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ObjectivesRheumatic immune-related adverse events (irAE) such as (poly)arthritis in patients undergoing immune checkpoint inhibitor (ICI) treatment pose a major clinical challenge. ICI therapy improves CD8+T cell (CD8) function, but CD8 contributes to chronic inflammation in autoimmune arthritis (AA). Thus, we investigated whether immune functional and metabolic changes in CD8 explain the development of musculoskeletal irAE in ICI-treated patients.MethodsPeripheral CD8 obtained from ICI-treated patients with and without arthritis irAEs and from AA patients with and without a history of malignancy were stimulated in media containing13C-labelled glucose with and without tofacitinib or infliximab. Changes in metabolism, immune-mediator release, expression of effector cell-surface molecules and inhibition of tumour cell growth were quantified.ResultsCD8 from patients with irAE showed significantly lower frequency and expression of cell-surface molecule characteristic for activation, effector-functions, homing, exhaustion and apoptosis and reduced release of cytotoxic and proinflammatory immune mediators compared with CD8 from ICI patients who did not develop irAE. This was accompanied by a higher glycolytic rate and ATP production. Gene-expression analysis of pre-ICI-treated CD8 revealed several differentially expressed transcripts in patients who later developed arthritis irAEs. In vitro tofacitinib or infliximab treatment did not significantly change the immune-metabolic profile nor the capacity to release cytolytic mediators that inhibit the growth of the human lung cancer cell line H838.ConclusionsOur study shows that CD8 from ICI-treated patients who develop a musculoskeletal irAE has a distinct immune-effector and metabolic profile from those that remain irAE free. This specific irAE profile overlaps with the one observed in CD8 from AA patients and may prove useful for novel therapeutic strategies to manage ICI-induced irAEs.

S2-007 cells were transfected with Plac8-CFP (left panel) or YFP (right panel). ROIs (white outlines) in individual cells were repeatedly photobleached using high-power laser intensities and fluorescent signals recorded in regular intervals for 20 min of total duration.

Summary of assays and effects from RPPA analyses.

Plac8/PrP immunofluorescence.

S2-007 cells were co-transfected with Plac8-CFP (left panels/green pseudocolor in overlay) and YFP (middle panels/red pseudocolor in overlay). At the 60 s time point, cells were treated with digitonin, and at the 400 s time point trypsin was added. Scale bars: 10 µm.

Legends for Supplementary Tables, Movies, and Figures.

S2-007 cells were transfected with Plac8-YFP and TIRF images of an individual cell recorded over a timeframe of 9 min.

Induced morphology changes of cells and organelles are by far the easiest way to determine precise protein sub-locations and organelle quantities in light microscopy. By using hypotonic solutions to swell mammalian cell organelles we demonstrate that precise membrane, lumen or matrix protein locations within the endoplasmic reticulum, Golgi and mitochondria can reliably be established. We also show the benefit of this approach for organelle quantifications, especially for clumped or intertwined organelles like peroxisomes and mitochondria. Since cell and organelle swelling is reversible, it can be applied to live cells for successive high-resolution analyses. Our approach outperforms many existing imaging modalities with respect to resolution, ease-of-use and cost-effectiveness without excluding any co-utilization with existing optical (super)resolution techniques.

Die Grundschulmathematik lässt die Spannung erleben, einerseits die Inhalte kindgerecht zu vermitteln, und das heißt in der Praxis leider meist schlicht, andererseits die kraftvollen Ideen der Mathematik in nuce anzulegen und sie in solcher Breite darzustellen, dass sie Öffnungen in diverse Richtungen ermöglichen. Die Geometrie wird häufig als das Stiefkind der Grundschulmathematik vernachlässigt, sie wird im Vergleich zur Arithmetik als unbedeutender abgetan. Aber sie stellt mit ihren Bezügen zur Alltagswirklichkeit ein kraftvolles Mittel dar, um vielfältige Beziehungen erleben zu lassen, die anschließend in den höheren Klassen angereichert und begrifflich ausdifferenziert werden. Im Folgenden soll daher an einigen, wenigen Beispielen aus der Geometrie verdeutlicht werden, wie sich aus den Grundschulaktivitäten kraftvolle mathematische Gedanken entwickeln lassen, oder mehr noch, wie diese weiterführenden Ideen bereits in der Grundschule mitthematisiert werden können.

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Microtubule nucleation was uncovered as a key principle of spindle assembly. However, the mechanistic details about microtubule nucleation and the organization of spindle formation and symmetry are currently being revealed. Here we describe the function of coiled-coil domain containing 61 (Ccdc61), a so far uncharacterized centrosomal protein, in spindle assembly and symmetry. Our data describe that Ccdc61 is required for spindle assembly and precise chromosome alignments in mitosis. Microtubule tip-tracking experiments in the absence of Ccdc61 reveal a clear loss of the intrinsic symmetry of microtubule tracks within the spindle. Furthermore, we show that Ccdc61 controls the centrosomal localization of centrosomal protein of 170 kDa (Cep170), a protein that was shown previously to localize to centrosomes as well as spindle microtubules and promotes microtubule organization and microtubule assembly. Interestingly, selective disruption of Ccdc61 impairs the binding between Cep170 and TANK binding kinase 1, an interaction that is required for microtubule stability. In summary, we have discovered Ccdc61 as a centrosomal protein with an important function in mitotic microtubule organization.

Der Beitrag versucht die Untersuchungs- und Erkenntnisfortschritte zu skizzieren, welche durch die Arbeiten von Marianne Nolte Ende des letzten Jahrhunderts angestoßen wurden und sich zu einem wachsenden Problem im Mathematikunterricht entwickelten. Es wird auf die sich durch geänderte gesellschaftliche Entwicklungen wachsenden Anforderungen eingegangen, die Lehrkräfte zu bewältigen haben, um Kinder mit heterogenen Sprachkompetenzen zu einem Bild von Mathematik zu führen. Das Verhältnis von Sprache und Mathematik, gemeinhin als konträr konzipiert, wird im Unterricht spannungsreich zusammengeführt. Hierauf muss Mathematikdidaktik Antworten parat haben, soll nicht chronisches Missverständnis das Ergebnis von Unterricht sein.

Der Diskussionsbeitrag bemuht sich, einige Begriffe genauer zu beleuchten, welche in der aktuellen mathematikdidaktischen Diskussion an Scharfe eingebust zu haben scheinen. Es scheint, dass unterschiedliche Ansatze wie stoffdidaktische Orientierung vs. kognitionspsychologische Fragestellungen erneut in eine ungute Opposition gelangen und mit der Formulierung „empirische Forschung erster und zweiter Art“ herrschaftliche Erklarungsanspruche angemeldet werden, wie E. Ch. Wittmann dies in einem GDM-Vortrag getan hat, anstatt sich wohltuend zu erganzen. Im Folgenden wird auf die Reprasentation von Wissen im Zusammenhang mit der dortigen Kritik an dem Zahlenstrahlkonzept eingegangen. Dies wird als Ausgangspunkt gesehen, die unterschiedlichen Geltungsbereiche der beiden Ansatze und die verschiedenen Zielsetzungen, die damit verbunden sind, zu beschreiben. Es wird fur eine „friedliche Koexistenz“ dieser Ansatze pladiert. Einen zentralen Punkt nimmt die Darstellung der Befunde zur Zahlenstrahlreprasentation („mental number line“) ein, die nicht nur eine Metapher fur die kognitive Verarbeitungsweise von Zahlen darstellt, sondern ihre Modellbrauchbarkeit durch eine Vielzahl von empirischen Befunden gestutzt wird und in den letzten Jahren ausdifferenziert wurde.

CDC14A codes for a conserved proline-directed phosphatase, and mutations in the gene are associated with autosomal-recessive severe to profound deafness, due to defective kinocilia. A role of CDC14A in cilia formation has also been described in other organisms. However, how human CDC14A impacts on cilia formation remains unclear. Here, we show that human RPE1 hCDC14A(PD) cells, encoding a phosphatase dead version of hCDC14A, have longer cilia than wild-type cells, while hCDC14A overexpression reduces cilia formation. Phospho-proteome analysis of ciliated RPE1 cells identified actin-associated and microtubule binding proteins regulating cilia length as hCDC14A substrates, including the actin-binding protein drebrin. Indeed, we find that hCDC14A counteracts the CDK5-dependent phosphorylation of drebrin at S142 during ciliogenesis. Further, we show that drebrin and hCDC14A regulate the recruitment of the actin organizer Arp2 to centrosomes. In addition, during ciliogenesis hCDC14A also regulates endocytosis and targeting of myosin Va vesicles to the basal body in a drebrin-independent manner, indicating that it impacts primary cilia formation in a multilayered manner.

The endoplasmic reticulum (ER), as a multifunctional organelle, plays crucial roles in lipid biosynthesis and calcium homeostasis as well as the synthesis and folding of secretory and membrane proteins. Therefore, it is of high importance to maintain ER homeostasis and to adapt ER function and morphology to cellular needs. Here, we show that signal peptide peptidase (SPP) modulates the ER shape through degradation of morphogenic proteins. Elevating SPP activity induces rapid rearrangement of the ER and formation of dynamic ER clusters. Inhibition of SPP activity rescues the phenotype without the need for new protein synthesis, and this rescue depends on a pre-existing pool of proteins in the Golgi. With the help of organelle proteomics, we identified certain membrane proteins to be diminished upon SPP expression and further show that the observed morphology changes depend on SPP-mediated cleavage of ER morphogenic proteins, including the SNARE protein syntaxin-18. Thus, we suggest that SPP-mediated protein abundance control by a regulatory branch of ER-associated degradation (ERAD-R) has a role in shaping the early secretory pathway.

Mutations in PINK1 and PARK2/Parkin are a main risk factor for familial Parkinson disease. While the physiological mechanism of their activation is unclear, these proteins have been shown in tissue culture cells to serve as a key trigger for autophagy of depolarized mitochondria. Here we show that ablation of the mitochondrial rhomboid protease PARL leads to retrograde translocation of an intermembrane space-bridging PINK1 import intermediate. Subsequently, it is rerouted to the outer membrane in order to recruit PARK2, which phenocopies mitophagy induction by uncoupling agents. Consistent with a role of this retrograde translocation mechanism in neurodegenerative disease, we show that pathogenic PINK1 mutants which are not cleaved by PARL affect PINK1 kinase activity and the ability to induce PARK2-mediated mitophagy. Altogether we suggest that PARL is an important intrinsic player in mitochondrial quality control, a system substantially impaired in Parkinson disease as indicated by reduced removal of damaged mitochondria in affected patients.

Zusammenfassung. Zunächst wird ein Einblick in die historische Entwicklung der Zahlen und Möglichkeiten des Rechnens gegeben. Die Entwicklung des Zählens und der Gebrauch der Zahlen beim Kinde schließen sich an. Die Verwendung der Finger ist normal. Wann stößt das „Fingerrechnen“ an seine Grenzen und sollte durch weiter tragende Strategien abgelöst werden, um einen gut verlaufenden Lernprozess der Mathematik zu begünstigen?

Background and Purpose— Detection and localization of the early phase of blood–brain barrier disruption (BBBD) in vivo during cerebral ischemia/reperfusion injury remain a major challenge but may be a relevant outcome parameter in stroke. Methods— We studied early BBBD in mice after transient middle cerebral artery occlusion by multimodal, high-field (9.4T) in vivo magnetic resonance imaging, including the contrast agent gadofluorineM as an albumin-binding tracer. GadofluorineM contrast-enhanced magnetic resonance imaging was performed to determine BBBD at 2, 6, and 24 hours after reperfusion. BBBD was confirmed and localized along the microvascular tree by using fluorescent gadofluorineM and immunofluorescence stainings (cluster of differentiation 31, ephrin type-B receptor 4, alpha smooth muscle actin, ionized calcium binding adaptor molecule 1). Results— GadofluorineM contrast-enhanced magnetic resonance imaging revealed a multifocal spatial distribution of early BBBD and its close association with the microvasculature at a resolution of 40 μm. GadofluorineM leakage was closely associated with ephrin type-B receptor 4-positive but not alpha smooth muscle actin-positive vessels. The multifocal pattern of early BBBD (already at 2 hours after reperfusion) thus occurred in the distal capillary and venular microvascular bed. These multifocal zones showed distinct imaging signs indicative of early vasogenic edema. The total volume of multifocal early BBBD accurately predicted infarct size at 24 hours after reperfusion. Conclusions— Early BBBD in focal cerebral ischemia initiates multifocally in the distal capillary and venular bed of the cerebral microvasculature. It is closely associated with perimicrovascular vasogenic edema and microglial activation and predicts the extent of final infarction.

SSX2IP promotes centrosome maturation and maintenance at the onset of vertebrate development, preserving centrosome integrity and mitosis during rapid cleavage divisions and in somatic cells., Meiotic maturation in vertebrate oocytes is an excellent model system for microtubule reorganization during M-phase spindle assembly. Here, we surveyed changes in the pattern of microtubule-interacting proteins upon Xenopus laevis oocyte maturation by quantitative proteomics. We identified the synovial sarcoma X breakpoint protein (SSX2IP) as a novel spindle protein. Using X. laevis egg extracts, we show that SSX2IP accumulated at spindle poles in a Dynein-dependent manner and interacted with the γ-tubulin ring complex (γ-TuRC) and the centriolar satellite protein PCM-1. Immunodepletion of SSX2IP impeded γ-TuRC loading onto centrosomes. This led to reduced microtubule nucleation and spindle assembly failure. In rapidly dividing blastomeres of medaka (Oryzias latipes) and in somatic cells, SSX2IP knockdown caused fragmentation of pericentriolar material and chromosome segregation errors. We characterize SSX2IP as a novel centrosome maturation and maintenance factor that is expressed at the onset of vertebrate development. It preserves centrosome integrity and faithful mitosis during the rapid cleavage division of blastomeres and in somatic cells.

Ribosome stalling during translation can potentially be harmful, and is surveyed by a conserved quality control pathway that targets the associated mRNA and nascent polypeptide chain (NC). In this pathway, the ribosome-associated quality control (RQC) complex promotes the ubiquitylation and degradation of NCs remaining stalled in the 60S subunit. NC stalling is recognized by the Rqc2/Tae2 RQC subunit, which also stabilizes binding of the E3 ligase, Listerin/Ltn1. Additionally, Rqc2 modifies stalled NCs with a carboxy-terminal, Ala- and Thr-containing extension—the 'CAT tail'. However, the function of CAT tails and fate of CAT tail-modified ('CATylated') NCs has remained unknown. Here we show that CATylation mediates formation of detergent-insoluble NC aggregates. CATylation and aggregation of NCs could be observed either by inactivating Ltn1 or by analyzing NCs with limited ubiquitylation potential, suggesting that inefficient targeting by Ltn1 favors the Rqc2-mediated reaction. These findings uncover a translational stalling-dependent protein aggregation mechanism, and provide evidence that proteins can become specifically marked for aggregation. DOI: http://dx.doi.org/10.7554/eLife.11794.001, eLife digest Cells use molecular machines called ribosomes to build proteins by connecting amino acids – the building blocks of proteins – together in a particular sequence. The chain of amino acids gradually lengthens as the protein forms, yet remains attached to the ribosome until the protein is complete. While this process is underway, cells can check that a newly forming chain is not abnormal or damaged. If it is, a cell then essentially ‘decides’ on whether to correct or eliminate it. Such protein quality control processes are important for ensuring the health and fitness of cells and organisms. Recently, a new protein quality control mechanism was discovered that senses when a ribosome becomes jammed as it produces a new protein. This mechanism recycles the ribosome so it can make more new proteins. It also disposes of the stalled protein using a cell complex, called the ribosome-associated quality control complex, which is found in all eukaryotic organisms including yeast and humans. This protein complex consists of three subunits; one of which, called Rcq2, tags ribosome-stalled proteins with a “tail” that contains the amino acids alanine and threonine. However, the purpose of this tag was not clear. Yonashiro, Tahara et al. now show that the tagging of ribosome-stalled proteins by Rqc2 in yeast cells induces the tagged proteins to clump together. This clumping probably prevents these proteins from inadvertently interfering with other molecules or processes within the cell. The formation of these clumps also correlates with the activation of a stress response in the cell, indicating that these clumps create a signal that prompts the cell to protect itself in response to the accumulation of more abnormal proteins. Mutations in one subunit of the ribosome-associated quality control complex in mice cause a condition that resembles a neurological disease in humans, called amyotrophic lateral sclerosis or ALS for short. A future challenge is therefore to understand how much Rqc2-mediated tagging and clumping of ribosome-stalled protein has a role in this and other neurodegenerative diseases. DOI: http://dx.doi.org/10.7554/eLife.11794.002

Intramembrane proteolysis is a conserved mechanism that regulates a variety of cellular processes ranging from transcription control to signaling. In mitochondria, the inner membrane rhomboid protease PARL has been implicated in the control of life span and apoptosis by a so far uncharacterized mechanism. Here, we show that PARL cleaves human Pink1, which is implicated in Parkinson's disease, within its conserved membrane anchor. Mature Pink1 is then free to be released into the cytosol or the mitochondrial intermembrane space. Upon depolarization of the mitochondrial membrane potential, the canonical import of Pink1 and PARL-catalyzed processing is blocked, leading to accumulation of the Pink1 precursor. As targeting of this precursor to the outer mitochondrial membrane has been shown to trigger mitophagy, we suggest that the PARL-catalyzed removal of the Pink1 signal sequence in the canonical import pathway acts as a cellular checkpoint for mitochondrial integrity. Furthermore, we show that two Parkinson's disease-causing mutations decrease the processing of Pink1 by PARL, with attendant implications for pathogenesis.

Tail-anchored (TA) proteins are post-translationally targeted to and inserted into the endoplasmic reticulum (ER) membrane through their single C-terminal transmembrane domain. Membrane insertion of TA proteins in mammalian cells is mediated by the ATPase TRC40/Asna1 (Get3 in yeast) and a receptor in the ER membrane. We have identified tryptophan-rich basic protein (WRB), also known as congenital heart disease protein 5 (CHD5), as the ER membrane receptor for TRC40/Asna1. WRB shows sequence similarity to Get1, a subunit of the membrane receptor complex for yeast Get3. Using biochemical and cell imaging approaches, we demonstrate that WRB is an ER-resident membrane protein that interacts with TRC40/Asna1 and recruits it to the ER membrane. We identify the coiled-coil domain of WRB as the binding site for TRC40/Asna1 and show that a soluble form of the coiled-coil domain interferes with TRC40/Asna1-mediated membrane insertion of TA proteins. The identification of WRB as a component of the TRC (Get) pathway for membrane insertion of TA proteins raises new questions concerning the proposed roles of WRB (CHD5) in congenital heart disease, and heart and eye development.

We introduce Map3-2D, a freely available software to accurately project up to five-dimensional (5D) fluorescence microscopy image data onto full-content 2D maps. Similar to the Earth’s projection onto cartographic maps, Map3-2D unfolds surface information from a stack of images onto a single, structurally connected map. We demonstrate its applicability for visualization and quantitative analyses of spherical and uneven surfaces in fixed and dynamic live samples by using mammalian and yeast cells, and giant unilamellar vesicles. Map3-2D software is available at http://www.zmbh.uni-heidelberg.de//Central_Services/Imaging_Facility/Map3-2D.html.

We introduce Map3-2D, a freely available software to accurately project up to five-dimensional (5D) fluorescence microscopy image data onto full-content 2D maps. Similar to the Earth’s projection onto cartographic maps, Map3-2D unfolds surface information from a stack of images onto a single, structurally connected map. We demonstrate its applicability for visualization and quantitative analyses of spherical and uneven surfaces in fixed and dynamic live samples by using mammalian and yeast cells and giant unilamellar vesicles. Map3-2D software is available at http://www.zmbh.uni-heidelberg.de//Central_Services/Imaging_Facility/Map3-2D.html.

Pancreatic ductal adenocarcinoma (PDAC) carries the most dismal prognosis of all solid tumors and is generally strongly resistant to currently available chemo- and/or radiotherapy regimens, including targeted molecular therapies. Therefore, unraveling the molecular mechanisms underlying the aggressive behavior of pancreatic cancer is a necessary prerequisite for the development of novel therapeutic approaches. We previously identified the protein placenta-specific 8 (PLAC8, onzin) in a genome-wide search for target genes associated with pancreatic tumor progression and demonstrated that PLAC8 is strongly ectopically expressed in advanced preneoplastic lesions and invasive human PDAC. However, the molecular function of PLAC8 remained unclear, and accumulating evidence suggested its role is highly dependent on cellular and physiologic context. Here, we demonstrate that in contrast to other cellular systems, PLAC8 protein localizes to the inner face of the plasma membrane in pancreatic cancer cells, where it interacts with specific membranous structures in a temporally and spatially stable manner. Inhibition of PLAC8 expression strongly inhibited pancreatic cancer cell growth by attenuating cell-cycle progression, which was associated with transcriptional and/or posttranslational modification of the central cell-cycle regulators CDKN1A, retinoblastoma protein, and cyclin D1 (CCND1), but did not impact autophagy. Moreover, Plac8 deficiency significantly inhibited tumor formation in genetically engineered mouse models of pancreatic cancer. Together, our findings establish PLAC8 as a central mediator of tumor progression in PDAC and as a promising candidate gene for diagnostic and therapeutic targeting. Cancer Res; 76(1); 96–107. ©2015 AACR.

Drosophila melanogaster embryogenesis begins with 13 nuclear division cycles within a syncytium. This produces >6,000 nuclei that, during the next division cycle, become encased in plasma membrane in the process known as cellularization. In this study, we investigate how the secretory membrane system becomes equally apportioned among the thousands of syncytial nuclei in preparation for cellularization. Upon nuclear arrival at the cortex, the endoplasmic reticulum (ER) and Golgi were found to segregate among nuclei, with each nucleus becoming surrounded by a single ER/Golgi membrane system separate from adjacent ones. The nuclear-associated units of ER and Golgi across the syncytial blastoderm produced secretory products that were delivered to the plasma membrane in a spatially restricted fashion across the embryo. This occurred in the absence of plasma membrane boundaries between nuclei and was dependent on centrosome-derived microtubules. The emergence of secretory membranes that compartmentalized around individual nuclei in the syncytial blastoderm is likely to ensure that secretory organelles are equivalently partitioned among nuclei at cellularization and could play an important role in the establishment of localized gene and protein expression patterns within the early embryo.

Understanding the cell biology of many proteins requires knowledge of their in vivo topological distribution. Here we describe a new fluorescence-based technique, fluorescence protease protection (FPP), for investigating the topology of proteins and for localizing protein subpopulations within the complex environment of the living cell. In the FPP assay, adapted from biochemical protease protection assays, GFP fusion proteins are used as noninvasive tools to obtain details of protein topology and localization within living cells in a rapid and straightforward manner. To demonstrate the broad applicability of FPP, we used the technique to define the topology of proteins localized to a wide range of organelles including the endoplasmic reticulum (ER), Golgi apparatus, mitochondria, peroxisomes and autophagosomes. The success of the FPP assay in characterizing the topology of the tested proteins within their appropriate compartments suggests this technique has wide applicability in studying protein topology and localization within the cell.

Die Entstehung der inzwischen landerubergreifenden und flachendeckenden Vergleichsarbeiten (VERA) wird beschrieben und die dabei auftretenden Schweierigkeiten der Durchfuhrung und Akzeptanz in der Schulwirklichkeit. Die Ziele des «Systemmonitoring» und der Hilfen fur die Selbstevaluation im Sinne der Qualitatsentwicklung entlang der vereinbarten Standards werden entfaltet. Die Geschichte von VERA muss summarisch als durchaus erfolgreich eingeschatz werden, wobei es sich um einen langwierigen Prozess handelt, in dem es zu einer engen Kooperation zwischen praxis und Wissenschaft kommt.

Der Wahrscheinlichkeitsbegriff, schon fur Erwachsene schwierig genug, scheint dem Grundschul- oder gar Vorschulkind verschlossen. Dem ist aber keineswegs so. Der Begriff entwickelt sich bereits im fruhen Alter, auch wenn noch keine formale Begrifflichkeit zur Verfugung steht. Es lassen sich aber Entwicklungslinien identifizieren, Aspekte im Umgang mit stochastischen Situationen, in denen das Kind seine individuelle Sicht auf Zufallsereignisse preisgibt. Es wird in dem Beitrag diesen verschiedenen Perspektiven von Kindern nachgegangen, die nicht als Entwicklungsabfolge verstanden werden konnen, sondern als miteinander verwobene, sich unterschiedlich lange haltende und sich revidierende Ansichten auf Situationen mit unsicherem Ausgang.

The grossing-up method and the chain-ladder method, both with canonical weights, are two famous methods of loss reserving. In the present paper it is shown that the predictors of the ultimate aggregate claims provided by these methods are identical; moreover, under the assumptions of the multiplicative model these predictors can be interpreted as estimators of the expected ultimate aggregate claims and are shown to be identical with the marginal-sum estimators.