Graph colouring approaches for a satellite range scheduling problem

A goal of this paper is to efficiently adapt the best ingredients of the graph colouring techniques to an NP-hard satellite range scheduling problem, called MuRRSP. We propose two new heuristics for the MuRRSP, where as many jobs as possible have to be scheduled on several resources, while respecting time and capacity constraints. In the permutation solution space, which is widely used by other researchers, a solution is represented by a permutation of the jobs, and a schedule builder is needed to generate and evaluate a feasible schedule from the permutation. On the contrary, our heuristics are based on the solution space which contains all the feasible schedules. Based on the similarities between the graph colouring problem and the MuRRSP, we show that the latter solution space has significant advantages. A tabu search and an adaptive memory algorithms are designed to tackle the MuRRSP. These heuristics are derived from efficient graph colouring methods. Numerical experiments, performed on large, realistic, and challenging instances, showed that our heuristics are very competitive, robust, and outperform algorithms based on the permutation solution spac

Rapid selection of specific MAP kinase-binders from designed ankyrin repeat protein libraries

We describe here the rapid selection of specific MAP-kinase binders from a combinatorial library of designed ankyrin repeat proteins (DARPins). A combined in vitro/in vivo selection approach, based on ribosome display and the protein fragment complementation assay (PCA), yielded a large number of different binders that are fully functional in the cellular cytoplasm. Ribosome-display selection pools of four successive selection rounds were examined to monitor the enrichment of JNK2-specific DARPins. Surprisingly, only one round of ribosome display with subsequent PCA selection of this pool was necessary to isolate a first specific binder with micromolar affinity. After only two rounds of ribosome-display selection followed by PCA, virtually all DARPins showed JNK2-specific binding, with affinities in the low nanomolar range. The enrichment factor of ribosome display thus approaches 105 per round. In a second set of experiments, similar results were obtained with the kinases JNK1 and p38 as targets. Again, almost all investigated DARPins obtained after two rounds of ribosome display showed specific binding to the targets used, JNK1 or p38. In all three selection experiments the identified DARPins possess very high specificity for the target kinase. Taken together, the combination of ribosome display and PCA selections allowed the identification of large pools of binders at unparalleled speed. Furthermore, DARPins are applicable in intracellular selections and immunoprecipitations from the extract of eukaryotic cell

Rapid selection of specific MAP kinase-binders from designed ankyrin repeat protein libraries

We describe here the rapid selection of specific MAP-kinase binders from a combinatorial library of designed ankyrin repeat proteins (DARPins). A combined in vitro/in vivo selection approach, based on ribosome display and the protein fragment complementation assay (PCA), yielded a large number of different binders that are fully functional in the cellular cytoplasm. Ribosome-display selection pools of four successive selection rounds were examined to monitor the enrichment of JNK2-specific DARPins. Surprisingly, only one round of ribosome display with subsequent PCA selection of this pool was necessary to isolate a first specific binder with micromolar affinity. After only two rounds of ribosome-display selection followed by PCA, virtually all DARPins showed JNK2-specific binding, with affinities in the low nanomolar range. The enrichment factor of ribosome display thus approaches 105 per round. In a second set of experiments, similar results were obtained with the kinases JNK1 and p38 as targets. Again, almost all investigated DARPins obtained after two rounds of ribosome display showed specific binding to the targets used, JNK1 or p38. In all three selection experiments the identified DARPins possess very high specificity for the target kinase. Taken together, the combination of ribosome display and PCA selections allowed the identification of large pools of binders at unparalleled speed. Furthermore, DARPins are applicable in intracellular selections and immunoprecipitations from the extract of eukaryotic cell

Drug Export Pathway of Multidrug Exporter AcrB Revealed by DARPin Inhibitors

The multidrug exporter AcrB is the inner membrane component of the AcrAB-TolC drug efflux system in Escherichia coli and is responsible for the resistance of this organism to a wide range of drugs. Here we describe the crystal structure of the trimeric AcrB in complex with a designed ankyrin-repeat protein (DARPin) inhibitor at 2.5-Å resolution. The three subunits of AcrB are locked in different conformations revealing distinct channels in each subunit. There seems to be remote conformational coupling between the channel access, exit, and the putative proton-translocation site, explaining how the proton motive force is used for drug export. Thus our structure suggests a transport pathway not through the central pore but through the identified channels in the individual subunits, which greatly advances our understanding of the multidrug export mechanism

Effects of dignity therapy on psychological distress and wellbeing of palliative care patients and family caregivers – a randomized controlled study

Background This study extended the original Dignity Therapy (DT) intervention by including partners and family caregivers (FCs) of terminally-ill cancer patients with the overall aim of evaluating whether DT can mitigate distress in both patients nearing the end of life and their FCs. Methods In this multicenter, randomized controlled trial (RCT), a total of 68 patients with life expectancy < 6 months and clinically-relevant stress levels (Hospital Anxiety Depression total score; HADS$_{tot}$ ≥ 8) including their FCs were randomly assigned to DT, DT + (including their FCs), or standard palliative care (SPC) in a 1:1:1 ratio. Study participants were asked to complete a set of questionnaires pre- and post-intervention. Results The coalesced group (DT and DT +) revealed a significant increase in patients’ perceived quality of life (FACIT-Pal-14) following the intervention (mean difference 6.15, SD = 1.86, p < 0.01). We found a statistically significant group-by-time interaction effect: while the HADS$_{tot}$ of patients in the intervention group remained stable over the pre-post period, the control group’s HADS$_{tot}$ increased (F = 4.33, df = 1, 82.9; p < 0.05), indicating a protective effect of DT. Most patients and their FCs found DT useful and would recommend it to other individuals in their situation. Conclusions The DT intervention has been well-received and shows the potential to increase HRQoL and prevent further mental health deterioration, illness burden and suffering in terminally-ill patients. The DT intervention holds the potential to serve as a valuable tool for facilitating end-of-life conversations among terminally-ill patients and their FCs. However, the implementation of DT within the framework of a RCT in a palliative care setting poses significant challenges. We suggest a slightly modified and less resource-intensive version of DT that is to provide the DT inventory to FCs of terminally-ill patients, empowering them to ask the questions that matter most to them over their loved one’s final days. Trial registration This study was registered with Clinical Trial Registry (ClinicalTrials.gov -Protocol Record NCT02646527; date of registration: 04/01/2016). The CONSORT 2010 guidelines were used for properly reporting how the randomized trial was conducted

Reporting quality of clinical trial protocols: a repeated cross-sectional study about the Adherence to SPIrit Recommendations in Switzerland, CAnada and GErmany (ASPIRE-SCAGE)

OBJECTIVES Comprehensive protocols are key for the planning and conduct of randomised clinical trials (RCTs). Evidence of low reporting quality of RCT protocols led to the publication of the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) checklist in 2013. We aimed to examine the quality of reporting of RCT protocols from three countries before and after the publication of the SPIRIT checklist. DESIGN Repeated cross sectional study. SETTING Swiss, German and Canadian research ethics committees (RECs). PARTICIPANTS RCT protocols approved by RECs in 2012 (n=257) and 2016 (n=292). PRIMARY AND SECONDARY OUTCOME MEASURES The primary outcomes were the proportion of reported SPIRIT items per protocol and the proportion of trial protocols reporting individual SPIRIT items. We compared these outcomes in protocols approved in 2012 and 2016, and built regression models to explore factors associated with adherence to SPIRIT. For each protocol, we also extracted information on general trial characteristics and assessed whether individual SPIRIT items were reported RESULTS: The median proportion of reported SPIRIT items among RCT protocols showed a non-significant increase from 72% (IQR, 63%-79%) in 2012 to 77% (IQR, 68%-82%) in 2016. However, in a preplanned subgroup analysis, we detected a significant improvement in investigator-sponsored protocols: the median proportion increased from 64% (IQR, 55%-72%) in 2012 to 76% (IQR, 64%-83%) in 2016, while for industry-sponsored protocols median adherence was 77% (IQR 72%-80%) for both years. The following trial characteristics were independently associated with lower adherence to SPIRIT: single-centre trial, no support from a clinical trials unit or contract research organisation, and investigator-sponsorship. CONCLUSIONS In 2012, industry-sponsored RCT protocols were reported more comprehensively than investigator-sponsored protocols. After publication of the SPIRIT checklist, investigator-sponsored protocols improved to the level of industry-sponsored protocols, which did not improve

Nonregistration, discontinuation, and nonpublication of randomized trials: A repeated metaresearch analysis

BACKGROUND We previously found that 25% of 1,017 randomized clinical trials (RCTs) approved between 2000 and 2003 were discontinued prematurely, and 44% remained unpublished at a median of 12 years follow-up. We aimed to assess a decade later (1) whether rates of completion and publication have increased; (2) the extent to which nonpublished RCTs can be identified in trial registries; and (3) the association between reporting quality of protocols and premature discontinuation or nonpublication of RCTs. METHODS AND FINDINGS We included 326 RCT protocols approved in 2012 by research ethics committees in Switzerland, the United Kingdom, Germany, and Canada in this metaresearch study. Pilot, feasibility, and phase 1 studies were excluded. We extracted trial characteristics from each study protocol and systematically searched for corresponding trial registration (if not reported in the protocol) and full text publications until February 2022. For trial registrations, we searched the (i) World Health Organization: International Clinical Trial Registry Platform (ICTRP); (ii) US National Library of Medicine (ClinicalTrials.gov); (iii) European Union Drug Regulating Authorities Clinical Trials Database (EUCTR); (iv) ISRCTN registry; and (v) Google. For full text publications, we searched PubMed, Google Scholar, and Scopus. We recorded whether RCTs were registered, discontinued (including reason for discontinuation), and published. The reporting quality of RCT protocols was assessed with the 33-item SPIRIT checklist. We used multivariable logistic regression to examine the association between the independent variables protocol reporting quality, planned sample size, type of control (placebo versus other), reporting of any recruitment projection, single-center versus multicenter trials, and industry versus investigator sponsoring, with the 2 dependent variables: (1) publication of RCT results; and (2) trial discontinuation due to poor recruitment. Of the 326 included trials, 19 (6%) were unregistered. Ninety-eight trials (30%) were discontinued prematurely, most often due to poor recruitment (37%; 36/98). One in 5 trials (21%; 70/326) remained unpublished at 10 years follow-up, and 21% of unpublished trials (15/70) were unregistered. Twenty-three of 147 investigator-sponsored trials (16%) reported their results in a trial registry in contrast to 150 of 179 industry-sponsored trials (84%). The median proportion of reported SPIRIT items in included RCT protocols was 69% (interquartile range 61% to 77%). We found no variables associated with trial discontinuation; however, lower reporting quality of trial protocols was associated with nonpublication (odds ratio, 0.71 for each 10% increment in the proportion of SPIRIT items met; 95% confidence interval, 0.55 to 0.92; p = 0.009). Study limitations include that the moderate sample size may have limited the ability of our regression models to identify significant associations. CONCLUSIONS We have observed that rates of premature trial discontinuation have not changed in the past decade. Nonpublication of RCTs has declined but remains common; 21% of unpublished trials could not be identified in registries. Only 16% of investigator-sponsored trials reported results in a trial registry. Higher reporting quality of RCT protocols was associated with publication of results. Further efforts from all stakeholders are needed to improve efficiency and transparency of clinical research

CD4-Specific Designed Ankyrin Repeat Proteins Are Novel Potent HIV Entry Inhibitors with Unique Characteristics

Here, we describe the generation of a novel type of HIV entry inhibitor using the recently developed Designed Ankyrin Repeat Protein (DARPin) technology. DARPin proteins specific for human CD4 were selected from a DARPin DNA library using ribosome display. Selected pool members interacted specifically with CD4 and competed with gp120 for binding to CD4. DARPin proteins derived in the initial selection series inhibited HIV in a dose-dependent manner, but showed a relatively high variability in their capacity to block replication of patient isolates on primary CD4 T cells. In consequence, a second series of CD4-specific DARPins with improved affinity for CD4 was generated. These 2nd series DARPins potently inhibit infection of genetically divergent (subtype B and C) HIV isolates in the low nanomolar range, independent of coreceptor usage. Importantly, the actions of the CD4 binding DARPins were highly specific: no effect on cell viability or activation, CD4 memory cell function, or interference with CD4-independent virus entry was observed. These novel CD4 targeting molecules described here combine the unique characteristics of DARPins—high physical stability, specificity and low production costs—with the capacity to potently block HIV entry, rendering them promising candidates for microbicide development

The trispecific DARPin ensovibep inhibits diverse SARS-CoV-2 variants

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.1 and BA.2. In Roborovski dwarf hamsters infected with SARS-CoV-2, ensovibep reduced fatality similarly to a standard-of-care monoclonal antibody (mAb) cocktail. When used as a single agent in viral passaging experiments in vitro, ensovibep reduced the emergence of escape mutations in a similar fashion to the same mAb cocktail. These results support further clinical evaluation of ensovibep as a broad variant alternative to existing targeted therapies for Coronavirus Disease 2019 (COVID-19)

Selection of binders and inhibitors from designed ankyrin repeat protein libraries

Antikörper sind unersetzliche Bindemoleküle in der Forschung, der Diagnose und der Therapie. Trotzdem sind Antikörper mit verschiedenen Problemen behaftet, so wie teure Produktion und limitierte Stabilität, vor allem in der Zelle. Deshalb wären bessere Moleküle von grossem Wert. Diese Arbeit beschreibt die erste erfolgreiche Selektion von Bindemolekülen und Inhibitoren aus "Designed Ankyrin Repeat Protein" (DARPin) Bibliotheken. DARPins verbinden hochaffines und hochspezifisches Binden, mit guter Expression und hoher Stabilität, auch unter reduzierenden Bedingungen. Die Selektionen von Bindern wurden mittels "Ribosome Display" bewerkstelligt. Diese in vitro Selektionstechnologie ist ideal um aus Proteinbibliotheken Binder zu selektionieren. Eine Vielzahl von Zielmolekülen wurde für die Selektion gewählt, unter anderem Maltose-binde-Protein (MBP) von Escherichia coli (E. coli), aminoglycoside phosphotransferase (APH), die eukaryotischen protein kinasen p38, JNK1, JNK2 und AMPK und Sec YEG, ein Membranprotein aus E. coli, verantwortlich für den Proteinexport. Gegen sämtliche dieser Zielmoleküle wurden spezifische Binder generiert, was die Qualität der DARPin Proteinbibliothek unterstreicht. Der nächste Schritt war die Demonstration der intrazellulären Funktionalität der DARPins. Zu diesem Zweck wurde APH als Zielmolekül ausgewählt. APH ist eine bakterielle Kinase, die pathogenen Bakterien Kanamycin Resistenz vermittelt. Die potentesten Inhibitoren erwirkten beim Bakterium einen Phänotyp, wie er bei Abwesenheit des APH Gens auftritt. Die Struktur eines Inhibitors zeigt, dass der DARPin eine nicht aktive Konformation des Enzyms stabilisiert. Diese Resultate zeigen klar auf, dass es uns gelungen ist eine vielversprechende Alternative zu Antikörpern zu generieren, welche diese in wichtigen Punkten sogar übertrifft. Antibodies are indispensable binding molecules in research, diagnostics and therapy. Nevertheless, antibodies are afflicted with problems, such as poor expression and limited stability, especially in the intracellular milieu. Thus, superior alternatives would be of great value. This thesis describes the first successful selections of binding molecules and inhibitors from designed ankyrin repeat protein (DARPin) libraries. DARPins combine high affinity and specificity in target binding with high expression levels and high stabilities in all conditions. The selections of DARPins binding to the given target proteins were achieved by ribosome display, an in vitro selection technology, ideally suited to identify or evolve binding molecules from combinatorial protein libraries. Several different target proteins were chosen, including maltose binding protein (MBP) of Escherichia coli (E. coli), aminoglycoside phosphotransferase (APH), the eukaryotic protein kinases p38, JNK1, JNK2 and AMPK and Sec YEG, a membrane protein of E. coli responsible for protein export. Against these target proteins, specific binding molecules were obtained, demonstrating the power of DARPin libraries. The intracellular functionality of DARPins was demonstrated by the selection of inhibitors against APH. APH is a bacterial kinase and mediates kanamycin resistance in different pathogenic bacteria. The most potent of the selected inhibitors mediated a phenotype comparable to the APH gene knock out. The structure of the inhibitor in complex with APH showed that the DARPin bound and stabilized the kinase in an unproductive conformation, thus causing inhibition. In conclusion, this thesis demonstrates that DARPins are a true alternative to antibodies, surpassing them in several biophysical properties