The rapid growing of information technology (IT) motivates and makes competitive advantages in health care industry. Nowadays, many hospitals try to build a successful customer relationship management (CRM) to recognize target and potential patients, increase patient loyalty and satisfaction and finally maximize their profitability. Many hospitals have large data warehouses containing customer demographic and transactions information. Data mining techniques can be used to analyze this data and discover hidden knowledge of customers. This research develops an extended RFM model, namely RFML (added parameter: Length) based on health care services for a public sector hospital in Iran with the idea that there is contrast between patient and customer loyalty, to estimate customer life time value (CLV) for each patient. We used Two-step and K-means algorithms as clustering methods and Decision tree (CHAID) as classification technique to segment the patients to find out target, potential and loyal customers in order to implement strengthen CRM. Two approaches are used for classification: first, the result of clustering is considered as Decision attribute in classification process and second, the result of segmentation based on CLV value of patients (estimated by RFML) is considered as Decision attribute. Finally the results of CHAID algorithm show the significant hidden rules and identify existing patterns of hospital consumers. Customer relationship management (CRM) denotes that managerial efforts to business processes and technologies that designed to understand the customers of a firm . RecentlUnderstanding more about high-value and potential patients, including the hospital departments and services that they utilize, will help hospitals appropriately design resource allocation strategies for health care services . For disBackgroundCustomer relationship management (CRM) CRM is a philosophy that anticipates customer needs with the purpose of providing the target customers with the right product, at the right time, in the right place . EnterprCLV, RFM , wRFM and eRFM definitionsCustomer lifetime value (CLV) is typically used to identify profitable customers and to develop strategies to target customers . General1) Recency of the last purchase (R): refers to the interval between the time that the latest consuming behavior happens and present. The shorter the interval is, the bigger R is.2) Frequency of the purchases (F): refers to the number of transactions in a particular period, for example, two times of one year, two times of one quarter or two times of one month. The many the frequency is, the bigger F is.3) Monetary value of the purchases (M): refers to consumption money amount in a particular period. The much the monetary is, the bigger M is.Wu & Lin  in theirRRcj+ WFFcj+ WMMcj, where WR, WF, WM are the relative importance of the RFM variables. wRFM integrates analytic hierarchy process method (AHP) and data mining to recommend products based on customer lifetime value. The wRFM based method employs association rule mining to identify recommendation rules from customer groups that are clustered according to weighted RFM values. There are two opposing views about weights of the three variables of RFM in the literature that they are identical or not. But some research indicated that recency, frequency and monetary are different in the importance due to the characteristic of the industry  Can produce understandable rules;2) Perform tasks without much computing3) Can handle continuous and categorical variables4) Can learn which attributes are important for classification .Decision tree algorithm has been used in several studies for the purpose of customer database segmentation , 46. A CClustering: K-means applicationClustering is the process of grouping a set of physical or abstract objects into groups of similar objects . A clustResearch frameworkconstraints in the health care industryA patient is considered as a customer in health care industry in the literatures. This means a health care organization is equal to a business. So health care services marketing is an important concern of decision makers and stockholders. However there are some differences between patient going to a hospital and a customer going to a shop for buying goods or services. The major differences are:1- Customer needs considered as permanent needs such as food and other things that need for living and comfort. But patient needs considered as case based temporary needs that may occur suddenly.2- There is negative value of customer life time value in health care industry. Because patients preferred to improve as soon as possible. In other words short customer life is preferable. This is in contrast with traditional customer life time value.3- Most of time patients are referred to the special hospital for various reasons such as: doctor prescription or order, lack of adequate equipment for specific disease or hospitalization and emergency department or triage referring and etc. These differences are obstacles to behave patient as customer in marketing strategies in health care services but many researchers don’t assist them. This paper tried to minimum this conflict, therefore considers three conditions for analyzing patients information. First, only outpatient data  are extracted. Second, a longer period is considered for extracted data from HIS (from 2009 to 2012) that finally, causes the large data records of patients for evaluating . In these circumstances we can see to a patient as a customer but even may be the cases that didn’t satisfy the conditions which the long period time and large data decrease this contrast.Empirical study Shohadaye Tajrish Educational Hospital  founded in 1966 on Tehran, Iran has more than twenty specialist clinics and four hundred beds considered as a big public sector hospital. Data records are extracted from hospital information system (HIS). Due to the nature of treatment industry and contrast between customer loyalty and patient loyalty, we select outpatient data records  to analyze patients’ behavior to detect all kind of the hospital customers and level of the loyalty. The time period taken in this study is four years. Period of time has long been considered as possible to increase transparency of patient behavior. The number of total outpatient transactions is 768,864 from September 2008 to October 2012. The study collects the data set of 234,690 patients who visited these emergency clinics in four years period.Weighted RFMLR, WF, WM and WL. We used questioner and interview to determine efficient factors on patient loyalty that add to RFM model and weights of the extended model from administrative managers and medical directors of ST Hospital . According to the assessments, the relative weights of the RFML variables are 4.5, 8.3, 8.2 and 4.9 respectively . The RFM model has been applied to various industries but it is rarely used in the context of the health care industry . Some reStructure of the modelStructure of the model is shown as flowchart in Step 1. Data records collection : The study selected data fields or attributes based on the RFML model from outpatient datasets of the HIS. These required features of the patients include: ID, age, gender, date of referring or admission, type of used services and the amount paid. In the first, datasets of ST Hospital are preprocessed to extract the customer transactions. 768,864 data records that belonged to 234,690 patients are collected. Each record contains information about each patient admission or transaction.Step 2. Data records preparation (ready data): Data preprocessing is an inevitable step in knowledge discovery process. This step first, eliminates records which include noisy, inaccurate and missing values. In this case more than 150,000 records are deleted. These records include incorrect values such patient referrals with zero payment or invalid date and etc. Second, some features of data records are transformed into a format to be ready for next step. For example date of admission transformed to the number. Finally, RFML variables are calculated based on all transactions of each patient that make a table with 234,690 records. Also according to the assessments obtained by the AHP, the relative weights of the RFML variables are determined.Step 4. Data normalization.We normalize R, F, M and L variables to be consistent in terms of numerical unit. So they are standardized to the interval . The equations for normalization are shown below : i / Max (Vi) that V={R, F, M, L}V= VStep 5. Determination of the cluster number (K)Despite the classification algorithms, the segments are unknown in the clustering algorithm. But the number of segments must be defined by user in some algorithms such K-means. In this study Two step method is used to predict the optimum K or number of clusters. Two step algorithm don’t need the exact number of clusters which was defined by user while it finds the optimum number of cluster in a range which defined by user . Here, tStep 5-1. Patient ClusteringThe general purpose of clustering attempts to maximize the inter-cluster distance (to ensure the clusters are well separated) while minimizing the intra-cluster distance (to ensure compactness of the clusters) .According to quantitative value of normalized R, F, M and L attributes for each patient, data partitioned into 5 clusters using the K-means algorithm (based on two ways: non weighted and weighted parameters). Step 5-2. Estimation of CLVThe proposed optimum K is used to categorize all patients by customer value or CLV to K segments. The output of this segmentation will be used as decision field for classification. In the first, CLV is calculated based on weighted RFML method for each customer by equation 3. Relative importance of the variables is 4.5, 8.3, 8.2 and 4.9 respectively. Ci= NRci*WR + NFci*WF+ NMci*WM+ NLci*WL (equation 3)CLVSecond, patients are categorized into 5 segments  based on CLV of each patient. Now we have loyalty attribute for each patient that show customer class based on CLV value. Finally, this attribute is considered as decision field for classification in the step 5-2-2.Step5-1-1 & Step 5-2-1. Data discretization For improving the classification accuracy and solving the challenge of generating a large number of decision rules in the segmentation models, attributes are granulated . ApplyinStep5-1-2 classification by clusters (approach 1) Decision tree algorithm (CHAID) is used to generate the decision rules set from the experimental dataset for classifying ST hospital patients. Practically, a classification model is first built on a training set. Afterwards, the model is validated using previously unseen data, the test set . The expStep 5-2-2 classification by CLV (approach 2)In the approach, customers are classified with CHAID algorithm based on CLV categories as decision field (step 5-2). Step 8. Compare & extract rulesOrder of variables importance in the both methods is the same. This shows recency and length respectively have the greatest impact on identify target customers in health care services. Results of output fields of two approaches by comparing actual and predicted class  displayed the first approach, classification by k-means, has more accuracy (89.69%) than classification by CLV (80.98%). This experiment was examined by C5 and neural network algorithms for evaluations. Performance evaluations results are shown in Findingsclustering analysis Extract and interpret the rules CHAID method based on importance degree of input variables creates a decision tree. Decision tree in this experiment has depth of 4 that Recency_TILE5, Length_TILE5, Frequency_TILE5 and Monetary_TILE5 variables are entered respectively . All patients that last visit were about 947 days ago (about 3 years ago- first year of the considered period time of this study) definitely fall in cluster 1 or churn customers. The number of churn customers are considerable (41.17%). But the hospital loses them and must spend a lot of money for acquisition of this market segment. Hospital management should try to compensate this by improving quality of the services and strong customer relationship to cause less churn rate from now.Patients that last visit were about 1.5 years ago (second year of the considered period time of this study) with low and medium monetary (<M5), despite the low value of frequency and length attributes can be valuable customers as low value patients. The number of this segment is considerable (42.47%). Due to temporary needs of people to health care services this market segment may return and use the hospital services when they need. Due to the large number of this patients and likelihood of readmission, the administrators can guarantee significant profitability in the future by spending money to build a strong relationship with these customers.Patients that last visit were about 6-7 month ago with long time activation (L5), upward monetary (>=M4) and minimum number of visit 4 times (F>=F4) are very high value customers. This segment is profitable following of loyal customers but the number is more than loyal patients . The hospital management should operate on these patients has become loyal customers by introduce new, free and supplementary services.Patients that last visit were about 1 year ago with long term activation (L5) and frequency more than 2 times (>=F3) are high value customers. The result shows monetary of this patient are significant. Ranking of all segment based on meaning rules extracted from decision algorithm (CHAID) are: loyal segment, very high value, high value, medium value and churn group in ascending order that the number of each segment are: 1.45%, 5.99%, 8.92%, 42.47% and 41.17% respectively. The administrators should adopt appropriate strategies to suit each group in order to further profit for the hospital.conclusion and suggestion for marketing development Nowadays, health care industry like other industries should interact with patients as customers and implement strong marketing strategies for equation and retention of them. There are some differences between patient and customer needs and requirements which must be considered for assessment them. This research develops a classification method based on weighted eRFM (RFML) with clustering and customer life time value (CLV) as two approaches. The results show classification based on clustering (k-means) has more accuracy than CLV method. The outcomes of the study identify all patients’ classes of ST hospital and behavior of them. More revenue of the hospital generated from less than 1.5% all customers as loyal patients, so the managers should adopt special and custom relations to hold and retention of the profitable market. A large number of patients placed in churn group (41.17%) that shows such shortcoming or low quality of hospital services. Also the result shows there is a significant group (42.47%) that recently visited with medium monetary and low frequency and length attributes who is a new opportunity for the hospital. The hospital administrators should establish excellent relationship and implement marketing strategies to improve satisfaction of this new market. This article highlighted the role of Customer Relationship Management in the hospitals and proposed a classification model for identifying all kind of patients to improve marketing strategies. Further research will focus on the effective factors and reasons on choosing hospital by patients with aim of services quality improvement.


Introduction: Anterior mini-thoracotomy approach is a good alternative to median sternotomy in Atrial Septal Defect (ASD) repair. Our purpose is to explain the details of our technique and peresent the results.Methods: Seventy five patients with ASD  were operated with anterior mini-thoracotomy approach in our tertiary center between March 2012 and March 2014. The mean age was 14±10 ranged from 2 to 42 years. Outcomes were defined according to cardiopulmonary and aortic cross-clump time, intensive care unit stay time, morbidity, mortality, the size of incision, the amount of post-operative bleeding, need for blood transfusion and reoperation.Results: Mean Cardiopulmonary bypass time was 49.62 minutes (26 to 105 minutes) and mean aortic cross clamp time was 22.29±6.77 minutes (11 to 47 minutes). The mean amount of blood transfusion was 47.49± 62.22 mm (0 to 200 cc) and the mean chest tube drainage after surgery was 80.17 ±121.06 mm (0 to 600 cc). One patient re-operated for dehiscence of ASD surgical sutures and there was no reoperation for surgical bleeding or tamponade drainage in these patients. In 74 cases the defect was secundum type, in 2 patients it was sinus venosus type and in one with associated partial Anomalous repair.Conclusion: Anterior thoracotomy approach is safe and may be the surgical technique of choice for secundum ASD repair in all age groups and we can utilize this technique also for more complicated kinds of surgery for instance, sinus venosus type ASD with or without Partial Anomalous Defect. Per-cutaneous closure of defect with amplatzer is the technique of choice in the most frequent form of ASD (secundum type) but if this modality cannot be used because of the lack of borders in the foramen or failing the technique, invasive surgery is recommended. In the most traditional form of surgery midline sternotomy and use of total Cardiopulmonary Bypass (CBP) with ascending aorta and Superior Vena Cava (SVC) and Inferior Vena Cava (IVC) cannulation is recommended. Recently anterolateral thoracotomy techniques with peripheral cannulation of aorta , IVC and SVC is discussed by some authors, but peripheral cannulation needs one more incision and it is more complicated in very low weight children or in fat peoples and has its own risks of peripheral vessels damage. Also in these modalities the need for long and narrow cannulas and anesthesiologists help and for cervical cannulation of SVC and also the need for negative pressure pump reservoir is mandatory that would cost more. Also device closure of secundum ASDs is sometimes needed in young children; however, little is known about the safety and outcome of this procedure in infants.In this study, the safety and efficacy of secundum ASD closure with anterior thoracotomy approach was evaluated. Since the trends of these young groups of patients are for minimal incisions, we would choose a technique by right anterior mini-thoracotomy approach with single sub-mammillary incision and central cannulation of ascending aorta, SVC and IVC. Surgical techniques and our 2 years of experiences and results are discussed in details.ASD repair was attempted in 75 patients through anterior mini-thoracotomy approach in our tertiary research center between March 2012 and March 2014. All patients were operated by a single cardiac surgeon. Age ranged from 2 to 42 years; mean 14.06±10.49, 52 were female (69.3%).Their weights ranged from 7 to 82 kg and their heights from 76 to 180 cm.th or 5th intercostals space, exactly in the mid part position between right clavicle and lower part of the anterior ribs was performed with minimal rib spreading. First, we prefer to open the chest with a 2 cm incision and estimate the position of pericardium in small sized patients, heart and diaphragm and if it is in the mid-portion between diaphragm and ascending aorta, then the incision can be expanded, otherwise we can go one space up or down to achieve the best exposure to both inferior vena cava and ascending aorta. A rib retractor should be employed and the right lung should be retracted posteriorly with a wet long gaze or a gaze suitable to the size of the patients. We open Pericardium 2 cm above phrenic nerve and in the cases with very large right thymus lobe; a partial thymectomy can be utilized for better exposure, Then we can cut a piece of pericardium for subsequent closure of ASD, and the stitch sutures can be used for retraction of pericardium for better exposure. Central cannulations of aorta were performed in all except 3 and the superior vena cava and inferior vena cava were cannulated from this single incision in all patients. We put the aortic cannula in the top of incision and the two venous cannulas in the lower part of our incision; there would be no interfering with good exposure.Single lumen endotracheal intubation was performed for all the patients. One longitudinal pad was used under right shoulder of patients with about 20 degree elevation for better exposure. The skin incision (4-8 cm length) in right anterior 4The incision is single anterior thoracotomy and we perform this kind of cannulation for all patients from 12 months (6 kg) child with a narrow femoral artery and vein to a 100 kg weighed 40 years old lady with very bad femoral exposure. After the systemic administration of the heparin, cannulation of ascending aorta with downward traction of aorta by a simple clump superior vena cava after expose it above the reflection of pericardium and right atrium near inferior vena can be done. We used to use minimally invasive instruments, although simple instruments are enough for use. We generally cannulate ascending aorta directly by 10 to 18 French Edwards Life Science Fem Flex Femoral Arterial Cannula depending on patient’s weight .2, ascending aorta was cross clamped with a DeBakey type Trans-thoracic aortic clamp through the third intercostals space in the mid-axillary line. This incision could be used for later chest tube insertion but we clamped the aorta from our thoracotomy incision directly in most of the cases.Superior vena cava would be surrounded with tape for ligation before we cannulate it and we surround inferior vena cava with tape after beginning of CBP and stopping ventilation. Superior vena cava and Inferior vena cava cannulate by malleable venous cannulas. After starting CBP and stopping ventilation and sending a tape around the inferior vena cava in normothermia and without the use of CoMyocardial protection would be achieved with ante grade cardioplegic solution infusion through an angiocath at the ascending aorta, then right atrium opene and stay sutures are used for better exposure. After examination of both mitral and tricuspid valves we can repair the defect with pericardial patch. In first cases, we closed ASD directly but because we had a case with detachment of ASD closure in suture lines, now we almost always use a pericardial patch for the closure of ASD. After de-airing of left atrium from the last suture, atrium closure and de-airing of ascending aorta from the insertion point of cardioplegia and de-clamping of Aorta, we can close this point with a simple stitch. After discontinuation of CPB, de-cannulation, administration of protamine, hemostasis, and chest tube insertion, the incision can be closed in 4 layers’ of soft tissue and sub-coetaneous stitches for skin closure . We did th post-operative day and because of epilepsy this patient stayed in intensive care unit for 6 days after reoperation.We had no early postoperative mortality. CPB time was 26 to 105 minutes (mean 49.62±15.71 minutes). Aortic cross-clamp time can also imply the difficulty of a cardiovascular surgery and were from 11 to 47 minutes (mean 22.29±6.77 minutes). Incision sizes were from 4 to 8 cm (mean 4.98±1.01 cm). Intensive Care Unit stay time was from 2 to 6 days (mean 2.57 ±0.79 days) and the mean amounts of blood transfusion during and after operation were 47.49±62.22 milliliters range from 0 to 200 cc and mean post-operative chest tube drainage were 80.17±121.06 ranges between 0 to 600 cc . There w4 Izzat et al. used mini-thoracotomy approach in ASD closure.5 Percutaneous approach was first introduced by Bennhagen et al. in 2001.6 Butera et al. compared per-coetaneous closure of ASD with surgical closure and described their indication.7 Chang et al. compared sternotomy with thoracotomy approach in ASD closure.8 Mishra et al. compared mini-thoracotomy with trans-catheter approach in ASD closure.9 We used mini-thoracotomy approach in 76 cases in last 2 years. we approached all our ASD cases from anterior mini-thoracotomy in the last 2 years and there were no sternotomy cases for comparison. In Mishra group, lateral thoracotomy with peripheral cannulation was used and our incision is more acceptable because of aesthetics reasons and especially in ladies it can be hidden behind breast. All our patients were operated by one incision and we cannulated all cases from this incision from 12 months (6 kg) child with a narrow femoral artery and vein to a 100 kg weighed 40 years old lady with very bad femoral exposure. The only complication was a case of dehiscence of sutures that was operated by primary closure of ASD and after this complication, we preferred to use pericardial patch for all our cases for reducing tension on suture lines. CPB time is a useful operative measure in cardiac surgery and aortic cross-clamp time which can also imply the difficulty of a cardiovascular surgery, was both acceptable and a little longer than Mishra group, but we closed all Foramens with a pericardial patch in our experience that needs a little more time and our incisions were shorter which requires us to be more precise in operation (mean 4.98 cm in our group and 8 cm in Mishra group). There was no case of surgical drainage or blood or fluid induces tamponade in our experience. Our first cases complained of chest wall pain and nipple numbness but after discontinuation of rib suture usage, these complaints discontinued. Now we close chest-wall fascia in 2 layers and sub-coetaneous and skin in 2 other layers.Lancaster et al. compared thoracotomy with sternotomy in surgical closure of ASD.Anterior mini-thoracotomy approach for repair of ASD brings benefits to patients and is very practical if designed correctly, even though it is a more complex surgical procedure with longer CPB and aortic cross-clamp time, it can be the preferred approach if the surgeon has had enough experiences .In our point of view, anterior thoracotomy approach is safe and may be the surgical technique of choice for secundum ASD repair in all age groups and we can utilize this technique also for more complicated kinds of surgery for instance, sinus venosus type ASD with or without partial anomalous defect. After all, this approach neither needs additional instruments nor more expenses.This study was approved by our local Ethics Committee.Authors declare no conflict of interest in this study.


The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins.DOI:http://dx.doi.org/10.7554/eLife.11897.001 Human, yeast and other eukaryotic cells contain compartments called mitochondria. These compartments are surrounded by two membranes and are most famous for their essential role in supplying the cell with energy. While mitochondria can make a few of their own proteins, the vast majority of mitochondrial proteins are produced elsewhere in the cell and are subsequently imported into mitochondria. During the import process, most proteins need to cross both mitochondrial membranes.Many mitochondrial proteins are transported across the inner mitochondrial membrane by a molecular machine called the TIM23 complex. The complex forms a channel in the inner membrane and contains an import motor that drives the movement of mitochondrial proteins across the membrane. However, it is not clear how the channel and import motor are coupled together. There is some evidence that a protein within the TIM23 complex called Tim44 – which is made of two sections called the N-terminal domain and the C-terminal domain – is responsible for this coupling. It has been suggested that mainly the N-terminal domain of Tim44 is required for this role.Banerjee et al. used biochemical techniques to study the role of Tim44 in yeast. The experiments show that both the N-terminal and C-terminal domains are essential for its role in transporting mitochondrial proteins. The N-terminal domain interacts with the import motor, whereas the C-terminal domain interacts with the channel and the mitochondrial proteins that are being moved.Banerjee et al. propose a model of how the TIM23 complex works, in which the import of proteins into mitochondria is driven by rearrangements in the two domains of Tim44. A future challenge is to understand the nature of these rearrangements and how they are influenced by other components of the TIM23 complex.DOI:http://dx.doi.org/10.7554/eLife.11897.002 Mitochondria perform a number of essential cellular functions ranging from production of ATP and diverse other metabolic intermediates to initiation of apoptosis. It is thus not very surprising that disturbances in mitochondrial function are associated with a number of human diseases, including neurodegenerative disorders, diabetes, and various forms of cancer . An esseThe TIM23 complex mediates translocation of presequence-containing precursor proteins into the matrix as well as their lateral insertion into the inner membrane. The latter process requires the presence of an additional, lateral insertion signal. After initial recognition on the intermembrane space side of the inner membrane by the receptors of the TIM23 complex, Tim50 and Tim23, precursor proteins are transferred to the translocation channel in the inner membrane in a membrane-potential dependent step . The traThe translocation channel and the import motor of the TIM23 complex are thought to be coupled by Tim44, a peripheral inner membrane protein exposed to the matrix . Like otTim44 binds to the Tim17-Tim23 core of the translocation channel . Tim44 aTim44 is composed of two domains, depicted as N- and C-terminal domains . Recent in trans, can support, although poorly, growth of yeast cells, giving us a tool to dissect the role of the C-terminal domain in vivo. We identify the C-terminal domain of Tim44 as the domain of Tim44 that is in contact with translocating proteins and that directly interacts with Tim17, a component of the translocation channel. Our data suggest that intricate rearrangements of the two domains of Tim44 are required during transfer of translocating precursor proteins from the channel in the inner membrane to the ATP-dependent motor at the matrix face.We report here that the function of the full-length Tim44 cannot be rescued by its N-terminal domain extended to include membrane-recruitment helices of the C-terminal domain, demonstrating an unexpected essential function of the core of the C-terminal domain. Surprisingly, we observed that the two domains of Tim44, when expressed URA plasmid carrying the wild-type, full-length copy of Tim44, no viable cells were obtained  with that of the strain having two Tim44 domains, both containing A1 and A2 helices, expressed and 30°C . Still, We isolated mitochondria from FL and N+C strains grown on fermentable medium and compared their mitochondrial protein profiles. Immunostaining with antibodies raised against full-length Tim44 detected no full-length protein in N+C mitochondria but rather two faster migrating bands . Based oWe conclude that Tim44 can be split into its two domains that are sufficient to support the function of the full-length protein, although only poorly.in vivo △DHFR, and pSu9(1–69)DHFR, were drastically reduced in N+C mitochondria when compared to wild type. Import of presequence-containing precursor of Oxa1 that contains multiple transmembrane segments was similarly impaired. Likewise, precursor proteins that are laterally inserted into the inner membrane by the TIM23 complex, such as pDLD1 and pcytb2, were imported with reduced efficiency into N+C mitochondria. In agreement with the established role of Tim44 in import of precursors of a number of components of respiratory chain complexes and their assembly factors, we observed a slightly reduced membrane potential in N+C mitochondria as compared to wild type △DHFR as a TOM-TIM23-spanning intermediate. Briefly, this precursor protein consists of the first 167 residues of yeast cytochrome b2, with a 19 residue deletion in its lateral insertion signal, fused to the passenger protein dihydrofolate reductase. In the presence of methotrexate, that stabilizes folded DHFR, the b2 part reaches the matrix, whereas the DHFR moiety remains on the mitochondrial surface resulting in an intermediate that spans both TOM and TIM23 complexes. The association of Tim44 and its domains with the arrested precursor protein was analyzed by chemical crosslinking followed by immunoprecipitation with antibodies to full-length Tim44 and its individual domains. In wild-type mitochondria, all three antibodies precipitated a crosslinking adduct of Tim44 to the arrested precursor protein, demonstrating that they are all able to immunoprecipitate the respective antigens , suggesting that the yeast system is more robust. This observation is in agreement with the notion that mutations that would severely affect the function of the TIM23 complex would likely be embryonically lethal in humans. Still, the disease caused by a mutation in the C-terminal domain of human Tim44 speaks for an important role of this domain in the function of the entire TIM23 complex. Furthermore, the mutation maps to the short loop between A3 and A4 helices in the C-terminal domain of Tim44. Based on the crystal structure of Tim44, it was previously suggested that the mutation could affect the conformational flexibility of the A1 and A2 helices  containing a full-length TIM44 followed by replacement of the chromosomal copy of TIM44 with a HIS3 cassette by homologous recombination. For complementation analyzes, endogenous promoter, mitochondrial presequence (residues 1–42) and the 3’-untranslated region of TIM44 were cloned into centromeric yeast plasmids pRS315 (LEU marker) and pRS314 (TRP marker) and obtained plasmids subsequently used for cloning of various Tim44 constructs. The following constructs were used in the analyzes: Tim44(43–209), Tim44(43–262), Tim44(264–431), and Tim44(210–431). The constructs encompassing the N- and the C-terminal domains of Tim44 were cloned into pRS315 and pRS314 plasmids, respectively. Plasmids carrying the full-length copy of TIM44 were used as positive controls and empty plasmids as negative ones. A Tim44 plasmid shuffling yeast strain was transformed with two plasmids simultaneously and selected on selective glucose medium lacking respective markers. Cells that lost the wild-type copy of Tim44 on the URA plasmid were selected on medium containing 5-fluoroorotic acid at 30°C.Wild-type haploid yeast strain YPH499 was used for all genetic manipulations. A Tim44 plasmid shuffling yeast strain was made by transforming YPH499 cells with a pVT-102U plasmid  , Tim44(43–209) (referred to as N in E. coli BL21(DE3) at 37°C and purified using affinity chromatography on NiNTA-agarose beads  followed by gel filtration on Superdex 75 column . Unless otherwise indicated, the His6-tags were removed by incubation with the TEV protease. The purified proteins were stored at -80oC in 20 mM HEPES/KOH, 200 mM KCl, 5 mM MgCl2, pH 7.5, until use.DNA sequences coding for various segments of Tim44 were cloned into bacterial expression vector pET-Duet1 introducing a TEV cleavage site between the HisoC. After three washing steps, specifically bound proteins were eluted with Laemmli buffer. Samples were analyzed by SDS–PAGE and immunoblotting.Purified proteins were coupled to CNBr-Sepharose beads  according to manufacturer's instructions and stored at 4°C. The beads were used for purification of domain-specific antibodies from the serum raised in rabbits against recombinantly expressed full-length Tim44. For direct binding analysis, mitochondria isolated from wild-type yeast cells were solubilized with 0.5% Triton X-100 in 20 mM Tris/HCl, pH 8.0, 80 mM KCl, 10% glycerol at 1 mg/mL and incubated with Tim44 constructs coupled to CNBr-Sepharose beads for 30 min at 4Thermal stabilities of wild type and P282Q mutant form of Tim44 were analyzed by fluorescence thermal shift assay . RecombiPreviously published procedures were used for protein import into isolated mitochondria, crosslinking, coimmunoprecipitations and arrest of mitochondrial precursor proteins as TOM-TIM23 spanning intermediates followed by crosslinking and immunoprecipitation under denaturing conditions . review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and three reviewers, one of whom, Klaus Pfanner, is a member of our Board of Reviewing Editors. The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.Thank you for submitting your work entitled "Rearrangements of the two domains of Tim44 drive protein translocation into mitochondria" for consideration by Summary:Mitochondrial import of matrix proteins requires the mitochondrial import motor to drive protein translocation. The manuscript by Banerjee et al. investigates the structure function relation of Tim44, an essential subunit of the mitochondrial import motor. To dissect the N- and C-terminal domains functionally, the authors expressed these domains in trans and find that they suffice to confer viability to yeast cells lacking TIM44. Their analyses reveal that mitochondria with "split" Tim44 display transport defects along the presequence import route. Their analyses suggest that the N- and C-terminal domains engage with different modules of the presequence translocase. In comparison to full length Tim44, the split protein causes reorganization within the presequence translocase, implicating Tim44 in translocase/motor communication.This is an interesting analysis that provides new and unexpected insight into how the import motor constituent Tim44 engages with the presequence translocase. Moreover, the data provides evidence for a link between motor dynamics and translocase organization. Thus, the manuscript addresses a long standing question on how the dynamic of the import motor is linked to translocase function.Essential revisions:1) Since rearrangements of the two domains of Tim44 are not directly demonstrated, the title should be modified to better reflect the main message of the paper.2) The observed import defects of sorted and matrix targeted precursors is surprising. Why are sorted proteins affected that should not depend on the ATP driven motor and thus not on Tim44 function? The authors should assess the membrane potential directly to exclude indirect effects. Since the membrane potential-dependence of precursors varies a lot, AAC import alone might be misleading. In addition, carrier transport should be analyzed also by BN PAGE.3) Ting et al. (2014) reported that the N-terminal domain of Tim44 interacts with Tim23 as well as motor components by using crosslinking in intact mitochondria. Using purified Tim44 domains and mitochondria lysed with detergent, the authors confirmed the interaction of the N-terminal domain with motor components, however, not with Tim23. They speculate that crosslinking of the N-terminal Tim44 domain to Tim23 requires a specific conformation of Tim23 only adopted when Tim23 is bound to Tim17 in the inner membrane (i.e. in intact mitochondria). Since the authors demonstrate the interaction of the C-terminal domain of Tim44 with Tim17 by using the purified domains and lysed mitochondria, but use crosslinking in intact mitochondria for showing the interaction with precursor proteins, the two approaches should be compared more directly. A suitable approach would be to analyze the interaction of the C-terminal domain of Tim44 with Tim17 in intact mitochondria by crosslinking. They may use radiolabeled Tim17 imported into mitochondria and their domain-specific Tim44 antibodies to address this topic.4) The purification experiments shown in 5) 6) The conclusion that the TIM23 complex adopts an "altered conformation" in N+C mitochondria is based on crosslinking experiments. It would be good to support this conclusion by an independent approach, such as native gel electrophoresis or differential protease sensitivity of the TIM23 components.Moreover, the term "altered conformational flexibility" is not fully fitting to the results presented here. The authors rather look at an altered subunit composition and not protein conformations. The text should be changed.7) The part on the human patient mutation mimicked in yeast Tim44 is interesting, but not required for the rest of the story. Does the mutation in Tim44 affect the TIM23 complex or protein import in yeast or humans? As it stands it is not very informative. The authors should either remove that part or perform a functional analysis, i.e. test mutant cells/mitochondria for the functionality of the Tim44 C-terminal domain in the binding of Tim17 and precursor binding during mitochondrial import. 1) Since rearrangements of the two domains of Tim44 are not directly demonstrated, the title should be modified to better reflect the main message of the paper.The title was modified to now read “Two domains of Tim44 have different roles in the presequence translocase during transport of proteins into mitochondria”. We agree that the title may have been somewhat misleading as we have indeed not directly demonstrated rearrangements of the two domains of Tim44.2) The observed import defects of sorted and matrix targeted precursors is surprising. Why are sorted proteins affected that should not depend on the ATP driven motor and thus not on Tim44 function? The authors should assess the membrane potential directly to exclude indirect effects. Since the membrane potential-dependence of precursors varies a lot, AAC import alone might be misleading. In addition, carrier transport should be analyzed also by BN PAGE.We would not agree that the import defects of TIM23 substrates we observed in N+C mitochondria are in general surprising. Tim44 has been identified over twenty years ago as an essential component of the TIM23 complex required for transport of presequence-containing precursors into mitochondria. Over the years, several temperature-sensitive mutants of Tim44 have been identified that specifically affected transport along the TIM23 pathway. We would thus rather argue that it would be surprising if in N+C mitochondria, which contain only a split version of this protein, no import defect was observed. We were certainly surprised to see that the N+C cells are viable at all. The point that we observed no difference between sorted and matrix targeted precursors may potentially be seen as surprising. However, it should also be said that the role of Tim44 in the differential sorting of proteins by the TIM23 complex has not been carefully analyzed, at least to the best of our knowledge, in contrast to mtHsp70 and the J proteins Tim14/Pam18 and Tim16/Pam16. While the latter proteins are very likely directly involved in the ATP-dependent steps of protein transport, the exact role of Tim44 is far less clear. Indeed the data presented in this manuscript would suggest a role of Tim44 also at an earlier stage of protein transport, which could affect both sorted as well as matrix-targeted precursors. Also, we have previously seen that Tim44 can be crosslinked to laterally sorted precursors , even when their transport does not depend on the ATPase activity of the import motor. However, as differential sorting of precursors by the TIM23 complex is not a focus of this manuscript, we removed this point from the text to simplify it for non-specialists.We measured the membrane potential in FL and N+C mitochondria and, not surprisingly, observed its slight reduction in N+C mitochondria, in agreement with the very strong growth defect of N+C cells and with the established essential role of Tim44 in transport of a number of components of respiratory chain complexes and their assembly factors into mitochondria. In this respect, it is also worth noting that even in glucose medium at either 24°C or 30°C, N+C cells grow about three times slower than FL. In addition, rather than analyzing import of AAC by another method, we analyzed import of another TIM23-independent substrate, Tim23 itself. Like with AAC, we also observed no major difference in its import efficiency in FL and N+C mitochondria. These data are now included in 3) Ting et al. (2014) reported that the N-terminal domain of Tim44 interacts with Tim23 as well as motor components by using crosslinking in intact mitochondria. Using purified Tim44 domains and mitochondria lysed with detergent, the authors confirmed the interaction of the N-terminal domain with motor components, however, not with Tim23. They speculate that crosslinking of the N-terminal Tim44 domain to Tim23 requires a specific conformation of Tim23 only adopted when Tim23 is bound to Tim17 in the inner membrane (i.e. in intact mitochondria). Since the authors demonstrate the interaction of the C-terminal domain of Tim44 with Tim17 by using the purified domains and lysed mitochondria, but use crosslinking in intact mitochondria for showing the interaction with precursor proteins, the two approaches should be compared more directly. A suitable approach would be to analyze the interaction of the C-terminal domain of Tim44 with Tim17 in intact mitochondria by crosslinking. They may use radiolabeled Tim17 imported into mitochondria and their domain-specific Tim44 antibodies to address this topic.We agree that it would be excellent to demonstrate a direct Tim17-Tim44 interaction also by another assay such as crosslinking. However, to the best of our knowledge, no crosslinks of Tim17 to any other component of the TIM23 complex have ever been seen in wild type mitochondria, radiolabeled or not, not even to Tim23, even though their interaction was demonstrated by several different assays. The only crosslinks to Tim17 observed so far were upon a very comprehensive cysteine-scanning mutagenesis of Tim23 followed by cysteine-specific crosslinking  and the above mentioned paper by Ting et al. that used site-specific UV crosslinking to demonstrate an interaction between Tim17 and Pam17. Since the surfaces of either Tim17 or Tim44 involved in this interaction remain currently unknown, we feel that undertaking such a comprehensive site-specific approach would be beyond the scope of this manuscript.4) The purification experiments shown in  All the experiments presented in this manuscript are representatives of experiments repeated at least three times. In addition, all the critical experiments were not only performed with at least three biological replicates but were also repeated by at least two different researchers to make sure that all the observed effects are reproducible.Following the suggestion of the reviewers, we included a panel containing quantifications of binding of mtHsp70, as a representative of the import motor components, and of Tim17 to the various beads that were obtained in three different experiments. In addition, we added to the Figure legend the information that input contains 4.5% of the material used for binding. This information was unfortunately missing in the original submission.The original 5) The new 6) The conclusion that the TIM23 complex adopts an "altered conformation" in N+C mitochondria is based on crosslinking experiments. It would be good to support this conclusion by an independent approach, such as native gel electrophoresis or differential protease sensitivity of the TIM23 components.Moreover, the term "altered conformational flexibility" is not fully fitting to the results presented here. The authors rather look at an altered subunit composition and not protein conformations. The text should be changed.We greatly appreciate this suggestion. We analyzed the TIM23 complex by blue native PAGE and indeed observed a changed running behavior of both Tim17 and Tim23 in N+C mitochondria as compared to FL. Since this alteration mainly affected the 90 kDa complex whose only known components are Tim17 and Tim23, this finding provides further support for our notion that the conformation, rather than the composition, of the TIM23 complex is affected in N+C mitochondria. This new finding is now included in As also discussed above, since the coimmunoprecipitation experiments revealed no major changes in the composition of the complex and both crosslinking and BN-PAGE support the notion of the changed conformation of the complex, we think that the term “altered conformational flexibility” is fitting our results and is supported by them.7) The part on the human patient mutation mimicked in yeast Tim44 is interesting, but not required for the rest of the story. Does the mutation in Tim44 affect the TIM23 complex or protein import in yeast or humans? As it stands it is not very informative. The authors should either remove that part or perform a functional analysis, i.e. test mutant cells/mitochondria for the functionality of the Tim44 C-terminal domain in the binding of Tim17 and precursor binding during mitochondrial import.We agree that the patient mutation mimicked in yeast Tim44 is not required to understand the rest of the story and have therefore removed this finding from the Abstract. As we also previously stated in the Discussion, we have performed initial analyses of yeast cells carrying this mutation and we observed neither an obvious growth nor an obvious in vivo import defect in these cells. Both these observations are in agreement with the relatively mild effect seen in the affected patients . We did however observe that the mutation partially destabilizes the protein and we feel that this is an important observation as it may provide a clue as to what goes wrong in the affected patients and may stimulate studies with patient cell lines, which are clearly beyond the scope of this manuscript. We would thus prefer to keep this finding in the manuscript but are happy to leave the final decision to the editors.


In eukaryotes, intra-chromosomal recombination generates DNA circles, but little is known about how cells react to them. In yeast, partitioning of such circles to the mother cell at mitosis ensures their loss from the population but promotes replicative ageing. Nevertheless, the mechanisms of partitioning are debated. In this study, we show that the SAGA complex mediates the interaction of non-chromosomal DNA circles with nuclear pore complexes (NPCs) and thereby promotes their confinement in the mother cell. Reciprocally, this causes retention and accumulation of NPCs, which affects the organization of ageing nuclei. Thus, SAGA prevents the spreading of DNA circles by linking them to NPCs, but unavoidably causes accumulation of circles and NPCs in the mother cell, and thereby promotes ageing. Together, our data provide a unifying model for the asymmetric segregation of DNA circles and how age affects nuclear organization.DOI:http://dx.doi.org/10.7554/eLife.03790.001 Budding yeast is a microorganism that has been widely studied to understand how it and many other organisms, including animals, age over time. This yeast is so named because it proliferates by ‘budding’ daughter cells out of the surface of a mother cell. For each daughter cell that buds, the mother cell loses some fitness and eventually dies after a certain number of budding events. This process is called ‘replicative ageing’, and it also resembles the way that stem cells age. In contrast, the newly formed daughters essentially have their age ‘reset to zero’ and grow until they turn into mother cells themselves.Several molecules or factors have been linked to replicative ageing. These are retained in the mother cell during budding, rather than being passed on to the daughters. Non-chromosomal DNA circles, for example, are rings of DNA that detach from chromosomes during DNA repair and that accumulate inside the ageing mother cell over time. How the mother cells retain these circles of DNA is an on-going topic of debate.Similar to plants and animals, chromosomes in yeast cells are confined in a membrane-bound structure known as the cell nucleus. The nuclear membrane is perforated by channels called nuclear pore complexes that ensure the transport of molecules into, and out of, the nucleus. Now, Denoth-Lippuner et al. establish that for the non-chromosomal DNA circles to be efficiently confined in the mother cell, the DNA circles must be anchored to the nuclear pore complexes.Denoth-Lippuner et al. next asked how the DNA circles were anchored to these complexes; and found that another complex of proteins known as SAGA is involved. When components of the SAGA complex were deleted in budding yeast cells, non-chromosomal DNA circles spread into the daughters as well. On the other hand, artificially anchoring these DNA circles to the nuclear pore complex alleviated the need for the SAGA complex, in order to retain these molecules in the mother cell.Denoth-Lippuner et al. also show that SAGA-dependent attachment of the DNA circles to the nuclear pore complexes causes these complexes to remain in the mother cell. As a consequence, these nuclear pore complexes accumulate in the mother cells as they age. The number of nuclear pore complexes in the daughter cells, however, remained fairly constant. Together these data raise the question of whether the effects of DNA circles on the number and activity of the nuclear pores might account for their contribution to ageing, perhaps by affecting the workings of the nucleus.DOI:http://dx.doi.org/10.7554/eLife.03790.002 Homologous recombination plays an important role in DNA repair in all organisms and acts through the formation of a Holliday junction between the damaged molecule and its repair template. When recombination involves two homologous chromosomes, the resolution of the Holliday junction leads to a cross-over, the exchange of arms between two chromosomes, in 50% of the cases . HoweverSaccharomyces cerevisiae, extrachromosomal rDNA circles (ERCs) form spontaneously but fail to accumulate in the population, owing to their highly asymmetric segregation upon cell division . Since division . Likewisdivision . Converse longer . Interese longer . Thus, nOne approach to characterize the effects of DNA circles on cellular physiology is to study the mechanism of their segregation. Two models have been proposed to explain the retention of circular DNA molecules in the mother cells . The morThe main difference between these models is whether confinement of the circle within the mother cell is purely passive or relies on mechanisms that are able to distinguish non-chromosomal DNA circles from bona-fide chromosomes to promote their specific anchorage and asymmetric segregation. However, no such mechanism is known yet. Whether DNA circles passively diffuse or are recognized by the cell would be predicted to have distinct consequences on the localization of the circles and their effects on nuclear organization. A passive model predicts that DNA circles do not interact specifically with any nuclear structure. Therefore, their accumulation should have little impact on nuclear organization. On the other hand, if cells recognize DNA circles, accumulating circles would increasingly interact with the corresponding structure and should progressively affect its size and organization. Thus, in order to better understand whether and how DNA circles are recognized by the cell, and to shed light on how they interfere with cellular physiology, we investigated how accumulating DNA circles localize and whether they affect nuclear organization.URA3 located between the two recombination-sites to select against accidental centromere excision and LEU2 on the residual backbone, allowing selection for the plasmid after centromere excision. In budding yeast, all centromeres co-localize with spindle pole bodies (SPBs) throughout the cell cycle  contained no plasmid, consistent with the plasmids segregating asymmetrically and accumulating in only few retaining cells. A small fraction of the cells (1%) contained few plasmids visualized as small foci as above, whereas the rest of the cells (6%) carried one large and intense signal, 8-fold above background . ConsistIn order to characterize this structure in more detail, the accumulated plasmids were visualized in wild-type cells co-expressing TetR-mCherry with different nuclear markers fused to GFP. The nucleoporin Nup82 tagged with three copies of a super-folder GFP (Nup82-3sfGFP) was used to visualize the morphology of the nuclear envelope; Spc42-GFP indicated the position of the SPB and Fob1-GFP labeled the nucleolus. In most cases, the accumulated DNA circles localized adjacent to, but were excluded from, the nucleolus . In wild-type cells, non-centromeric circles rarely passed to the daughter cells  complex, showed a clear increase in plasmid propagation , similar to the defect observed in bud6∆ mutant cells and fitting well with propagation frequencies predicted by the passive model. The SAGA complex regulates chromatin organization of actively transcribed genes and their recruitment to the nuclear periphery through anchorage to NPCs . Thus, SAGA and its acetyl–transferase activity, which are known to link chromatin to NPCs, promote the retention of DNA circles in the mother cell.Using this assay, no effect on circle retention was observed in cells lacking any of the sirtuin proteins, Sir1-4, which are involved in chromatin silencing and telomere anchorage to the inner nuclear membrane , in cell to NPCs . All SAGn5-E173A  led to aADE2 gene  of the wild-type cells, all ERCs that had accumulated in the mother cell remained in the mother cell, as demonstrated by the fact that their daughters lacked the ADE2 marker and formed a red colony  in wildmethods, ). This psgf73∆ and gcn5∆ mutant mother cells after 11 and 14 generations using the mother enrichment program , indicating that the effects of inactivating SAGA and TREX-2 were not additive. We conclude that the SAGA complex and the NPC component TREX-2 act together in a pathway mediating the targeted retention of non-chromosomal circles in the mother cell.Therefore, we next asked whether SAGA's function in tethering chromatin to nuclear pores was directly relevant for its role in circle retention. SAGA's function in chromatin anchorage to NPCs involves TREX-2, a multiprotein complex that localizes to nuclear pores, facilitates mRNA export, and physically interacts with SAGA . Thus, womplexes , and delgcn5∆ mutant cells) or both SAGA and TREX-2 function . To determine whether the plasmids localizing to the periphery co-localized with the nuclear pores, Nup82-3sfGFP intensity traces (N > 50 cells) were measured along the nuclear envelope in equatorial focal sections of the nuclei containing a single labeled acentric DNA circle at the rim. High LED-based illumination and fast image acquisition minimized blur due to NPC movement. Aligning all traces with the position of the DNA circle showed that the average Nup82-3sfGFP intensity increased in the vicinity of the circle compared to elsewhere . Thus, we conclude that SAGA indeed promotes the interaction of non-chromosomal DNA circles to nuclear pores.Thus, we next wondered whether TREX-2 and SAGA promoted the linkage of DNA circles to NPCs  and Sus1 . Thus, the main function of Sgf73 and Sus1 in plasmid retention is to physically link SAGA to nuclear pores. These data establish that SAGA needs to be at NPCs in order to function in circle retention.To investigate how direct the effect of SAGA on circle anchorage to NPCs might be, we next asked whether a physical link between SAGA and NPCs was required for SAGA's function in mediating the anchorage of circles to NPCs and whether SAGA interacted with the DNA circles themselves. To address the first question, we generated fusion proteins to covalently link the SAGA complex to the TREX-2 complex (Gcn5-Sac3) or directly to NPCs (Spt7-Nup49) and asked whether this was sufficient to restore the retention of DNA circle in cells lacking Sgf73 or Sus1. Expression of either of these two fusion proteins had no effect on plasmid retention in otherwise wild-type cells  than in those on the opposite side  and at opposite side of the nucleus (Io) in wild-type cells loaded with circles, with both measurement regions equidistant from the bleached spot, the signal decayed slower and to a lesser extent in regions associated with the circles . Thus, SAGA-dependent attachment of non-centromeric DNA circles substantially, and specifically, reduces the mobility of the corresponding NPCs. These data establish two points. First, in circle-free exponentially growing cells only very few NPCs, if any, are stably attached to and slowed down by chromatin. Second, NPCs that are attached to DNA circles show very slow dynamics, possibly explaining their enhanced retention in the mother cell during mitosis.We could not perform the same experiment in SAGA deficient cells because circles did not cluster to one focus and the cells formed no NPC cap. NPC distribution remained uniform over the nuclear surface, and non-centromeric circles were scattered throughout the nucleoplasm . We concConsistent with the attachment of circles to NPCs affecting each other's dynamics, SAGA also slowed down the non-centromeric DNA circles. In time-lapse movies , the average speed of the DNA circles was significantly higher in cells lacking Gcn5  than in wild-type cells , and faster in the sir2∆ mutant mother cells  than sgf73∆ (44 generations). Therefore, Sgf73 also contributes to ageing by additional mechanisms beyond reducing ERC formation. To test whether Sgf73 promotes ageing via the retention of ERCs and NPCs in the ageing cell, we asked whether targeting SAGA back to NPCs restored ageing in the sgf73∆ mutant cells. Expression of the fusion proteins Gcn5-Sac3 and Spt7-Nup49 did not alter the longevity of the wild-type cells . HoweveWe show that SAGA specifies the fate of non-chromosomal circles by promoting their attachment to NPCs and thereby their high-fidelity confinement into the mother cell during mitosis. This prevents the propagation of these molecules in the population. We discovered that, as a consequence of this retention mechanism, NPCs cluster around DNA circles and are concomitantly retained in the mother cell. Thus, both ERCs and NPCs accumulate in old cells, promoting ageing. These findings allow making the following points.yku70∆) and mutations that weaken the nuclear diffusion barrier without prolonging anaphase (bud6∆) both affect circle retention. Second, we propose that tight confinement of the circles by the diffusion barrier requires the attachment of the circles to NPCs. Indeed, what the barrier efficiently retains in the mother cell is neither NPCs alone nor the unbound circles but rather the circle-bound NPCs. Accordingly, anchorage of the circles to NPCs strongly enhances the retention of both NPCs and circles.Our data provide a unifying model for how non-chromosomal DNA circles are retained Model . First, This finding helps solving a debate in the field. Although NPC attachment had already been proposed to mediate the retention of DNA circles , the obsTwo types of mechanisms may underlie the selective retention of circle-bound NPCs. First, it might result from the reduced mobility of the circle-bound NPCs, which necessarily reduces the frequency at which they approach the barrier, and therefore their chance of passing it. This reduced mobility may be due to the drag that attached plasmids might oppose to NPCs. In addition, it might be exacerbated by the propensity of circle-bound NPCs to cluster, by a yet unknown mechanism. Alternatively, post-translational modifications of the NPC, perhaps by SAGA, may affect the interaction of the NPC with myosin and actin  or with Strikingly, the immobilization and segregation effects observed upon circle attachment to NPCs are reminiscent of the phenotypes caused by removing the nucleoporin Nsp1  or exprebud6∆ and the sgf73∆ mutant cells being remarkably long-lived. This large impact on longevity for apparently small defects in retention is fully in line with the idea that high-fidelity retention is a crucial parameter, as predicted by in silico simulations  or were manually created as described -hphNT1-GCN5(3′UTR) and pYB1739 containing SPT7-linker-NUP49-NUP49(3′UTR)hphNT1-SPT7(3′UTR) were created by homologous recombination into pRS314. PCR fragments containing the fusions were treated with DpnI and gel-purified before integration into yeast. Integrations were verified by PCR. For all fusion proteins the linker sequence is as described in Yeast strains used in this study are listed in e Pillus . GFP andescribed . Cells wescribed  and pYB1. Berger . gcn5-E1For microscopy, cells were resuspended in non-fluorescent medium (SD–TRP) and either mounted on a glass cover slip (for still images or movies of max. 5 min duration) or immobilized on a 2% agar pad containing non-fluorescent medium.rec-CEN4-URA3-rec) and to select for the presence of the plasmid. After 16–18 hr, a Deltavision microscope (Applied Precision) equipped with a CCD HQ2 camera (Roper), 250W Xenon laps, Softworx software (Applied Precision), and a temperature chamber set to 30°C was used to visualize the plasmids and the depicted proteins tagged with GFP. Time lapse movies of 5 min, intervals for 1h taking 20 Z-stacks of 0.4-μm spacing, were recorded with a 100×/1.4 NA objective and 2x2 binning. To determine the total NPC fluorescence, integrated density was measured of the nucleus and background in sum projections using Image J . After subtraction of the background, the fluorescence was normalized by the median fluorescence of cells without plasmids. To determine fluorescence intensity, the mean gray value of the NPC area adjacent to the accumulated circles (Ic) and the residual NPC area (Ir) was measured and normalized by the median intensity of Ir. For the percentage fluorescence segregated to the mother cell, the integrated density of the nucleus in the mother and the bud was measured after nuclear division, and after subtraction of the background the fluorescence in the mother was divided by the total fluorescence. The nuclear area depicts the 2D area of the nucleus in a sum projection in the mother and daughter of telophase cells. The mean intensity is the total intensity divided by the nuclear area. Box plots represent the distribution of the measurements in single cells whereas whiskers reach from min to max, the box covers 50% of the data and the line depicts the median.To visualize, the accumulated plasmids cells were grown on SD–URA to maintain the centromere on the plasmid, then shifted to SD -LEU containing β-Estradiol  to induce excision of the centromere , and the TillVision software (TillPhotonics). Images were acquired with a 100x / 1.4 NA objective, 2 × 2 binning, and stacks of 20 focal slices. Maximal intensity projections were used to determine plasmid propagation (described below ‘plasmid retention assay’).To determine individual plasmid localization, accumulated plasmid co-localization with Gcn5 and Sgf73, nuclear geometry, and anaphase duration; a Deltavision microscope was used (described above). Prior to analysis, the images were deconvolved. For plasmid localization, images were taken with an Olympus 100×/1.4 NA objective and 1 × 1 binning. 15 focal sections with 0.3-µm spacing were acquired. The focal plane in which the plasmid was the brightest was used to determine whether the plasmid was at the nuclear rim or at a clearly resolvable distance from the nuclear periphery. Nuclear geometry was analyzed using an Olympus 100×/1.4 NA objective, 1 × 1 binning, and a 1.6× auxiliary magnification. Stacks of 15 sections and 0.2-µm spacing were obtained. Nuclear length and width was measured as illustrated in To investigate the enrichment of NPCs close to non-centromeric plasmids, a Core Deltavision containing a CCD HQ camera (Roper) and solid state LEDs was used. An Olympus 100×/1.4 NA objective, 1 × 1 binning was used to create stacks of 15 focal sections and 0.2-µm spacing. Using ImageJ fluorescence intensity was measured along the nuclear rim. Nup82-3sfGFP intensity was normalized by its average intensity and TetR-mCherry intensity was normalized by its highest intensity in each cell. Afterwards, all obtained traces were aligned by the peak of TetR-mCherry (plasmid) intensity.To determine the speed of the non-centromeric plasmids, cells were imaged 3h after centromere excision using the same Deltavision with LEDs. First a Z-stack of 16 sections and 0.3-μm spacing was recorded in CFP and YFP to visualize both the plasmid and the SPBs in order to select for the non-centromeric plasmids. Then the plasmid was followed in a time lapse movie, taking the same stacks but only in GFP every 3 s for 3 min. The plasmids were tracked using Imaris (Bitplane) to determine their three dimensional coordinates. The coordinates were then used to determine the distance covered by the plasmid for each time point, which was deviated by three to determine the distance per second. All speed values were averaged for every plasmid, then averaged over all non-centromeric plasmids recorded and plotted in the graph.70 was defined as the time after which 70% of the initial intensity was still recorded. Finally, the average t70 of several cells was calculated for both Ic and Io and shown in the graph. The same settings were used to investigate the fluorescence decay in the whole nucleus, whereas the whole nuclear area was measured and set to 100% prior to bleaching. The mean and standard deviation of individual cells was plotted in the graph.Both FLIP and FRAP experiments were essentially done as described in To determine the strength of the nuclear diffusion barrier, a Zeiss LSM 510 microscope was used. Early anaphase cells were selected and photobleaching was applied on the depicted areas. To analyze the FLIP experiments, the total integrated fluorescent density in both the mother and daughter part of the nucleus was measured. After subtracting the average fluorescent loss determined by 5 neighboring cells, the fluorescent signal of the mother and daughter part was set to 100% at the beginning of the experiment. All experiments were pooled and analyzed using Prism to fit a one-phase decay curve. The barrier index was defined as the ratio of the times needed to lose 40% of the initial fluorescent signal in the bud over the mother compartment.15 was defined as the time to recover 15% fluorescence. The average of t15 of all measured cells was calculated and is shown in the graph.For the FRAP experiments, the whole bud compartment of early anaphase nuclei was bleached and fluorescent recovery was measured in a constant area located in the bud. After subtracting the average fluorescent loss by imaging based on three neighboring cells, the fluorescence after photobleaching was set to 0%. The fluorescence recovery was fitted to a one-phase association curve for each cell using Prism. T4√c)/(x+2y+4z), where a, b, and c being the percentages of cells retaining all plasmids in the mother cell and x, y, and z being the number of cells counted for one, two, or four plasmids, respectively.Late anaphase or telophase cells containing one, two, or four plasmids that did not co-localize with the spindle pole body were analyzed. Propagation frequency (pf) was calculated as; 100- and recovered for 2hr at 30°C prior to the addition of β-Estradiol . After either 26 hr and 27.5 hr or 34 hr and 35.5 hr (for wt and mutant cells) cells were washed, diluted in PBS, and incubated with 25 µl streptavidin-coated magnetic beads  for 30 min at 4°C before purification using LS MACS columns (Miltenyi Biotec). Cells were eluted using 1× PBS containing 2 mM EDTA, pelleted, and split into two fractions: 1) cells were incubated in 20 µg/ml calcofluor white and bud scars were counted using fluorescence microscopy; 2) cells were lysed and DNA was purified using standard methods. DNA content was measured performing qPCR amplifying ACT1 as follows: genomic DNA from wt non-aged cells was used to generate a dilution curve. Simultaneously, three qPCRs for two different dilutions were run for each genomic DNA sample derived from the aged wt, gcn5∆ and sgf73∆ mutant cells. The average of the six qPCR values obtained for each strain was used to calculate the volume loaded on a 0.6% agarose gel containing ethidium bromide. The gel was run in TBE containing ethidium bromide for 24 hr at 50 V at 4°C. The gel was blotted to transfer membrane  using standard protocols. Membranes were hybridized with a 32P-labeled probe specific to the rDNA as described .Cells containing one copy of the ChIP analysis was essentially done as described : Cells wChrV: GAACAAGGTTACAAATCC and CCGATTTGTGAGATTCTTCCTARS: AGTAAAGATTTCGTGTTCATGCAG and ATAGTGAAGGAGCATGTTCGG non-coding sequence (NCS1): GATACCTGAGTATTCCCACAG and CACACCGCATATGGTGCACTC non-coding sequence (NCS2): CCGCTTACCGGATACCTGTC and ATCCTGTTACCAGTGGCTGCFold enrichment was calculated as IP/Input of protein X normalized by the same ratio measured in wt cells, where no protein was tagged with GFP to correct for unspecific binding.sir2Δ cells) and after 48 hr and 64 hr for normal or long-lived strains using a mercury lamp as a light source . Stacks of 14 slices covering a range of 7 μm were captured every 15 min for 2 hr, to follow the nuclear division. Nuclei of cells of different ages were analyzed using sum projections of the fluorescence images from both time points (48 hr and 64 hr). Total fluorescence (integrated density) was measured in the mother and daughter cell after nuclear division using ImageJ. After background subtraction, the values were grouped by the indicated age categories of the mother cells, normalized by the median of the daughter cells in the youngest age category, and plotted using Prism. Alternatively, nuclear fluorescence in the mother cells was divided by the total fluorescence (mother plus daughter nuclei) for each cell and plotted against the age of the mother cell to visualize the increase in asymmetry with age. This data were used to fit a one-phase association curve, which is shown in the graph. Additionally, 10 values were grouped age dependently and plotted showing the average age and percentage of fluorescence segregated to the mother cell as well as the standard deviations. For G1 cells, the total amount of NPCs (integrated density) was measured, plotted against their age, and an exponential curve was fitted using Prism. The values were then normalized by intersection of the curve with the Y-axis and set to 1. The dots on the graph were created from 10 values as described for the dividing cells. Similarly, the mean fluorescence intensity (integrated density divided by area) was plotted as well as the radius of the G1 nuclei, measured drawing a line through the nucleus at its broadest area using ImageJ, dividing it by two and plotting it against the age of the cell.Pore segregation during ageing was investigated using the microfluidics dissection platform  with conPedigree analysis was done as described : freshlyOne-way ANOVA followed by Dunnett's multiple comparison or an unpaired two-tailed t-test was used to test for significance except the lifespan curves, which were compared using Log-rank (Mantel–Cox) tests and the ChIP experiment, for which a paired t-test (within the same experiment) was used. review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , a guest Reviewing editor, and 2 reviewers.Thank you for sending your work entitled "Role of SAGA in the asymmetric segregation of DNA circles during yeast ageing" for consideration at The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.gcn5∆ sac3∆ (or ada5∆sac3∆– better) measurements to properly test that there is no additive effect in eliminating TREX and SAGA.The paper is interesting and worthy of publication. The reviewers agree with the conclusions that, as yeast cells age, ERCs interact with the NPC through a mechanism that requires SAGA and that this alters the behavior  of NPCs. However the reviewers also agree that the authors have overstated the mechanistic contribution of SAGA in the linkage of circular DNAs to the NPC. This latter conclusion is unsubstantiated without quite a bit more experimental evidence. To conclude that SAGA is really mediating the interaction with the NPC, the authors need to better define this mechanism. Does SAGA bind equally throughout the plasmid after eviction of the CEN? How does the E173A mutation impact this binding? How different is the acetylation of the histones associated with centromeric vs the non-centromeric plasmid? Therefore, it is recommended that the authors may proceed if they remove these claims. Nevertheless, even to make the case for the SAGA linkage at all, there are couple of things that must be addressed, some with simple straightforward experiments: 1) In the plasmid retention assay, perform the double mutant bud6∆ mutant should be shown in 2) The quantitative data for NPC cap formation in a GCN5 and SPT20 are required for the RLS extension caused by deleting FOB1, possibly because the gcn5∆ mutant already has reduced ERC accumulation (as shown in 3) In  The paper is interesting and worthy of publication. The reviewers agree with the conclusions that, as yeast cells age, ERCs interact with the NPC through a mechanism that requires SAGA and that this alters the behavior  of NPCs. However the reviewers also agree that the authors have overstated the mechanistic contribution of SAGA in the linkage of circular DNAs to the NPC.We agree with the reviewers’ comment that we don’t know precisely how SAGA promotes the interaction of DNA circles with NPCs. SAGA might be part of a physical linker or might act by modifying chromatin or pore components to promote interaction between the circles and the NPCs. We have edited the manuscript to make sure that there is no confusion about this point and added a paragraph in the Discussion section to address the different possibilities of how SAGA would promote attachment of DNA circles to NPCs.1) In the plasmid retention assay, perform the double mutant gcn5∆ sac3∆ (or ada5∆sac3∆ – better) measurements to properly test that there is no additive effect in eliminating TREX and SAGA.ada5Δ sac3Δ double mutant cells were very sick. Therefore, we analyzed the gcn5Δ sac3Δ double mutant cells. Simultaneously deleting GCN5 and SAC3 does not lead to additive effects compared to the single mutants, supporting the hypothesis that SAGA and TREX-2 function together in the same pathway for what concerns DNA circle retention in the mother cell. These data were added to the manuscript in To address the reviewers’ suggestion we created both double mutant strains. Unfortunately, in our hands the 2) The quantitative data for NPC cap formation in a bud6∆ mutant should be shown inAs requested by the reviewers, we added these results to 3) In, another interpretation of the data is that GCN5 and SPT20 are required for the RLS extension caused by deleting FOB1, possibly because the gcn5∆ mutant already has reduced ERC accumulation (as shown in).gcn5∆ mutant cells do not show a reduced frequency of rDNA excision; if anything it is rather slightly increased. Thus, the only thing that we can conclude from these data is that in both the gcn5Δ single and gcn5Δ fob1Δ double mutant cells ERC accumulation is no longer the factor limiting longevity.We agree with the reviewers that this is a possibility; however this is unlikely to be due to a reduced rate of ERC formation. Indeed, we are now adding data on the effect of SAGA inactivation on recombination in the rDNA. As we show now, the gcn5∆, we confirmed that indeed, cells lacking Sgf73 show a reduction (2 folds) in ERC formation, similar to the cells lacking Fob1. However, the sgf73∆ mutant cells show a much more prolonged lifespan compared to the fob1∆ mutant cells. Thus, the effect of the sgf73∆ mutation on longevity probably involved a combination of effects, such as a reduction of ERC formation and the lack of retention of the residual ERCs. This interpretation is still fully supported by the fact that restoring SAGA linkage to NPCs in these cells restores ageing.Our new data also permit to extend the discussion opened by a recent study by


With the increased availability of regional reference networks, Precise Point Positioning (PPP) can achieve fast ambiguity resolution (AR) and precise positioning by assimilating the satellite fractional cycle biases (FCBs) and atmospheric corrections derived from these networks. In such processing, the atmospheric corrections are usually treated as deterministic quantities. This is however unrealistic since the estimated atmospheric corrections obtained from the network data are random and furthermore the interpolated corrections diverge from the realistic corrections. This paper is dedicated to the stochastic modelling of atmospheric corrections and analyzing their effects on the PPP AR efficiency. The random errors of the interpolated corrections are processed as two components: one is from the random errors of estimated corrections at reference stations, while the other arises from the atmospheric delay discrepancies between reference stations and users. The interpolated atmospheric corrections are then applied by users as pseudo-observations with the estimated stochastic model. Two data sets are processed to assess the performance of interpolated corrections with the estimated stochastic models. The results show that when the stochastic characteristics of interpolated corrections are properly taken into account, the successful fix rate reaches 93.3% within 5 min for a medium inter-station distance network and 80.6% within 10 min for a long inter-station distance network. Precise Point Positioning (PPP) has the capability of providing centimeter or even millimeter positioning accuracy using a single dual-frequency global navigation satellite system (GNSS) receiver . PPP hasIFL) ambiguities can be decomposed into wide-lane (WL) and narrow-lane (NL) ambiguities. However, the process still needs a few tens of minutes in order to reliably fix the ambiguities [Traditional PPP is based on real-valued ambiguity solutions and typically suffers from long convergence times. In order to shorten the convergence time, ambiguity resolution (AR) is necessary, and several approaches have been developed for this purpose in recent years ,10,11,12iguities . If a reiguities . In suchiguities .The essential difference between PPP and network RTK (NRTK) stems from the distinct types of corrections generated at the network level: processing of network data into corrections in the state space representation (SSR) enables PPP implementation, while the network correction given in the observation state representation (OSR) leads to NRTK implementation. Compared to NRTK, several major advantages of regional augmented PPP can be identified . First, The proper determination of a mathematical model for atmospheric corrections is essential for regional augmented PPP, which includes both functional and stochastic models. As to the functional model, several examples have been developed, including the linear combination method (LCM) , linear i.e., the ambiguities that can be confidently fixed are left unfixed [Several studies have considered the stochastic characteristics of atmospheric corrections. For networks with inter-station distances around 50 km or less, the atmospheric corrections generated from the network are usually treated as deterministic quantities without any consideration of their uncertainties , which,  unfixed .This paper aims to improve the performance of regional augmented PPP ambiguity resolution by strengthening its float solutions with properly estimated stochastic characteristics of the atmospheric corrections generated from the regional network. The method starts with the generation of the satellite FCBs, ionospheric and tropospheric delays for the user stations based on the observations of the regional reference network. Instead of determining the empirical variances of atmospheric corrections as a linear function of the average distance amongst the reference stations as in the traditional strategy, we determine the variances of interpolated atmospheric corrections by considering both their random uncertainties and their discrepancy against the actual atmospheric correction at the user station. The variance of the first component (random) is estimated by the error propagation law, while the second component (discrepancy) comes from the regional network. The interpolated atmospheric corrections are used as the pseudo-observations together with the estimated variance-covariance matrix to describe the strength level of the constraint. Two experiments from different sizes of networks are processed to demonstrate the superior PPP AR performance of the proposed method.jth frequency can be written as:r denotes the receiver; n satellites are simultaneously tracked, g; 1L with Since the coordinates of all network reference stations and the precise satellite orbits are given, the single-epoch undifferenced GPS code and phase observations on the We define:j. Collecting single-epoch dual-frequency observations essed as :(3)Qs=dIn order to remove ionospheric delays, ionosphere-free observations are usually used for PPP. The ionosphere-free observation can be expressed as follows:Ambiguity resolution is conducted first in wide-lane, and then in narrow-lane. The float WL ambiguities bination ,30,31 ofMeanwhile the ZTD n stations tracking m satellites. The float undifferenced ambiguities at each station are estimated as iB. For all the float ambiguity, we have an observation as follows:Let us assume that we have a network of iR all elements of one column are –1 and all other entries are zero. For matrix iS each line has one element of 1, the other entries are zero. in is the undifferenced integer ambiguity vector for station i.In matrix Under the condition that all the integer ambiguities are known and that one receiver FCB is fixed to zero, the satellite and receiver FCBs can be estimated by means of a least square from the following observation Equation (7):We assume that the receiver FCB at the arbitrarily selected station is zero. Then the nearest integers of the ambiguities at this station are the integer ambiguities and the fractional parts are estimates of the corresponding satellite FCBs. When applying these satellite FCBs to the common satellites of the next station, the corrected ambiguities should have a very similar fractional part. The mean fractional parts of all the common satellites correspond to the receiver FCB. With this receiver FCB, FCBs of the newly appearing satellites at the station can be estimated. Repeating this procedure for all stations, we can obtain the approximate FCBs for all receivers and satellites.After correcting the ambiguities with the FCBs, the integer property of ambiguity can be recovered, thus ambiguity-fixing can be attempted. By replacing integer ambiguity parameters with their fixed values in Equation (7), the remaining parameters can be estimated. The FCB estimates are improved and will in turn help to resolve more integer ambiguities. The above procedure can be done iteratively until no more integer ambiguities can be fixed.The approach described above is first applied to all the float undifferenced WL ambiguities, so that the WL FCBs and integer WL ambiguities are estimated. With the integer WL ambiguities estimated, the float NL ambiguities are derived from the ionosphere-free ambiguities. Afterwards, the same approach is used to estimate the NL FCBs from the float NL ambiguities. For more details about the above FCB and ambiguity estimation process, one refers to .Once the WL and NL FCBs are determined, L1 and L2 FCBs can be also recovered as follows:Thus, we can derive the ionospheric delays based on the phase observations as follows:As stated in Teunissen and Khodabandeh , the ionThe slant ionospheric and ZTD corrections at reference stations are used to interpolate the atmospheric correction at user station by using inverse distance-based interpolation :(10a)τuAfter interpolation, the atmospheric correction for the user stations can be defined as l [The covariance matrix of interpolated atmospheric corrections has been usually determined using empirical functions. Taking the ionospheric delay as the example, it is common that the STD of the ionospheric corrections is modeled as a linear function of the baseline length l :(11)σι=Similarly, the linear function can also be employed to determine the atmospheric stochastic model for network augmented PPP because undifferenced and double-differenced data processing are only different implementations (utilization) of the network data. Odijk  suggestechapelle  suggesteThe uncertainties of the interpolated ionospheric and ZTD corrections theoretically depend on two uncorrelated error sources. One source is the error caused by the multipath and noises in the estimated corrections at the reference stations, called Source I error in the sequel. The covariance matrix corresponding to this error source is expressed as cov , (10a) and (10b):Since the computational procedure is the same for ZTDs and slant ionospheric delays, only the variance determination for the Source II ionospheric error is described which includes a two-step procedure. The first step is to select a group of reference stations near the user station to interpolate the ionospheric errors at those selected stations and the second step is to apply those interpolated ionospheric errors at those selected stations to interpolate the ionospheric error at the user station. Details are given below:Step 1: Determination of interpolated ionospheric errors at selected reference stationsrn − 1 selected stations  average of rn − 1 reference stations.As the ionospheric effects are proportional to the baseline length in real situation, the interpolated ionospheric error at a user station  the estimated variance adaptively varies with the inter-station distances of the network to describe the uncertainties of the ionospheric corrections, e.g., larger in the sparse network, and smaller when in a dense network; (2) the estimated variance can better capture the complicated ionospheric variability which differs from time to time during one day than empirical values; (3) In the kinematic mode, the estimated variance can reflect the ionospheric variability caused by spatial variations.G is the design matrix of the unknown position increment vector Moving all the known terms to the left side, the satellite FCBs can be eliminated while the receiver code and phase biases remain which however can be assimilated by the receiver code and phase clocks. Therefore, such systematic biases have no effect on the ionospheric corrections at the user station. Finally the observation equations at the user station read as:cov x˜n filter :(25a)x^We denote the updated solutions using the same symbols as for standard Kalman filter solutions. Two test networks of different spatial extent are analyzed, one with medium inter-station distances of 41.4–65.3 km and the other with longer inter-station distances of 65.2–114.7 km. The test networks are constructed by using several GPS stations of the USA Continuously Operating Reference Stations CORS, . Ten staFour schemes for determining the variance of ionospheric delays are used for the purpose of comparison. Scheme 1 treats atmospheric correction as deterministic quantities. Scheme 2 uses the accuracy of ZTD ended by  and the 57 mm/km . Scheme 74 mm/km . Scheme The true ionospheric delays and ZTD at a user station, which is used for assessing the interpolated corrections, can be computed by processing user station as a reference station. The real accuracies of these atmospheric corrections are represented as the difference between the true and interpolated atmospheric corrections. The estimated accuracies of atmospheric corrections can be derived from the covariance matrix determined with Equations (12)–(20).The new version of the LAMBDA software is applied to conduct partial ambiguity resolution. Both ratio test and success rate are applied to validate the ambiguities. The ratio threshold value is taken w.r.t different ambiguity dimensions as 2 for 2-dimension, 1.5 for 3/4-dimension, 1.3 for 5 to 7-dimension and 1.2 for higher than 7-dimension . The sucAs claimed in As shown in Taking the undifferenced ionospheric and tropospheric delays retrieved at the four reference stations, the accuracies of atmospheric correction determined by different schemes are presented in The real accuracies of interpolated ionospheric errors reach 1.8–3.5 cm with mean of 2.8 cm. The accuracies of ionospheric errors determined using the Schemes 2–4 are from 2.1 to 2.8 cm with the mean of 2.6 cm, 2.8–3.7 cm with the mean of 3.4 cm and 1.9–3.6 cm with mean of 2.6 cm. It can be seen that both Schemes 2 and 4 better represent the real ionospheric errors over all.For a user station with an average distance of 45 km to the reference stations, the empirical accuracies of interpolated ionospheric corrections determined by Scheme 2 and Scheme 3 are 2.6 cm and 3.3 cm during one day. However, these values are not realistic for all real situation. For the medium inter-station distance network, the cumulative distributions of time-to-first-fixed (TTFF), defined as the time needed for successful AR are shown in The corresponding empirical fix rate and success rate with different schemes are shown in The results reveal that Scheme 4 obtains the highest success rate of 93.3% with 5-min observation accumulation. Scheme 1 fixes most of ambiguities faster compared to the other schemes, as 77.2% of the ambiguities can be fixed within 1 min. After 5 min, the fix rate reaches 94.1%. However, the success fix rate is only 86.9%, which indicates unreliable ambiguity fixing. As to Schemes 2 and 3, the success rates of 5-min solution are improved to 91.7% and 89.3% by empirically considering the stochastic model of atmospheric correction. With the estimated stochastic model of atmospheric corrections, the success rate can be further improved to 93.3% with Scheme 4. Most importantly, Scheme 4 achieves the lowest incorrect fix rate with 3.3%.The interpolated ZTD errors still remain smaller than 1.5 cm. The interpolated ionospheric correction error become larger compared to those using a medium inter-station distance network. The accuracy of the ionospheric corrections by using Schemes 4 and 3 is the most close to the actual one for stations SG01 and SG47. It can be seen that lower success rates are obtained compared to the medium inter-station distance network. This is because larger variances of atmospheric corrections have been assigned to reduce the model strength and subsequently to avoid potential biases in the float solution. As a result, Scheme 1 can successfully fix 49.0% of ambiguities with 2-min observation time. 19.4%, 9.7% and 42.8% of ambiguities can be fixed using Schemes 2–4 with the same observation period. However, the success rate with scheme 1 does not significantly increase with increase of the observation time as those with other schemes. This is because that Scheme 1 treats ionospheric correction as deterministic. Scheme 1 can obtain a fixed solution successfully only if the ionospheric corrections are adequately small, but it fails to fix most ambiguities. For the other schemes, the average success rate is the highest for Scheme 4, which is 80.6% with 10 min observation time. The success fix rate, and fix rate with different schemes are shown in We have developed a method for regional augmented PPP by using atmospheric corrections and their stochastic model derived from regional reference networks. The experimental results reveal that the proposed method can improve the float solutions so that ambiguities can be fixed quickly and reliably by users within the network coverage. For a medium inter-station distance network, the ZTD correction errors are less than 1 cm on average, while the ionospheric correction errors are less than 3 cm. The performance of the proposed regional augmented PPP can be comparable to NRTK since the ambiguities can be fixed with a success fix rate of 93.3% within 5 min. For the long inter-station distance network, both interpolated ZTD and ionospheric correction errors will increase to 2 cm and 5 cm, respectively. The ambiguity success fix rate with the proposed method suffers a degradation compared to the other schemes, but it still achieves a 80.6% rate. This study has only discussed the stochastic model based on a distance-based linear interpolation model. The development of stochastic models for atmospheric corrections based on the other functional models will be further investigated in future work.