Using External Information for More Precise Inferences in General Regression Models.

Empirical research usually takes place in a space of available external information, like results from single studies, meta-analyses, official statistics or subjective (expert) knowledge. The available information ranges from simple means and proportions to known relations between a multitude of variables or estimated distributions. In psychological research, external information derived from the named sources may be used to build a theory and derive hypotheses. In addition, techniques do exist that use external information in the estimation process, for example prior distributions in Bayesian statistics. In this paper, we discuss the benefits of adopting generalized method of moments with external moments, as another example for such a technique. Analytical formulas for estimators and their variances in the multiple linear regression case are derived. An R function that implements these formulas is provided in the supplementary material for general applied use. The effects of various practically relevant moments are analyzed and tested in a simulation study. A new approach to robustify the estimators against misspecification of the external moments based on the concept of imprecise probabilities is introduced. Finally, the resulting externally informed model is applied to a dataset to investigate the predictability of the premorbid intelligence quotient based on lexical tasks, leading to a reduction of variances and thus to narrower confidence intervals.

In models we trust: preregistration, large samples, and replication may not suffice.

Despite discussions about the replicability of findings in psychological research, two issues have been largely ignored: selection mechanisms and model assumptions. Both topics address the same fundamental question: Does the chosen statistical analysis tool adequately model the data generation process? In this article, we address both issues and show, in a first step, that in the face of selective samples and contrary to common practice, the validity of inferences, even when based on experimental designs, can be claimed without further justification and adaptation of standard methods only in very specific situations. We then broaden our perspective to discuss consequences of violated assumptions in linear models in the context of psychological research in general and in generalized linear mixed models as used in item response theory. These types of misspecification are oftentimes ignored in the psychological research literature. It is emphasized that the above problems cannot be overcome by strategies such as preregistration, large samples, replications, or a ban on testing null hypotheses. To avoid biased conclusions, we briefly discuss tools such as model diagnostics, statistical methods to compensate for selectivity and semi- or non-parametric estimation. At a more fundamental level, however, a twofold strategy seems indispensable: (1) iterative, cumulative theory development based on statistical methods with theoretically justified assumptions, and (2) empirical research on variables that affect (self-) selection into the observed part of the sample and the use of this information to compensate for selectivity.

Arf1 coordinates fatty acid metabolism and mitochondrial homeostasis.

Lipid mobilization through fatty acid ß-oxidation is a central process essential for energy production during nutrient shortage. In yeast, this catabolic process starts in the peroxisome from where ß-oxidation products enter mitochondria and fuel the tricarboxylic acid cycle. Little is known about the physical and metabolic cooperation between these organelles. Here we found that expression of fatty acid transporters and of the rate-limiting enzyme involved in ß-oxidation is decreased in cells expressing a hyperactive mutant of the small GTPase Arf1, leading to an accumulation of fatty acids in lipid droplets. Consequently, mitochondria became fragmented and ATP synthesis decreased. Genetic and pharmacological depletion of fatty acids phenocopied the arf1 mutant mitochondrial phenotype. Although ß-oxidation occurs in both mitochondria and peroxisomes in mammals, Arf1's role in fatty acid metabolism is conserved. Together, our results indicate that Arf1 integrates metabolism into energy production by regulating fatty acid storage and utilization, and presumably organelle contact sites.

A common mechanism of Sec61 translocon inhibition by small molecules.

The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural and synthetic small molecules specifically inhibit Sec61, generating cellular effects that are useful for therapeutic purposes, but their inhibitory mechanisms remain unclear. Here we present near-atomic-resolution structures of human Sec61 inhibited by a comprehensive panel of structurally distinct small molecules-cotransin, decatransin, apratoxin, ipomoeassin, mycolactone, cyclotriazadisulfonamide and eeyarestatin. All inhibitors bind to a common lipid-exposed pocket formed by the partially open lateral gate and plug domain of Sec61. Mutations conferring resistance to the inhibitors are clustered at this binding pocket. The structures indicate that Sec61 inhibitors stabilize the plug domain in a closed state, thereby preventing the protein-translocation pore from opening. Our study provides the atomic details of Sec61-inhibitor interactions and the structural framework for further pharmacological studies and drug design.

Retrograde transport of CDMPR depends on several machineries as analyzed by sulfatable nanobodies.

Retrograde protein transport from the cell surface and endosomes to the TGN is essential for membrane homeostasis in general and for the recycling of mannose-6-phosphate receptors (MPRs) for sorting of lysosomal hydrolases in particular. We used a nanobody-based sulfation tool to more directly determine transport kinetics from the plasma membrane to the TGN for the cation-dependent MPR (CDMPR) with and without rapid or gradual inactivation of candidate machinery proteins. Although knockdown of retromer (Vps26), epsinR, or Rab9a reduced CDMPR arrival to the TGN, no effect was observed upon silencing of TIP47. Strikingly, when retrograde transport was analyzed by rapamycin-induced rapid depletion (knocksideways) or long-term depletion by knockdown of the clathrin adaptor AP-1 or of the GGA machinery, distinct phenotypes in sulfation kinetics were observed, suggesting a potential role of GGA adaptors in retrograde and anterograde transport. Our study illustrates the usefulness of derivatized, sulfation-competent nanobodies, reveals novel insights into CDMPR trafficking biology, and further outlines that the selection of machinery inactivation is critical for phenotype analysis.

Small disulfide loops in peptide hormones mediate self-aggregation and secretory granule sorting.

Unlike constitutively secreted proteins, peptide hormones are stored in densely packed secretory granules, before regulated release upon stimulation. Secretory granules are formed at the TGN by self-aggregation of prohormones as functional amyloids. The nonapeptide hormone vasopressin, which forms a small disulfide loop, was shown to be responsible for granule formation of its precursor in the TGN as well as for toxic fibrillar aggregation of unfolded mutants in the ER. Several other hormone precursors also contain similar small disulfide loops suggesting their function as a general device to mediate aggregation for granule sorting. To test this hypothesis, we studied the capacity of small disulfide loops of different hormone precursors to mediate aggregation in the ER and the TGN. They indeed induced ER aggregation in Neuro-2a and COS-1 cells. Fused to a constitutively secreted reporter protein, they also promoted sorting into secretory granules, enhanced stimulated secretion, and increased Lubrol insolubility in AtT20 cells. These results support the hypothesis that small disulfide loops act as novel signals for sorting into secretory granules by self-aggregation.

RABEP1/Rabaptin5: a link between autophagy and early endosome homeostasis.

Selective autophagy of damaged organelles assures maintenance of cellular homeostasis in eukaryotes. While the mechanisms by which cells selectively remove dysfunctional mitochondria, lysosomes, endoplasmic reticulum and other organelles has been well characterized, little is known about specific autophagy of damaged early endosomes. In our recent study, we uncovered a new role for RABEP1/Rabaptin5, a long-established regulator of early endosome function, in targeting the autophagy machinery to early endosomes damaged by chloroquine or by internalized Salmonella via interaction with RB1CC1/FIP200 and ATG16L1.

Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1.

Selective autophagy of damaged organelles is important to maintain cellular homeostasis. The mechanisms how autophagy selects specific targets is often poorly understood. Rabaptin5 was previously known as a major regulator of early endosome identity and maturation. Here, we identify two novel Rabaptin5 interactors: FIP200, a subunit of the ULK1 autophagy initiator complex, and ATG16L1, a central component of the E3-like enzyme in LC3 lipidation. Autophagy of early endosomes damaged by chloroquine or monensin treatment requires Rabaptin5 and particularly a short sequence motif that binds to the WD domain of ATG16L1. Rabaptin5 and its interaction with ATG16L1 further contributes to the autophagic elimination of Salmonella enterica early after infection, when it resides in phagosomes with early endosomal characteristics. Our results demonstrate a novel function of Rabaptin5 in quality control of early endosomes in the selective targeting of autophagy to damaged early endosomes and phagosomes.

Shared and specific functions of Arfs 1-5 at the Golgi revealed by systematic knockouts.

ADP-ribosylation factors (Arfs) are small GTPases regulating membrane traffic in the secretory pathway. They are closely related and appear to have overlapping functions, regulators, and effectors. The functional specificity of individual Arfs and the extent of redundancy are still largely unknown. We addressed these questions by CRISPR/Cas9-mediated genomic deletion of the human class I (Arf1/3) and class II (Arf4/5) Arfs, either individually or in combination. Most knockout cell lines were viable with slight growth defects only when lacking Arf1 or Arf4. However, Arf1+4 and Arf4+5 could not be deleted simultaneously. Class I Arfs are nonessential, and Arf4 alone is sufficient for viability. Upon Arf1 deletion, the Golgi was enlarged, and recruitment of vesicle coats decreased, confirming a major role of Arf1 in vesicle formation at the Golgi. Knockout of Arf4 caused secretion of ER-resident proteins, indicating specific defects in coatomer-dependent ER protein retrieval by KDEL receptors. The knockout cell lines will be useful tools to study other Arf-dependent processes.

Efficient integration of transmembrane domains depends on the folding properties of the upstream sequences.

The topology of most membrane proteins is defined by the successive integration of α-helical transmembrane domains at the Sec61 translocon. The translocon provides a pore for the transfer of polypeptide segments across the membrane while giving them lateral access to the lipid. For each polypeptide segment of ∼20 residues, the combined hydrophobicities of its constituent amino acids were previously shown to define the extent of membrane integration. Here, we discovered that different sequences preceding a potential transmembrane domain substantially affect its hydrophobicity requirement for integration. Rapidly folding domains, sequences that are intrinsically disordered or very short or capable of binding chaperones with high affinity, allow for efficient transmembrane integration with low-hydrophobicity thresholds for both orientations in the membrane. In contrast, long protein fragments, folding-deficient mutant domains, and artificial sequences not binding chaperones interfered with membrane integration, requiring higher hydrophobicity. We propose that the latter sequences, as they compact on their hydrophobic residues, partially folded but unable to reach a native state, expose hydrophobic surfaces that compete with the translocon for the emerging transmembrane segment, reducing integration efficiency. The results suggest that rapid folding or strong chaperone binding is required for efficient transmembrane integration.

Cotranslational Targeting and Posttranslational Translocation can Cooperate in Spc3 Topogenesis.

Secretory and membrane proteins follow either the signal recognition particle (SRP)-dependent cotranslational translocation pathway or the SRP-independent Sec62/Sec63-dependent posttranslational pathway for their translocation across the endoplasmic reticulum (ER). However, increasing evidence suggests that most proteins are cotranslationally targeted to the ER, suggesting mixed mechanisms. It remains unclear how these two pathways cooperate. Previous studies have shown that Spc3, a signal-anchored protein, requires SRP and Sec62 for its biogenesis. This study investigated the targeting and topogenesis of Spc3 and the step at which SRP and Sec62 act using in vivo and in vitro translocation assays and co-immunoprecipitation. Our data suggest that Spc3 reaches its final topology in two steps: it enters the ER lumen head-first and then inverts its orientation. The first step is partially dependent on SRP, although independent of the Sec62/Sec63 complex. The second step is mediated by the Sec62/Sec63 complex. These data suggest that SRP and Sec62 act on a distinct step in the topogenesis of Spc3.

Genetic forms of neurohypophyseal diabetes insipidus.

In the majority of cases, hereditary neurohypophyseal diabetes insipidus (DI) is a monogenic disorder caused by mutations in the AVP gene. Dominant transmission is by far the most common form. In these patients, symptoms develop gradually at various ages during childhood, progressing with complete penetrance to polyuria and polydipsia that is usually severe. In autosomal dominant neurohypophyseal DI (ADNDI), the mutant prohormone is folding deficient and consequently retained in the ER, where it forms amyloid-like fibrillar aggregates. Degradation by proteasomes occurs, but their clearance capacity appears to be insufficient. Postmortem studies in affected individuals suggest a neurodegenerative process confined to vasopressinergic neurons. Other forms of genetic neurohypophyseal DI include the very rare autosomal recessive type, also caused by mutations in the AVP gene, and complex multiorgan disorders, such as Wolfram syndrome. In all individuals where a congenital form of DI is suspected, including nephrogenic types, genetic analysis should be performed.

Generalized estimating equations to estimate the ordered stereotype logit model for panel data.

By modeling the effects of predictor variables as a multiplicative function of regression parameters being invariant over categories, and category-specific scalar effects, the ordered stereotype logit model is a flexible regression model for ordinal response variables. In this article, we propose a generalized estimating equations (GEE) approach to estimate the ordered stereotype logit model for panel data based on working covariance matrices, which are not required to be correctly specified. A simulation study compares the performance of GEE estimators based on various working correlation matrices and working covariance matrices using local odds ratios. Estimation of the model is illustrated using a real-world dataset. The results from the simulation study suggest that GEE estimation of this model is feasible in medium-sized and large samples and that estimators based on local odds ratios as realized in this study tend to be less efficient compared with estimators based on a working correlation matrix. For low true correlations, the efficiency gains seem to be rather small and if the working covariance structure is too flexible, the corresponding estimator may even be less efficient compared with the GEE estimator assuming independence. Like for GEE estimators more generally, if the true correlations over time are high, then a working covariance structure which is close to the true structure can lead to considerable efficiency gains compared with assuming independence.

Amyloid-like aggregation of provasopressin.

The antidiuretic hormone vasopressin is synthesized as a longer precursor protein. After folding in the endoplasmic reticulum (ER), provasopressin is transported through the secretory pathway, forms secretory granules in the trans-Golgi network (TGN), is processed, and finally secreted into the circulation. Mutations in provasopressin cause autosomal dominant diabetes insipidus. They prevent native protein folding and cause fibrillar, amyloid-like aggregation in the ER, which eventually results in cell death. Secretory granules of peptide hormones were proposed to constitute functional amyloids and thus might be the cause of amyloid formation of misfolded mutant protein in the ER. Indeed, the same two segments in the precursor-vasopressin and a C-terminal glycopeptide-were found to be responsible for pathological aggregation in the ER and physiological aggregation in granule formation in the TGN. Furthermore, even wild-type provasopressin tends to aggregate in the ER, but is controlled by ER-associated degradation. When essential components thereof, Sel1L or Hrd1, were inactivated, wild-type provasopressin accumulated as fibrillar aggregates in vasopressinergic neurons in mice, causing diabetes insipidus. Evolution of amyloidogenic sequences for granule formation thus made provasopressin dependent on ER quality control mechanisms. These principles may similarly apply to other peptide hormones.

Model-based goodness-of-fit tests for the ordered stereotype model.

This paper presents two new model-based goodness-of-fit tests for the ordered stereotype model applied to an ordinal response variable. The proposed tests are based on the Lipsitz test, which partitions the subjects into G groups following the popular Hosmer-Lemeshow test for binary data. The tests construct an alternative model where group effects are added into the null model. If the model fits the data well then the null model is correct, and there should be no group effects. One of the main advantages of the ordered stereotype model is that it allows us to determine a new uneven spacing of the ordinal response categories, dictated by the data. The two proposed tests use this new adjusted spacing. One test uses the form of the original ordered stereotype model, and the other uses an ordinary linear model. We demonstrate the performance of both tests under a variety of scenarios. Finally, the results of the application in three examples are presented.

Role of protein aggregation and degradation in autosomal dominant neurohypophyseal diabetes insipidus.

This review focuses on the cellular and molecular aspects underlying familial neurohypophyseal diabetes insipidus (DI), a rare disorder that is usually transmitted in an autosomal-dominant fashion. The disease, manifesting in infancy or early childhood and gradually progressing in severity, is caused by fully penetrant heterozygous mutations in the gene encoding prepro-vasopressin-neurophysin II, the precursor of the antidiuretic hormone arginine vasopressin (AVP). Post mortem studies in affected adults have shown cell degeneration in vasopressinergic hypothalamic nuclei. Studies in cells expressing pathogenic mutants and knock-in rodent models have shown that the mutant precursors are folding incompetent and fail to exit the endoplasmic reticulum (ER), as occurs normally with proteins that have entered the regulated secretory pathway. A portion of these mutants is eliminated via ER-associated degradation (ERAD) by proteasomes after retrotranslocation to the cytosol. Another portion forms large disulfide-linked fibrillar aggregates within the ER, in which wild-type precursor is trapped. Aggregation capacity is independently conferred by two domains of the prohormone, namely the AVP moiety and the C-terminal glycopeptide (copeptin). The same domains are also required for packaging into dense-core secretory granules and regulated secretion, suggesting a disturbed balance between the physiological self-aggregation at the trans-Golgi network and avoiding premature aggregate formation at the ER in the disease. The critical role of ERAD in maintaining physiological water balance has been underscored by experiments in mice expressing wild-type AVP but lacking critical components of the ERAD machinery. These animals also develop DI and show amyloid-like aggregates in the ER lumen. Thus, the capacity of the ERAD is exceeded in autosomal dominant DI, which can be viewed as a neurodegenerative disorder associated with the formation of amyloid ER aggregates. While DI symptoms develop prior to detectable cell death in transgenic DI mice, the eventual loss of vasopressinergic neurons is accompanied by autophagy, but the mechanism leading to cell degeneration in autosomal dominant neurohypophyseal DI still remains unknown.

Hereditary Neurohypophyseal Diabetes Insipidus.

Neurohypophyseal diabetes insipidus (DI) is most often caused by trauma, including operations, and infiltrating processes in the hypothalamic-pituitary region. Irradiation, ischemia, infections, or autoimmunity can also underlie the disease. Since the middle of the nineteenth century, familial forms of neurohypophyseal DI have been described. Most commonly, the disease is transmitted in an autosomal dominant fashion; very rarely, autosomal recessive inheritance has been observed. Hereditary neurohypophyseal DI is caused by mutations in the gene encoding the antidiuretic hormone vasopressin (AVP) and its carrier protein neurophysin II (NPII). Symptoms result from the lack of hormone, or from the inability of mutant AVP to activate its renal receptor, and respond to treatment with desmopressin (DDAVP). Dominant mutations cause retention of the hormone precursor in the endoplasmic reticulum (ER) of vasopressinergic neurons in the hypothalamus, resulting in cellular dysfunction and eventually neuronal death. This so-called neurotoxicity hypothesis was initially established on the basis of autopsy studies in affected humans and has been supported by heterologous cell culture expression experiments and murine knock-in models. Current data show that retained mutants fail to be eliminated by the cell's quality control system and accumulate in fibrillar aggregations within the ER. Autosomal dominant neurohypophyseal DI may thus be viewed as a neurodegenerative disease confined to vasopressinergic neurons.

Rethinking the Interpretation of Item Discrimination and Factor Loadings.

Factor loadings and item discrimination parameters play a key role in scale construction. A multitude of heuristics regarding their interpretation are hardwired into practice-for example, neglecting low loadings and assigning items to exactly one scale. We challenge the common sense interpretation of these parameters by providing counterexamples and general results which altogether cast doubt on our understanding of these parameters. In particular, we highlight the counterintuitive way in which the best prediction of a test taker's latent ability depends on the factor loadings. As a consequence, we emphasize that practitioners need to shift their focus from interpreting item discrimination parameters by their relative loading to an interpretation which incorporates the structure of the model-based latent ability estimate.

Quantitative proteomics reveals reduction of endocytic machinery components in gliomas.

BACKGROUND: Gliomas are the most frequent and aggressive malignancies of the central nervous system. Decades of molecular analyses have demonstrated that gliomas accumulate genetic alterations that culminate in enhanced activity of receptor tyrosine kinases and downstream mediators. While the genetic alterations, like gene amplification or loss, have been well characterized, little information exists about changes in the proteome of gliomas of different grades. METHODS: We performed unbiased quantitative proteomics of human glioma biopsies by mass spectrometry followed by bioinformatic analysis. FINDINGS: Various pathways were found to be up- or downregulated. In particular, endocytosis as pathway was affected by a vast and concomitant reduction of multiple machinery components involved in initiation, formation, and scission of endocytic carriers. Both clathrin-dependent and -independent endocytosis were changed, since not only clathrin, AP-2 adaptins, and endophilins were downregulated, but also dynamin that is shared by both pathways. The reduction of endocytic machinery components caused increased receptor cell surface levels, a prominent phenotype of defective endocytosis. Analysis of additional biopsies revealed that depletion of endocytic machinery components was a common trait of various glioma grades and subclasses. INTERPRETATION: We propose that impaired endocytosis creates a selective advantage in glioma tumor progression due to prolonged receptor tyrosine kinase signaling from the cell surface. FUND: This work was supported by Grants 316030-164105 (to P. Jenö), 31003A-162643 (to M. Spiess) and PP00P3-176974 (to G. Hutter) from the Swiss National Science Foundation. Further funding was received by the Department of Surgery from the University Hospital Basel.

Membrane Protein Integration and Topogenesis at the ER.

Most membrane proteins are composed of hydrophobic α-helical transmembrane segments and are integrated into the lipid bilayer of the endoplasmic reticulum by the highly conserved Sec61 translocon. With respect to the integration mechanism, three types of transmembrane segments can be distinguished-the signal, the stop-transfer sequence, and the re-integration sequence-which in linear succession can account for all kinds of membrane protein topologies. The transmembrane orientation of the initial signal and to a weaker extent also of downstream transmembrane segments is affected by charged flanking residues according to the so-called positive-inside rule. The main driving force for transmembrane integration is hydrophobicity. Systematic analysis suggested thermodynamic equilibration of each peptide segment in the translocon with the membrane as the underlying mechanism. However, there is evidence that integration is not entirely sequence-autonomous, but depends also on the sequence context, from very closely spaced transmembrane segments to the folding state and properties of neighboring sequences. Topogenesis is even influenced by accessory proteins that appear to act as intramembrane chaperones.