Chemometrics in Protein Formulation: Stability Governed by Repulsion and Protein Unfolding.

Therapeutic proteins can be challenging to develop due to their complexity and the requirement of an acceptable formulation to ensure patient safety and efficacy. To date, there is no universal formulation development strategy that can identify optimal formulation conditions for all types of proteins in a fast and reliable manner. In this work, high-throughput characterization, employing a toolbox of five techniques, was performed on 14 structurally different proteins formulated in 6 different buffer conditions and in the presence of 4 different excipients. Multivariate data analysis and chemometrics were used to analyze the data in an unbiased way. First, observed changes in stability were primarily determined by the individual protein. Second, pH and ionic strength are the two most important factors determining the physical stability of proteins, where there exists a significant statistical interaction between protein and pH/ionic strength. Additionally, we developed prediction methods by partial least-squares regression. Colloidal stability indicators are important for prediction of real-time stability, while conformational stability indicators are important for prediction of stability under accelerated stress conditions at 40 °C. In order to predict real-time storage stability, protein-protein repulsion and the initial monomer fraction are the most important properties to monitor.

Self-Interactions of Two Monoclonal Antibodies: Small-Angle X-ray Scattering, Light Scattering, and Coarse-Grained Modeling.

Using light scattering (LS), small-angle X-ray scattering (SAXS), and coarse-grained Monte Carlo (MC) simulations, we studied the self-interactions of two monoclonal antibodies (mAbs), PPI03 and PPI13. With LS measurements, we obtained the osmotic second virial coefficient, B22, and the molecular weight, Mw, of the two mAbs, while with SAXS measurements, we studied the mAbs' self-interaction behavior in the high protein concentration regime up to 125 g/L. Through SAXS-derived coarse-grained representations of the mAbs, we performed MC simulations with either a one-protein or a two-protein model to predict B22. By comparing simulation and experimental results, we validated our models and obtained insights into the mAbs' self-interaction properties, highlighting the role of both ion binding and charged patches on the mAb surfaces. Our models provide useful information about mAbs' self-interaction properties and can assist the screening of conditions driving to colloidal stability.

PICH: a DNA translocase specially adapted for processing anaphase bridge DNA.

The Plk1-interacting checkpoint helicase (PICH) protein localizes to ultrafine anaphase bridges (UFBs) in mitosis alongside a complex of DNA repair proteins, including the Bloom's syndrome protein (BLM). However, very little is known about the function of PICH or how it is recruited to UFBs. Using a combination of microfluidics, fluorescence microscopy, and optical tweezers, we have defined the properties of PICH in an in vitro model of an anaphase bridge. We show that PICH binds with a remarkably high affinity to duplex DNA, resulting in ATP-dependent protein translocation and extension of the DNA. Most strikingly, the affinity of PICH for binding DNA increases with tension-induced DNA stretching, which mimics the effect of the mitotic spindle on a UFB. PICH binding also appears to diminish force-induced DNA melting. We propose a model in which PICH recognizes and stabilizes DNA under tension during anaphase, thereby facilitating the resolution of entangled sister chromatids.

A new species of viviparous brotula genus Pseudogilbia (Ophidiiformes: Dinematichthyidae) from Brazilian reefs, with an updated diagnosis of the genus.

In this study, a new species of Pseudogilbia Møller, Schwarzhans & Nielsen 2004 is described based on two male specimens (40-44 mm LS ) from shallow reefs of Bahia, Brazil. Pseudogilbia australis sp. nov. is distinguished from its only congener, Pseudogilbia sanblasensis Møller, Schwarzhans & Nielsen 2004 from Caribbean Panama, by having: two lower preopercular pores (vs. one); dorsal-fin rays 65-67 (vs. 69); anal-fin rays 51-53 (vs. 56); pectoral-fin rays 18 (vs. 20); caudal vertebrae 27-28 (vs. 30); pectoral-fin length 15.0%-15.9% LS (vs. 14.3); pelvic-fin length 13.5% LS (vs. 16.4) and a different morphology of the male copulatory organ. Pseudogilbia australis sp. nov. is the only dinematichthyid so far recorded in the South Atlantic. An updated diagnosis for the genus is also provided.

Personalized treatment selection in routine care: Integrating machine learning and statistical algorithms to recommend cognitive behavioral or psychodynamic therapy.

Objective: This study aims at developing a treatment selection algorithm using a combination of machine learning and statistical inference to recommend patients' optimal treatment based on their pre-treatment characteristics. Methods: A disorder-heterogeneous, naturalistic sample of N = 1,379 outpatients treated with either cognitive behavioral therapy or psychodynamic therapy was analyzed. Based on a combination of random forest and linear regression, differential treatment response was modeled in the training data (n = 966) to indicate each individual's optimal treatment. A separate holdout dataset (n = 413) was used to evaluate personalized recommendations. Results: The difference in outcomes between patients treated with their optimal vs. non-optimal treatment was significant in the training data, but non-significant in the holdout data (b = -0.043, p = .280). However, for the 50% of patients with the largest predicted benefit of receiving their optimal treatment, the average percentage of change on the BSI in the holdout data was 52.6% for their optimal and 38.4% for their non-optimal treatment (p = .017; d = 0.33 [0.06, 0.61]). Conclusion: A treatment selection algorithm based on a combination of ML and statistical inference might improve treatment outcome for some, but not all outpatients and could support therapists' clinical decision-making.

The cell-cell junctions of mammalian testes. III. Absence of an endothelial cell layer covering the peritubular wall of the seminiferous tubules-an immunocytochemical correction of a 50-year-old error in the literature.

In the molecular biological and ultrastructural studies of the peritubular wall cells encasing the seminiferous tubules of mammalian testes, we found it necessary to characterize the outermost cell layer bordering on the interstitial space in detail. For half a century, the extremely thin cells of this monolayer have in the literature been regarded as part of a lymphatic endothelium, in particular in rodents. However, our double-label immunofluorescence microscopical results have shown that in all six mammalian species examined, including three rodent ones (rat, mouse, guinea pig), this classification is not correct: the very attenuated cells of this monolayer are not of lymphatic endothelial nature as they do not contain established endothelial marker molecules. In particular, they do not contain claudin-5-positive tight junctions, VE-cadherin-positive adherens junctions, "lymph vessel endothelium hyaluronan receptor 1" (LYVE-1), podoplanin, protein myozap and "von Willebrand Factor" (vWF). By contrast and as controls, all these established marker molecules for the lymphatic endothelial cell type are found in the endothelia of the lymph and-partly also-blood vessels located nearby in the interstitial space. Thus, our results provide evidence that the monolayer cells covering the peritubular wall do not contain endothelial marker molecules and hence are not endothelial cells. We discuss possible methodological reasons for the maintenance of this incorrect cell type classification in the literature and emphasize the value of molecular analyses using multiple cell type-specific markers, also with respect to physiology and medical sciences.

Striatin is a novel modulator of cell adhesion.

The adherens junctions (AJs) and tight junctions (TJs) provide critical adhesive contacts between neighboring epithelial cells and are crucial for epithelial adhesion, integrity, and barrier functions in a wide variety of tissues and organisms. The striatin protein family, which are part of the striatin interaction phosphatases and kinases complex, are multidomain scaffolding proteins that play important biologic roles. We have previously shown that striatin colocalizes with the tumor suppressor protein adenomatous polyposis coli in the TJs of epithelial cells. Here we show that striatin affects junction integrity and cell migration, probably through a mechanism that involves the adhesion molecule E-cadherin. Cells engaged in cell-cell adhesion expressed a high MW-modified form of striatin that forms stable associations with detergent-insoluble, membrane-bound cellular fractions. In addition, striatin has recently been suggested to be a target of the poly (ADP-ribose) polymerases Tankyrase 1, and we have found that striatin interacts with Tankyrase 1 and is subsequently poly-ADP-ribosylated. Taken together, our results suggest that striatin is a novel cell-cell junctional protein that functions to maintain correct cell adhesion and may have a role in establishing the relationship between AJs and TJs that is fundamental for epithelial cell-cell adhesion.-Lahav-Ariel, L., Caspi, M., Nadar-Ponniah, P. T., Zelikson, N., Hofmann, I., Hanson, K. K., Franke, W. W., Sklan, E. H., Avraham, K. B., Rosin-Arbesfeld, R. Striatin is a novel modulator of cell adhesion.

The Effect of Point Mutations on the Biophysical Properties of an Antimicrobial Peptide: Development of a Screening Protocol for Peptide Stability Screening.

Therapeutic peptides and proteins show enormous potential in the pharmaceutical market, but high costs in discovery and development are limiting factors so far. Single or multiple point mutations are commonly introduced in protein drugs to increase their binding affinity or selectivity. They can also induce adverse properties, which might be overlooked in a functional screen, such as a decreased colloidal or thermal stability, leading to problems in later stages of the development. In this study, we address the effect of point mutations on the stability of the 4.4 kDa antimicrobial peptide plectasin, as a case study. We combined a systematic high-throughput biophysical screen of the peptide thermal and colloidal stability using dynamic light scattering and differential scanning calorimetry with structure-based methods including small-angle X-ray scattering, analytical ultracentrifugation, and nuclear magnetic resonance spectroscopy. Additionally, we applied molecular dynamics simulations to link obtained protein stability parameters to the protein's molecular structure. Despite their predicted structural similarities, all four plectasin variants showed substantially different behavior in solution. We observed an increasing propensity of plectasin to aggregate at a higher pH, and the introduced mutations influenced the type of aggregation. Our strategy for systematically assessing the stability and aggregation of protein drugs is generally applicable and is of particular relevance, given the increasing number of protein drugs in development.

Advancing Therapeutic Protein Discovery and Development through Comprehensive Computational and Biophysical Characterization.

Therapeutic protein candidates should exhibit favorable properties that render them suitable to become drugs. Nevertheless, there are no well-established guidelines for the efficient selection of proteinaceous molecules with desired features during early stage development. Such guidelines can emerge only from a large body of published research that employs orthogonal techniques to characterize therapeutic proteins in different formulations. In this work, we share a study on a diverse group of proteins, including their primary sequences, purity data, and computational and biophysical characterization at different pH and ionic strength. We report weak linear correlations between many of the biophysical parameters. We suggest that a stability comparison of diverse therapeutic protein candidates should be based on a computational and biophysical characterization in multiple formulation conditions, as the latter can largely determine whether a protein is above or below a certain stability threshold. We use the presented data set to calculate several stability risk scores obtained with an increasing level of analytical effort and show how they correlate with protein aggregation during storage. Our work highlights the importance of developing combined risk scores that can be used for early stage developability assessment. We suggest that such scores can have high prediction accuracy only when they are based on protein stability characterization in different solution conditions.

The cell-cell junctions of mammalian testes: II. The lamellar smooth muscle monolayer cells of the peritubular wall are laterally connected by vertical adherens junctions-a novel architectonic cell-cell junction system.

The testes of sexually mature males of six mammalian species (men, bulls, boars, rats, mice, guinea pigs) have been studied using biochemical as well as light and electron microscopical techniques, in particular immunolocalizations. In these tissues, the peritubular walls represent lamellar encasement structures wrapped around the seminiferous tubules as a bandage system of extracellular matrix layers, alternating with monolayers of very flat polyhedral "lamellar smooth muscle cells" (LSMCs), the number of which varies in different species from 1 to 5 or 6. These LSMCs are complete SMCs containing smooth muscle α-actin (SMA), myosin light and heavy chains, α-actinin, tropomyosin, smoothelin, intermediate-sized filament proteins desmin and/or vimentin, filamin, talin, dystrophin, caldesmon, calponin, and protein SM22α, often also cytokeratins 8 and 18. In the monolayers, the LSMCs are connected by adherens junctions (AJs) based on cadherin-11, in some species also with P-cadherin and/or E-cadherin, which are anchored in cytoplasmic plaques containing ß-catenin and other armadillo proteins, in some species also striatin family proteins, protein myozap and/or LUMA. The LSMC cytoplasm is rich in myofilament bundles, which in many regions are packed in paracrystalline arrays, as well as in "dense bodies," "focal adhesions," and caveolae. In addition to some AJ-like end-on-end contacts, the LSMCs are laterally connected by numerous vertical AJ-like junctions located in variously sized and variously shaped, overlapping (alter super alterum) lamelliform cell protrusions. Consequently, the LSMCs of the peritubular wall monolayers are SMCs sensu stricto which are laterally connected by a novel architectonic system of arrays of vertical AJs located in overlapping cell protrusions.

A novel TPR-BEN domain interaction mediates PICH-BEND3 association.

PICH is a DNA translocase required for the maintenance of chromosome stability in human cells. Recent data indicate that PICH co-operates with topoisomerase IIα to suppress pathological chromosome missegregation through promoting the resolution of ultra-fine anaphase bridges (UFBs). Here, we identify the BEN domain-containing protein 3 (BEND3) as an interaction partner of PICH in human cells in mitosis. We have purified full length PICH and BEND3 and shown that they exhibit a functional biochemical interaction in vitro. We demonstrate that the PICH-BEND3 interaction occurs via a novel interface between a TPR domain in PICH and a BEN domain in BEND3, and have determined the crystal structure of this TPR-BEN complex at 2.2 Å resolution. Based on the structure, we identified amino acids important for the TPR-BEN domain interaction, and for the functional interaction of the full-length proteins. Our data reveal a proposed new function for BEND3 in association with PICH, and the first example of a specific protein-protein interaction mediated by a BEN domain.

A de Novo-Designed Monomeric, Compact Three-Helix-Bundle Protein on a Carbohydrate Template.

De novo design and chemical synthesis of proteins and of other artificial structures that mimic them is a central strategy for understanding protein folding and for accessing proteins with new functions. We have previously described carbohydrates that act as templates for the assembly of artificial proteins, so-called carboproteins. The hypothesis is that the template preorganizes the secondary structure elements and directs the formation of a tertiary structure, thus achieving structural economy in the combination of peptide, linker, and template. We speculate that the structural information from the template could facilitate protein folding. Here we report the design and synthesis of three-helix-bundle carboproteins on deoxyhexopyranosides. The carboproteins were analyzed by CD, analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and NMR spectroscopy, and this revealed the formation of the first compact and folded monomeric carboprotein, distinctly different from a molten globule. En route to this carboprotein we observed a clear effect originating from the template on protein folding.

Striatins as plaque molecules of zonulae adhaerentes in simple epithelia, of tessellate junctions in stratified epithelia, of cardiac composite junctions and of various size classes of lateral adherens junctions in cultures of epithelia- and carcinoma-derived cells.

Proteins of the striatin family (striatins 1-4; sizes ranging from 90 to 110 kDa on SDS-polyacrylamide gel electrophoresis) are highly homologous in their amino acid sequences but can differ in their cell-type-specific gene expression patterns and biological functions. In various cell types, we have found one, two or three polypeptides of this evolutionarily old and nearly ubiquitous family of proteins known to serve as scaffold proteins for diverse protein complexes. Light and electron microscopic immunolocalization methods have revealed striatins in mammalian cell-cell adherens junctions (AJs). In simple epithelia, we have localized striatins as constitutive components of the plaques of the subapical zonulae adhaerentes of cells, including intestinal, glandular, ductal and urothelial cells and hepatocytes. Striatins colocalize with E-cadherin or E-N-cadherin heterodimers and with the plaque proteins α- and ß-catenin, p120 and p0071. In some epithelia and carcinomas and in cultured cells derived therefrom, striatins are also seen in lateral AJs. In stratified epithelia and in corresponding squamous cell carcinomas, striatins can be found in plaques of some forms of tessellate junctions. Moreover, striatins are major plaque proteins of composite junctions (CJs; areae compositae) in the intercalated disks connecting cardiomyocytes, colocalizing with other CJ molecules, including plectin and ankyrin-G. We discuss the "multimodulator" scaffold roles of striatins in the initiation and regulation of the formation of various complex particles and structures. We propose that striatins are included in the diagnostic candidate list of proteins that, in the CJs of human hearts, can occur in mutated forms in the pathogeneses of hereditary cardiomyopathies, as seen in some types of genetically determined heart damage in boxer dogs.

Non-radial oscillation modes with long lifetimes in giant stars.

Towards the end of their lives, stars like the Sun greatly expand to become red giant stars. Such evolved stars could provide stringent tests of stellar theory, as many uncertainties of the internal stellar structure accumulate with age. Important examples are convective overshooting and rotational mixing during the central hydrogen-burning phase, which determine the mass of the helium core, but which are not well understood. In principle, analysis of radial and non-radial stellar oscillations can be used to constrain the mass of the helium core. Although all giants are expected to oscillate, it has hitherto been unclear whether non-radial modes are observable at all in red giants, or whether the oscillation modes have a short or a long mode lifetime, which determines the observational precision of the frequencies. Here we report the presence of radial and non-radial oscillations in more than 300 giant stars. For at least some of the giants, the mode lifetimes are of the order of a month. We observe giant stars with equally spaced frequency peaks in the Fourier spectrum of the time series, as well as giants for which the spectrum seems to be more complex. No satisfactory theoretical explanation currently exists for our observations.

Geosynthetics in geoenvironmental engineering.

Geosynthetics are planar polymeric products, which are used in connection with soil, rock or other soil-like materials to fulfill various functions in geoenvironmental engineering. Geosynthetics are of ever-growing importance in the construction industry. Sealing of waste storage facilities to safely prevent the emission of wastewater, landfill gas and contaminated dust as well as the diffusion of pollutants into the environment and coastal protection against storms and floods and reconstruction after natural disaster are important fields of application. We will give an overview of the various geosynthetic products. Two examples of the material problems related to geosynthetics are discussed in detail: the effect of creep on the long-term performance of geocomposite drains and the numerical simulation of the interaction of soil with geogrids. Both issues are of importance for the use of these products in landfill capping systems. The various functions, which geosynthetics may fulfill in the protection of coastal lines, are illustrated by case studies. The geosynthetic market is evaluated and economical and environmental benefits, as well as environmental side effects related to the use of geosynthetics, are discussed.

Feedback and therapist effects in the context of treatment outcome and treatment length.

OBJECTIVE: This study estimates feedback and therapist effects and tests the predictive value of therapists' and patient attitudes toward psychometric feedback for treatment outcome and length. METHODS: Data of 349 outpatients and 44 therapists in private practices were used. Separate multilevel analyses were conducted to estimate predictors and feedback and therapist effects. RESULTS: Around 5.88% of the variability in treatment outcome and 8.89% in treatment length were attributed to therapists. There was no relationship between the average effectiveness of therapists and the average length of their treatments. Initial impairment, early alliance, number of diagnoses, feedback as well as therapists' and patients' attitudes toward feedback were significant predictors of treatment outcome. Treatments tended to be longer for patients with a higher number of approved sessions by the insurance company, with higher levels of interpersonal distress at intake, and for those who developed negatively (negative feedback) over the course of their treatment. CONCLUSIONS: Therapist effects on treatment outcome and treatment length in routine care seem to be relevant predictors in the context of feedback studies. Therapists' attitudes toward and use of feedback as well as patients' attitudes toward feedback should be further investigated in future research on psychometric feedback.

[Typical patterns of depressive disorders during outpatient psychotherapy and their prediction]. / Verlaufsmuster depressiver Störungen bei ambulanten psychotherapeutischen Behandlungen und deren Vorhersage.

OBJECTIVES: Are there typical patterns of outpatient psychotherapy among depressed patients? What characterizes patients with different patterns? METHODS: We examined N= 548 patients with primary depressive disorders using a naturalistic design. Using a latent-state-mixture model and depression measures at baseline, therapy end and 1-year follow-up we found a total of five patterns. Subgroups were compared with respect to sociodemographic and treatment-related variables. RESULTS: Responders with moderate depressive symptoms at baseline and responders with severe symptoms at baseline were most common (54% and 25% of the sample, respectively) compared to late responders (9 %), small-response patients (9 %) and recidivists (4 %). Patterns of change were related to symptom intensity at baseline and ratings of perceived helpfulness at the end of treatment. CONCLUSIONS: Since psychometric scales better predicted change pattern than sociodemographic characteristics, primary and secondary diagnoses, psychometric assessments and feedback systems could be a useful supplement to traditional quality assurance procedures.

Benefits and challenges in practice-oriented psychotherapy research in Germany: the TK and the QS-PSY-BAY projects of quality assurance in outpatient psychotherapy.

OBJECTIVE: Two patient-focused long-term research projects performed in the German outpatient psychotherapy system are focused on in this article. The TK (Techniker Krankenkasse) project is the first study to evaluate a quality assurance and feedback system with regard to its practical feasibility in German routine care. The other study ("Quality Assurance in Outpatient Psychotherapy in Bavaria"; QS-PSY-BAY) was designed to test a new approach for quality assurance in outpatient psychotherapy using electronic documentation of patient characteristics and outcome parameters. In addition this project provides the opportunity to analyze data on health-related costs for the patients undergoing outpatient psychotherapy. METHOD: Both projects and their results indicating high effect sizes are briefly described. RESULTS: From the perspectives of the research teams, advisory boards and other stakeholders, the experiences with these projects are discussed focusing on obstacles, challenges, difficulties, and benefits in developing and implementing the studies. The triangle collaboration of therapists, researchers, and health insurance companies/health service institutions turned out to be fruitful in both studies. CONCLUSIONS: Despite some controversies between the partners the experiences indicate the importance of practiced-research collaborations to provide relevant information about the delivery of outpatient psychotherapy in the health system.

Mice with cardiac-restricted overexpression of Myozap are sensitized to biomechanical stress and develop a protein-aggregate-associated cardiomyopathy.

The intercalated disc (ID) is a major component of the cell-cell contact structures of cardiomyocytes and has been recognized as a hot spot for cardiomyopathy. We have previously identified Myozap as a novel cardiac-enriched ID protein, which interacts with several other ID proteins and is involved in RhoA/SRF signaling in vitro. To now study its potential role in vivo we generated a mouse model with cardiac overexpression of Myozap. Transgenic (Tg) mice developed cardiomyopathy with hypertrophy and LV dilation. Consistently, these mice displayed upregulation of the hypertrophy-associated and SRF-dependent gene expression. Pressure overload (transverse aortic constriction, TAC) caused exaggerated cardiac hypertrophy, further loss of contractility and LV dilation. Similarly, a physiological stimulus (voluntary running) also led to significant LV dysfunction. On the ultrastructural level, Myozap-Tg mouse hearts exhibited massive protein aggregates composed of Myozap, desmoplakin and other ID proteins. This aggregate-associated pathology closely resembled the alterations observed in desmin-related cardiomyopathy. Interestingly, desmin was not detectable in the aggregates, yet was largely displaced from the ID. Molecular analyses revealed induction of autophagy and dysregulation of the unfolded protein response (UPR), associated with apoptosis. Taken together, cardiac overexpression of Myozap leads to cardiomyopathy, mediated, at least in part by induction of Rho-dependent SRF signaling in vivo. Surprisingly, this phenotype was also accompanied by protein aggregates in cardiomyocytes, UPR alteration, accelerated autophagy and apoptosis. Thus, this mouse model may also offer additional insight into the pathogenesis of protein-aggregate-associated cardiomyopathies and represents a new candidate gene itself.

Protein LUMA is a cytoplasmic plaque constituent of various epithelial adherens junctions and composite junctions of myocardial intercalated disks: a unifying finding for cell biology and cardiology.

In a series of recent reports, mutations in the gene encoding a protein called LUMA (or TMEM43), widely speculated to be a tetraspan transmembrane protein of the nuclear envelope, have been associated with a specific subtype of cardiomyopathy (arrhythmogenic cardiomyopathies) and cases of sudden death. However, using antibodies of high specificity in immunolocalization experiments, we have discovered that, in mammals, LUMA is a component of zonula adhaerens and punctum adhaerens plaques of diverse epithelia and epithelial cell cultures and is also located in (or in some species associated with) the plaques of composite junctions (CJs) in myocardiac intercalated disks (IDs). In CJs, LUMA often colocalizes with several other CJ marker proteins. In all these cells, LUMA has not been detected in the nuclear envelope. Surprisingly, under certain conditions, similar CJ localizations have also been seen with some antibodies commercially available for some time. The identification of LUMA as a plaque component of myocardiac CJs leads to reconsiderations of the molecular composition and architecture, the development, the functions, and the pathogenic states of CJs and IDs. These findings now also allow the general conclusion that LUMA has to be added to the list of mutations of cardiomyocyte junction proteins that may be involved in cardiomyopathies.