An ultra-high-throughput screen for the evaluation of peptide HLA-Binder interactions.

Peptide human leukocyte antigen (pHLA) targeting therapeutics like T-cell receptor based adoptive cell therapy or bispecific T cell engaging receptor molecules hold great promise for the treatment of cancer. Comprehensive pre-clinical screening of therapeutic candidates is important to ensure patient safety but is challenging because of the size of the potential off-target space. By combining stabilized peptide-receptive HLA molecules with microarray printing and screening, we have developed an ultra-high-throughput screening platform named ValidaTe that enables large scale evaluation of pHLA-binder interactions. We demonstrate its potential by measuring and analyzing over 30.000 binding curves for a high-affinity T cell Engaging Receptor towards a large pHLA library. Compared to a dataset obtained by conventional bio-layer interferometry measurements, we illustrate that a massively increased throughput (over 650 fold) is obtained by our microarray screening, paving the way for use in pre-clinical safety screening of pHLA-targeting drugs.

Nanocellulose Composites as Smart Devices With Chassis, Light-Directed DNA Storage, Engineered Electronic Properties, and Chip Integration.

The rapid development of green and sustainable materials opens up new possibilities in the field of applied research. Such materials include nanocellulose composites that can integrate many components into composites and provide a good chassis for smart devices. In our study, we evaluate four approaches for turning a nanocellulose composite into an information storage or processing device: 1) nanocellulose can be a suitable carrier material and protect information stored in DNA. 2) Nucleotide-processing enzymes (polymerase and exonuclease) can be controlled by light after fusing them with light-gating domains; nucleotide substrate specificity can be changed by mutation or pH change (read-in and read-out of the information). 3) Semiconductors and electronic capabilities can be achieved: we show that nanocellulose is rendered electronic by iodine treatment replacing silicon including microstructures. Nanocellulose semiconductor properties are measured, and the resulting potential including single-electron transistors (SET) and their properties are modeled. Electric current can also be transported by DNA through G-quadruplex DNA molecules; these as well as classical silicon semiconductors can easily be integrated into the nanocellulose composite. 4) To elaborate upon miniaturization and integration for a smart nanocellulose chip device, we demonstrate pH-sensitive dyes in nanocellulose, nanopore creation, and kinase micropatterning on bacterial membranes as well as digital PCR micro-wells. Future application potential includes nano-3D printing and fast molecular processors (e.g., SETs) integrated with DNA storage and conventional electronics. This would also lead to environment-friendly nanocellulose chips for information processing as well as smart nanocellulose composites for biomedical applications and nano-factories.

Heterosubtypic, cross-reactive immunity to human Cytomegalovirus glycoprotein B.

Cytomegalovirus (CMV) genome is highly variable and heterosubtypic immunity should be considered in vaccine development since it can enhance protection in a cross-reactive manner. Here, we developed a protein array to evaluate heterosubtypic immunity to CMV glycoprotein B (gB) in natural infection and vaccination. DNA sequences of four antigenic domains (AD1, AD2, AD4/5, and AD5) of gB were amplified from six reference and 12 clinical CMV strains, and the most divergent genotypes were determined by phylogenetic analysis. Assigned genotypes were in vitro translated and immobilized on protein array. Then, we tested immune response of variable serum groups (primarily infected patients, reactivated CMV infections and healthy individuals with latent CMV infection, as well gB-vaccinated rabbits) with protein in situ array (PISA). Serum antibodies of all patient cohorts and gB-vaccinated rabbits recognized many genetic variants of ADs on protein array, including but not limited to the subtype of infecting strain. High-grade cross-reactivity was observed. In several patients, we observed none or neglectable immune response to AD1 and AD2, while the same patients showed high antibody response to AD4/5 and AD5. Among the primary infected patients, AD5 was the predominant AD, in antibody response. The most successful CMV vaccine to date contains gB and demonstrates only 50% efficacy. In this study, we showed that heterosubtypic and cross-reactive immunity to CMV gB is extensive. Therefore, the failure of CMV gB vaccines cannot be explained by a highly, strain-specific immunity. Our observations suggest that other CMV antigens should be addressed in vaccine design.

A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients binds to the ACE2-RBD interface and is tolerant to most known RBD mutations.

The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19.

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface.

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a new recently emerged sarbecovirus. This virus uses the human ACE2 enzyme as receptor for cell entry, recognizing it with the receptor binding domain (RBD) of the S1 subunit of the viral spike protein. We present the use of phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve antibody gene libraries HAL9/10 and subsequent identification of 309 unique fully human antibodies against S1. 17 antibodies are binding to the RBD, showing inhibition of spike binding to cells expressing ACE2 as scFv-Fc and neutralize active SARS-CoV-2 virus infection of VeroE6 cells. The antibody STE73-2E9 is showing neutralization of active SARS-CoV-2 as IgG and is binding to the ACE2-RBD interface. Thus, universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovering patients in a pandemic situation.

Digital DNA microarray generation on glass substrates.

In this work we show how DNA microarrays can be produced batch wise on standard microscope slides in a fast, easy, reliable and cost-efficient way. Contrary to classical microarray generation, the microarrays are generated via digital solid phase PCR. We have developed a cavity-chip system made of a PDMS/aluminum composite which allows such a solid phase PCR in a scalable and easy to handle manner. For the proof of concept, a DNA pool composed of two different DNA species was used to show that digital PCR is possible in our chips. In addition, we demonstrate that DNA microarray generation can be realized with different laboratory equipment (slide cycler, manually in water baths and with an automated cartridge system). We generated multiple microarrays and analyzed over 13,000 different monoclonal DNA spots to show that there is no significant difference between the used equipment. To show the scalability of our system we also varied the size and number of the cavities located in the array region up to more than 30,000 cavities with a volume of less than 60 pL per cavity. With this method, we present a revolutionary tool for novel DNA microarrays. Together with new established label-free measurement systems, our technology has the potential to give DNA microarray applications a new boost.

KOFFI and Anabel 2.0-a new binding kinetics database and its integration in an open-source binding analysis software.

The kinetics of featured interactions (KOFFI) database is a novel tool and resource for binding kinetics data from biomolecular interactions. While binding kinetics data are abundant in literature, finding valuable information is a laborious task. We used text extraction methods to store binding rates (association, dissociation) as well as corresponding meta-information (e.g. methods, devices) in a novel database. To date, over 270 articles were manually curated and binding data on over 1705 interactions was collected and stored in the (KOFFI) database. Moreover, the KOFFI database application programming interface was implemented in Anabel (open-source software for the analysis of binding interactions), enabling users to directly compare their own binding data analyses with related experiments described in the database.

How to copy and paste DNA microarrays.

Analogous to a photocopier, we developed a DNA microarray copy technique and were able to copy patterned original DNA microarrays. With this process the appearance of the copied DNA microarray can also be altered compared to the original by producing copies of different resolutions. As a homage to the very first photocopy made by Chester Charlson and Otto Kornei, we performed a lookalike DNA microarray copy exactly 80 years later. Those copies were also used for label-free real-time kinetic binding assays of apo-dCas9 to double stranded DNA and of thrombin to single stranded DNA. Since each DNA microarray copy was made with only 5 µl of spPCR mix, the whole process is cost-efficient. Hence, our DNA microarray copier has a great potential for becoming a standard lab tool.

Protein Microarray Copying: Easy on-Demand Protein Microarray Generation Compatible with Fluorescence and Label-Free Real-Time Analysis.

Protein microarrays are essential to understand complex protein interaction networks. Their production, however, is a challenge and renders this technology unattractive for many laboratories. Recent developments in cell-free protein microarray generation offer new opportunities, but are still expensive and cumbersome in practice. Herein, we describe a cost-effective and user-friendly method for the cell-free production of protein microarrays. From a polydimethylsiloxane (PDMS) flow cell containing an expressible DNA microarray, proteins of interest are synthesised by cell-free expression and then immobilised on a capture surface. The resulting protein microarray can be regarded as a "copy" of the DNA microarray. 2 His6 - and Halo-tagged fluorescent reference proteins were used to demonstrate the functionality of nickel nitrilotriacetic acid (Ni-NTA) and Halo-bind surfaces in this copy system. The described process can be repeated several times on the same DNA microarray. The identity and functionality of the proteins were proven during the copy process by their fluorescence and on the surface through a fluorescent immune assay. Also, single-colour reflectometry (SCORE) was applied to show that, on such copied arrays, real-time binding kinetic measurements were possible.

Anabel: An Online Tool for the Real-Time Kinetic Analysis of Binding Events.

Anabel (Analysis of binding events + l) is an open source online software tool (www.skscience.org/anabel) for the convenient analysis of molecular binding interactions. Currently, exported datasets from Biacore (surface plasmon resonance [SPR]), FortéBio (biolayer interference [BLI]), and Biametrics (single color reflectometry [SCORE]) can be uploaded and evaluated in Anabel using 2 different evaluation methods. Moreover, a universal data template format is provided to upload any other binding dataset to Anabel. This enables an easier comparison of different analysis methods for all users. Furthermore, a guide was established in Anabel to help inexperienced users to obtain optimal results. In addition, expert features can be used to optimize and control the fit of the binding model to the measured data. We tried to make the process of fitting and evaluating as easy as possible through the use of an intuitive user interface. At the end of every analysis, a single excel file, containing all results and graphs of the performed analysis, can be downloaded.

Multiplexed antibody detection from blood sera by immobilization of in vitro expressed antigens and label-free readout via imaging reflectometric interferometry (iRIf).

The detection of antibodies from blood sera is crucial for diagnostic purposes. Miniaturized protein assays in combination with microfluidic setups hold great potential by enabling automated handling and multiplexed analyses. Yet, the separate expression, purification, and storage of many individual proteins are time consuming and limit applicability. In vitro cell-free expression has been proposed as an alternative procedure for the generation of protein assays. We report the successful in vitro expression of different model proteins from DNA templates with an optimized expression mix. His10-tagged proteins were specifically captured and immobilized on a Ni-NTA coated sensor surface directly from the in vitro expression mix. Finally, the specific binding of antibodies from rabbit-derived blood sera to the immobilized proteins was monitored by imaging reflectometric interferometry (iRIf). Antibodies in the blood sera could be identified by binding to the respective epitopes with minimal cross reactivity. The results show the potential of in vitro expression and label-free detection for binding assays in general and diagnostic purposes in specific.

Microfluidic Proximity Ligation Assay for Profiling Signaling Networks with Single-Cell Resolution.

The proximity ligation assay (PLA) is a technique that can be used to characterize proteins, protein-protein interactions, and protein modifications at the single-cell level. Image-based in situ detection of proteins using PLA is a quantitative method with a high degree of sensitivity and specificity. The miniaturization and parallelization of the PLA onto a microfluidic chip and concurrent use of an automated cell-culture system increase the throughput of this technology. Here, we describe the performance of PLA on a microfluidic chip. We provide protocols for on-chip cell culture, time-shifted cell stimulation and fixation, PLA implementation, and computational image analysis in order to achieve single-cell resolution. As a proof of concept, we studied the phosphorylation of Akt in response to stimulation with platelet-derived growth factor.

A peptide-functionalized polymer as a minimal scaffold protein to enhance cluster formation in early T cell signal transduction.

In cellular signal transduction, scaffold proteins provide binding sites to organize signaling proteins into supramolecular complexes and act as nodes in the signaling network. Furthermore, multivalent interactions between the scaffold and other signaling proteins contribute to the formation of protein microclusters. Such microclusters are prominent in early T cell signaling. Here, we explored the minimal structural requirement for a scaffold protein by coupling multiple copies of a proline-rich peptide corresponding to an interaction motif for the SH3 domain of the adaptor protein GADS to an N-(2-hydroxypropyl)methacrylamide polymer backbone. When added to GADS-containing cell lysates, these scaffolds (but not individual peptides) promoted the binding of GADS to peptide microarrays. This can be explained by the cross-linking of GADS into larger complexes. Furthermore, following import into Jurkat T cell leukemia cells, this synthetic scaffold enhanced the formation of microclusters of signaling proteins.

Improving health status and reduction of institutionalization in long-term care--Effects of the Resident Assessment Instrument-Home Care by degree of implementation.

A cluster randomized controlled trial showed that the Resident Assessment Instrument (RAI) could not improve or stabilize the health status of people in need of long-term care or reduce the rate of institutionalization in Germany among clients of home care agencies. The aim of this article is to investigate whether the effect of RAI depends on the degree of implementation. A factor analysis was used to distinguish between those agencies that implemented RAI intensively and those that did not. The clients of home care agencies working intensively with RAI were significantly less hospitalized (P = 0.0284) and fared slightly better according to activities of daily living (ADL, instrumental ADL (IADL)), cognitive skills (Mini-Mental Status Test (MMST)) and quality of life (EuroQol (EQ-5D)) compared with the control group. In contrast, those not working intensively with RAI had worse outcomes (IADL, MMST, EQ-5D) than the control group (not significant). It is important to guarantee a successful implementation of RAI.

Real-time stability testing of air-dried primers and fluorogenic hydrolysis probes stabilized by trehalose and xanthan.

A method for conserving primers and differently labeled fluorogenic hydrolysis (i.e., TaqMan) probes at ambient conditions is presented. Primers and hydrolysis probes with four different fluorophore-quencher combinations (6- FAM-BHQ1, HEX-BHQ1, ROX-BHQ650, and Cy5-BHQ2) were mixed with trehalose and xanthan at final concentrations of 56 mM and 2.78 mM, respectively. Mixtures were air-dried at 23°C for 30 min on strips composed of cyclo olefin polymer (COP), a material widely used in the manufacturing of in vitro diagnostic (IVD) test carriers. After one year of storage, the functionality of the primers and fluorophore-quencher combinations was validated by real-time polymerase chain reaction (real-time PCR), confirming their stability when stored in the presence of stabilizers, with the best results achieved using trehalose. This approach could be of great benefit for manufacturing IVD systems, for example, for genotyping applications based on multiplexing using different fluorescent dyes.

The long and bumpy road to outcome-oriented management of long-term care in Germany: implementation of the Resident Assessment Instrument in home-care services.

OBJECTIVE: Although the quality of long-term care has improved, many problems still remain, and better processes seem to be necessary. Hence, outcome-oriented management is of particular importance. The Resident Assessment Instrument (RAI) is a tool that has been used successfully in many countries to improve quality of care. However, there are problems of implementation and it lacks information on the conditions of successful or failing information of the RAI. The aim of this article is to find out to what extent technical/qualification requirements help to introduce or lead to failure of the implementation of an assessment instrument like RAI. METHODS: Therefore, a cluster randomized controlled trial showed services using RAI intensively tend to have better outcomes after 12 months. But the effects depend on the success of the implementation. Using a factor analysis, an index was built to divide the care providers into "optimal" and "suboptimal" RAI users. RESULTS: Some factors that seem to lead to a rather successful implementation could be detected: A higher proportion of qualified staff, a lower perceived quantitative workload, a small size of care providers, the type of ownership (for-profit) and a late entry in study [Correction made here after initial online publication.]. CONCLUSION: The success or failure of the implementation of an outcome-oriented control instrument is determined by professional, organizational restrictions. The results show that a better implementation leads to better outcomes for clients.

A quantitative assessment of costimulation and phosphatase activity on microclusters in early T cell signaling.

T cell signaling is triggered through stimulation of the T cell receptor and costimulatory receptors. Receptor activation leads to the formation of membrane-proximal protein microclusters. These clusters undergo tyrosine phosphorylation and organize multiprotein complexes thereby acting as molecular signaling platforms. Little is known about how the quantity and phosphorylation levels of microclusters are affected by costimulatory signals and the activity of specific signaling proteins. We combined micrometer-sized, microcontact printed, striped patterns of different stimuli and simultaneous analysis of different cell strains with image processing protocols to address this problem. First, we validated the stimulation protocol by showing that high expression levels CD28 result in increased cell spreading. Subsequently, we addressed the role of costimulation and a specific phosphotyrosine phosphatase in cluster formation by including a SHP2 knock-down strain in our system. Distinguishing cell strains using carboxyfluorescein succinimidyl ester enabled a comparison within single samples. SHP2 exerted its effect by lowering phosphorylation levels of individual clusters while CD28 costimulation mainly increased the number of signaling clusters and cell spreading. These effects were observed for general tyrosine phosphorylation of clusters and for phosphorylated PLCγ1. Our analysis enables a clear distinction between factors determining the number of microclusters and those that act on these signaling platforms.

Centrifugal gas-phase transition magnetophoresis (GTM)--a generic method for automation of magnetic bead based assays on the centrifugal microfluidic platform and application to DNA purification.

Transportation of magnetic beads between different reagents plays a crucial role in many biological assays e.g. for purification of biomolecules or cells where the beads act as a mobile solid support. Therefore, usually a complex set-up either for fluidic processing or for manipulation of magnetic beads is required. To circumvent these drawbacks, we present a facile and automated method for the transportation of magnetic beads between multiple microfluidic chambers on a centrifugal microfluidic cartridge "LabDisk". The method excels by requiring only one stack of stationary permanent magnets, a specific microfluidic layout without actively controlled valves and a predefined frequency protocol for rotation of the LabDisk. Magnetic beads were transported through three fluidically separated chambers with a yield of 82.6% ± 3.6%. Bead based DNA purification from a dilution series of a Listeria innocua lysate and from a lambda phage DNA standard was demonstrated where the three chambers were used for binding, washing and elution of DNA. Recovery of L. innocua DNA was up to 68% ± 24% and for lambda phage DNA 43% ± 10% compared to manual reference purification in test tubes. Complete purification was conducted automatically within 12.5 min. Since all reagents can be preloaded onto the LabDisk prior to purification, no further hands-on steps are required during processing. Due to its modular and generic character, the presented method could also be adapted to other magnetic bead based assays e.g. to immunoassays or protein affinity purification, solely requiring the adjustment of number and volumes of the fluidic chambers.

Centrifugo-dynamic inward pumping of liquids on a centrifugal microfluidic platform.

We present a method to pump liquids in a centrifugal microfluidic spinning disk from a radial outward position to a radial inward position. Centrifugal forces are applied to compress air in a cavity, this way storing pneumatic energy. The cavity is connected to an outlet channel having a lower hydraulic resistance compared to the inlet channel. The stored pneumatic energy is quickly released by fast reduction of rotational frequency. This way liquid is transported mainly through the channel with lower resistance, directing the liquid radially inwards. Pump efficiencies of >75% per pump cycle have been demonstrated for water, ethanol, a highly viscous lysis buffer and whole blood. By employing three pump cycles, water has been pumped radially inwards with an efficiency of >90%. The inward pumping requires centrifugation only, which is intrinsically available on every centrifugal microfluidic platform.

Mediator probe PCR: a novel approach for detection of real-time PCR based on label-free primary probes and standardized secondary universal fluorogenic reporters.

BACKGROUND: The majority of established techniques for monitoring real-time PCR amplification involve individual target-specific fluorogenic probes. For analysis of numerous different targets the synthesis of these probes contributes to the overall cost during assay development. Sequence-dependent universal detection techniques overcome this drawback but are prone to detection of unspecific amplification products. We developed the mediator probe PCR as a solution to these problems. METHODS: A set of label-free sequence-specific primary probes (mediator probes), each comprising a target-specific region and a standardized mediator tag, is cleaved upon annealing to its target sequence by the polymerases' 5' nuclease activity. Release of a mediator triggers signal generation by cleavage of a complementary fluorogenic reporter probe. RESULTS: Real-time PCR amplification of human papillomavirus 18 (HPV18), Staphylococcus aureus, Escherichia coli, and Homo sapiens DNA dilution series showed exceptional linearity when detected either by novel mediator probes (r(2) = 0.991-0.999) or state-of-the-art hydrolysis probes (TaqMan probes) (r(2) = 0.975-0.993). For amplification of HPV18 DNA the limits of detection were 78.3 and 85.1 copies per 10-µL reaction when analyzed with the mediator probe and hydrolysis probe, respectively. Duplex amplification of HPV18 target DNA and internal standard had no effects on back calculation of target copy numbers when quantified with either the mediator probe PCR (r(2) = 0.998) or the hydrolysis probe PCR (r(2) = 0.988). CONCLUSIONS: The mediator probe PCR has equal performance to hydrolysis probe PCR and has reduced costs because of the use of universal fluorogenic reporters.