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.

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.

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.

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.

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.

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.

Natural killer cell signal integration balances synapse symmetry and migration.

Natural killer (NK) cells discern the health of other cells by recognising the balance of activating and inhibitory ligands expressed by each target cell. However, how the integration of activating and inhibitory signals relates to formation of the NK cell immune synapse remains a central question in our understanding of NK cell recognition. Here we report that ligation of LFA-1 on NK cells induced asymmetrical cell spreading and migration. In contrast, ligation of the activating receptor NKG2D induced symmetrical spreading of ruffled lamellipodia encompassing a dynamic ring of f-actin, concurrent with polarization towards a target cell and a "stop" signal. Ligation of both LFA-1 and NKG2D together resulted in symmetrical spreading but co-ligation of inhibitory receptors reverted NK cells to an asymmetrical migratory configuration leading to inhibitory synapses being smaller and more rapidly disassembled. Using micropatterned activating and inhibitory ligands, signals were found to be continuously and locally integrated during spreading. Together, these data demonstrate that NK cells spread to form large, stable, symmetrical synapses if activating signals dominate, whereas asymmetrical migratory "kinapses" are favoured if inhibitory signals dominate. This clarifies how the integration of activating and inhibitory receptor signals is translated to an appropriate NK cell response.

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.

Reelin signals through apolipoprotein E receptor 2 and Cdc42 to increase growth cone motility and filopodia formation.

Lipoprotein receptor signaling regulates the positioning and differentiation of postmitotic neurons during development and modulates neuronal plasticity in the mature brain. Depending on the contextual situation, the lipoprotein receptor ligand Reelin can have opposing effects on cortical neurons. We show that Reelin increases growth cone motility and filopodia formation, and identify the underlying signaling cascade. Reelin activates the Rho GTPase Cdc42, known for its role in neuronal morphogenesis and directed migration, in an apolipoprotein E receptor 2-, Disabled-1-, and phosphatidylinositol 3-kinase-dependent manner. We demonstrate that neuronal vesicle trafficking, a Cdc42-controlled process, is increased after Reelin treatment and further provide evidence that the peptidergic VIP/PACAP38 system and Reelin can functionally interact to promote axonal branching. In conclusion, Reelin-induced activation of Cdc42 contributes to the regulation of the cytoskeleton of individual responsive neurons and converges with other signaling cascades to orchestrate Rho GTPase activity and promote neuronal development. Our data link the observation that defects in Rho GTPases and Reelin signaling are responsible for developmental defects leading to neurological and psychiatric disorders.

Protein expression from exogenous mRNA: uptake by receptor-mediated endocytosis and trafficking via the lysosomal pathway.

Insertional mutagenesis and the inherent risk of malignancy compromise the clinical use of DNA-based therapies. Being a transient copy of genetic material, mRNA is a safe alternative, overcoming this limitation. As a prerequisite for the development of efficient mRNA-based therapies, we investigated the cellular uptake and intracellular fate of mRNA for the first time. To this end we determined cell-type, dose and energy dependence of mRNA internalisation. Moreover, we employed markers for uptake pathways and cellular compartments to analyse the route of mRNA internalisation and its intracellular destination. Finally, we addressed the involvement of receptors and their nature using a competitor-based approach. We found that all cell types tested were amenable to uptake and expression of naked mRNA. Internalisation mainly occurred via caveolae/lipid raft-rich membrane domains and involved scavenger-receptor(s). Following endocytosis, mRNA eventually accumulated in lysosomes, while part of it escaped into the cytosol giving rise to protein synthesis. Taken together, our findings provide unprecedented insights into the internalisation and trafficking of exogenous mRNA, greatly facilitating the development of effective mRNA-based therapies in the future.

Microfluidic lab-on-a-chip platforms: requirements, characteristics and applications.

This critical review summarizes developments in microfluidic platforms that enable the miniaturization, integration, automation and parallelization of (bio-)chemical assays (see S. Haeberle and R. Zengerle, Lab Chip, 2007, 7, 1094-1110, for an earlier review). In contrast to isolated application-specific solutions, a microfluidic platform provides a set of fluidic unit operations, which are designed for easy combination within a well-defined fabrication technology. This allows the easy, fast, and cost-efficient implementation of different application-specific (bio-)chemical processes. In our review we focus on recent developments from the last decade (2000s). We start with a brief introduction into technical advances, major market segments and promising applications. We continue with a detailed characterization of different microfluidic platforms, comprising a short definition, the functional principle, microfluidic unit operations, application examples as well as strengths and limitations of every platform. The microfluidic platforms in focus are lateral flow tests, linear actuated devices, pressure driven laminar flow, microfluidic large scale integration, segmented flow microfluidics, centrifugal microfluidics, electrokinetics, electrowetting, surface acoustic waves, and dedicated systems for massively parallel analysis. This review concludes with the attempt to provide a selection scheme for microfluidic platforms which is based on their characteristics according to key requirements of different applications and market segments. Applied selection criteria comprise portability, costs of instrument and disposability, sample throughput, number of parameters per sample, reagent consumption, precision, diversity of microfluidic unit operations and the flexibility in programming different liquid handling protocols (295 references).

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.

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.

Centrifugal microfluidic system for primary amplification and secondary real-time PCR.

Pre-amplification is a basis for numerous polymerase chain reaction (PCR) protocols but bears severe contamination risks due to handling of high-copy DNA samples. Therefore we developed a self-contained centrifugal microfluidic system comprising pre-stored reagents; it enables pre-amplification of specific DNA sequences prior to automated aliquoting and real-time PCR in a modified commercial thermocycler.

Continuous microfluidic DNA extraction using phase-transfer magnetophoresis.

This paper reports a novel microfluidic-chip based platform using "phase-transfer magnetophoresis" enabling continuous biomolecule processing. As an example we demonstrate for the first time continuous DNA extraction from cell lysate on a microfluidic chip. After mixing bacterial Escherichia coli culture with superparamagnetic bead suspension, lysis and binding buffers, DNA is released from cells and captured by the beads. These DNA carrying beads are continuously transported across the interfaces between co-flowing laminar streams of sample mixture, washing and elution buffer. Bead actuation is achieved by applying a time-varying magnetic field generated by a rotating permanent magnet. Flagella-like chains of magnetic beads are formed and transported along the microfluidic channels by an interplay of fluid drag and periodic magnetic entrapment. The turnover time for DNA extraction was approximately 2 minutes with a sample flow rate of 0.75 µl s(-1) and an eluate flow rate of 0.35 µl s(-1). DNA recovery was 147% (on average) compared to bead based batch-wise extraction in reference tubes within a dilution series experiment over 7 orders of magnitude. The novel platform is suggested for automation of various magnetic bead based applications that require continuous sample processing, e.g. continuous DNA extraction for flow-through PCR, capture and analysis of cells and continuous immunoassays. Potential applications are seen in the field of biological safety monitoring, bioprocess control, environmental monitoring, or epidemiological studies such as monitoring the load of antibiotic resistant bacteria in waste water from hospitals.

Microfluidic lab-on-a-foil for nucleic acid analysis based on isothermal recombinase polymerase amplification (RPA).

For the first time we demonstrate a self-sufficient lab-on-a-foil system for the fully automated analysis of nucleic acids which is based on the recently available isothermal recombinase polymerase amplification (RPA). The system consists of a novel, foil-based centrifugal microfluidic cartridge including prestored liquid and dry reagents, and a commercially available centrifugal analyzer for incubation at 37 degrees C and real-time fluorescence detection. The system was characterized with an assay for the detection of the antibiotic resistance gene mecA of Staphylococcus aureus. The limit of detection was <10 copies and time-to-result was <20 min. Microfluidic unit operations comprise storage and release of liquid reagents, reconstitution of lyophilized reagents, aliquoting the sample into < or = 30 independent reaction cavities, and mixing of reagents with the DNA samples. The foil-based cartridge was produced by blow-molding and sealed with a self-adhesive tape. The demonstrated system excels existing PCR based lab-on-a-chip platforms in terms of energy efficiency and time-to-result. Applications are suggested in the field of mobile point-of-care analysis, B-detection, or in combination with continuous monitoring systems.

Microstructuring of polymer films for sensitive genotyping by real-time PCR on a centrifugal microfluidic platform.

We present a novel process flow enabling prototyping of microfluidic cartridges made out of polymer films. Its high performance is proven by implementation of a microfluidic genotyping assay testing 22 DNA samples including clinical isolates from patients infected by methicilin-resistant Staphylococcus aureus (MRSA). The microfluidic cartridges (disks) are fabricated by a novel process called microthermoforming by soft lithography (microTSL). Positive moulds are applied allowing for higher moulding precision and very easy demoulding when compared to conventional microthermoforming. High replication accuracies with geometric disk-to-disk variations of less than 1% are typical. We describe and characterise fabrication and application of microfluidic cartridges with wall thicknesses <188 microm thus enabling efficient thermocycling during real-time polymerase chain reaction (PCR). The microfluidic cartridges are designed for operation in a slightly modified commercial thermocycling instrument. This approach demonstrates new opportunities for both microfluidic developments and well-established laboratory instruments. The microfluidic protocol is controlled by centrifugal forces and divides the liquid sample parallely into independent aliquots of 9.8 microl (CV 3.4%, N = 32 wells). The genotyping assays are performed with pre-stored primers and probes for real-time PCR showing a limit of detection well below 10 copies of DNA per reaction well (N = 24 wells in 3 independent disks). The system was evaluated by 44 genotyping assays comprising 22 DNA samples plus duplicates in a total of 11 disks. The samples contained clinical samples of seven different genotypes of MRSA as well as positive and negative controls. The results are in excellent agreement with the reference in microtubes.

Peptide microarrays for the profiling of cytotoxic T-lymphocyte activity using minimum numbers of cells.

The identification of epitopes that elicit cytotoxic T-lymphocyte activity is a prerequisite for the development of cancer-specific immunotherapies. However, especially the parallel characterization of several epitopes is limited by the availability of T cells. Microarrays have enabled an unprecedented miniaturization and parallelization in biological assays. Here, we developed peptide microarrays for the detection of CTL activity. MHC class I-binding peptide epitopes were pipetted onto polymer-coated glass slides. Target cells, loaded with the cell-impermeant dye calcein, were incubated on these arrays, followed by incubation with antigen-expanded CTLs. Cytotoxic activity was detected by release of calcein and detachment of target cells. With only 200,000 cells per microarray, CTLs could be detected at a frequency of 0.5% corresponding to 1,000 antigen-specific T cells. Target cells and CTLs only settled on peptide spots enabling a clear separation of individual epitopes. Even though no physical boundaries were present between the individual spots, peptide loading only occurred locally and cytolytic activity was confined to the spots carrying the specific epitope. The peptide microarrays provide a robust platform that implements the whole process from antigen presentation to the detection of CTL activity in a miniaturized format. The method surpasses all established methods in the minimum numbers of cells required. With antigen uptake occurring on the microarray, further applications are foreseen in the testing of antigen precursors that require uptake and processing prior to presentation.

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.