The study presents a multi-channel patient-independent neonatal seizure detection system based on the Support Vector Machine (SVM) classifier.A machine learning algorithm (SVM) is used as a classifier to discriminate between seizure and non-seizure EEG epochs. Two post-processing steps are proposed to increase both the temporal precision and the robustness of the system. The resulting system is validated on a large clinical dataset of 267 h of EEG data from 17 full-term newborns with seizures.The performance of the system using event-based metrics is reported. The system showed the best up-to-date performance of a neonatal seizure detection system. The system was able to achieve an average good detection rate of ∼89% with one false seizure detection per hour, ∼96% with two false detections per hour, or ∼100% with four false detections per hour. An analysis of errors revealed sources of misclassification in terms of both missed seizures and false detections.The results obtained with the proposed SVM-based seizure detection system allow for its practical application in neonatal intensive care units.The proposed SVM-based seizure detection system can greatly assist clinical staff, in a neonatal intensive care unit, to interpret the EEG. The system allows control of the final decision by choosing different confidence levels which makes it flexible for clinical needs. The obtained results may provide a reference for future seizure detection systems. The incidence of clinically apparent neonatal seizures is generally reported as around 3 per 1000 and under certain circumstances, such as in very preterm babies, 50 per 1000 . In realSeizures are missed because they are very difficult to detect clinically; unlike older children and adults, babies do not always exhibit obvious clinical signs during seizures. EEG is the only available method to detect all seizures in babies. Most neonatal intensive care units (NICUs) lack the expertise required to interpret multi-channel EEG results, particularly on a 24/7 basis. Instead they often rely on amplitude integrated EEG systems (aEEG) that employ one or two channels of EEG and interpretation of a compressed and highly filtered output. Seizure detection using aEEG systems is heavily dependent on the experience of the user, the frequency and amplitude of seizures and their location and duration. It is not surprising therefore that seizure detection rates ranging from 12% to 76% have been reported using aEEG . We and A system that could automatically detect and annotate seizures on the neonatal EEG would be extremely useful to clinicians in the neonatal intensive care unit. Although a number of methods and algorithms have been proposed previously in an attempt to automatically detect neonatal seizures, to date their transition to clinical use has been limited due to poor performance.Numerous approaches have been proposed to identify and quantify the increase in periodicity of the EEG seen during neonatal seizures. Spectral analysis , autocorA method to mimic a human observer using a detector designed to identify spike-train like seizures and a second detector looking for oscillatory seizures has recently been proposed by Instead of using a set of heuristic rules and thresholds, several approaches utilise a classifier – a data-driven set of thresholds automatically trained on the data. A system based on a multilayer perceptron to classify neonatal EEG into one of six background states or two seizure states has been proposed by one group . Our groRecent work on statistical machine learning has shown the advantages of discriminative classifiers such as Support Vector Machines (SVM) in a range of applications , includiIn this study, a multi-channel patient-independent neonatal seizure detection system was designed, based on an SVM classifier and retrospectively evaluated on a large clinical dataset . By vary22.1In this study we used a dataset of recordings from 17 newborns in the Neonatal Intensive Care Unit (NICU) of Cork University Maternity Hospital, Cork, Ireland recorded over a 2 year period. During this period, 55 full-term babies with Hypoxic Ischaemic Encephalopathy (HIE) were recruited and EEGs were recorded for up to 72 h; 17 had seizures. Further details regarding the dataset can be found in Seizure is not always present in all EEG channels. Therefore in order to train a system for detection of neonatal seizures, it is necessary to know the channels involved in the seizure. For this purpose, a 2-min file with seizure activity was also created for each patient from the above-described dataset. The individual channel annotations were then produced for each file. This seizure data was then used for training the classifier model as explained in Section 2.22.2.1The outline of the system is shown in 2.2.2The EEG is down-sampled from 256 to 32 Hz with an anti-aliasing filter set at 12.8 Hz. It has been shown that frequencies of neonatal EEG seizures range between 0.5 and 13 Hz and the dominant frequencies of seizures vary between 0.5 and 6 Hz . The EEG2.2.3The features which have been shown to be useful for neonatal seizure detection in a number of papers are extracted for each epoch . The freIn total, 55 features are extracted. Despite the fact that some features may be redundant, preliminary experiments confirmed that the SVM was not very sensitive to their presence which has also been shown in the literature . Indeed,2.2.4The extracted feature vectors are classified using a SVM classifier. The SVM is a discriminative model classification technique that mainly relies on two assumptions . First, Classification consists of two steps – training and testing. The leave-one-out cross-validation method is used to assess the performance of the system for patient-independent seizure detection. This way, all but one patients’ data is used for training and the remaining patient’s data is used for testing. This procedure is repeated until each patient has been a test subject and the mean result is reported. The leave-one-out method is known to be an almost unbiased estimation of the true generalization error; that is, the performance reported with the leave-one-out method is the most similar to the performance this system would show on an unseen test dataset of infinite length once it is trained on all available data .M * 2 min per patient for seizures involved in M channels. For example, if a training dataset consists of 16 patients for which 2 min of seizure are transcribed and on average four channels are involved in every seizure, then for an epoch length of 8 s with an overlap of 4 s, the seizure class of training data will consist of 16 patients * (120 s/4 s) * 4 channels = 1920 epochs. It may be more or less depending on the number of channels involved in every seizure for every patient. For the non-seizure class, 10,000 epochs of the non-seizure data are randomly selected from all channels of all patients in training dataset. The features extracted from each epoch are then fed to train one SVM classifier. The training data for the SVM classifier are first normalized anisotropically by subtracting the mean and dividing by the standard deviation to assure commensurability of various features. The obtained normalizing template is then applied to the testing data. In this work, a Gaussian kernel is employed. For model selection , fivefold cross-validation on training data is used. Once the optimal pair of parameters is found, it is used to train the final model on all the training data.In the training stage, seizure and non-seizure epochs are labelled 1 and −1, respectively, for each channel. For the seizure class, the training dataset consists of 2 min of EEG per patient for which individual channel annotations are available, which sum up to In the testing stage, the obtained classifier is applied separately to each channel of the testing data and the decisions are post-processed and fused as described below.2.2.5Post-processing is a technique which is applied to the classifier outputs. As can be seen in f is the distance to the separating hyperplane, i.e. the output of the SVM classifier, A and B are the parameters of the sigmoid function estimated on the training dataset using the method described by f  signal, 2N + 1 is the order of the filter (the number of points used in the moving average). Essentially, the moving average filter is a convolution of the input signal with a rectangular pulse of unit area. It has been noted in A central linear moving average filter (MAF) is applied to the time sequence of probabilities in each channel c. It is The averaged value is then compared to a threshold from the interval [0 1]  is defined as the percentage of seizure events correctly identified by the system as labelled by an expert in neonatal EEG. If a seizure was detected any time between the start and end of a labelled seizure this was considered a good detection . The othAdditionally, an analysis of errors is performed in this work in order to gain further insight into the performance of the system. Specifically, missed seizures were analysed by grouping seizure events into four subsets according to their duration. Also every false alarm obtained by setting the system to a specific operating point was analysed to determine the nature of the EEG patterns that resulted in false detections. The false detections were visually grouped into three classes: artifact-free background activity, artifact contaminated EEG, and seizure-like activity.3The overall performance of our system is seen in 3.1In order to gain further insight into the performance of the system, missed seizures were analysed. Seizure events were grouped into four subsets according to the duration of the seizure event and the percentage of seizures detected is shown in It should be noted that in this study only full-term babies with HIE were included and generally seizures in this group are over 1 min in duration. Some seizures in neonates can be of short duration particularly in preterm babies. In clinical practice, a single short duration seizure in a neonate lasting 10 s or a little longer would rarely be treated.An example of a longer seizure, missed by the presented automated neonatal seizure detection system, from patient 16 is shown in 3.2The EEG was reviewed for each of the 277 false alarms obtained by setting the system to 1 FD/h operating point  to determine the nature of the EEG patterns that resulted in false detections. The false detections were visually grouped into three classes as shown in From Some false alarms were seen during periods of usual background activity. Delta activity was found to trigger false alarms when the background patterns became more rhythmic or sharp. An example of a background pattern incorrectly classified as a seizure is shown in The most prevalent artifacts causing false detections were electrode-disconnect, respiration and high amplitude activity caused by movement or handling of the patient. Electrode detachment was found to be the most predominant cause of false detections among artifacts. An example of electrode detachment artifact is shown in An example of the respiration artifact is shown in An example of the movement artifact from patient 12 which causes high amplitude patterns in the EEG is presented in It was found that approximately 5% of false detections occurred from short runs of seizure activity (10–15 s) that had not been annotated as seizures by the neurophysiologists. In all cases, these short duration seizures occurred during periods of status epilepticus or frequent seizures.When we went back to the neurophysiologists they agreed that these should be labelled as seizures but felt that they were not significant enough to warrant annotation during the first assessment in view of the surrounding longer seizures . HoweverThe integration of the feedback outlined in the analysis of errors to further improve the robustness and performance of the neonatal seizure detector is the scope of the future work. It is not trivial to figure out why the algorithm responded to certain characteristics of the signal: whether it happened because certain EEG activity was underrepresented in the training data or whether there is a certain feature (s) which considered this activity close enough to seizure and produced a ‘seizure’ response to this activity. For these reasons, we would like to avoid coming up with simple, untested and thus unreliable solutions to the identified problems but instead would rather perform a more thorough investigation.4In this paper we have presented a novel approach to detect neonatal seizures based on a set of diverse features, which capture energy, frequency and structural nature of the EEG signal, and a statistical machine learning algorithm – SVM – to derive a data-driven non-linear function to discriminate between seizure and non-seizure. The system showed the best performance (up to this date) of a neonatal seizure detection system. The resultant methodology is patient-independent, that is, the model trained has not seen the testing patient beforehand. The patient-independent model produced is robust to a seizure-channel connection usually observed in patient-dependent seizure detectors which implies that the system does not expect a seizure to happen in a particular channel . AdditioIn The future work will be devoted to addressing the limitations of the system. In particular, the most common sources and reasons for misclassification outlined in Sections 5An SVM-based multi-channel neonatal seizure detection system has been presented. The system has been validated on a large clinical dataset of babies with seizures and the results have been reported. The proposed analysis of errors has overviewed common sources and reasons of misclassification with several examples shown. The proposed SVM-based seizure detection system allows for control of the final decision by choosing different confidence levels which makes the proposed system flexible for clinical needs. In future work we will focus on integrating the feedback outlined in the analysis of errors to further improve the robustness and performance of the neonatal seizure detector.


In this work an Intrinsic Fabry-Perot Interferometer (IFPI) based on an air-microcavity is presented. Here the air microcavity, with silica walls, is formed at a segment of a hollow core photonic crystal fiber (HCPCF), which is fusion spliced with a single mode fiber (SMF). Moreover, the spectral response of the IFPI is experimentally characterized and some results are provided. Finally, the viability to use the IFPI to implement a simple, compact size, and low cost refractive index sensor is briefly analyzed. Here a standard arc fusion splicer was used to splice two different optical fibers (HCPCF and single mode fiber) and to form the air microcavity. Consequently, thermal effects can affect the properties of the physical joint  which ca2.2.1.The fusion splicing of standard single mode fiber (SMF28) with a segment of HCPCF  is a compulsory step in the air microcavity fabrication, which is basically the main part of our IFPI. Here, it was necessary to form a splice joint with a non-collapsed region. In 2.2.L from the fusion splicer electrodes  Apply the splicer clean process; (e) Apply the pre-fusion program of the fusion splicer. Here it is important to mention that one part of the HCPCF will collapse forming a silica wall and therefore a quantity of air will be confined between this wall and the SMF; (f) Apply another set of arc discharges to change the length of the air microcavity.After the splice joint with a non-collapsed region was formed, the next step consisted on the air microcavity fabrication. This was achieved by performing the following procedure: (a) Horizontally displace the splice joint a distance ectrodes . Here itL) and the number of arc discharges (ND) are some of the main parameters in the fabrication process and therefore it is important to characterize their effect over the final IFPI properties. In this way we fabricated some IFPI using different L distances to analyse its effects over the air microcavity fabrication process. From these experiments we found that for L < 100 μm the hollow core fiber completely collapsed, making impossible the air microcavity formation. Moreover, for L > 350 μm the HCPCF do not collapsed uniformly and therefore was not possible to form the air microcavity. For this reason and according to experimental results we considered for the IFPI fabrication an operating range of 100 < L < 350 μm.As can be appreciated in both procedures described above the arc fusion splicer is used only to form the splice joint and the air microcavity. However the fusion splicer alignment procedure was not used at all. Here the offset distance  and the rest is transmitted into the air microcavity. In this cavity the medium is air (n2 ≈ 1) and the light will travel a distance l1 until it reaches the silica wall, which was formed by the collapsed HCPCF cladding. At this interface a part of the light is reflected (R2) and the rest is transmitted into the silica wall with refractive index is n1 ≈ 1.4682 and thickness l2. Finally at the end of the silica wall another reflection (R3) will occur at the interface formed by the silica wall and the medium around the IFPI tip with refractive index n3.The IFPI mirrors are formed by the boundaries between the air microcavity and the optical fibers (SMF and HCPCF). Since our IFPI will be physically formed at the tip of the SMF see  thereforAt this point it is important to mention that at the splice joint some birefringence effects can be present since there are changes in the form of the propagation path and consequently some polarization changes can occur. However, this effect it is neither studied nor characterized in this work since we focused on the IFPI fabrication process and on the air microcavity characterization.4.4.1.To characterize the IFPI optical response the experimental setup shown in 4.2.L distances were fabricated in order to characterize its effects over the spectral response. For practical reason we used L = 100, 150, 200 and 250 μm since these are within the operating range of 100 < L < 350 μm. Moreover, for all these IFPIs the same number of arc discharges (ND = 7) was used. Here during the fabrication of these IFPIs the length of the air microcavity was measured using the fusion splicer and in average obtained that this was approximately l1 = 48 μm. Moreover, also using the fusion splicer it was possible to measure the thickness of the silica wall and in average, for the four IFPIs, was approximately l2 = 70 μm. Using these values it is possible to determine the free spectral range of the IFPI fabricated by using the next formula [λ0 is the reference wavelength, Δλ is the spectral separation between two consecutive fringes, n is the refractive index of the FPI cavity (in our case is air with n = 1) and l is the air microcavity length (l = l1). Using these values we obtained that the free spectral range for our FPIs should be approximately Δλ = 21.90 nm. In λ = 26.48 nm which is very close to the physically measured with the fusion splicer. Moreover, based on these results can be determined that the cavity length of the air microcavity is not changed in a sensitive way if 100 < L < 350 μm and ND = 7. However as was stated before, for L values out of this range the air microcavity could not be formed for the way as the HCPCF collapses.Some IFPIs considering different λ ≈ 10.82 nm, this considering that the silica wall has a refractive index n1 ≈ 1.4682 and thickness l2 = 70 μm. This can be appreciated clearly in Furthermore it is possible to expect that another FPI reflection spectrum can be present due to multiple reflections occurring within the silica wall. This FPI pattern should have Δ4.3.L was fixed to 200 μm while for one we IFPI a ND = 8 was used and for the other one ND = 12 was used. The measured spectra of these two IFPIs are shown in λ ≈ 22 nm while for the second one is Δλ ≈ 7 nm. This means that the length of the microcavity can be varied by applying different number of arc discharges (using the splicer program 2) to the HCPCF during the fabrication process. Moreover the fringe contrast is reduced when the optical distance is increased, which can be explained by the fact that the beam exiting from the SMF is diverging and this will strongly affect the contrast and the shape of the FPI fringes [In order to characterize the effects of applying different number of arc discharges (ND) on the spectral response other two IFPI were fabricated. For this two  fringes –26.5.3n) and its effect on the spectral response characterized. For instance a detail of the measured reflection fringe pattern, from 1,380 to 1,400 nm, is shown in 3n was varied however the fringe contrast is affected. In our case the measured fringe contrast variation was in the order of 4.7 dBm when the RI was changed within a range from 1 to 1.473 (see Some setups based on the FPI have been proposed to measure RI –30 which.473 see .n = 1.37) balanced with distillated water (n = 1.33) and the respective measured spectra from 1,360 to 1,460 was analysed (see In order to characterize the response of the IFPI to different concentrations of the same material some mixtures were prepared. For instance an IFPI was immersed in a mixture of 25%, 50% and 75% ethyl alcohol (ysed see . In this6.In this work a method to fabricate and IFPI based on an air microcavity was presented. It was shown that the fabrication method proposed provides flexibility to change the length of the air microcavity as well as it is low cost since requires the use of standard equipment. Moreover the spectral response of the IFPI and the air microcavity were characterized. Moreover it was shown that the spectral response of the fabricated IFPI is affected by changes of the medium around it. This is very important since it can be used to implement a simple and low cost refractive index sensor.


Today more than two million people with Turkish migration background live in Germany making them the largest ethnic minority in the country. Data concerning language skills and the perception of medical information in hospitalised patients with Turkish migration background (T) are scarce. Our study is the first to gather quantitative information on this important subject.T and hospitalised German patients without migration background (G) of our university hospital were prospectively included into a cross-sectional study and completed a questionnaire - each group in the appropriate language .121 T and 121 G were included. Groups significantly differed in age  and proportion of males  but not regarding the proportion of college graduates . The majority of T was born in Turkey (71%) and is of Turkish nationality (66%). 74% of T speak mainly Turkish at home; however, 73% speak German at work. 74.4% of T self-rated their German linguistic proficiency as “average” or better while 25.6% reported it as “very bad” or “bad”. 10.7% of T need translation in order to pursue everyday activities. T were significantly less satisfied with the physician’s information on disease and estimated to understand significantly less of what the physician told them: 46.3% of T estimated their reception of the physician’s information to be “average” or worse. 43.3% of T had the impression that it would have helped them “much” or “very much” to be aided by an interpreter at the hospital. The information transmitted while giving informed consent to invasive medical procedure was judged to be “mostly” or “completely” sufficient by the majority of T (76%) and G (89.8%). In this setting 37 of 96 T (38.5%) reported being helped by an interpreter – in most cases (64.9%) a family member.Although the majority of patients with Turkish migration background have spent most of their lives in Germany (28.94 ± 10.41y) a large part of this population has limited German language skills and difficulties obtaining medical information when hospitalised. One of the principles of modern Western medicine is the physician acting to the patient’s benefit after obtaining informed consent. The physician thus has to make sure that the patient has the ability to understand the relevant information, to grasp the medical consequences of the situation, to weigh up different treatment options and to communicate a choice . EnsurinLanguage barriers between patients and their care providers inflict an array of problems. Insufficient language abilities may lead to discrimination by a healthcare system , to an iTo overcome language barriers physicians have to rely on the help of interpreters. Professional medical interpreters would be most suitable for this purpose . HoweverOur hospital is located in the Ruhr-Area in the German state North Rhine – Westphalia (NRW). This region had been dominated by a thriving and labor-intensive coal mining and steel industry until the 1980s. Following a bilateral agreement between Turkey and Germany in 1961 many Turkish workers emigrated to this region . In 1996In our hospital patients with Turkish migration background (T) make up the largest ethnic minority. Currently a list of Turkish-speaking members of the hospital staff is available to find an interpreter more quickly. Courses in “medical Turkish” are offered for free to every employee. But further efforts to ameliorate the situation of Turkish speaking patients are to be taken since the above-mentioned measures have not lead to a perceptible improvement in everyday practice. However, it is difficult to overcome the language barrier with a tailored solution as long as the extent of the problem is not known. Our study is the first to evaluate and quantify language skills and perception of medical information in patients with a migration background in a German hospital.We conducted a prospective study of hospitalised patients at the University Hospital Marienhospital Herne, Germany. 121 T and 121 German patients without Turkish migration background (G) filled in a questionnaire during their hospital stay.The University Hospital Marienhospital Herne is part of the medical centre of the Ruhr – University Bochum, Germany. It is a 575 bed tertiary hospital with more than 20,000 admissions per year. The study was carried out at the larger of the two sites of the hospital with 362 beds. The following medical departments are located at this site: Internal Medicine, Nephrology, Pulmonology and Sleep Medicine, Gastroenterology, Cardiology and Angiology, Anaesthesiology, Oncology and Haematology, Geriatrics and Early Rehabilitation, General- and Abdominal Surgery, Vascular Surgery, Hand Surgery, Radiotherapy, Radiology, Gynaecology and Obstetrics including a unit specialised in diseases of the breast, the Newborn-Ward, the Interdisciplinary Intensive Care Unit, Central Patients Admission Unit and Palliative Care.Adult patients (aged ≥18 years) were prospectively included into the study between the second and third inpatient day. Informed consent was a precondition for participation. Patients were defined as having a Turkish migration background if they had migrated from Turkey to the current territory of the Federal Republic of Germany after 1949 or were born in Germany as Turkish citizens or were born in Germany as German Citizens but had at least one parent who migrated from Turkey after 1949 or was born as a Turkish Citizen in Germany  accordinA specific questionnaire was developed for a reliable quantification of the interaction between patients and physicians. If applicable the items of the questionnaire were shaded in 5 progressive grades to allow a more precise quantification and better comparability . Two bilingual translators converted the questionnaire from the German into the Turkish language. After a third translator had consented to it the questionnaire was tested with regard to its comprehensibility in a pilot test with patients from each group  and optimized thereafter. The following features were included in both versions of the questionnaire: migration background, demographic features and school education. Other items vary according to the patient’s ethnicity: 3 (T) items assessed language preferences during different activities of daily life (see Table After obtaining informed consent for study participation the questionnaire was completed by the study participants in the German (G) or Turkish (T) language depending on which group they belonged to. A bilingual person was available to help physically disabled or illiterate persons fill in the test.Analyses were conducted using SPSS . To compare numerical data the Student T test was chosen. Categorical data were compared using the Fisher's exact test. A binary logistic regression analysis was performed to assess the effect of different explanatory variables on the quality of the reception of medical information by T (Question: “How well did you understand what the physician told you?”). For the purpose of the regression analysis we divided each of the relevant explanatory variables into two categories  itself was categorised into “not at all”, “a little” and “mediocre” vs. “well” and “very well”. The study was approved by the ethics committee of the medical faculty of the Ruhr-University Bochum, Germany.121 T and 121 G were included in the study during 7 months between July 2011 and March 2012. During this period 13,034 patients were admitted to the hospital, 877 6.7%) of which were T according to an onomastic analysis. The demographic features and school education of both groups are shown in Table .7% of whTable The self-rated ability to communicate in German or Turkish (spoken and written or read) is depicted in Table Interaction between hospitalised patients and their physicians essentially takes place during daily rounds. Some of the questions used in our study are aimed at evaluating the patients’ perception of important aspects of the interaction with the physician. Table Obtaining informed consent to invasive medical procedure  is mandatory in many countries. In Germany the legislation obliges the physician to make sure that the patient has fully understood all relevant information before consenting to the procedure. In general, information on medical procedures is done verbally with the help of a standard brochure that will be individualised to the patient’s situation with schematic drawings and handwritten comments and signed by patient and physician. In our study 96 T and 108 G reported having been informed about a relevant diagnostic or therapeutic procedure. The responses to our questions concerning patient information are summarised in Table Among the 96 T that had been informed about a relevant diagnostic or therapeutic procedure (giving informed consent) 37 T reported that this was done with the help of an interpreter. Table Among 121 T 76 (62.8%) were female and 45 (37.2%) were male. Between T female and T male there was no statistical difference in terms of age , proportion of immigrants  and lifetime spent in Germany . Furthermore no statistically significant difference could be found concerning language preferences (German only vs. Turkish or other), self-rated German and Turkish linguistic proficiency and literacy, interaction between patient and physician in charge and being able to give informed consent.Of the 121 T 86 were born in Turkey (T born T), 35 were born in Germany (T born G). We did a subgroup analysis to differentiate between T depending on their place of birth. Language preferences of T depending on their birthplace are summarised in Table For the practice of medicine a sufficient exchange of information between physician and patient is crucial. Not only does the physician have to be able to comprehend and correctly interpret the patient but he is also obliged to sufficiently inform his patient in order to allow him making his decisions in self-determination. A language barrier is one of many obstacles in such a therapeutic relationship. Problems arising from language barriers have been described in many countries -16. TheySince our study is the first of its kind among German hospitalised patients it remains uncertain whether the ethnic composition of our sample is representative for other hospitals and situations. However, the findings of our current study go well with two German representative surveys among people with Turkish migration background conducted nationwide  and in tsine qua non for a successful therapy that is irrefutable from a legal point of view. It seems evident that overcoming language problems would be a first and important step toward better migrant health in Germany. In the current study the majority of T (50.4%) responding to that question would appreciate the help of a professional medical interpreter. Even though translation by a professional medical interpreter might also have its limitations [A previous study that did not assess language capacities has shown that Turkish migration background is an independent predictor for an unsuccessful rehabilitation treatment of patients with psychological or psychosomatic disorders in Germany . The woritations  it is thity of T 0.4% respity of T 0.4% respitations . Secondlitations . A promiitations . The resThe subgroup analysis comparing T depending on their place of birth (Turkey or Germany) depicted in Table There is evidence that gender has an influence on the integration of T into the German society. A transnational marriage to a T residing in Germany has been the most common motive for immigration among female T. Female Turkish immigrants are reported to reach a lower level of school education and the self-rated knowledge of the German language was lower among female T responding to a representative German national survey . In a reConducting a study among patients of different ethnic background and talking different languages brings along some challenges that we tried to overcome with our study design. We opted for a questionnaire in Turkish for T and one in German for G. As a bilingual person was always available and as practically all T spoke Turkish we never experienced any language difficulties concerning the administration of the questionnaire. Even though we put a lot of care and expertise in the generation and translation of the questions – especially the grading of the answers – we are conscious of the fact that answers to the same questions in different languages may not be suited for a direct comparison . Repetit55.4% of hospitalised patients with Turkish migration background (T) self-rated their German linguistic proficiency to be “average” or worse. Asked how well they understood what the physician told them, 46.3% of T respond “mediocre” or worse. The ability to comprehend medical information correlates well with the self-rated German language proficiency. The majority (50.4%) of T would appreciate the help of a professional interpreter “much” or “very much”. T born in Germany generally have less difficulties communicating in the German language and estimate to have a better capacity to cope with all other situations in the hospital they were asked about compared to T born in Turkey.The authors declare that they have no competing interests.AG conceived the study and its design, coordinated the study, drafted and revised the manuscript, performed the statistical analysis and contributed substantially to the interpretation of data. MU helped designing the study, translated the interview into the Turkish language, carried out the interviews, and was responsible for the acquisition of data and critical reading of the manuscript. HU participated in the conception and design of study as well as in the interpretation of data and critical revision of the manuscript, and translated the questionnaire into the Turkish language. SB contributed to the interpretation of data and the critical revision of the manuscript. BH participated in the conception of the study as well as its design and coordination and helped to draft 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/1472-6963/13/196/prepubQuestionnaire used in the evaluation of language competences in hospitalised patients with Turkish migration background in Germany and in native German controls.Click here for file


Panulirus argus) is an important economic activity throughout the Wider Caribbean Region both as a source of income and employment for the local population as well as foreign exchange for national governments. Due to the high unit prices of the product, international lobster trade provides a way to improve the livelihoods of fisheries-dependent populations. The specie harvested is identical throughout the region and end market prices are roughly similar. In this paper we wish to investigate to which extent lobster fishers’ job satisfaction differs in three countries in the Caribbean and how these differences can be explained by looking at the national governance arrangements.Lobster fishing (targeting the spiny lobster  A variety of researchers have carried out job satisfaction studies in North American fisheries during the last decades  is an important economic activity throughout the Wider Caribbean Region, both as a source of income and employment for the local population as well as foreign exchange for national governments. At present 50,000 lobster fishers are estimated to be active in the Wider Caribbean Region, with an additional 200,000 people working in positions related to the lobster fishery  the majority of respondents are small-scale. Only 4 respondents in Nicaragua employ industrial type methods. The industrial trapping boats are steel vessels with large freezer holds that go out to sea for 45 days at a time.The Likert scale was used to rank the responses to the 27 questions in the survey. When responding to a Likert questionnaire item, fishers specify their level of agreement according to a five-point scale ranging from (1) very unsatisfied, (2) unsatisfied, (3) neutral, to (4) satisfied and (5) very satisfied. In addition three overall questions on job satisfaction were added relating to whether a fisher would enter a job outside of fishing, move to another type of fishing and whether he or she would advise a young adult to enter the fishery.In Jamaica the survey was administered to a sample of 26 trap fishers and divers in six communities: Crawford, Black River, Alligator Pond, Whitehouse, Gallon beach, and Treasure Beach. These communities are located in Westmoreland parish. Westmoreland is one of four parishes with the highest production of lobster in Jamaica. Not all the fishers limit their activities to the inshore zone; some go for 8–9-day fishing trips to Pedro Bank, located some 80 km away from the mainland. The researchers approached fishers randomly at the landing beaches where they would arrive in the afternoon (in the case of day fishers) and in the night/morning (in the case of Pedro Bank fishers).In Nicaragua, the survey was carried out on Corn Island. Corn Island is responsible for half of the lobster landings in Nicaragua. Three fishing groups are available on the island: small-scale divers, small-scale trappers and industrial trappers. The industrial diving fleet, which is also of importance in the northern zone of Nicaragua, is not present in this area and therefore not represented in the survey. The researchers administered the survey to a sample of 26 fishers, 18 small-scale trap fishers, 4 industrial trap fishers and 4 small-scale divers. The researchers interviewed a random sample at the different lobster buying locations where fishers would sell their catch in the afternoon, or at the central ‘park’ on the island. Industrial fishers were approached at the piers of processing plants and the public pier where the industrial boats are stationed.In Belize, the survey was administered to a sample of trap and dive fishers in two locations. Twenty fishers were interviewed in Caye Caulker and eleven in Belize City. All lobster divers are stationed in Belize City. Caye Caulker is one of four coastal communities where day fishers use traps to catch lobster. A small cooperative associated with one of the big fishing cooperatives in Belize City is based in Caye Caulker. In both locations, the researchers approached fishers at the landing site Fig. .Fig. 1CuTable Table Fishing in Jamaica has long been a source of both food and employment. The fishery makes a significant contribution to the Jamaican economy accounting for 0.4 % of the GDP and 7.5 % of the output of the Agriculture, Forestry and Fishing sector combined Van Riel  see Tab. The secFinfish, both in volume and value, is the most important marine export product; conch is second, followed by spiny lobster and shrimp. On average 700,000 tons of lobster tails are exported a year. However, this is only 15 percent of the total catch as the majority of the lobster catch goes to local hotels and restaurants Venema .The current fishing industry is primarily small-scale. There are over 14,000 registered fishers, but estimates indicate that more than 20,000 Jamaicans are actually engaged in fishing FAO   said yes, 11 % said they were unsure and only 4 % said no. These responses indicate an ambivalence concerning the occupation, but the large percentage reporting that they would advise a young person to fish reflects positively on the desirability of fishing as an occupation.We first examine these responses in relation to background social variables Tables , 4, 5. FWillingness to change is expected to be related to levels of job satisfaction—the higher the satisfaction the less willing a fisher should be to change fishing type or leave the occupation of fishing, and the more willing they should be to advise a young person to fish. Since we are predicting the direction of the relationship, one-tailed statistical tests of significance are used. Results indicate that those more satisfied with management are less likely to say they would change their fishing type Table . This reFishermen scoring lower on the self actualization and nature categories of job satisfaction are more likely to report that they would leave the occupation of fishing Table  and thosLobster, shrimp and finfish occupy the 6th, 13th and 15th place in terms of the most valuable export products of Nicaragua Rivera , and theUntil the 1950s, exploitation of the lobster resource on the Caribbean coast of Nicaragua was limited, owing mainly to the difficulty of access to international markets  none of the other correlations are statistically significant Table . This isIn the Nicaraguan sample, only one fisher was willing to change fishing type or leave the occupation. The majority (69 %) reacted positively with regard to advising a young person to enter the occupation. Due to the extremely small number of fishers giving a positive response to the first two general questions, we are only able to conduct statistical analyses on responses concerning advising a young person to fish. However, the low level of responses to willingness to change type of fishing or leave the occupation and high numbers of those advising a young person to enter the occupation could relate to the fact the surveys are undertaken in an area where there are hardly any options other than lobster fishing. The economy of Corn Island depends highly on fishing, and alternatives are few.Willingness to change is expected to be related to levels of job satisfaction—the higher the satisfaction the more willing they should be to advise a young person to fish. One-tailed statistical tests of significance are used to test this relationship. Table We examined the responses in relation to background social variables but the only statistically significant difference is that those with more education (mean 6.7 years) are more likely than those with less education (mean 3.9 years) to advise a young person to enter the occupation of fishing Table . The relBelize fisheries cater to domestic consumption as well as export markets and provide a large measure of livelihood and employment. The export of sea products is an important source of foreign exchange. The Belize barrier reef supports a variety of fisheries of which the spiny lobster, queen conch and pink shrimp are among the most important commercial species caught. During the last 40 years however it has been the spiny lobster fishery that has dominated the fishing industry. Over 500,000 tons is exported annually, mainly to the USA FAO  see Tab.P. argus), conch (Strombus gigas) and a variety of finfish for the lucrative US and Caribbean markets . More often than not, fishers go out a few days a week and often combine fishing with other economic activities such as tourism. These fishers usually do not take ice to sea as the harvested lobsters are not killed until they reach shore.The Belize government has established a set of restrictions and regulations regarding the lobster fishery. There are laws stipulating a closed season and prohibiting the catch of undersized and molting lobsters, and berried females. The level of overexploitation of the lobster resource in Belize is disputed see FAO .The results of the job satisfaction study in Belize shows that fishers score high on the social needs category, followed by self actualization, basic needs, nature and management Fig. . Most scThe high scores on social needs and self-actualization could relate to the fishers’ autonomous position in the lobster fishery. They are often independent workers. Middlemen are present but fishers mostly sell to the cooperative to which they belong. None of the fishers spend more time away from their family than 9 days and if a fisher is not a captain he will at times skip a week to spend more time with the family. In addition, fishers work no more than approximately 8 h a day. In the evenings they listen to the radio and play games like domino. Day trap fishers are home every day with their family and friends and therefore do not miss spending time with them.The basic needs category scores high as well. This is related to the fact Belize is a multi-species fishery. During the closed season for lobster, fishers go out for finfish or conch; during the closed season for conch they target lobster and finfish. Because the fishery is organized around two fishing cooperatives which hold exclusive rights over seafood exports, fishers are able to reap some of the highest fishing incomes in the region Huitric . All proHowever, analyses of the general questions show that 32 % of the Belizean sample states they would be willing to leave their current fishing type, 61 % say they would leave fishing for another occupation and only 29 % point out that they would advise a young person to enter the occupation of fishing. In view of the high values recorded in Fig. Results show that there are no significant relationships between the background variables  and willingness to change occupation or fishing type Tables , 13. HowWillingness to change could also be expected to correlate with levels of job satisfaction—the higher the satisfaction the less willing a fisher should be to change fishing type or leave the occupation of fishing, and the more willing he should be to advise a young person to fish. Mean values on job satisfaction categories in relation to responses to these questions are examined in Tables Since this paper is predicting the direction of the relationship, one-tailed statistical tests of significance are used. The analysis presented in Table The three countries show distinct patterns in terms of job satisfaction outcome. Mean values for all scores, except management, fall above the mid-point of 3 indicating general satisfaction with the other four categories of job satisfaction. Table Belize and Nicaragua show a relatively similar ranking pattern except for the category of management. Belize scores very low on management whereas Nicaragua shows higher results. The reverse would actually be expected. Further research is needed to explain this discrepancy. The graphs show clearly that in all countries the category management scores lowest. Within this category fishers are especially dissatisfied with the performance of government officials Table . NonetheNicaragua also scores higher on the nature category than Belize. Jamaica, however, scores highest. Although the exact levels of overexploitation are unknown in Jamaica they are believed to be relatively high in comparison to Belize and at least equal to that of Nicaragua. According to the FAO  the lobsThis could be related to the fact the national tourist industry is also a large buyer of lobsters. Even though Jamaican fishers might be fishing undersized lobster they still have a market whereas this market is much smaller in Nicaragua and to a lesser extent in Belize.The categories of basic needs, social needs, and self-actualization scores are very low in Jamaica in comparison to the other two countries. Although fishers are relatively positive towards the level of exploitation they are much less satisfied with the extent to which their basic needs, social needs and self-actualization needs are met. Additional research is required to explain these results.Table There are no statistically significant differences between married and single fishers on the five job satisfaction categories Table . FishingA little less than half of the fishers, 47 % in all the countries said they would be willing to change fishing type. A higher number of fishers expressed that they would be willing to leave the occupation, a majority said they would advise a young person to enter the occupation of fishing.These responses indicate ambivalence. The analysis of the distributions in Tables Additional data shows that fishers with more education and smaller household sizes are less in favor of changing fishing type. In addition fishers with larger households are more likely to say they would leave the occupation of fishing. This is possibly in relation to their greater need for stable and predictable income. Finally, fishers who would advise a young person to enter the occupation are younger, have more years of education, and have fished fewer years. These fishers have obviously only recently made this decision themselves. These results are therefore to be expected. Older fishers have seen the resource decline over the last decades which would probably make them less inclined to advise a young person to enter the fishery.Thirty-one percent of the single fishers versus 56 % of married fishers say they would change fishing type, 44 % of the trap fishers and 40 % of the dive fishers report they would change fishing type—a difference that is not statistically significant. Finally, 43 % of the captains and 50 % of the crew say they would change type, a difference that is also not statistically significant.Willingness to change is expected to be related to levels of job satisfaction—the higher the satisfaction the less willing a fisher should be to change fishing type or leave the occupation of fishing, and the more willing they should be to advise a young person to fish. Mean values on job satisfaction categories in relation to responses to these questions are examined in Tables Table It is clear from the results that in all three countries, fishers like their occupation. The lobster fishers show a general satisfaction with their occupation. They highly value their occupation and despite an overall declining resource a majority said they would advise a young person to enter the occupation of fishing.As the resource is declining throughout the area, a question concerning characteristics of the occupation of fishing that make it attractive arises. The data show that the categories basic needs, social needs and self-actualization score high . Consequently, if one wishes to provide an alternative occupation to fishers, it will need to be attractive to them on those accounts.Across all three countries fishers are least satisfied with management. This is shared in all job satisfaction studies carried out, where fishers targeted government officers as the object of their greatest dissatisfaction (Bavinck and Monnereau


Three-dimensional (3D) structure determination by single particle electron cryomicroscopy (cryoEM) involves the calculation of an initial 3D model, followed by extensive iterative improvement of the orientation determination of the individual particle images and the resulting 3D map. Because there is much more noise than signal at high resolution in the images, this creates the possibility of noise reinforcement in the 3D map, which can give a false impression of the resolution attained. The balance between signal and noise in the final map at its limiting resolution depends on the image processing procedure and is not easily predicted. There is a growing awareness in the cryoEM community of how to avoid such over-fitting and over-estimation of resolution. Equally, there has been a reluctance to use the two principal methods of avoidance because they give lower resolution estimates, which some people believe are too pessimistic. Here we describe a simple test that is compatible with any image processing protocol. The test allows measurement of the amount of signal and the amount of noise from overfitting that is present in the final 3D map. We have applied the method to two different sets of cryoEM images of the enzyme beta-galactosidase using several image processing packages. Our procedure involves substituting the Fourier components of the initial particle image stack beyond a chosen resolution by either the Fourier components from an adjacent area of background, or by simple randomisation of the phases of the particle structure factors. This substituted noise thus has the same spectral power distribution as the original data. Comparison of the Fourier Shell Correlation (FSC) plots from the 3D map obtained using the experimental data with that from the same data with high-resolution noise (HR-noise) substituted allows an unambiguous measurement of the amount of overfitting and an accompanying resolution assessment. A simple formula can be used to calculate an unbiased FSC from the two curves, even when a substantial amount of overfitting is present. The approach is software independent. The user is therefore completely free to use any established method or novel combination of methods, provided the HR-noise test is carried out in parallel. Applying this procedure to cryoEM images of beta-galactosidase shows how overfitting varies greatly depending on the procedure, but in the best case shows no overfitting and a resolution of ~6 Å. (382 words) •A new method to validate 3D cryoEM maps of biological structures is described.•High-resolution noise substitution is a tool to measure the amount of overfitting of noise in single particle cryoEM.•A reliable, unbiased resolution estimation can be obtained even when some overfitting is present.•Structure of beta-galactosidase at ~6 Å resolution is determined by cryoEM. The field began with the pioneering work of Dubochet and colleagues, who developed conditions for rapidly freezing thin films of biological macromolecules in amorphous ice As the field has grown, a need for quality control criteria and validation methods has become more important. In a recent report summarising the current consensus These needs are most vital for single particle cryoEM, because the Fourier transforms of the raw images and the final three-dimensional (3D) map are continuous. They do not have the empty space that is found between the diffraction spots in 2D crystals or between the layer lines for helical assemblies, which can then be used to estimate signal-to-noise ratio. Progress at the first level of overall validation at lower resolution has been made through the use of tilt pair validation In this paper, we focus on the middle level of validation, which concerns the inter-related problems of overfitting and resolution estimation. In conventional single particle cryoEM analysis, the density in a single 3D map is derived from many 2D images of identical structures in different orientations, whose geometrical relationship to the 3D model are deduced by iterative refinement of five parameters (two for position and three for orientation) for each 2D image. If there were accurate prior knowledge of these 5 parameters for each 2D image, the resulting 3D map would consist of a 3D image of the true structure plus random noise arising purely from the statistical nature of the contributing low-dose images. In practice, however, the positions and orientations must be iteratively refined against the gradually improving 3D model. Since the raw images contain much more random noise than real features at high resolution, the positions and orientations are biased slightly by fitting of the 2D noise to features in the 3D map. As the cycles of iteration proceed, some noise features in the 3D map gradually build up and bias the 2D particle parameters so that these false features are locked into the 3D map. The final global convergence of 3D map and 2D parameters thus creates a 3D map that consists of a 3D image of the true structure, plus genuine random noise, plus a third component that consists of noise arising from overfitted parameters. In this paper, we use the terms overfitted noise, overfitted parameters and overfitting in general to describe this phenomenon. The term over-fitting was first used in a similar context by Stewart and Grigorieff The problems of both overfitting and resolution estimation have been the subject of a great deal of discussion over the last few years, so it is useful to review what has been said before. We then describe our proposed tool to allow definitive measurement of any overfitting, however large or small, that is associated with the processing of a particular data set, and to validate the estimate of the overall global resolution of a 3D map. We deliberately aimed to develop a procedure that avoided using atomic coordinates for validation, since once a structure reaches the stage of being interpretable in terms of a model, such as when α-helices or β-sheets are recognisable, the FSC between map and model is a reliable way to evaluate both the procedure used and the final map.Our proposal is to process, alongside the original data set, a data set in which the raw images are modified by high resolution noise (HR-noise) substitution, and to compare the resulting FSC curves. This procedure can be used for validation whether or not an atomic interpretation is proposed or available. The HR-noise substitution procedure may also be useful to measure undesirable resolution artefacts due to masking that may affect the FSC. The procedure we describe does not test the correctness of any low-resolution starting model: another procedure such as tilt pair validation is needed for that. Nor does it test whether the final map retains any initial model bias.1.1The Fourier transforms of single particles are continuous, so it is not as easy to determine signal-to-noise ratio as a function of resolution as it is with 2D crystals The early introduction of the Fourier Shell Correlation (FSC) function These procedures and the accompanying resolution criteria worked adequately for structures calculated from images of negatively stained particles, where signal-to-noise ratio is high. However, problems appeared when 3D structures were calculated from cryoEM images of ice-embedded single particles, caused by the lower contrast and higher noise, resulting in the need to combine many more single particle images. One of the first papers to note the new problems posed by the intrinsically lower signal-to-noise ratio Grigorieff showed 3, in Frealign so that the alignment would be guided only by regions of the molecular transform where diffraction was strong and would ignore regions where the noise was dominant. In addition to using this weighting scheme, they also maximised the modulus of CC rather than CC itself, so that some of the very noisy images with large negative values of CC would be subtracted from the 3D average and so counteract the noise accumulation. These two improvements, implemented in Frealign There have been a number of attempts to improve the accuracy of alignment of the individual images against the 3D model during the iterative refinement procedure, and to minimise the build-up of overfitted noise. For example, Stewart and Grigorieff In EMAN2 In other words, although it has been known for at least 12 years that overfitting, with its accompanying overestimation of resolution, can be avoided by the strict quarantine of the two half data sets throughout the refinement procedure, this separation has not been widely adopted. It is still common practice to carry out the iterative refinement against a common 3D reference and then, at the very end, to split the data set into two halves and calculate the FSC, which as a result cannot be used to distinguish genuine structure from overfitted noise. The problems of reliable resolution estimation and overfitting of noise have therefore become intertwined.It has also been shown A variant on the exclusion of the highest resolution shells of reciprocal space during particle alignment was suggested by Shaikh et al. The use of these above two procedures for alignment, namely the splitting of data into two completely independent halves, or exclusion of the highest resolution (and weakest) zone have thus been known for many years to allow structure analysis that avoids overfitting and gives reliable resolution estimates. However, neither has been popular for two reasons, one logical and one cosmetic. The logical reason is that the exclusion of any data that could help orientation determination must reduce the accuracy of a structure determination. The two independent 3D maps, each calculated from half the data, must be less accurate, and therefore the particle orientations will be less accurate and therefore the final structure calculated by averaging the two maps will be less accurate. The cosmetic reason is that any overfitting that cannot be distinguished from a genuinely higher resolution makes the results of a research project appear better than they are, thus  impressing colleagues, referees and grant-awarding bodies.A good understanding of how this situation arose is apparent from a recent publication that discusses the definition and estimation of resolution in single particle reconstructions The main argument for using all the images to calculate the best 3D map at each cycle and using all the diffraction data out to the highest possible resolution for the orientation determination is that the orientations obtained must be more accurate if the 3D reference contains all the information available. So it is understandable that there has been a reluctance to split the data into two halves and keep them completely separate, or to impose a resolution cut-off in data used for orientation determination. Both these procedures, in different ways, use less than the complete available information for parameter refinement.The Protein Data Bank organised a meeting of an electron microscopy validation task force (EMVTF) around these topics to discuss the future development of validation criteria and try to extract some kind of consensus Many image processing packages, as an integral part of the process, subject the 3D map at each cycle to a low pass filter that removes the high-resolution data that is most susceptible to overfitting before it is used as a 3D reference for the next cycle of orientation determination, sometimes increasing the resolution as the refinement proceeds In a recent advance, Scheres and Chen 1.2t when carried out with the original data set and FSCn for the HR-noise data set. Any non-zero FSCn values beyond the resolution where the HR-noise was introduced is then due to overfitting and the true resolution obtained can be determined by a comparison of FSCt with FSCn. Since the experimenter is allowed to apply any 3D reconstruction method or combination of methods, including new algorithms or tricks of their choice, we hope it will become a popular as well as foolproof method of measuring overfitting and validating resolution. Since the HR-noise data set contains very slightly less information about the structure than does the original data set, it is possible that the particle orientations obtained may be very slightly less accurate, but this should not affect the value of FSCn at high resolution, where only noise is being fitted. We estimate, using Scheres' estimate of the contribution of different frequencies to orientability The core idea we propose to get round all these problems is therefore to create a second stack of single particle images that are identical in every way to the original raw particle images, except that the structure factors  beyond a certain chosen resolution are substituted by pure high-resolution noise (HR-noise). This second data set is then subjected to the identical image processing procedure used for the original experimental data, followed by calculation and comparison of the FSC between the two halves of the data. We refer to the two FSC curves as FSCThe build-up of noise after many cycles of 3D reconstruction and orientation refinement depends on many parameters including the detector resolution and particle masking, both of which affect inter-pixel correlation. Therefore, to ensure that the substituted HR-noise data set responds in the same way to the 3D reconstruction procedure as does the original data, the high frequency noise should have the same power spectrum as the raw data, and preferably also the same inter-pixel correlation. The raw image data can come from detectors with different point spread functions and correspondingly different modulation transfer functions and from specimens prepared in different ways sometimes on a carbon substrate with an underlying non-zero noise contribution. Consequently, the simplest way to obtain noise with exactly the same power spectrum and inter-pixel reciprocal-space correlation as found in the particles is to use an adjacent featureless area of the image close to each particle. Since picking the noise regions can be tedious and may be a barrier to its ease of use, we have compared the substitution of high-resolution noise from an area in each micrograph that is adjacent to each particle with the simpler procedure of randomising the phases beyond the chosen resolution. Since we find no difference in outcome between these two methods of introduction of HR-noise, we recommend high-resolution phase randomisation as the preferred procedure in practice. We hope this will be useful to evaluate established procedures as well as future innovations.2E.coli beta-galactosidase  in 30 mM phosphate buffer at pH 7.0 to Quantifoil grids (1.2 μm holes), followed by blotting for 5 s and plunge freezing into liquid ethane at near liquid nitrogen temperature. Grids were transferred into an FEI Polara G2 electron microscope and low-dose images recorded either on film at 80 keV and 39,000x magnification or on a Falcon II CMOS direct electron detector at 300 keV and 80,240x magnification . The pixel size was 6 μm on film and 14 μm on the Falcon detector, which translates into a sampling rate of ~1.5 Å/pixel with film and ~1.75 Å/pixel with the Falcon. Both nominal pixel sizes required later refinement by up to 3% against an atomic model from X-ray crystallography Specimens were prepared by application of 3 μl drops of a 1 mg/ml solution of Two complete data sets were collected. The first consisted of 52 images on film. The second consisted of 89 images on the FEI Falcon II CMOS detector. Stacks of particles were picked by hand using Ximdisp E. coli beta-galactosidase structure in vacuo, another map with a B-factor of 2000 and a scale factor of 0.8. This solvent correction affects only the low-resolution structure: its effect falls to about a third at 20 Å and is negligible by 10 Å. The 80% factor is a rough approximation for the amount of electron scattering by ice compared with protein although a slightly lower value of 72% was estimated previously A starting reference map was calculated from the atomic coordinates of an makestack_HRnoise, which substitutes either the amplitudes and phases from an accompanying stack of featureless areas selected from adjacent regions of the same images, or alternatively randomises the phases of the particle structure factors beyond a chosen resolution. The first option creates high-resolution noise with the same image statistics including adjacent inter-pixel correlation. The second option retains the original particle amplitudes but removes any phase correlations between adjacent pixels. Exploring both types of noise substitution was designed to ensure that the HR-noise test represented an information-free control.The high-resolution noise substitution was performed by a programme named t, and the FSC for the HR-noise substituted stack FSCn. If we assume that the amount of overfitted noise in FSCt is similar to the amount in FSCn and that the difference represents the true correlation of genuine structural features, we can deduce the value of FSCtrue as follows.Image processing of the particle stacks to produce 3D maps was carried out using the standard single particle iterative procedure of parameter determination and 3D structure calculation, following the normal protocols in each case, as for example with Frealign S represents a Fourier component of the structure, Nof represents overfitted noise and Nran represents random noise, then the signal-to-noise ratio (SNR) and FSC are given byIf t and SNRn, but both overfitted and random noise appear in the denominator of SNRtrue.Note that only random noise appears in the denominator of SNRIt is easiest to prove the following equation is true by substituting true has been calculated from FSCt and FSCn.Eq. 3A small region of a typical image of beta-galactosidase is shown in t and FSCn, shaded in pink, shows the amount of true signal from real features of the structure. It is clear that, from 6733 particle images on film at 80 keV, the resolution reaches about 13 Å. If the orientation parameters are refined using only data to 17 Å, The particle images recorded on film were processed using Frealign and the results are shown in 2, effectively amplifying the contribution of the high resolution information to the correlation function . Using the recommended value of RB-factor (0 Å2) gave less overfitting, whereas a larger value (−300 Å2) gave more overfitting. true from FSCt and FSCn, is plotted in purple. This shows that the resolution of the final 3D map was 6.9 Å. The same resolution was obtained when the RB-factor used was 0 Å2. Since this resolution is slightly beyond the 7.0 Å resolution used for orientation refinement, it is unaffected by the HR-noise control. In the region between 7.0 Å and 10.0 Å resolution, the effect of overfitting is fairly small, but without carrying out the HR-noise control, we would not have known its extent. It is also clear that the quality of the 300 keV/Falcon experimental single particle images is significantly better than for those obtained at 80 keV on film.A data set was then collected at 300 keV using a backthinned Falcon CMOS direct electron detector. The result of processing 43758 particle images from 89 micrographs using Frealign is shown in The 300 keV data set was then processed using Relion The HR-noise substitution test is also valuable for program development. The procedure we describe can be used to improve the algorithm in any new procedure with the goal of minimising overfitting and maximising resolution. We have therefore tested it on a new, unpublished 3D reconstruction procedure in the process of being developed by one of us (GM), and the results are shown in t and FSCn of 3D masking at three increasingly stringent levels is shown in t and FSCn is similar and the same Eq. Although the HR-noise substitution procedure was designed to show the effect of overfitting random noise in cryoEM images and its propagation into the final 3D map, we discovered that the HR-noise map can also be used to measure the effect of different types of mask on the FSC curves. The effect on FSCLastly, in The best resolution between map and flexibly-fitted model shown in t and FSCn curves shown in Finally, 4R-factor in X-ray crystallography Rfree and Rwork are both calculated. Rwork always decreases since it refers to the data being used in refinement, but Rfree decreases only if the model is genuinely improving and by a greater amount when the improvement is greater. The difference between Rfree and Rwork measures the amount of overfitting, whereas the value of Rfree gives an unbiased estimate of how well the model agrees with the observations as a function of resolution. In our case, no data are excluded from the structure determination; in this sense it differs from the free R-factor approach. Instead, in the parallel set of HR-noise substituted data, noise replaces the data beyond a resolution where the real structural information is sufficiently small in total power (estimated to be less than 5%) that it has a negligible effect on the accuracy of orientation determination in the HR-noise control data set. Any non-zero value of the FSCn above the resolution cut-off used in the HR-noise test gives a measure of the amount of overfitting, whereas the difference between the two curves (FSCt and FSCn) gives an unbiased estimation of resolution. As we have shown, it is also possible to deconvolute the unbiased part of the FSCt curve using FSCn to give FSCtrue.There is some similarity between our proposed resolution and overfitting test and the use of the free Rfree and Rwork, for example more than 5%. Resolution is estimated from the point at which Rfree exceeds a threshold value that is agreed by the community based on experience, normally around 30-35%. In cryoEM, there is no consensus yet on an acceptable amount of overfitting and what threshold value of FSC should be used in resolution estimation. We would argue that it is simplest to use one of the established procedures [e.g. 10] to obtain a map with no overfitting and to determine resolution using the 0.143 criterion. However, clearly a small proportion of overfitting of, for example, less than one third of the total power in a particular resolution shell simply introduces some extra noise into the 3D map that can be dealt with by using less sharpening or more damping. Consequently, we argue that any method of cryoEM structure analysis can be used even when it is known to be susceptible to overfitting, provided it is accompanied by the HR-noise test. The HR-noise test is also compatible with the proposed introduction of single particle Wiener filters that restrict the FSC to the region of the 3D map occupied by the particle t by the mask will also appear in FSCn, thus maintaining the integrity of the HR-noise test, as shown in In the X-ray case, overfitting becomes apparent as a wider gap between true would then be the correct one to use in calculating signal-to-noise weighting of the map using the Cref formula, sometimes called figure-of-merit (FOM) weighting, proposed by Rosenthal and Henderson true gives an reasonable resolution estimate of 6.5 Å and because the power at high resolution was very weak, the map was not unduly noisy.Note that the appearance of a large degree of overfitting, as seen for example in Note also that the slightly different resolutions obtained using the different programme packages after application of Eq. Stewart and Grigorieff used a related approach to the one we have introduced here when exploring noise bias. In their 2003 paper t falls to 75 or 80%.There is one important parameter in the HR-noise test: the resolution beyond which HR-noise should be introduced. If it is too low, then the particle orientations will be degraded in the HR-noise comparison and the difference between the two FSC curves will give a false reassurance about resolution. If the resolution is too high, then all the HR-noise will be beyond the resolution where the FSC becomes weak, and the exercise will be uninformative. It is therefore important to choose a resolution where the signal from the structure is sufficiently weak that it contributes little or nothing to the accuracy of the orientation determinations, yet is strong enough to be distinguished from noise in the 3D map. The tilt pair approach showed that most of the information which determines orientation in current images is at low or medium resolution, below 15 Å. Scheres The procedure we describe represents a new tool that can be used alongside others to help the experimenter to judge the quality of a 3D single particle structure. It can also measure the robustness of an existing or new protocol in protecting against any intrinsic tendency to overfitting. We hope it will be generally useful both for programmers and experimentalists.5In single particle electron cryomicroscopy, there are two accepted ways to prevent overfitting. One way is to compare maps obtained by dividing the data in half, and processing each set in a completely independent way throughout the refinement; the resolution obtained by comparing how well the two maps agree is then used to filter a map obtained using all the data. The other way is to limit the resolution for determination of orientation parameters. Some individuals may prefer other methods that have some susceptibility to overfitting, but may not know whether their resulting structure is compromised. The method of HR-noise substitution described in this paper can measure overfitting and validate the map resolution with any image processing procedure. It is also useful in deconvoluting the effect of applying a molecular envelope or mask.6Chen prepared cryo-specimens and carried out the electron microscopy data collection. Faruqi and McMullan helped to develop the detector used in collecting the 300 keV images. Scheres performed computer analysis using the Relion and Xmipp software packages, used in


Immunosuppression in breast cancer has been reported in women and in the highly metastatic mouse mammary tumor model 4 T1. The immunosuppressive environment complicates the use of the humoral response against the tumor as an immunodiagnostic tool. IgM has not been used in immunodiagnostic in part because its antitumor responses, both innate and adaptive, have not been studied in function of time in breast cancer.We show a new approach to analyzing the mouse humoral immune response, and compare the evolution with time of IgG and IgM responses against the antigens of 4 T1 cells.The study is based on 2-dimensional immunoblotting detection of antigens from 4 T1 cells by the IgG and IgM antibodies in the serum of female mice injected with 4 T1 cells.There was a high variability in the intra-and inter-mouse response. Variability in the IgM response was manifested as a pattern of spots that could become a multibinomial variable of 0 and 1, which could represent a signature of the immune response. Different numbers of spots was found in the IgG and IgM responses from week 1 to 5. On average, the IgM had more but the IgG response decrease with the time. The natural IgM at t = 0 responds stronger than w1; the adaptive response of both IgM and IgG were elicited where, with the former being stronger better than the latter. Antigens that are recognized by some female mice in the first week are also recognized by other female mice at time 0. Contamination of the natural IgM makes difficult use the adaptive IgM as a tool for immunodiagnostic.IgM and IgG response varied with the time and individuals. Spot variation in 2D pattern for the natural IgM could be expressed as a binomial signature, which opens up the way to correlate a particular pattern with resistance or susceptibility. This uncovers a battery of IgMs for each individual to confront cancer or infections. The possibility to differentiate between adaptive IgM antibodies from the natural IgM will allow investigation of the adaptive IgM for early immunodiagnosis. During a lifetime, human beings are permanently exposed to the development of transformed cells, which arise spontaneously or by contact with trigger factors. Immune surveillance of cancer leads to the generation of anti-tumor antibodies from the early stages of the disease. Once a The images in Figure + cells in the apparent absence of antigen stimulation, which constitutes most of the circulating IgM-1-propanesulfonate; SDS: Sodium dodecyl sulfate; HCl: Hydrochloric acid; KCl: Potassium chloride; NaOH: Sodium hydroxide; ∑: Summation; t = 0: Time 0.The authors declare that they have no competing interests.MDZ carried out aspects of the mouse experiments and 2D Immunoblots, reviewed the manuscript its scientific veracity. RH participated in the execution of the mouse studies and participated in all experiments. POS conceived, designed and analyzed the collective results of all the experiments, and wrote the manuscript. All the authors read and approved the final manuscript.MDZ has a Doctoral fellowship from the Consejo Nacional de Ciencia yTecnología, México. RH is a Master of Science and has an academic position at the Instituto de Investigaciones Biomédicas. POS is a PhD in Biochemistry and is a Researcher at the Instituto de Investigaciones Biomédicas. All work at the Universidad Nacional Autónoma de México.


Gila cypha (humpback chub) in the regulated Colorado River and the unregulated tributary and primary spawning area, the Little Colorado River. We develop eight multistate models for the population based on three movement hypotheses, in which states are defined in terms of fish size classes and river locations. We fit these models to mark–recapture data collected in 2009–2012. We compare survival and growth estimates between the Colorado River and Little Colorado River and calculate abundances for all size classes. The best model supports the hypotheses that larger adults spawn more frequently than smaller adults, that there are residents in the spawning grounds, and that juveniles move out of the Little Colorado River in large numbers during the monsoon season (July–September). Monthly survival rates for G. cypha in the Colorado River are higher than in the Little Colorado River in all size classes; however, growth is slower. While the hypothetical life histories of life-long residents in the Little Colorado River and partial migrants spending most of its time in the Colorado River are very different, they lead to roughly similar fitness expectations when we used expected number of spawns as a proxy. However, more research is needed because our study period covers a period of years when conditions in the Colorado River for G. cypha are likely to have been better than has been typical over the last few decades.While the ecology and evolution of partial migratory systems (defined broadly to include skip spawning) have been well studied, we are only beginning to understand how partial migratory populations are responding to ongoing environmental change. Environmental change can lead to differences in the fitness of residents and migrants, which could eventually lead to changes in the frequency of the strategies in the overall population. Here, we address questions concerning the life history of the endangered  Partial migration, in which a portion of a population is migratory and the rest is sedentary, is a widespread phenomenon observed in a variety of taxa, including fish  found in the headwaters of the Bitterroot River (Montana) was not directly related to presence of the dam, but rather to changes in factors such as predation or temperature in the main river (hereafter mainstem).River systems are among the most modified ecosystems worldwide  was widely distributed in the Colorado River  and slow-speed boat electrofishing . Sampling in the LCR consisted of mark–recapture events spanning the whole LCR study site and mark-only events over a more limited spatial extent Table . Mark-onUpon capture, chub were measured, scanned for prior marks, and, if appropriate, marked. All chub >100 mm total length (TL) that did not already have a 134.2 kHz passive integrated transponder (PIT) tag received a PIT tag with a unique number allowing future identification of individual fish. Juvenile chub (40–100 mm TL) received a visible implant elastomer (VIE) mark that identified the current sampling trip and spatial location. This occurred regardless of whether a fish was previously marked, so juveniles accumulate marks if they are caught on multiple trips until they grow to greater than 100 mm TL. As a consequence, each juvenile carries its capture history with it (in the form of VIE marks); however, information on individual covariates is lost. Juveniles that were captured or recaptured in the LCR study site during spring sampling received no mark, and these captures were not included in our analysis.We use multistate models conditioned on first capture Arnason  to test Our study period is from April 2009 and October 2012, and we assume a monthly time step for modeling. Of a possible 43 months, the Colorado River study site is sampled 15 times and the LCR is sampled 23 times Table . In othel at time t survives from month t to t + 1, regardless of whether the individual transitions into another state during the same interval. All the models examined in this paper assume that survival is constant through time  growth conditional on survival and (2) movement conditional on survival and growth. Growth was assumed to be constant over time, to vary between those states in which growth was possible , and to be the same for a given size class found in either of the two Colorado River locations. As such, we considered eight growth probabilities, γi, where the first four parameters  correspond to the probability of transitioning out of size classes one through four in the LCR, and the next four  are defined as the probabilities of growing out of size classes one through four in the Colorado River.In our model survival, τ, of chub within the Colorado River are found within the Colorado River study site, regardless of size class. This assumption should be approximately true provided that vital rates in the Colorado River study site are similar to elsewhere in the Colorado River. Based on these assumptions, we decomposed movement in a vector or probabilities, t and t + 1. For chub moving from the LCR into the Colorado River, τ to express movement into the Colorado River study site and multiplied by (1 − τ) for movement into areas outside of the Colorado River study site. For states associated with size classes 2 and 3 , ω7 = ω12). Lastly, because no instances of juvenile fish moving from the Colorado River to the LCR were observed in our data set, the associated parameters (ω6) were fixed at zero, for all t.Movement probabilities were constrained based on two assumptions. First, we assumed that movement of chub between the Colorado River study site and other parts of the Colorado River is negligible. We base this assumption on studies over both short and long temporal scales that suggest that chub exhibit strong patterns of spatial fidelity (outside of migration) when they are in the Colorado River, restricting their movements to an average linear extent of around 500 m  to state 6 (juvenile in the Colorado River study site) can be expressed as follows:We incorporated the joint effects of survival and transition probabilities in an array, φ1), the probability of not growing into state 2 (1−γ1), the probability of moving from the LCR into the Colorado River τ).which states that the probability of surviving and moving from state 1 to state 6 is the product of the probability of surviving in state 1  show that VIE loss rate is higher for individuals growing quickly in warmer water, we also include a monthly mark loss rate (π) of 0.03 for VIE tagged fish in the LCR where juvenile growth rates are higher.We assume that when states were observed, they were observed perfectly . We account for the possibility that chub could be in a particular state and be unobserved using a vector of parameters, Hypothesis 1 (H1): Larger adults spawn more frequently than smaller adults, so chub in state nine (size class four) will have lower movement rates  and thus more likely to remain in the LCR in the future than other adults . In other words, movement of potential adult residents out of the LCR  will be less than movements of migrants, especially in the 2 months after spawning , the probability of surviving from July of a fish's first year to the larger mature size class (size class 5), and the expected life span of size class 5 chub conditional on them having survived to that size . As a measure of the relative fitness of each strategy, we also derive the expected number of spawns as a large adult for a fish that survives to July of its first year and then adopts either the migratory or the resident strategy by multiplying the probability of surviving to size class 5 under each strategy, by the expected number of spawns after reach size class 5. We also derived estimates of abundance for different states and for the overall adult abundance using the Horvitz–Thompson estimator (see Appendix S3 for details).The best model according to QAIC supported all three movement hypotheses Table  and had Monthly survival rates are higher in the Colorado River than in the LCR for all size classes Fig. A. In addτ as 0.18 (95% CI: 0.13–0.24), within the range we predicted a priori. Derived estimates of juvenile abundance in the Colorado River study site increased each year between July and September consistent with our juvenile movement hypothesis , ignore substantial uncertainty, and occurred during a period when conditions in the Colorado River were favorable. For example, average water temperatures in the regulated Colorado River were ∼1°C colder during the 1980s through early 2000s as compared to our study period (and presumably chub growth rates were likewise lower). Furthermore, salmonid abundances near the confluence were relatively low during the study period compared with estimates from other times in the last few decades . The increase in abundances between July and September of 2010 (and between August and September 2009), in contrast, is much smaller, suggesting either that the export rate is not constant or that there was a smaller population of juveniles in the LCR to begin with in July. Interestingly, there was substantially more flooding during the monsoon season in 2011 and 2012 than in 2009 and 2010, suggesting a potential link between export and LCR flood frequency. Unfortunately, we are not able to estimate juvenile abundances in the LCR during July, and the relatively small number of marks put into the LCR during July does not allow us to estimate year-specific rates of outmigration over the study period. Ongoing research is increasing effort in July to obtain abundance estimates and allow for yearly estimates of outmigration rates.τ) currently resides in the Colorado River during the nonspawning season, and vital rates under the environmental conditions experienced in 2009–2012 suggest that juveniles can successfully rear to adulthood in the Colorado River mainstem . As LCR residents would have a quicker generation time, they should also increase (or decrease) more quickly. On the other hand, 82% of the adult population . Moreover, as system dynamics are relatively slow, conditions in the Colorado River will likely have to persist for long periods of time to change adult population sizes. Vital rates and abundances of smaller size classes will provide a leading indicator of future adult population size. However, given the long periods of time spent in these vulnerable life stages, a year or two of beneficial conditions can be easily negated by a change in these conditions. Future work will focus on exploring annual variation in vital rates, particularly as salmonid abundances in the Colorado River may be increasing.One explanation is that under current environmental conditions, both strategies have roughly similar fitness's and are contributing to a fairly stable or slightly increasing adult population; however, when environmental conditions in the Colorado River decline, the migrating population in the Colorado River may become a population sink (sensu Pulliam