Determination of Binding Sites on Trastuzumab and Pertuzumab to Selective Affimers Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a method to probe the solvent accessibility and conformational dynamics of a protein or a protein-ligand complex with respect to exchangeable amide hydrogens. Here, we present the application of HDX-MS to determine the binding sites of Affimer reagents to the monoclonal antibodies trastuzumab and pertuzumab, respectively. Intact and subunit level HDX-MS analysis of antibody-affimer complexes showed significant protection from HDX in the antibody Fab region upon affimer binding. Bottom-up HDX-MS experiments including online pepsin digestion revealed that the binding sites of the affimer reagents were mainly located in the complementarity-determining region (CDR) 2 of the heavy chain of the respective antibodies. Three-dimensional models of the binding interaction between the affimer reagents and the antibodies were built by homology modeling and molecular docking based on the HDX data.

Aggregation Time Machine: A Platform for the Prediction and Optimization of Long-Term Antibody Stability Using Short-Term Kinetic Analysis.

Monoclonal antibodies are the fastest growing class of therapeutics. However, aggregation limits their shelf life and can lead to adverse immune responses. Assessment and optimization of the long-term antibody stability are therefore key challenges in the biologic drug development. Here, we present a platform based on the analysis of temperature-dependent aggregation data that can dramatically shorten the assessment of the long-term aggregation stability and thus accelerate the optimization of antibody formulations. For a set of antibodies used in the therapeutic areas from oncology to rheumatology and osteoporosis, we obtain an accurate prediction of aggregate fractions for up to three years using the data obtained on a much shorter time scale. Significantly, the strategy combining kinetic and thermodynamic analysis not only contributes to a better understanding of the molecular mechanisms of antibody aggregation but has already proven to be very effective in the development and production of biological therapeutics.

A pragmatic randomised controlled trial referring to a Personalised Self-management SUPport Programme (P-SUP) for persons enrolled in a disease management programme for type 2 diabetes mellitus and/or for coronary heart disease.

BACKGROUND: Type 2 diabetes mellitus (T2DM) and coronary heart disease (CHD) are two chronic diseases that cause a tremendous burden. To reduce this burden, several programmes for optimising the care for these diseases have been developed. In Germany, so-called disease management programmes (DMPs), which combine components of Disease Management and the Chronic Care Model, are applied. These DMPs have proven effective. Nevertheless, there are opportunities for improvement. Current DMPs rarely address self-management of the disease, make no use of peer support, and provide no special assistance for persons with low health literacy and/or low patient activation. The study protocol presented here is for the evaluation of a programme that addresses these possible shortcomings and can be combined with current German DMPs for T2DM and CHD. This programme consists of four components: 1) Meetings of peer support groups 2) Personalised telephone-based health coaching for patients with low literacy and/or low patient activation 3) Personalised patient feedback 4) A browser-based web portal METHODS: Study participants will be adults enrolled in a DMP for T2DM and/or CHD and living in North Rhine-Westphalia, a state of the Federal Republic of Germany. Study participants will be recruited with the assistance of their general practitioners by the end of June 2021. Evaluation will be performed as a pragmatic randomised controlled trial with one intervention group and one waiting control group. The intervention group will receive the intervention for 18 months. During this time, the waiting control group will continue with usual care and the usual measures of their DMPs. After 18 months, the waiting control group will also receive a shortened intervention. The primary outcome is number of hospital days. In addition, the effects on self-reported health-state, physical activity, nutrition, and eight different psychological variables will be investigated. Differences between values at month 18 and at the beginning will be compared to judge the effectiveness of the intervention. DISCUSSION: If the intervention proves effective, it may be included into the DMPs for T2DM and CHD. TRIAL REGISTRATION: The study was registered in the German Clinical Trials Registry (Deutsches Register Klinischer Studien (DRKS)) in early 2019 under the number 00020592. This registry has been affiliated with the WHO Clinical Trials Network ( https://www.drks.de/drks_web/setLocale_EN.do ) since 2008. It is based on the WHO template, but contains some additional categories for which information has to be given ( https://www.drks.de/drks_web/navigate.do?navigationId=entryfields&messageDE=Beschreibung%20der%20Eingabefelder&messageEN=Description%20of%20entry%20fields ). A release and subsequent number assignment only take place when information for all categories has been given.

Enhanced CHO Clone Screening: Application of Targeted Locus Amplification and Next-Generation Sequencing Technologies for Cell Line Development.

Early analytical clone screening is important during Chinese hamster ovary (CHO) cell line development of biotherapeutic proteins to select a clonally derived cell line with most favorable stability and product quality. Sensitive sequence confirmation methods using mass spectrometry have limitations in throughput and turnaround time. Next-generation sequencing (NGS) technologies emerged as alternatives for CHO clone analytics. We report an efficient NGS workflow applying the targeted locus amplification (TLA) strategy for genomic screening of antibody expressing CHO clones. In contrast to previously reported RNA sequencing approaches, TLA allows for targeted sequencing of genomic integrated transgenic DNA without prior locus information, robust detection of single-nucleotide variants (SNVs) and transgenic rearrangements. During clone selection, TLA/NGS revealed CHO clones with high-level SNVs within the antibody gene and we report in another case the utility of TLA/NGS to identify rearrangements at transgenic DNA level. We also determined detection limits for SNVs calling and the potential to identify clone contaminations by TLA/NGS. TLA/NGS also allows to identify genetically identical clones. In summary, we demonstrate that TLA/NGS is a robust screening method useful for routine clone analytics during cell line development with the potential to process up to 24 CHO clones in less than 7 workdays.

A new quantum speed-meter interferometer: measuring speed to search for intermediate mass black holes.

The recent discovery of gravitational waves (GW) by Advanced LIGO (Laser Interferometric Gravitational-wave Observatory) has impressively launched the novel field of gravitational astronomy and allowed us to glimpse exciting objects about which we could previously only speculate. Further sensitivity improvements at the low-frequency end of the detection band of future GW observatories must rely on quantum non-demolition (QND) methods to suppress fundamental quantum fluctuations of the light fields used to readout the GW signal. Here we present a novel concept of how to turn a conventional Michelson interferometer into a QND speed-meter interferometer with coherently suppressed quantum back-action noise. We use two orthogonal polarizations of light and an optical circulator to couple them. We carry out a detailed analysis of how imperfections and optical loss influence the achievable sensitivity. We find that the proposed configuration significantly enhances the low-frequency sensitivity and increases the observable event rate of binary black-hole coalescences in the range of 1 0 2 - 1 0 3 M ⊙ by a factor of up to ~300.

Chronic care model strategies in the United States and Germany deliver patient-centered, high-quality diabetes care.

Improving the quality of care for chronic diseases is an important issue for most health care systems in industrialized nations. One widely adopted approach is the Chronic Care Model (CCM), which was first developed in the late 1990s. In this article we present the results from two large surveys in the United States and Germany that report patients' experiences in different models of patient-centered diabetes care, compared to the experiences of patients who received routine diabetes care in the same systems. The study populations were enrolled in either Geisinger Health System in Pennsylvania or Barmer, a German sickness fund that provides medical insurance nationwide. Our findings suggest that patients with type 2 diabetes who were enrolled in the care models that exhibited key features of the CCM were more likely to receive care that was patient-centered, high quality, and collaborative, compared to patients who received routine care. This study demonstrates that quality improvement can be realized through the application of the Chronic Care Model, regardless of the setting or distinct characteristics of the program.

Variation in the calpain-10 gene is not associated with gestational diabetes mellitus.

BACKGROUND: In several large studies an association between certain single-nucleotide polymorphisms (SNP) of the calpain-10 gene (CAPN10) with type 2 diabetes mellitus (T2D) has been identified. Since T2D and gestational diabetes mellitus (GDM) seem to be linked pathophysiologically, we examined the frequencies of CAPN10-polymorphisms in women with GDM. METHODS: By using real-time PCR assisted melting curve analysis samples of 204 women with GDM and 297 controls were tested for variations of SNP-43, -44, -63 and Indel-19 of CAPN10. RESULTS: Since the genotype frequencies found in SNP-44 among the controls did not meet the Hardy-Weinberg-Equilibrium, the further analysis was performed with SNP-43, -63 and Indel-19 only. Herein, the distribution of neither genotype nor allele nor haplogenotype-combination nor haplotype showed a significant difference between both groups. CONCLUSIONS: Variations of SNP-43, -63 and Indel-19 of CAPN10 were not associated with an increased risk of developing GDM.

Differences in conformational dynamics within the Hsp90 chaperone family reveal mechanistic insights.

The molecular chaperones of the Hsp90 family are essential in all eukaryotic cells. They assist late folding steps and maturation of many different proteins, called clients, that are not related in sequence or structure. Hsp90 interaction with its clients appears to be coupled to a series of conformational changes. Using hydrogen exchange mass spectrometry (HX-MS) we investigated the structural dynamics of human Hsp90ß (hHsp90) and yeast Hsp82 (yHsp82). We found that eukaryotic Hsp90s are much more flexible than the previously studied Escherichia coli homolog (EcHtpG) and that nucleotides induce much smaller changes. More stable conformations in yHsp82 are obtained in presence of co-chaperones. The tetratricopeptide repeat (TPR) domain protein Cpr6 causes a different amide proton protection pattern in yHsp82 than the previously studied TPR-domain protein Sti1. In the simultaneous presence of Sti1 and Cpr6, protection levels are observed that are intermediate between the Sti1 and the Cpr6 induced changes. Surprisingly, no bimodal distributions of the isotope peaks are detected, suggesting that both co-chaperones affect both protomers of the Hsp90 dimer in a similar way. The cochaperones Sba1 was found previously in the crystal structure bound to the ATP hydrolysis-competent conformation of Hsp90, which did not allow to distinguish the mode of Sba1-mediated inhibition of Hsp90's ATPase activity by stabilizing the pre- or post-hydrolysis step. Our HX-MS experiments now show that Sba1 binding leads to a protection of the ATP binding lid, suggesting that it inhibits Hsp90's ATPase activity by slowing down product release. This hypothesis was verified by a single-turnover ATPase assay. Together, our data suggest that there are much smaller energy barriers between conformational states in eukaryotic Hsp90s than in EcHtpG and that co-chaperones are necessary in addition to nucleotides to stabilize defined conformational states.

Length sensing and control of a Michelson interferometer with power recycling and twin signal recycling cavities.

The techniques of power recycling and signal recycling have proven as key concepts to increase the sensitivity of large-scale gravitational wave detectors by independent resonant enhancement of light power and signal sidebands within the interferometer. Developing the latter concept further, twin signal recycling was proposed as an alternative to conventional detuned signal recycling. Twin signal recycling features the narrow-band sensitivity gain of conventional detuned signal recycling but furthermore facilitates the injection of squeezed states of light, increases the detector sensitivity over a wide frequency band and requires a less complex detection scheme for optimal signal readout. These benefits come at the expense of an additional recycling mirror, thus increasing the number of degrees of freedom in the interferometer which need to be controlled.In this article we describe the development of a length sensing and control scheme and its successful application to a tabletop-scale power recycled Michelson interferometer with twin signal recycling. We were able to lock the interferometer in all relevant longitudinal degrees of freedom and thus laid the foundation for further investigations of this interferometer configuration to evaluate its viability for the application in gravitational wave detectors.

[Evaluating the effectiveness of a disease management program diabetes in the German Statutory Health Insurance: first results and methodological considerations]. / Evaluation der Effektivität eines Disease Management Programms Diabetes Mellitus in der GKV - Erste Ergebnisse und methodische Überlegungen.

Disease management programs (DMPs) were implemented in the German Statutory Health Insurance (SHI) in a nationwide rollout in 2002. The explicit goal of the programs is to improve coordination and quality of care for the chronically ill (Sect. 137f, SGB V). To reach this goal extensive quality assurance measures in the programs are mandatory, enrolment and coordination of care rests with the primary care or DMP physician, treatment is based on evidence-based care guidelines, and patients are offered diabetes education classes to support self-management. The present study evaluates the DMP diabetes mellitus type II, a nationwide program offered by the BARMER, a German health insurance company. To minimize selection bias we formed a control group of administrative data using a propensity score matching approach. In comparison to the control group DMP participants have a significantly lower mortality rate, and their average drug and hospital costs are reduced. Enrolled patients also had a lower mean number of hospital stays and shorter hospital stays. These results indicate that the programs meet the initial goal of improving the quality of care for the chronically ill.

Patients with type 2 diabetes benefit from primary care-based disease management: a propensity score matched survival time analysis.

This study aimed to assess the impact of a nationwide German diabetes mellitus disease management program (DMP) on survival time and costs in comparison to routine care. The authors conducted a retrospective observational cohort study using routine administration data from Germany's largest sickness fund to identify insured suffering from diabetes in 2002. A total of 95,443 insured with type 2 diabetes mellitus who were born before January 1, 1962 met the defined inclusion criteria, resulting in 19,888 pairs of DMP participants and nonparticipants matched for socioeconomic and health status using propensity score matching methods. This is the first time propensity score matching has been used to evaluate a survival benefit of DMPs. In the time frame analyzed (3 years), mean survival time for the DMP group was 1045 days vs. 985 days for the routine care group (P<0.001). Mean daily hospital and total costs (including DMP administration and medical costs) were lower for the DMP group in the case of deceased insureds (92€ vs. 139€ and 122€ vs. 169€, respectively) as well as for censored observations (6€ vs. 7€ and 12.9€ vs. 13.4€, respectively). Mean daily drug costs were slightly lower for deceased insured in the DMP group (difference 0.6€), while no identifiable difference was found for censored observations. In this study, insured who were enrolled in a DMP for diabetes mellitus in the German Statutory Health Insurance showed a significant benefit in survival time. They also incurred lower costs compared to propensity score matched insured in routine care.

Dynamics of the regulation of Hsp90 by the co-chaperone Sti1.

In eukaryotic cells, Hsp90 chaperones assist late folding steps of many regulatory protein clients by a complex ATPase cycle. Binding of clients to Hsp90 requires prior interaction with Hsp70 and a transfer reaction that is mediated by the co-chaperone Sti1/Hop. Sti1 furthers client transfer by inhibiting Hsp90's ATPase activity. To better understand how Sti1 prepares Hsp90 for client acceptance, we characterized the interacting domains and analysed how Hsp90 and Sti1 mutually influence their conformational dynamics using hydrogen exchange mass spectrometry. Sti1 stabilizes several regions in all three domains of Hsp90 and slows down dissociation of the Hsp90 dimer. Our data suggest that Sti1 inhibits Hsp90's ATPase activity by preventing N-terminal dimerization and docking of the N-terminal domain with the middle domain. Using crosslinking and mass spectrometry we identified Sti1 segments, which are in close vicinity of the N-terminal domain of Hsp90. We found that the length of the linker between C-terminal dimerization domain and the C-terminal MEEVD motif is important for Sti1 association rates and propose a kinetic model for Sti1 binding to Hsp90.

Structural frustration and occupational disorder: the rare earth metal polysulfides Tb8S(14.8), Dy8S(14.9), Ho8S(14.9), and Y8S(14.8).

Dark red crystals of Y8S(14.8), Tb8S(14.8), Dy8S(14.9), and Ho8S(14.9) have been obtained following different reaction routes. The isostructural title compounds adopt the Gd8Se15 type, a 24-fold superstructure of the ZrSSi-type and can be described in space group A112 (non standard setting of C121, no. 5) with lattice parameter of a = 11.505(1) Å, b = 15.385(1) Å, c = 15.726(1) Å, and γ = 90.21(2)° for Y8S(15-x); a = 11.660(1) Å, b = 15.468(2) Å, c = 15.844(2) Å, and γ = 90.19(2)° for Tb8S(15-x); a = 11.584(1) Å, b = 15.340(2) Å, c = 15.789(2) Å, and γ = 90.34(2)° for Dy8S(15-x); and a = 11.538(1) Å, b = 15.288(2) Å, c = 15.740(2) Å, and γ = 90.23(1)° for Ho8S(15-x), respectively. The structure consists of an alternating stacking of puckered [RES] (RE, rare-earth metals) double slabs and planar sulfur layers along [001]. The planar sulfur layers have a complex arrangement of S2²â» dinuclear dianions, isolated S²â» ions, and vacancies. All compounds contain trivalent rare-earth metal ions, for Tb8S(15-x) and Dy8S(15-x) antiferromagnetic order was found at T(N) = 5.4(2) K and 3.8(1) K, respectively. Short wavelength cutoff optical band gaps of 1.6 to 1.7 eV were determined.

Lipids trigger a conformational switch that regulates signal recognition particle (SRP)-mediated protein targeting.

Co-translational protein targeting to the membrane is mediated by the signal recognition particle and its receptor (FtsY). Their homologous GTPase domains interact at the membrane and form a heterodimer in which both GTPases are activated. The prerequisite for protein targeting is the interaction of FtsY with phospholipids. However, the mechanism of FtsY regulation by phospholipids remained unclear. Here we show that the N terminus of FtsY (A domain) is natively unfolded in solution and define the complete membrane-targeting sequence. We show that the membrane-targeting sequence is highly dynamic in solution, independent of nucleotides and directly responds to the density of anionic phospholipids by a random coil-helix transition. This conformational switch is essential for tethering FtsY to membranes and activates the GTPase for its subsequent interaction with the signal recognition particle. Our results underline the dynamics of lipid-protein interactions and their importance in the regulation of protein targeting and translocation across biological membranes.

German diabetes management programs improve quality of care and curb costs.

This paper reports the results of a large-scale analysis of a nationwide disease management program in Germany for patients with diabetes mellitus. The German program differs markedly from "classic" disease management in the United States. Although it combines important hallmarks of vendor-based disease management and the Chronic Care Model, the German program is based in primary care practices and carried out by physicians, and it draws on their personal relationships with patients to promote adherence to treatment goals and self-management. After four years of follow-up, overall mortality for patients and drug and hospital costs were all significantly lower for patients who participated in the program compared to other insured patients with similar health profiles who were not in the program. These results suggest that the German disease management program is a successful strategy for improving chronic illness care.

Deuteration distribution estimation with improved sequence coverage for HX/MS experiments.

MOTIVATION: Time-resolved hydrogen exchange (HX) followed by mass spectrometry (MS) is a key technology for studying protein structure, dynamics and interactions. HX experiments deliver a time-dependent distribution of deuteration levels of peptide sequences of the protein of interest. The robust and complete estimation of this distribution for as many peptide fragments as possible is instrumental to understanding dynamic protein-level HX behavior. Currently, this data interpretation step still is a bottleneck in the overall HX/MS workflow. RESULTS: We propose HeXicon, a novel algorithmic workflow for automatic deuteration distribution estimation at increased sequence coverage. Based on an L(1)-regularized feature extraction routine, HeXicon extracts the full deuteration distribution, which allows insight into possible bimodal exchange behavior of proteins, rather than just an average deuteration for each time point. Further, it is capable of addressing ill-posed estimation problems, yielding sparse and physically reasonable results. HeXicon makes use of existing peptide sequence information, which is augmented by an inferred list of peptide candidates derived from a known protein sequence. In conjunction with a supervised classification procedure that balances sensitivity and specificity, HeXicon can deliver results with increased sequence coverage. AVAILABILITY: The entire HeXicon workflow has been implemented in C++ and includes a graphical user interface. It is available at http://hci.iwr.uni-heidelberg.de/software.php. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Possible beneficial effect of bisphosphonates in osteonecrosis of the knee.

UNLABELLED: Osteonecrosis (ON) in the knee occurs as a localized inflammatory disease in relation to spontaneous or non-traumatic ON. Conservative treatment possibilities are limited, and prognosis appears to be poor; in most cases, ON results in knee arthroplasty. Bisphosphonates are suggested to prevent bone resorption and collapse of necrotic bone. In this observational, prospective study we investigated the effect of bisphosphonate treatment in patients with spontaneous or arthroscopy-induced ON of the knee. Twenty-eight patients with osteonecrotic lesions and bone marrow oedema in the knee were included. In 22 patients (80%), ON was identified after arthroscopic surgery of the knee; six patients were diagnosed with spontaneous ON. Patients were initially given pamidronate 120 mg i.v. divided in 3-4 perfusions over 2 weeks, followed by oral bisphosphonate treatment with alendronate 70 mg weekly for 4-6 months. Bisphosphonate treatment resulted in a rapid pain relief, VAS decreasing from 8.2 ± 1.2 at baseline to 5.02 ± 0.6 after 4-6 weeks (p < 0.001). After 6 months, the VAS decreased by 80% (p < 0.001). At the 6-month follow-up, symptoms had resolved completely in 15 patients out of 28; in 6 patients, minimal symptoms (VAS 1-2) remained. In two patients, treatment effect was unsatisfactory, and surgical intervention was needed (arthroplasty). Bone marrow oedema on MRI resolved completely in 18 patients out of 28 with substantial reduction in the remaining. Furthermore, osteonecrotic area resolved completely or demarcation with sclerotic changes of the necrotic area could be observed. Bisphosphonate treatment in patients with osteonecrosis of the knee was associated with a rapid improvement in pain score and radiological consolidation of the area of osteonecrosis. Further randomized, controlled trials are warranted to confirm the potential beneficial role of bisphosphonates in the treatment of osteonecrosis of the knee. LEVEL OF EVIDENCE: observational study, level IV.

Insights into the conformational dynamics of the E3 ubiquitin ligase CHIP in complex with chaperones and E2 enzymes.

The dimeric E3 ubiquitin ligase CHIP binds with its tetratricopeptide repeat (TPR) domain the C-terminus of molecular chaperones Hsp70 and Hsp90 and with its U-box region E2 ubiquitin-conjugating enzymes. By ubiquitinating chaperone-bound polypeptides, CHIP thus links the chaperone machinery to the proteasomal degradation pathway. The molecular mechanism of how CHIP discriminates between folding and destruction of chaperone substrates is not yet understood. Two recently published crystal structures of mouse and zebrafish CHIP truncation constructs differ substantially, showing either an asymmetric assembly or a symmetric assembly with a highly ordered middle domain. To characterize the conformational properties of the intact full-length protein in solution, we performed amide hydrogen exchange mass spectrometry (HX-MS) with human CHIP. In addition, we monitored conformational changes in CHIP upon binding of Hsp70, Hsp90, and their respective C-terminal EEVD peptides, and in complex with the different E2 ubiquitin-conjugating enzymes UbcH5a and Ubc13. Solution HX-MS data suggest a symmetric dimer assembly with highly flexible parts in the middle domain contrasting both the asymmetric and the symmetric crystal structure. CHIP exhibited an extraordinary flexibility with a largely unprotected N-terminal TPR domain. Formation of a complex with intact Hsp70 and Hsp90 or their respective C-terminal octapeptides induced folding of the TPR domain to a defined, highly stabilized structure with protected amide hydrogens. Interaction of CHIP with two different E2 ubiquitin-conjugating enzymes, UbcH5a and Ubc13, had distinct effects on the conformational dynamics of CHIP, suggesting different roles of the CHIP-E2 interaction in the ubiquitination of substrates and interaction with chaperones.