallel  and that enables simultaneous monitoring at different locations (multi-site). In that study, a label-free, hybridization-based DNA sensor was integrated with CMOS technology.Complementary metal-oxide semiconductor (CMOS) technology is a powerful tool for biosensor implementation that is capable of having millions of transistors within system-on-chip and lab-on-chip potentials. Moreover, CMOS has the ability to scale up with tens of nanometers of critical feature size, meeting the requirements for most biosensing schemes . The lowectrodes ,97. In aet al. have presented miniaturized CMOS capacitive sensors for monitoring DA down to the sub-fM range. The 5×5 sensor array contains five parts composed of various electrode sizes, and each part group's five sensors. No additional lithographic steps were needed after the CMOS foundry process, and CMOS sensor can be used directly [et al. have developed a CMOS potentiostat circuit that can be scaled to a large array [et al. also presented interdigitated microelectrodes integrated with CMOS sensing circuitry. The device could be used as an immunosensor, with its sensitivity being enhanced by gold NPs.Furthermore, the detection of the NT, choline, was achieved by assembling the chip with biosensor microprobe arrays . Lu et adirectly . Ayers ege array ; two poset al. presented an active CMOS-based electrochemical biosensor array with high-throughput capability (100 electrodes) for on-chip amperometric measurement of NT release [Microelectrodes covered by a silicon dioxide layer were fabricated using a maskless post-CMOS process. When charged biomolecules bind to the functionalized oxide surface after immobilization, a capacitive change will be produced for the underlying electrodes . In anot release . The bioet al. [Nazari et al.  have alset al. presented a low-power, noise-immune CMOS-based neurochemical circuit for implantation purposes. Using electrochemical techniques, the device senses picoscale to microscale currents, corresponding to micro-molar NT concentrations. The low-noise characteristics of the device make it suitable for the noisy environment often encountered in vivo [CMOS technology can also be used for implantable neural prosthetics. Poustinchi  in vivo . The bioet al. used an edge plane pyrolytic graphite electrode modified with multi-walled carbon nanotubes (MWNTs/EPPGE) for the quantitative detection of EP in the body fluids of smokers and non-smokers using CV and SWV [et al. investigated the real-time assay of the EP using square-wave anodic stripping voltammetry (SWASV) with a modified carbon nanotube electrode (CNE). They used chelate DNA immobilized on a catalytic CNT surface. The method showed a lower detection limit than other modifications. Thus, it can be attuned to low-detection ranges for in vivo analysis and neuro-current detection. A CMOS circuit is also implemented to detect the redox current. Collection efficiency is significantly enhanced via reduced electrode separation [/Pt/Au stacks deposited and structured via lift-off techniques and plasma-enhanced vapor deposition of silicon oxides and nitrides, followed by sputtering of Pt to make electrodes [In another study, Goyal  and SWV . The vol and SWV . Ly et aparation . Overallectrodes ,104,105.ectrodes .5.in vitro experiments have been performed using artificial cerebrospinal fluid (ACSF) from Tocris Bioscience and microspheres to test the behaviour of the designed LOC [(DEP) is the movement of particles in a non-uniform electric field, which is due to the interaction of the particles' dipoles and the spatial gradient of the electric field. DEP has been applied to the manipulation of biological particles, such as bacteria, viruses, proteins, nucleic acids and other biomolecules. Its label-free nature, the simplicity of the instrumentation and the ability to induce both negative and positive forces, decrease electrode spacing and increase effective field strengths have been made DEP one of the most popular methods of particle manipulation in microsystems. It is also applicable to non-conducting particles and can be generated either by using a direct current (DC) or alternating current (AC) field. In addition, in the DEP technique, a lower voltage is needed than in electrophoresis, which is suitable for integrated LOC systems. The proposed platform offers a microfluidic system integrated with a microelectronics chip. Both parts are packaged by glass and PDMS followed incorporating into a biocompatible device  capacitive sensor and tested the device functionality by using various organic solvents with different dielectric constants . AdditioThe integrated potentiostats would also be suitable for the detection circuit of the implantable devices, because of the fast performance of the electrochemical techniques, high sensitivity and temporal resolution, as well as small probe size.6.et al. designed a CMOS capacitive sensor with sub-μM interdigitated microelectrodes covered by an inter-metal dielectric layer, having a 200 nM limit of detection for DA [et al. designed a 5 × 5 CMOS capacitive sensor array for the detection of DA in fM ranges [Because the plasma kinetics of most NTs is in nano-molar ranges –114, devn for DA . In anotM ranges . It has M ranges ,75,89,90M ranges ,56,66,747.in vivo. Because of the complications and cost effectiveness of the current devices, research in this area is conducted for simple and cost-efficient devices. In general, micro-devices for in vivo and in vitro detection of NTs would be promising in the future for the investigation of brain circuits, whereas, in parallel, their potential for neuroprosthetics applications would be valuable.Microelectronic biosensors have the potential to facilitate the development of inexpensive devices for molecular diagnostics. NTs, like most other biomolecules, have extra electrical charges or can be modified with redox-active tags. By applying simple electrochemical detection schemes and using the pre-defined electrical excitations, the monitoring of NTs is possible. This makes microelectronics and microsystems-based platforms useful for NT detection. Microelectronics offer high-performance features in terms of sensitivity, having low background noise, using cost-effective components and being able to perform measurements in turbid samples. In particular, the electrochemical sensor is a completely label-free detection platform, which is beneficial to portable sensors. Microtechnology can also be used as implantable micro-devices with the potential for neuroprosthetic applications, as well as for the investigation of brain circuits


Plasmodium yoelii, P. berghei ANKA (PbA) and P. chabaudi AS (PcAS). Although availability of these genomes made a significant impact on recent malaria research, these genomes were highly fragmented and were annotated with little manual curation. The fragmented nature of the genomes has hampered genome wide analysis of Plasmodium gene regulation and function.Rodent malaria parasites (RMP) are used extensively as models of human malaria. Draft RMP genomes have been published for PbA and PcAS, newly sequenced the virulent parasite P. yoelii YM genome, sequenced additional RMP isolates/lines and have characterized genotypic diversity within RMP species. We have produced RNA-seq data and utilised it to improve gene-model prediction and to provide quantitative, genome-wide, data on gene expression. Comparison of the RMP genomes with the genome of the human malaria parasite P. falciparum and RNA-seq mapping permitted gene annotation at base-pair resolution. Full-length chromosomal annotation permitted a comprehensive classification of all subtelomeric multigene families including the ‘Plasmodium interspersed repeat genes’ (pir). Phylogenetic classification of the pir family, combined with pir expression patterns, indicates functional diversification within this family.We have greatly improved the genome assemblies of Plasmodium biology. Genotypic diversity between P. chabaudi isolates makes this species an excellent parasite to study genotype-phenotype relationships. The improved classification of multigene families will enhance studies on the role of (variant) exported proteins in virulence and immune evasion/modulation.Complete RMP genomes, RNA-seq and genotypic diversity data are excellent and important resources for gene-function and post-genomic analyses and to better interrogate The online version of this article (doi:10.1186/s12915-014-0086-0) contains supplementary material, which is available to authorized users. Plasmodium berghei, P. yoelii, P. chabaudi and P. vinckei. Small differences exist in the biology of the different RMP in laboratory mice and this makes them particularly attractive models to investigate different aspects of human malaria. Specifically, P. chabaudi is a model to investigate mechanisms of drug resistances and immune evasion, in particular antigenic variation [P. berghei preferentially invades reticulocytes and usually produces infections in mice that induce severe pathology [P. yoelii is widely used in studies on the biology of liver stages and on innate and acquired immunity against liver stages [P. yoelii parasites of some lines are restricted to reticulocytes whereas others can invade all red blood cells and have been used to study receptors for erythrocyte binding [P. berghei and P. yoelii [Plasmodium gene function [Rodent malaria parasites (RMP) are used extensively as models of human malaria ,2. Four ariation ,4. It inr stages ,6. Blood binding ,8. The a. yoelii -11 and tfunction -14. For function .P. yoelii yoelii 17XNL [P. berghei ANKA (PbA) and P. chabaudi chabaudi AS (PcAS) in 2005 [P. falciparum and other primate malaria species defined a large set of core genes that are shared between RMPs and primate malarias [PbA and PcAS large-scale improvement of their existing genomes, with re-sequencing, re-analysis and manual re-annotation, and for P. y. yoelii a genome sequence was produced de novo from the virulent YM line using the latest sequencing technologies and computational algorithms. In addition, we have utilised comprehensive RNA-seq data derived from a number of life-cycle stages to both improve gene model prediction and to provide genome-wide, quantitative data on gene expression. By sequencing additional isolates/lines of P. berghei, P. yoelii and P. chabaudi (including the subspecies P. c. adami) we have documented genotypic diversity that exists within different RMP species. The availability of RMP reference genomes in combination with the RNA-seq and genotypic diversity data will serve as excellent resources for gene-function and post-genomic analyses and, therefore, better interrogation of Plasmodium biology and development of anti-malaria interventions.The first draft RMP genome was published in 2002 for ii 17XNL . This wamalarias -20. Althmalarias  and weremalarias ,10, thesPlasmodium interspersed repeat genes’ (pir) [Plasmodium species [pir genes and analyses of pir expression patterns in blood stages of P. berghei provide evidence of functional diversification within this gene family. The improved classification of multigene families will enhance studies on the role of (variant) exported proteins in virulence and evasion and modulation of the immune system.The genomes of RMP contain a number of multigene families located in the subtelomeric chromosomal regions. These include a large family of so-called ‘s’ (pir) , that ar species -23. Most species -26 and m species . As a rePbA, PcAS and P. y. yoelii YM (PyYM). This resulted in a significant reduction in contig number for the PbA and PcAS genomes , automated scaffolding, gap closure, error correction and annotation transfer, followed by manual inspection, we obtained highly accurate and almost complete reference genomes of es Table  comparedes Table . More thredicted  (see AddPcAS and PyYM. In PbA only a single copy (PBANKA_091920) is present, whereas the genomes of PcAS and PyYM contain multiple copies  are either located as clusters in the sequence gaps that still exist in the PbA subtelomeric regions or are located within the existing current 2.3 kb repeat-arrays. These 2.3 kb repeats contain telomeric repeat sequences and many contain a copy of a highly degenerate pir pseudogene  RMP proteins with orthologs in any of the primate malarias; (2) RMP-specific proteins with no orthologs in primate malarias; and (3) primate malaria-specific proteins with no orthologs in any of the RMP , P. c. chabaudi  and P. c. adami . In addition, we sequenced P. y. yoelii 17X , large differences exist in gene copy number of subtelomeric multigene families  non-subtelomeric genes having at least one SNP  in the P. c adami DK and DS isolates, respectively. Between different P. chabaudi isolates differences exist in virulence- and invasion phenotypes of blood stage infections  such as the Duffy-binding protein and reticulocyte binding proteins  [msp1 of all P. chabaudi isolates  ,33. Our PbA asexual blood stages  and from purified gametocytes and ookinetes. In addition, RNA-seq data was generated from multiple samples of blood stage trophozoites of PcAS and from blood stages of PyYM . Both the gametocyte and ookinete samples clustered separately from asexual blood stages, which reflects the different program of gene expression during sexual commitment and zygote development. Heat maps representing the expression of all PbA genes reveal clusters of genes with distinct expression patterns in the different life cycle stages  resolution for at least 89% of the genes. Of the 896 newly annotated protein-coding genes that were absent in the previous genome assemblies, 70% have primate malaria orthologs, 83% have expression evidence  and we could ascribe functions to 75% , a genome viewing tool . A compapir family is the most abundant multigene family (see next section) encoding exported proteins that lack a canonical PEXEL motif. The second largest gene family is the fam-a gene family, formerly identified as the pyst-a family in P. yoelii 17XNL and named as Pb-fam-1, Pc-fam-1 or fam-a [PbA fam-a proteins are exported into the host RBC and can be transported to the RBC surface membrane [pyst-b/pb-fam-3 and pyst-c genes [fam-b and fam-c, respectively (Table fam-b family is exclusively subtelomeric and is characterized by the presence of the pyst-b domain. Most members contain a transmembrane domain (58%), a signal peptide (75%) and PEXEL-motif (76%)  and six or more full length proteins  and ‘putative reticulocyte binding proteins’ Table  and 14. rozoites , are cleP. vivax  and the lciparum . These lg domain . The RMPPcAS several other expanded gene families are present in the subtelomeric regions. These include ‘putative lysophospholipases’, ‘erythrocyte membrane antigen 1’ (EMA1), and ‘putative haloacid dehalogenase-like hydrolases’  in gametocytes. Expression patterns are not only different between asexual and sexual stages but also between different asexual stages, for example distinct pir gene clusters are up-regulated in schizonts. Distinct transcription patterns in different life cycle stages of gene clusters may indicate functional differences between members of a single gene family. With the new genome assemblies we were able to determine the total number of pirs and their structure and spatial organization more precisely  is 194 and 100, respectively, whereas in PyYM this gene family is greatly expanded to 583 copies. In Additional files pirs is shown. Most pir genes share a similar structure across the different species with a short first exon, long second exon and a third exon encoding a trans-membrane domain. They lack a PEXEL-motif and all pirs are chromosomally arranged such that they are transcribed in a centromere to telomere direction except for several members of PcAS . A number of pirs have long low complexity regions in the predicted extracellular domain and a few Pc-pirs have a four exon structure  of pseudogenes and nearly half  of these Pb-pirs are contained within the 2.3 kb subtelomeric repeat described above  between pirs we undertook a detailed phylogenetic analysis of all RMP pirs (including predicted pseudogenes). Estimations of Maximum Likelihood (ML) phylogeny based on nucleotide sequences or amino acid sequences and an estimation of a Bayesian phylogeny resulted in a phylogenetic tree with a robust separation of ‘long-form’ and ‘short form’ pirs  with a mean length ranging from 786 to 952 aa. Most long-form pirs have an extended repetitive region located within the second exon, downstream of the core pir domain and upstream of the transmembrane region. All RMP species have both short- and long-form pirs, indicating that the presence or absence of an extended repetitive region has evolved once and defines a principle division in pir diversity. Many clades are dominated by pirs from one species, particularly PyYM  or PcAS (S1g) indicating that these lineages originate from the RMP ancestor and probably expanded after speciation. Both PcAS and PbA appear to have experienced their own specific expansions after speciation . The maintenance of orthology within clades, in the presence of frequent gene conversion  of the predicted RMP proteins have orthologs in primate malaria species. It is this high level of orthology between RMP and primate malaria genomes that strongly supports RMPs as models in experimental approaches to characterize the Plasmodium gene function. Similarly, the genome-wide RNA-seq data from different RMP developmental stages is a valuable resource to further analyse Plasmodium gene function and the regulatory networks underlying the multiple differentiation pathways of Plasmodium. The RNA-seq studies presented here provide information on gene expression at an unprecedented depth and breadth of coverage of multiple blood stages and ookinetes. Previously, only a few large-scale transcriptome (microarray) analyses of P. berghei blood stages and ookinetes had been performed [P. berghei genome and, therefore, expression data were only generated for about half of all P. berghei genes. In addition, important/valuable large scale transcriptome studies have been performed on RMP life-cycle stages, such as sporozoites and liver stages [By extensive re-sequencing and annotation we have generated three high quality RMP reference genomes with nearly all core genes as complete gene models and a much improved and almost complete representation of chromosomal subtelomeric regions. These reference genomes will greatly enhance the use of RMP as model organisms in malaria research. We provide full-length gene models for more than 98% of predicted protein-coding genes. The approximately 60% of genes with functional annotation is comparable to the percentage of functionally annotated genes in the erformed ,46. Thesr stages -49. ThesOur studies reveal that large scale changes in gene expression occur in ookinetes between 16 and 24 hours after fertilization, possibly required for the differentiation of (retort-form) zygotes into the mature ookinetes. The strong up-regulation in mature ookinetes of transcripts involved in ribosome biogenesis and protein translation suggest that the mature ookinete generates transcripts for proteins required after the ookinete has traversed the mosquito midgut wall and starts its rapid transition into the oocysts, possibly using mechanisms of translational repression similar to those in gametocytes ,51 and sP. berghei isolates was unexpected. Low sequence diversity of a limited number of genes of P. berghei isolates had been reported previously and it was proposed that this may result from cross-contamination of P. berghei isolates in the laboratory after isolation [P. berghei isolates we have sequenced were obtained from other laboratories  and they also show a similar lack of sequence polymorphism. In contrast, the isolates of P. chabaudi exhibit considerable genotypic diversity. These P. chabaudi isolates exhibit differences in virulence, RBC invasion, growth rates and immunogenic profiles [P. yoelii virulence has been associated with mutations in proteins involved in RBC invasion [P. chabaudi genes encoding such proteins. While much attention is given to the role of exported proteins of multigene families and virulence in both human and RMP, further analysis of RMP proteins that regulate invasion phenotypes may reveal novel mechanisms that underlie virulence.The additional sequence data from multiple RMP isolates will help to further unravel gene function and establish relationships between phenotypic traits and genotypic diversity. The near absence of polymorphisms within the genomes of solation ,55. Howeprofiles ,56-60 anprofiles ,61-63, wprofiles ,56,60,64invasion ,65,66. Ipir gene family, all three RMP contain an expanded gene family encoding exported proteins, fam-a, with orthology to a single-copy gene in primate malarias, which contains a START-domain . START- domain; . A STARTholipids . The sinhost RBC ,70. Combpir family is the largest RMP multigene family and is shared with human and non-human primate species P. vivax, P. knowlesi and P. cynomolgi [pirs is unknown and no functional domains have been identified so far. Recently, it has been shown that in P. chabaudi a change in virulence was associated with differential expression of members of the pir multi-gene family [Pfemp1 gene family of virulence factors in P. falciparum. However, for several P. berghei PIRs a direct role in RBC sequestration is unlikely since no evidence was found for their location on the RBC surface although they were exported into the RBC cytoplasm of both sequestering asexual blood stages and non-sequestering gametocytes [P. vivax PIRs it has been shown that different members have distinct subcellular locations in the infected RBC [PcAS pirs identified two distinct pir sub-families (A and B), which contain distinct amino acid sequence motifs [pirs from all three RMP species and resulted in the identification of a number of different clades. The presence of clearly distinguishable clades indicates that structural differentiation exists among pirs and that this evolved prior to the separation of the RMP species. Our observations of the stage-specific up- or down-regulation of expression of clusters of structurally different pirs in different blood stages supports the hypothesis that there is functional diversification within the pir family and that purifying selection plays a role in shaping this family. By including multiple species in the pir phylogeny it is clear that this gene family is subject to rapid turnover, that is, gene gain and loss, indicating the absence of strong selective forces that would result in distinct orthologous groups/clades that are shared and maintained in different species for functional reasons. Gain of pir genes in different species is evident in the multiple species-specific expansions of clades. Assuming that the common ancestor had a pir family equal in abundance and diversity, the relatively limited instances of orthology (12 clades) indicates significant losses of ancestral sequence types. A plausible explanation for both the abundance of species-specific sequences and the paucity of ancestral sequences is a continual process of gene turnover driven by gene conversion, a mechanism that has been proposed for pirs of P. chabaudi [pirs, which we propose, explains the presence of the (limited) orthology between pir clades of the different RMP species. The improved annotation and phylogeny demonstrating clusters of structurally different pirs in all RMP combined with expression profiles are powerful data that can help to further delineate function, the relationship of expression with virulence and how the (species-specific) expansion of the pirs is related to distinct selective pressures.The ynomolgi -23. PIR ynomolgi ,29,71,72e family . It has cted RBC . These oe motifs . Our phychabaudi  and whicP. berghei and P. chabaudi, comprehensively sequenced the P. yoelii YM genome, sequenced multiple RMP isolates and generated in-depth expression data from multiple RMP life-cycle stages. Comparison of the RMP and P. falciparum genomes and RNA-seq mapping permitted gene annotation at base-pair resolution and has defined the level of orthology between RMP and human parasite genomes. The very high level orthology between RMP and human malarias (both in genome structure and gene content) supports the use of highly tractable RMPs as experimental models to characterize the function of the very many Plasmodium genes that remain uncharacterised.To maximise the utility of RMP we have greatly improved the genome assemblies of P. berghei life-cycle stages had previously been performed. Moreover, these studies were based on highly fragmented draft RMP genomes and consequently, for example, for one of the most well studied RMP, P. berghei, gene expression data was only mapped to about half of all P. berghei genes that have now been characterised. The RNA-seq studies we present provide information on gene expression, at an unprecedented depth and breadth of coverage, of multiple life cycle stages and provide the foundational data needed for the performance of large-scale analyses of gene regulatory networks that underlie cellular differentiation.Only a few large-scale transcriptome (microarray) analyses of different P. chabaudi isolates making this species an excellent organism to study genotype-phenotype relationships. Differences in virulence red blood cell (RBC) invasion, growth rates and immunogenic profiles exist between parasites of these isolates. Therefore, studies, such as linkage or quantitative trait loci analysis, are now possible to help identify genes associated with these defined phenotypes. For RMP species there is evidence that differences in virulence are associated with differences in RBC invasion, and, indeed, we find extensive sequence polymorphism in P. chabaudi genes encoding proteins involved in RBC invasion. Much attention is given to the role of exported proteins of multigene families and virulence in both human and RMP , and analysis of differences between RMP proteins, that regulate invasion phenotypes, may reveal novel mechanisms that underlie virulence.We show that extensive genotypic diversity exists between Plasmodium species. Investigations with highly tractable RMP species can therefore be used to understand these mechanisms better.Full-length chromosomal annotation has permitted a comprehensive classification of all RMP subtelomeric multigene families. Our analyses indicate that most, if not all, RMP subtelomeric genes (apart from the RBP family) encode proteins exported out of the parasite; however, most lack a canonical PEXEL-motif. The presence of large numbers of PEXEL-negative exported proteins indicates alternative export mechanisms possibly common to all pirs, indicates functional diversification between members of the pir multigene family . Our new pir annotation and phylogeny demonstrates that clusters of structurally different pirs are differentially expressed. This is powerful data that can help to better understand their function, the relationship of pir expression with virulence and how the (species specific) pirs expansion is related to different selective pressures.Our analyses of the phylogeny and expression of the largest RMP multi-gene family, the P. yoelii YM and P. yoelii 17X: all animal work protocols were reviewed and approved by the Ethical Review Panel of the MRC National Institute for Medical Research and approved and licensed by the UK Home Office as governed by law under the Animals (Scientific Procedures) Act 1986 . Animals were handled in strict accordance with the ‘Code of Practice Part 1 for the housing and care of animals (21/03/05)’ available at [P. chabaudi DNA and RNA was carried out in strict accordance with the UK Animals (Scientific Procedures) Act 1986 and was approved by the Ethical Committee of the MRC National Institute for Medical Research, and the British Home Office (PPL: 80/2538).All animal experiments performed in the Leiden malaria Research Group were approved by the Animal Experiments Committee of the Leiden University Medical Center . The Ethics Statement for lable at . The numPbA the cloned reference line cl15cy1 of the ANKA isolate of P. berghei [PcAS the 2722 clone of the AS isolate of P. chabaudi chabaudi ; for PyYM the cloned 17XYM line of the YM line of P. yoelii yoelii [For sequencing of the RMP reference genomes the following were used: for  berghei ; for PcAi yoelii . In Addiab initio gene finding via Augustus [P. chabaudi isolates with the Bio::Align::DNAStatistics Perl module.Sequencing was performed using Sanger capillary, Illumina and 454 sequencing. Sequence assemblies were performed using different assemblers ,76, whicAugustus  and tranAugustus . Gene moAugustus  was usedAugustus . ClassifPbA, from PcAS blood stages (late trophozoites), isolated from different mice as described [PyYM late blood stages of two parasite lines (the cloned YM line and mutant PY01365-KO) [RNA was collected from multiple synchronized blood stages  and puriescribed  and from1365-KO) . RNA was1365-KO) ,44,87,881365-KO)  (version1365-KO) . AcceptiHeatmaps were generated with FPKM values of each gene and condition, using the heatmap.2 function of the gplots package. Correlation plots were done in R  and genpir coding sequences, including predicted pseudogenes, were used. Translated nucleotide sequences for 1,160 genes were aligned in ClustalW [pir nucleotide sequences . See also Additional file All full-length RMP ClustalW ; all mulClustalW  using a ClustalW . A BayesClustalW  with a GP. chabaudi chromosomes: LK022878-LK022893 and scaffolds: LK022855-LK022877, P. berghei chromosomes: LK023116-LK023131 and scaffolds: LK022894-LK022977; P. yoelii YM chromosomes: LK934629-LK934644 and scaffolds: LK023132-LK023312 and P. yoelii 17X chromosomes: LM993655-LM993670 and scaffolds: LK022978-LK023115.All the raw data used in the assemblies of the genome and the RNA-seq data have been deposited with accession numbers shown in Additional file