Repetitive Arrays Research Articles

Sir2 and Fun30 regulate ribosomal DNA replication timing via MCM helicase positioning and nucleosome occupancy.

The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well-known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae , the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA arrays (rDNA). We have previously reported that in the absence of SIR2 , a derepressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the nondisplaced MCMs. Furthermore, we found that both, activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30 . Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler.

Engineered Transcription Factor Binding Arrays for DNA-based Gene Expression Control in Mammalian Cells.

Manipulating gene expression in mammalian cells is critical for cell engineering applications. Here we explore the potential of transcription factor (TF) recognition element arrays as DNA tools for modifying free TF levels in cells and thereby controlling gene expression. We first demonstrate proof-of-concept, showing that Tet TF-binding recognition element (RE) arrays of different lengths can tune gene expression and alter gene circuit performance in a predictable manner. We then open-up the approach to interface with any TF with a known binding site by developing a new method called Cloning Troublesome Repeats in Loops (CTRL) that can assemble plasmids with up to 256 repeats of any RE sequence. Transfection of RE array plasmids assembled by CTRL into mammalian cells show potential to modify host cell gene regulation at longer array sizes by sequestration of the TF of interest. RE array plasmids built using CTRL were demonstrated to target both synthetic and native mammalian TFs, illustrating the ability to use these tools to modulate genetic circuits and instruct cell fate. Together this work advances our ability to assemble repetitive DNA arrays and showcases the use of TF-binding RE arrays as a method for manipulating mammalian gene expression, thus expanding the possibilities for mammalian cell engineering.

Protein Nanoparticles as Vaccine Platforms for Human and Zoonotic Viruses.

Vaccines are one of the most effective medical interventions, playing a pivotal role in treating infectious diseases. Although traditional vaccines comprise killed, inactivated, or live-attenuated pathogens that have resulted in protective immune responses, the negative consequences of their administration have been well appreciated. Modern vaccines have evolved to contain purified antigenic subunits, epitopes, or antigen-encoding mRNAs, rendering them relatively safe. However, reduced humoral and cellular responses pose major challenges to these subunit vaccines. Protein nanoparticle (PNP)-based vaccines have garnered substantial interest in recent years for their ability to present a repetitive array of antigens for improving immunogenicity and enhancing protective responses. Discovery and characterisation of naturally occurring PNPs from various living organisms such as bacteria, archaea, viruses, insects, and eukaryotes, as well as computationally designed structures and approaches to link antigens to the PNPs, have paved the way for unprecedented advances in the field of vaccine technology. In this review, we focus on some of the widely used naturally occurring and optimally designed PNPs for their suitability as promising vaccine platforms for displaying native-like antigens from human viral pathogens for protective immune responses. Such platforms hold great promise in combating emerging and re-emerging infectious viral diseases and enhancing vaccine efficacy and safety.

Self-Assembled Nanostructures Presenting Repetitive Arrays of Subunit Antigens for Enhanced Immune Response.

Infectious diseases pose persistent threats to public health, demanding advanced vaccine technologies. Nanomaterial-based delivery systems offer promising solutions to enhance immunogenicity while minimizing reactogenicity. We introduce a self-assembled vaccine (SAV) platform employing antigen-polymer conjugates designed to facilitate robust immune responses. The SAVs exhibit efficient cellular uptake by dendritic cells (DCs) and macrophages, which are crucial players in the innate immune system. The high-density antigen presentation of this SAV platform enhances the affinity for DCs through multivalent recognition, significantly augmenting humoral immunity. SAV induced high levels of immunoglobulin G (IgG), IgG1, and IgG2a, suggesting that mature DCs efficiently induced B cell activation through multivalent antigen recognition. Universality was confirmed by applying it to respiratory viruses, showcasing its potential as a versatile vaccine platform. Furthermore, we have also demonstrated strong protection against influenza A virus infection with SAV containing hemagglutinin, which is used in influenza A virus subunit vaccines. The efficacy and adaptability of this nanostructured vaccine present potential utility in combating infectious diseases.

Analysis of CENP-B Boxes as Anchor of Kinetochores in Centromeres of Human Chromosomes.

The kinetochore is a multiprotein structure that attaches at one end to DNA in the centromere and at the other end to microtubules in the mitotic spindle. By connecting centromere and spindle, the kinetochore controls the migration of chromosomes during cell division. The exact position where the kinetochore assembles on each centromere was uncertain because large sections of centromeric DNA had not been sequenced due to highly repetitive alpha-satellite arrays. Embedded in the arrays is a 17 bp consensus sequence, the so-called CENP-B box, which binds the CENP-B protein, the only protein that binds directly to centromeric DNA. Recently, the Telomere-to-Telomere Consortium published the complete centromeric DNA sequences of all chromosomes including their epigenetic modifications in the T2T-CHM13 map. I used data from the T2T-CHM13 map to locate the CENP-B boxes in the centromeres as anchor of kinetochores. Most of the CENP-B boxes in centromeric DNA are methylated with the exception of the so-called centromere dip region (CDR), where CENP-B protein dimers bind to adjacent unmethylated CENP-B boxes and interact with CENP-A and CENP-C proteins to assemble the kinetochore. The centromeres of all chromosomes combined have a size of 407 Mb of which the kinetochores account for 5.0 Mb or 1.2%. There is no correlation between centromere and kinetochore size (P = .77). While the number of CENP-B boxes varies 4-fold between chromosomes, their density (number/Kb) varies less than 2-fold with a mean of 2.61 ± 0.33. The narrow range ensures a uniform pull of the spindle on the centromeres. I illustrate the findings in a model of the human kinetochore anchored at unmethylated CENP-B boxes in the CDR and present circos plots of chromosomes to show the location of kinetochores in their respective centromeres.

High-Throughput Assembly of Compositionally Controlled 3D Cell Communities for Developmental Engineering.

Cell patterning for 3D culture has increased our understanding of how cells interact among themselves and with their environment during tissue morphogenesis. Building cell communities from the bottom up with size and compositional control is invaluable for studies of morphological transitions. Here, we detail Photolithographic DNA-programmed Assembly of Cells (pDPAC). pDPAC uses a photoactive polyacrylamide gel substrate to capture single-stranded DNA on a 2D surface in large-scale, highly resolved patterns using the photomask technology. Cells are then functionalized with a complementary DNA strand, enabling cells to be temporarily adhered to distinct locations only where their complementary strand is patterned. These temporary 2D patterns can be transferred to extracellular matrix hydrogels for 3D culture of cells in biomimetic microenvironments. Use of a polyacrylamide substrate has advantages, including a simpler photolithography workflow, lower non-specific cell adhesion, and lower stiction to ECM hydrogels during release of patterned hydrogels. The protocol is equally applicable to large (cm)-scale patterns and repetitive arrays of smaller-scale cell interaction or migration experiments.

Nanovaccines for Advancing Long-Lasting Immunity against Infectious Diseases.

Infectious diseases, particularly life-threatening pathogens such as small pox and influenza, have substantial implications on public health and global economies. Vaccination is a key approach to combat existing and emerging pathogens. Immunological memory is an essential characteristic used to evaluate vaccine efficacy and durability and the basis for the long-term effects of vaccines in protecting against future infections; however, optimizing the potency, improving the quality, and enhancing the durability of immune responses remains challenging and a focus for research involving investigation of nanovaccine technologies. In this review, we describe how nanovaccines can address the challenges for conventional vaccines in stimulating adaptive immune memory responses to protect against reinfection. We discuss protein and nonprotein nanoparticles as useful antigen platforms, including those with highly ordered and repetitive antigen array presentation to enhance immunogenicity through cross-linking with multiple B cell receptors, and with a focus on antigen properties. In addition, we describe how nanoadjuvants can improve immune responses by providing enhanced access to lymph nodes, lymphnode targeting, germinal center retention, and long-lasting immune response generation. Nanotechnology has the advantage to facilitate vaccine induction of long-lasting immunity against infectious diseases, now and in the future.

Safety and immunogenicity of a heterologous booster with an RBD virus-like particle vaccine following two- or three-dose inactivated COVID-19 vaccine

ABSTRACT LYB001 is an innovative recombinant SARS-CoV-2 vaccine that displays a repetitive array of the spike glycoprotein’s receptor-binding domain (RBD) on a virus-like particle (VLP) vector to boost the immune system, produced using Covalink plug-and-display protein binding technology. LYB001’s safety and immunogenicity were assessed in 119 participants receiving a booster with (1) 30 μg LYB001 (I-I-30 L) or CoronaVac (I-I-C), (2) 60 μg LYB001 (I-I-60 L) or CoronaVac in a ratio of 2:1 after two-dose primary series of inactivated COVID-19 vaccine, and (3) 30 μg LYB001 (I-I-I-30 L) after three-dose inactivated COVID-19 vaccine. A well-tolerated reactogenicity profile was observed for LYB001 as a heterologous booster, with adverse reactions being predominantly mild in severity and transient. LYB001 elicited a substantial increase in terms of the neutralizing antibody response against prototype SARS-CoV-2 28 days after booster, with GMT (95%CI) of 1237.8 (747.2, 2050.6), 554.3 (374.6, 820.2), 181.9 (107.6, 307.6), and 1200.2 (831.5, 1732.3) in the I-I-30 L, I-I-60 L, I-I-C, and I-I-I-30 L groups, respectively. LYB001 also elicited a cross-neutralizing antibody response against the BA.4/5 strain, dominant during the study period, with GMT of 201.1 (102.7, 393.7), 63.0 (35.1, 113.1), 29.2 (16.9, 50.3), and 115.3 (63.9, 208.1) in the I-I-30 L, I-I-60 L, I-I-C, and I-I-I-30 L groups, respectively, at 28 days after booster. Additionally, RBD-specific IFN-γ, IL-2, IL-4 secreting T cells dramatically increased at 14 days after a single LYB001 booster. Our data confirmed the favorable safety and immunogenicity profile of LYB001 and supported the continued clinical development of this promising candidate that utilizes the VLP platform to provide protection against COVID-19.

Chemically cross-linked hydrogels from repetitive protein arrays

Biomaterials for tissue regeneration must mimic the biophysical properties of the native physiological environment. A protein engineering approach allows the generation of protein hydrogels with specific and customised biophysical properties designed to suit a particular physiological environment. Herein, repetitive engineered proteins were successfully designed to form covalent molecular networks with defined physical characteristics able to sustain cell phenotype. Our hydrogel design was made possible by the incorporation of the SpyTag (ST) peptide and multiple repetitive units of the SpyCatcher (SC) protein that spontaneously formed covalent crosslinks upon mixing. Changing the ratios of the protein building blocks (ST:SC), allowed the viscoelastic properties and gelation speeds of the hydrogels to be altered and controlled. The physical properties of the hydrogels could readily be altered further to suit different environments by tuning the key features in the repetitive protein sequence. The resulting hydrogels were designed with a view to allow cell attachment and encapsulation of liver derived cells. Biocompatibility of the hydrogels was assayed using a HepG2 cell line constitutively expressing GFP. The cells remained viable and continued to express GFP whilst attached or encapsulated within the hydrogel. Our results demonstrate how this genetically encoded approach using repetitive proteins could be applied to bridge engineering biology with nanotechnology creating a level of biomaterial customisation previously inaccessible.

Effective Multivalent Oriented Presentation of Meningococcal NadA Antigen Trimers by Self-Assembling Ferritin Nanoparticles.

The presentation of viral antigens on nanoparticles in multivalent arrays has emerged as a valuable technology for vaccines. On the nanoparticle surface, highly ordered, repetitive arrays of antigens can mimic their geometric arrangement on virion surfaces and elicit stronger humoral responses than soluble viral antigens. More recently, bacterial antigens have been presented on self-assembling protein nanoparticles and have elicited protective antibody and effective T-helper responses, further supporting the nanoparticle platform as a universal approach for stimulating potent immunogenicity. Here, we present the rational design, structural analysis, and immunogenicity of self-assembling ferritin nanoparticles displaying eight copies of the Neisseria meningitidis trimeric adhesin NadA. We engineered constructs consisting of two different NadA fragments, head only and head with stalk, that we fused to ferritin and expressed in Escherichia coli. Both fusion constructs self-assembled into the expected nanoparticles as determined by Cryo electron microscopy. In mice, the two nanoparticles elicited comparable NadA antibody levels that were 10- to 100-fold higher than those elicited by the corresponding NadA trimer subunits. Further, the NadAferritin nanoparticles potently induced complement-mediated serum bactericidal activity. These findings confirm the value of self-assembling nanoparticles for optimizing the immunogenicity of bacterial antigens and support the broad applicability of the approach to vaccine programs, especially for the presentation of trimeric antigens.

Enrichment of centromeric DNA from human cells.

Centromeres are key elements for chromosome segregation. Canonical centromeres are built over long-stretches of tandem repetitive arrays. Despite being quite abundant compared to other loci, centromere sequences overall still represent only 2 to 5% of the human genome, therefore studying their genetic and epigenetic features is a major challenge. Furthermore, sequencing of centromeric regions requires high coverage to fully analyze length and sequence variations, and this can be extremely costly. To bypass these issues, we have developed a technique, named CenRICH, to enrich for centromeric DNA from human cells based on selective restriction digestion and size fractionation. Combining restriction enzymes cutting at high frequency throughout the genome, except within most human centromeres, with size-selection of fragments >20 kb, resulted in over 25-fold enrichment in centromeric DNA. High-throughput sequencing revealed that up to 60% of the DNA in the enriched samples is made of centromeric repeats. We show that this method can be used in combination with long-read sequencing to investigate the DNA methylation status of certain centromeres and, with a specific enzyme combination, also of their surrounding regions (mainly HSATII). Finally, we show that CenRICH facilitates single-molecule analysis of replicating centromeric fibers by DNA combing. This approach has great potential for making sequencing of centromeric DNA more affordable and efficient and for single DNA molecule studies.

Whole Numbers in Specified Arrays and Their Relationships in Multi – Dimensional Locales

The study details specified properties of whole numbers in conjunction with repetitive arrays and sequences. There prevails a common pattern for numbers when they exist in defined structures. The paper extends to the scope of progressions in regard to the specific number relationships and its reach in advanced mathematical studies. The properties of numbers enumerated have its scope in the field of recreational mathematical theories as well. Finding 01 proves the relationships between numbers in specific patterns, with exceptions in effect. The exceptions hold true only in the mentioned patterns of numbers. Findings 02 and 03 show the properties of numbers in Arithmetic Progression and the relationship of the numbers in specific arrangements with the Common Difference. Findings 04 and 05 work on the value of Determinants of matrices of which numbers are in Arithmetic and Geometric Progressions and its application in regard to positioning of multiple points in varying dimensions. Findings 06, 07 and 08 detail the unique properties of numbers when applied in addition, subtraction and multiplication and the ‘CONSTANCY’ in the end results. Findings 09 and 10 explain the relationships and properties of numbers with reference to the structures of regular polygons and the positioning of points in multiple dimensions.

Charged sequence motifs increase the propensity towards liquid-liquid phase separation.

Protein phase separation is a major governing factor in multiple cellular processes, such as RNA metabolism and those involving RNA-binding proteins. Despite many key observations, the exact structural characteristics of proteins involved in the phase separation process are still not fully deciphered. In this work, we show that proteins harbouring sequence regions with specific charged residue patterns are significantly associated with liquid-liquid phase separation. In particular, regions with repetitive arrays of alternating charges show the strongest association, whereas segments with generally high charge density and single α-helices also show detectable but weaker connections.

Characteristic Mode-Based Efficient Broadband Electromagnetic Scattering Analysis Method for Array Structures

A fast and accurate numerical method based on the theory of characteristic mode (TCM) is proposed for analyzing electromagnetic scattering from repetitive multiscale array structures. In order to overcome the low frequency breakdown problem when interacting at short distances between observation and source basis functions, the CMs are extracted from the impedance matrix generated by the augmented electric field integral equation (AEFIE), which is discretized by the method of moments (MoM). Then the CMs of a single unit are used as the global basis functions to reduce the unknowns of the array structures. In addition, the broadband multilevel fast multipole algorithm (MLFMA) based on approximate diagonalization of the Green’s function is implemented to accelerate the matrix-vector multiplications between the impedance matrix and CM vectors. Several numerical examples are given to demonstrate the high efficiency and accuracy of the proposed scheme.

Stabilization of the SARS-CoV-2 Spike Receptor-Binding Domain Using Deep Mutational Scanning and Structure-Based Design.

The unprecedented global demand for SARS-CoV-2 vaccines has demonstrated the need for highly effective vaccine candidates that are thermostable and amenable to large-scale manufacturing. Nanoparticle immunogens presenting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (S) in repetitive arrays are being advanced as second-generation vaccine candidates, as they feature robust manufacturing characteristics and have shown promising immunogenicity in preclinical models. Here, we used previously reported deep mutational scanning (DMS) data to guide the design of stabilized variants of the RBD. The selected mutations fill a cavity in the RBD that has been identified as a linoleic acid binding pocket. Screening of several designs led to the selection of two lead candidates that expressed at higher yields than the wild-type RBD. These stabilized RBDs possess enhanced thermal stability and resistance to aggregation, particularly when incorporated into an icosahedral nanoparticle immunogen that maintained its integrity and antigenicity for 28 days at 35-40°C, while corresponding immunogens displaying the wild-type RBD experienced aggregation and loss of antigenicity. The stabilized immunogens preserved the potent immunogenicity of the original nanoparticle immunogen, which is currently being evaluated in a Phase I/II clinical trial. Our findings may improve the scalability and stability of RBD-based coronavirus vaccines in any format and more generally highlight the utility of comprehensive DMS data in guiding vaccine design.

DNA replication, transcription, and H3K56 acetylation regulate copy number and stability at tandem repeats

Tandem repeats are inherently unstable and exhibit extensive copy number polymorphisms. Despite mounting evidence for their adaptive potential, the mechanisms associated with regulation of the stability and copy number of tandem repeats remain largely unclear. To study copy number variation at tandem repeats, we used two well-studied repetitive arrays in the budding yeast genome, the ribosomal DNA (rDNA) locus, and the copper-inducible CUP1 gene array. We developed powerful, highly sensitive, and quantitative assays to measure repeat instability and copy number and used them in multiple high-throughput genetic screens to define pathways involved in regulating copy number variation. These screens revealed that rDNA stability and copy number are regulated by DNA replication, transcription, and histone acetylation. Through parallel studies of both arrays, we demonstrate that instability can be induced by DNA replication stress and transcription. Importantly, while changes in stability in response to stress are observed within a few cell divisions, a change in steady state repeat copy number requires selection over time. Further, H3K56 acetylation is required for regulating transcription and transcription-induced instability at the CUP1 array, and restricts transcription-induced amplification. Our work suggests that the modulation of replication and transcription is a direct, reversible strategy to alter stability at tandem repeats in response to environmental stimuli, which provides cells rapid adaptability through copy number variation. Additionally, histone acetylation may function to promote the normal adaptive program in response to transcriptional stress. Given the omnipresence of DNA replication, transcription, and chromatin marks like histone acetylation, the fundamental mechanisms we have uncovered significantly advance our understanding of the plasticity of tandem repeats more generally.

Immunization with a Self-Assembled Nanoparticle Vaccine Elicits Potent Neutralizing Antibody Responses against EBV Infection.

Epstein-Barr virus (EBV) infection is a global health concern infecting over 90% of the population. However, there is no currently available vaccine. EBV primarily infects B cells, where the major glycoprotein 350 (gp350) is the main target of neutralizing antibodies. Given the advancement of nanoparticle vaccines, we describe rationally designed vaccine modalities presenting 60 copies of gp350 on self-assembled nanoparticles in a repetitive array. In a mouse model, gp350s on lumazine synthase (LS) and I3-01 adjuvanted with MF59 or aluminum hydroxide (Alhydrogel) elicited over 65- to 133-fold higher neutralizing antibody titers than the corresponding gp350 monomer to EBV. Furthermore, immunization with gp350D123-LS and gp350D123-I3-01 vaccine induced a Th2-biased response. For the nonhuman primate model, gp350D123-LS in MF59 elicited higher titers of total IgG and neutralizing antibodies than the monomeric gp350D123. Overall, these results support gp350D123-based nanoparticle vaccine design as a promising vaccine candidate for potent protection against EBV infection.

Tribological behavior of polymeric 3D-printed surfaces with deterministic patterns inspired in snake skin morphology

Surface engineering can rely on biological systems to mimic characteristics of living specimens in order to develop and fabricate deterministic elements to modify friction. In this work, the tribological response of bio-inspired patterns obtained by 3D-printing for polymer-metal contact in dry conditions are discussed. The repetitive array of elements called ‘fibrils’ in the ventral scale of Phython regius snake was used as an inspiration to fabricate the deterministic surfaces. The texturized samples were 3D-printed with Nylon filament and the counterpart was AISI 304 stainless steel. The results showed that the friction coefficient was consistently lower in the tests carried out with the texturized samples in comparison to those performed with stochastic surfaces. Lower friction values were clearly correlated to geometrical descriptors of the texture patters including the Feature Slenderness Ratio (FSR) and the Feature Aspect Ratio (FAR). The analysis of polymer’s worn surfaces revealed adhesive damage, localized melting and roll formation as the main wear mechanisms.

Loss of Smi1, a protein involved in cell wall synthesis, extends replicative lifespan by enhancing rDNA stability in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, replicative lifespan (RLS) is primarily affected by the stability of ribosomal DNA (rDNA). The stability of the highly repetitive rDNA array is maintained through transcriptional silencing by the NAD+-dependent histone deacetylase Sir2. Recently, the loss of Smi1, a protein of unknown molecular function that has been proposed to be involved in cell wall synthesis, has been demonstrated to extend RLS in S. cerevisiae, but the mechanism by which Smi1 regulates RLS has not been elucidated. In this study, we determined that the loss of Smi1 extends RLS in a Sir2-dependent manner. We observed that the smi1D mutation enhances transcriptional silencing at the rDNA locus and promotes rDNA stability. In the absence of Smi1, the stress-responsive transcription factor Msn2 translocates from the cytoplasm to the nucleus, and nuclear-accumulated Msn2 stimulates the expression of nicotinamidase Pnc1, which serves as an activator of Sir2. In addition, we observed that the MAP kinase Hog1 is activated in smi1D cells and that the activation of Hog1 induces the translocation of Msn2 into the nucleus. Taken together, our findings suggest that the loss of Smi1 leads to the nuclear accumulation of Msn2 and stimulates the expression of Pnc1, thereby enhancing Sir2-mediated rDNA stability and extending RLS in S. cerevisiae.

Loss of Smi1, a protein involved in cell wall synthesis, extends replicative life span by enhancing rDNA stability in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, replicative life span (RLS) is primarily affected by the stability of ribosomal DNA (rDNA). The stability of the highly repetitive rDNA array is maintained through transcriptional silencing by the NAD+-dependent histone deacetylase Sir2. Recently, the loss of Smi1, a protein of unknown molecular function that has been proposed to be involved in cell wall synthesis, has been demonstrated to extend RLS in S. cerevisiae, but the mechanism by which Smi1 regulates RLS has not been elucidated. In this study, we determined that the loss of Smi1 extends RLS in a Sir2-dependent manner. We observed that the smi1Δ mutation enhances transcriptional silencing at the rDNA locus and promotes rDNA stability. In the absence of Smi1, the stress-responsive transcription factor Msn2 translocates from the cytoplasm to the nucleus, and nuclear-accumulated Msn2 stimulates the expression of nicotinamidase Pnc1, which serves as an activator of Sir2. In addition, we observed that the MAP kinase Hog1 is activated in smi1Δ cells and that the activation of Hog1 induces the translocation of Msn2 into the nucleus. Taken together, our findings suggest that the loss of Smi1 leads to the nuclear accumulation of Msn2 and stimulates the expression of Pnc1, thereby enhancing Sir2-mediated rDNA stability and extending RLS in S. cerevisiae.