High-density Peptide Microarrays Research Articles

Antibody Cross-Reactivity in Antivenom Research.

Antivenom cross-reactivity has been investigated for decades to determine which antivenoms can be used to treat snakebite envenomings from different snake species. Traditionally, the methods used for analyzing cross-reactivity have been immunodiffusion, immunoblotting, enzyme-linked immunosorbent assay (ELISA), enzymatic assays, and in vivo neutralization studies. In recent years, new methods for determination of cross-reactivity have emerged, including surface plasmon resonance, antivenomics, and high-density peptide microarray technology. Antivenomics involves a top-down assessment of the toxin-binding capacities of antivenoms, whereas high-density peptide microarray technology may be harnessed to provide in-depth knowledge on which toxin epitopes are recognized by antivenoms. This review provides an overview of both the classical and new methods used to investigate antivenom cross-reactivity, the advantages and disadvantages of each method, and examples of studies using the methods. A special focus is given to antivenomics and high-density peptide microarray technology as these high-throughput methods have recently been introduced in this field and may enable more detailed assessments of antivenom cross-reactivity.

Zika Virus Specific Diagnostic Epitope Discovery.

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

High-density peptide microarray exploration of the antibody response in a rabbit immunized with a neurotoxic venom fraction

Polyvalent snakebite antivenoms derive their therapeutic success from the ability of their antibodies to neutralize venom toxins across multiple snake species. This ability results from a production process involving immunization of large mammals with a broad suite of toxins present in venoms. As a result of immunization with this wide range of toxins, many polyvalent antivenoms have a high degree of cross-reactivity to similar toxins in other snake venoms – a cross-reactivity which cannot easily be deconvoluted. As a proof of concept, we aimed at exploring the opposite scenario by performing a high-throughput evaluation of the extent of cross-reactivity of a polyclonal mixture of antibodies that was raised against only a single snake venom fraction. For this purpose, a venom fraction containing short neurotoxin 1 (SN-1; Uniprot accession number P01416, three-finger toxin (3FTx) family), which is the medically most important toxin from the notorious black mamba (Dendroaspis polylepis), was employed. Following immunization of a rabbit, a specific polyclonal antibody response was confirmed by ELISA and immunodiffusion. Subsequently, these antibodies were investigated by high-density peptide microarray to reveal linear elements of recognized epitopes across 742 3FTxs and 10 dendrotoxins. This exploratory study demonstrates in a single immunized animal that cross-reactivity between toxins of high similarity may be difficult to obtain when immunizing with a single 3FTx containing venom fraction. Additionally, this study explored the influence of employing different lengths of peptides in high-density peptide microarray experiments for identification of toxin epitopes. Using 8-mer, 12-mer, and 15-mer peptides, a single linear epitope element was identified in SN-1 with high precision.

Cross-recognition of a pit viper (Crotalinae) polyspecific antivenom explored through high-density peptide microarray epitope mapping.

Snakebite antivenom is a 120 years old invention based on polyclonal mixtures of antibodies purified from the blood of hyper-immunized animals. Knowledge on antibody recognition sites (epitopes) on snake venom proteins is limited, but may be used to provide molecular level explanations for antivenom cross-reactivity. In turn, this may help guide antivenom development by elucidating immunological biases in existing antivenoms. In this study, we have identified and characterized linear elements of B-cell epitopes from 870 pit viper venom protein sequences by employing a high-throughput methodology based on custom designed high-density peptide microarrays. By combining data on antibody-peptide interactions with multiple sequence alignments of homologous toxin sequences and protein modelling, we have determined linear elements of antibody binding sites for snake venom metalloproteases (SVMPs), phospholipases A2s (PLA2s), and snake venom serine proteases (SVSPs). The studied antivenom antibodies were found to recognize linear elements in each of the three enzymatic toxin families. In contrast to a similar study of elapid (non-enzymatic) neurotoxins, these enzymatic toxins were generally not recognized at the catalytic active site responsible for toxicity, but instead at other sites, of which some are known for allosteric inhibition or for interaction with the tissue target. Antibody recognition was found to be preserved for several minor variations in the protein sequences, although the antibody-toxin interactions could often be eliminated completely by substitution of a single residue. This finding is likely to have large implications for the cross-reactivity of the antivenom and indicate that multiple different antibodies are likely to be needed for targeting an entire group of toxins in these recognized sites.

Identification of three immunodominant motifs with atypical isotype profile scattered over the Onchocerca volvulus proteome.

Understanding the immune response upon infection with the filarial nematode Onchocerca volvulus and the mechanisms that evolved in this parasite to evade immune mediated elimination is essential to expand the toolbox available for diagnostics, therapeutics and vaccines development. Using high-density peptide microarrays we scanned the proteome-wide linear epitope repertoire in Cameroonian onchocerciasis patients and healthy controls from Southern Africa which led to the identification of 249 immunodominant antigenic peptides. Motif analysis learned that 3 immunodominant motifs, encompassing 3 linear epitopes, are present in 70, 43, and 31 of these peptides, respectively and appear to be scattered over the entire proteome in seemingly non-related proteins. These linear epitopes are shown to have an atypical isotype profile dominated by IgG1, IgG3, IgE and IgM, in contrast to the commonly observed IgG4 response in chronic active helminth infections. The identification of these linear epitope motifs may lead to novel diagnostic development but further evaluation of cross-reactivity against common co-infecting human nematode infections will be needed.

ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping.

Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity.B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope.

A novel approach to probe host-pathogen interactions of bovine digital dermatitis, a model of a complex polymicrobial infection.

BackgroundPolymicrobial infections represent a great challenge for the clarification of disease etiology and the development of comprehensive diagnostic or therapeutic tools, particularly for fastidious and difficult-to-cultivate bacteria. Using bovine digital dermatitis (DD) as a disease model, we introduce a novel strategy to study the pathogenesis of complex infections.ResultsThe strategy combines meta-transcriptomics with high-density peptide-microarray technology to screen for in vivo-expressed microbial genes and the host antibody response at the site of infection. Bacterial expression patterns supported the assumption that treponemes were the major DD pathogens but also indicated the active involvement of other phyla (primarily Bacteroidetes). Bacterial genes involved in chemotaxis, flagellar synthesis and protection against oxidative and acidic stress were among the major factors defining the disease.ConclusionsThe extraordinary diversity observed in bacterial expression, antigens and host antibody responses between individual cows pointed toward microbial variability as a hallmark of DD. Persistence of infection and DD reinfection in the same individual is common; thus, high microbial diversity may undermine the host’s capacity to mount an efficient immune response and maintain immunological memory towards DD. The common antigenic markers identified here using a high-density peptide microarray address this issue and may be useful for future preventive measures against DD.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3341-7) contains supplementary material, which is available to authorized users.

High-throughput immuno-profiling of mamba (Dendroaspis) venom toxin epitopes using high-density peptide microarrays.

Snakebite envenoming is a serious condition requiring medical attention and administration of antivenom. Current antivenoms are antibody preparations obtained from the plasma of animals immunised with whole venom(s) and contain antibodies against snake venom toxins, but also against other antigens. In order to better understand the molecular interactions between antivenom antibodies and epitopes on snake venom toxins, a high-throughput immuno-profiling study on all manually curated toxins from Dendroaspis species and selected African Naja species was performed based on custom-made high-density peptide microarrays displaying linear toxin fragments. By detection of binding for three different antivenoms and performing an alanine scan, linear elements of epitopes and the positions important for binding were identified. A strong tendency of antivenom antibodies recognizing and binding to epitopes at the functional sites of toxins was observed. With these results, high-density peptide microarray technology is for the first time introduced in the field of toxinology and molecular details of the evolution of antibody-toxin interactions based on molecular recognition of distinctive toxic motifs are elucidated.

High-Density Peptide Microarray Analysis of IgG Autoantibody Reactivities in Serum and Cerebrospinal Fluid of Multiple Sclerosis Patients

Intrathecal immunoglobulin G (IgG) synthesis and oligoclonal IgG bands in cerebrospinal fluid (CSF) are hallmarks of multiple sclerosis (MS), but the antigen specificities remain enigmatic. Our study is the first investigating the autoantibody repertoire in paired serum and CSF samples from patients with relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and other neurological diseases by the use of high-density peptide microarrays. Protein sequences of 45 presumed MS autoantigens (e.g.MOG, MBP, and MAG) were represented on the microarrays by overlapping 15mer peptides. IgG reactivities were screened against a total of 3991 peptides, including also selected viral epitopes. The measured antibody reactivities were highly individual but correlated for matched serum and CSF samples. We found 54 peptides to be recognized significantly more often by serum or CSF antibodies from MS patients compared with controls (pvalues <0.05). The results for RRMS and PPMS clearly overlapped. However, PPMS patients presented a broader peptide-antibody signature. The highest signals were detected for a peptide mapping to a region of the Epstein-Barr virus protein EBNA1 (amino acids 392-411), which is homologous to the N-terminal part of human crystallin alpha-B. Our data confirmed several known MS-associated antigens and epitopes, and they delivered additional potential linear epitopes, which await further validation. The peripheral and intrathecal humoral immune response in MS is polyspecific and includes antibodies that are also found in serum of patients with other diseases. Further studies are required to assess the pathogenic relevance of autoreactive and anti-EBNA1 antibodies as well as their combinatorial value as biomarkers for MS.

Peptide microarray patterning for controlling and monitoring cell growth

The fate of cells is influenced by their microenvironment and many cell types undergo differentiation when stimulated by extracellular cues, such as soluble growth factors and the insoluble extracellular matrix (ECM). Stimulating differentiation by insoluble or “immobilized” cues is a particularly attractive method because it allows for the induction of differentiation in a spatially-defined cohort of cells within a larger subpopulation. To improve the design of de novo screening of such insoluble factors, we describe a methodology for producing high-density peptide microarrays suitable for extended cell culture and fluorescence microscopy. As a model, we used a murine mammary gland cell line (NMuMG) that undergoes epithelial to mesenchymal transition (EMT) in response to soluble transforming growth factor beta (TGF-β) and surface-immobilized peptides that target TGF-β receptors (TGFβRI/II). We repurposed a well-established DNA microarray printing technique to produce arrays of micropatterned surfaces that displayed TGFβRI/II-binding peptides and integrin binding peptides. Upon long-term culture on these arrays, only NMuMG cells residing on EMT-stimulating areas exhibited growth arrest and decreased E-cadherin expression. We believe that the methodology created in this report will aid the development of peptide-decorated surfaces that can locally stimulate defined cell surface receptors and control EMT and other well-characterized differentiation events. Statement of SignificanceScope of work: This manuscript aims to accelerate the development of instructive biomaterials decorated with specific ligands that target cell-surface receptors and induce specific differentiation of cells upon contact. These materials can be used for practical applications, such as fabricating synthetic materials for large scale, stem cell culture, or investigating differentiation and asymmetric division in stem cells. Specifically, in this manuscript, we repurposed a DNA microarray printer to produce microarrays of peptide-terminated self-assembled monolayers (SAMs). To demonstrate the utility of these arrays in phenotypic assays with mammalian cells, we monitored the induction of epithelial to mesenchymal transition (EMT) in murine mammary epithelial cells using specific peptide ligands printed on these arrays. Novelty: We, and others, have published several strategies for producing peptide-based arrays suitable for long-term phenotypic assays. Many reports relied on patterning steps that made adaptation difficult. The use of a DNA microarray printer as the sole production tool simplified the production of peptide microarrays and increased the throughput of this technology. We confirmed that simplification in production did not compromise the performance of the array; it is still possible to study short-term adhesion, long-term growth, and complex phenotypic responses, such as EMT, in the cells. EMT was studied using immunofluorescent staining after four days of culture. Impact: This methodology will serve as a foundation for future screening of instructive biomaterials in our research group. As DNA printers are broadly available in academic institutions, we foresee rapid adaptation of this approach by academic researchers.

Abstract A156: Anti-p53 auto-antibody serum profiling using high-density peptide arrays

Abstract Cancer is a result of a number of genetic alterations that disturb normal, controlled cell growth and differentiation. Mutational events leading to the activation of oncogenes or the inactivation of tumor-suppressor genes have been linked causally to the formation of tumors. p53 is one of the most important regulators of transcription, cellular cycle, DNA repair and apoptosis detected to date. Anti-p53 antibodies have been detected in the serum of cancer patients. This immune response is probably due to a self-immunization process linked to the strong immunogenicity of the p53 protein, and is associated predominantly with p53 missense mutation and p53 accumulation in the tumor. Auto-antibodies have also been proposed as potential diagnostic biomarkers for early stage diagnosis of cancers, since an increase in serum levels of certain auto-antibodies has been shown to precede the development of disease symptoms and correlate with cancer incidence for various cancers including breast and lung cancer. Here we systematically evaluate reactivity of antibodies in p53 positive serum samples and identify reactive epitopes to normal and mutant peptides using a high-density (2.9 Million) peptide microarray. We assess the effect of linker length, peptide length, and flanking serines on antibody detection. We propose peptide array design parameters that can be applied to a whole proteome level to enable biomarker discovery and validation of novel auto-antibody epitopes associated with cancer. Citation Format: Ken C. Lo, John C. Tan, Eric Sullivan, Ryan Bannen, Todd Richmond, Florian Grupp, Stefan Weiser, Dieter Heindl, Klaus-Peter Stengele, Albert Thomas. Anti-p53 auto-antibody serum profiling using high-density peptide arrays. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A156.

Manufacturing of Peptide Microarrays Based on Catalyst-Free Click Chemistry.

Immobilization of peptides to a solid surface is frequently an important first step before they can be probed with a variety of biological samples in a heterogeneous assay format for research and clinical diagnostic purposes. Peptides can be derivatized in many ways to subsequently covalently attach them to an activated solid surface such as epoxy-functionalized glass slides. Here, we describe a clean, efficient, and reproducible fabrication process based on catalyst-free click chemistry compatible with the construction of low- to high-density peptide microarrays.

Solid-material-based coupling efficiency analyzed with time-of-flight secondary ion mass spectrometry

The coupling behavior of a microparticle embedded amino acid active-ester into a Poly(ethylene glycol)methacrylate-film, synthesized onto a silicon wafer by a grafting from approach, is characterized using dynamic time-of-flight secondary ion mass spectrometry (ToF-SIMS) to analyze the 3d distribution of the amino acids in the polymer film. Besides standard solid phase peptide synthesis, employing solubilized amino acids in a solvent, we used solid polymer microparticles, incorporating the amino acids. These microparticles were especially designed for a new technique to produce high-density combinatorial peptide microarrays: upon heating, the particles become viscous, which releases the embedded amino acids to diffuse and couple to the surface. In the scope of the development of this new particle-based application, ToF-SIMS is used to analyze a complex chemically modified polymer surface layer. Due to depth profile measurements, it is possible to investigate the particle-based coupling reaction not only on the surface, but also into the depth of the PEGMA film.

High-throughput sequencing enhanced phage display enables the identification of patient-specific epitope motifs in serum.

Phage display is a prominent screening technique with a multitude of applications including therapeutic antibody development and mapping of antigen epitopes. In this study, phages were selected based on their interaction with patient serum and exhaustively characterised by high-throughput sequencing. A bioinformatics approach was developed in order to identify peptide motifs of interest based on clustering and contrasting to control samples. Comparison of patient and control samples confirmed a major issue in phage display, namely the selection of unspecific peptides. The potential of the bioinformatic approach was demonstrated by identifying epitopes of a prominent peanut allergen, Ara h 1, in sera from patients with severe peanut allergy. The identified epitopes were confirmed by high-density peptide micro-arrays. The present study demonstrates that high-throughput sequencing can empower phage display by (i) enabling the analysis of complex biological samples, (ii) circumventing the traditional laborious picking and functional testing of individual phage clones and (iii) reducing the number of selection rounds.

Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants

Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens.

Automated High-Throughput Mapping of Linear B-Cell Epitopes Using a Statistical Analysis of High-Density Peptide Microarray Data.

Detailed information of antibodies' specificity is often missing or inadequate even for continuous (i.e., linear) epitopes. Recent developments in peptide microarray technology has enabled the synthesis of up to two million peptides per array thereby allowing linear peptide epitopes to be examined by a systematic amino acid substitution and positional scanning approach. This kind of analysis generates a very large body of data, which needs to be analyzed and interpreted in a robust and automated manner. Here, we describe a rational systematic approach to define linear antibody epitopes using ANOVA statistics to identify not only significant but also important residues involved in antibody recognition. This statistical approach can be used to perform a comprehensive linear epitope discovery. For polyclonal antibodies, this could be extended to entire proteins pinpointing critical residues for each epitope. We argue that the ANOVA analysis levels out issues of unknown peptide concentration/quality and unknown antibody titers leading to identification of epitopes that otherwise would be neglected if the evaluation was based merely on signal strength.

Surface Patterning via Thiol‐Yne Click Chemistry: An Extremely Fast and Versatile Approach to Superhydrophilic‐Superhydrophobic Micropatterns

An extremely fast and initiator-free approach to create superhydrophilic-superhydrophobic micropatterns via UV-induced sequential thiol-yne click chemistry is described. The photo­patterning can be performed at room temperature in a variety of solvents, including water, and can be also used for making high-density peptide or cell microarrays.

Identification and Mapping of Linear Antibody Epitopes in Human Serum Albumin Using High-Density Peptide Arrays

We have recently developed a high-density photolithographic, peptide array technology with a theoretical upper limit of 2 million different peptides per array of 2 cm2. Here, we have used this to perform complete and exhaustive analyses of linear B cell epitopes of a medium sized protein target using human serum albumin (HSA) as an example. All possible overlapping 15-mers from HSA were synthesized and probed with a commercially available polyclonal rabbit anti-HSA antibody preparation. To allow for identification of even the weakest epitopes and at the same time perform a detailed characterization of key residues involved in antibody binding, the array also included complete single substitution scans (i.e. including each of the 20 common amino acids) at each position of each 15-mer peptide. As specificity controls, all possible 15-mer peptides from bovine serum albumin (BSA) and from rabbit serum albumin (RSA) were included as well. The resulting layout contained more than 200.000 peptide fields and could be synthesized in a single array on a microscope slide. More than 20 linear epitope candidates were identified and characterized at high resolution i.e. identifying which amino acids in which positions were needed, or not needed, for antibody interaction. As expected, moderate cross-reaction with some peptides in BSA was identified whereas no cross-reaction was observed with peptides from RSA. We conclude that high-density peptide microarrays are a very powerful methodology to identify and characterize linear antibody epitopes, and should advance detailed description of individual specificities at the single antibody level as well as serologic analysis at the proteome-wide level.

An H3K36 Methylation-Engaging Tudor Motif of Polycomb-like Proteins Mediates PRC2 Complex Targeting

Polycomb repressive complex 2 (PRC2) regulates pluripotency, differentiation, and tumorigenesis through catalysis of histone H3 lysine 27 trimethylation (H3K27me3) on chromatin. However, the mechanisms that underlie PRC2 recruitment and spreading on chromatin remain unclear. Here we report that histone H3 lysine 36 trimethylation (H3K36me3) binding activity is harbored in the Tudor motifs of PRC2-associated polycomb-like (PCL) proteins PHF1/PCL1 and PHF19/PCL3. Ectopically expressed PHF1 induced Tudor-dependent stabilization of PRC2 complexes on bulk chromatin and mediated spreading of PRC2 and H3K27me3 into H3K36me3-containing chromatin regions. In murine pluripotent stem cells, we identified coexistence of H3K36me3, H3K27me3, and PHF19/PCL3 at a subset of poised developmental genes and demonstrated that PHF19/PCL3 Tudor function is required for optimal H3K27me3 and repression of these loci. Collectively, our data suggest that PCL recognition of H3K36me3 promotes intrusion of PRC2 complexes into active chromatin regions to promote gene silencing and modulate the chromatin landscape during development.