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Ionizing radiation has dramatic effects on living organisms, causing damage to proteins, DNA, and other cellular components. γ radiation produces reactive oxygen species (ROS) that damage biological macromolecules. Protein modification due to interactions with hydroxyl radical is one of the most common deleterious effects of radiation. The human eye lens is particularly vulnerable to the effects of ionizing radiation, as it is metabolically inactive and its proteins are not recycled after early development. Therefore, radiation damage accumulates and eventually can lead to cataract formation. Here we explore the impact of γ radiation on a long-lived structural protein. We exposed the human eye lens protein γS-crystallin (HγS) to high doses of γ radiation and investigated the chemical and structural effects. HγS accumulated many post-translational modifications (PTMs), appearing to gain significant oxidative damage. Biochemical assays suggested that cysteines were affected, with the concentration of free thiol reduced with increasing γ radiation exposure. SDS-PAGE analysis showed that irradiated samples form protein-protein cross-links, including nondisulfide covalent bonds. Tandem mass spectrometry on proteolytic digests of irradiated samples revealed that lysine, methionine, tryptophan, leucine, and cysteine were oxidized. Despite these chemical modifications, HγS remained folded past 10.8 kGy of γ irradiation as evidenced by circular dichroism and intrinsic tryptophan fluorescence spectroscopy.

Acta crystallographica / Section D 77(6), 820 - 834 (2021). doi:10.1107/S2059798321003855, Fixed-target serial crystallography has become an important method for the study of protein structure and dynamics at synchrotrons and X-ray free-electron lasers. However, sample homogeneity, consumption and the physical stress on samples remain major challenges for these high-throughput experiments, which depend on high-quality protein microcrystals. The batch crystallization procedures that are typically applied require time- and sample-intensive screening and optimization. Here, a simple protein crystallization method inside the features of the HARE serial crystallography chips is reported that circumvents batch crystallization and allows the direct transfer of canonical vapor-diffusion conditions to in-chip crystallization. Based on conventional hanging-drop vapor-diffusion experiments, the crystallization solution is distributed into the wells of the HARE chip and equilibrated against a reservoir with mother liquor. Using this simple method, high-quality microcrystals were generated with sufficient density for the structure determination of four different proteins. A new protein variant was crystallized using the protein concentrations encountered during canonical crystallization experiments, enabling structure determination from ∼55 µg of protein. Additionally, structure determination from intracellular crystals grown in insect cells cultured directly in the features of the HARE chips is demonstrated. In cellulo crystallization represents a comparatively un­explored space in crystallization, especially for proteins that are resistant to crystallization using conventional techniques, and eliminates any need for laborious protein purification. This in-chip technique avoids harvesting the sensitive crystals or any further physical handling of the crystal-containing cells. These proof-of-principle experiments indicate the potential of this method to become a simple alternative to batch crystallization approaches and also as a convenient extension to canonical crystallization screens., Published by Wiley, Bognor Regis

βγ-Crystallins are the primary structural and refractive proteins found in the vertebrate eye lens. Because crystallins are not replaced after early eye development, their solubility and stability must be maintained for a lifetime, which is even more remarkable given the high protein concentration in the lens. Aggregation of crystallins caused by mutations or post-translational modifications can reduce crystallin protein stability and alter intermolecular interactions. Common post-translational modifications that can cause age-related cataracts include deamidation, oxidation, and tryptophan derivatization. Metal ion binding can also trigger reduced crystallin solubility through a variety of mechanisms. Interprotein interactions are critical to maintaining lens transparency: crystallins can undergo domain swapping, disulfide bonding, and liquid-liquid phase separation, all of which can cause opacity depending on the context. Important experimental techniques for assessing crystallin conformation in the absence of a high-resolution structure include dye-binding assays, circular dichroism, fluorescence, light scattering, and transition metal FRET.

Cataract disease, a clouding of the eye lens due to precipitation of lens proteins, affects millions of people every year worldwide. The proteins that comprise the lens, the crystallins, show extensive post-translational modifications (PTMs) in aged and cataractous lenses, most commonly deamidation and oxidation. Although surface-exposed glutamines and asparagines show the highest rates of deamidation, multiple modifications can accumulate over time in these long-lived proteins, even for buried residues. Both deamidation and oxidation have been shown to promote crystallin aggregation in vitro; however, it is not clear precisely how these modified crystallins contribute to insolubilization. Here, we report six novel crystal structures of a major human lens protein, γS-crystallin (γS): one of the wild-type in a monomeric state, and five of deamidated γS variants, ranging from three to nine deamidation sites, after varying degrees of sample aging. Consistent with previous work that focused on single-to triple-site deamidation, the deamidation mutations do not appear to drastically change the fold of γS; however, increasing deamidation leads to accelerated oxidation and disulfide bond formation. Successive addition of deamidated sites progressively destabilized protein structure as evaluated by differential scanning fluorimetry. Light scattering showed the deamidated variants display an increased propensity for aggregation compared to the wild-type protein. The results suggest the deamidated variants are useful as models for accelerated aging; the structural changes observed over time provide support for redox activity of γS-crystallin in the human lens.HighlightsNovel structures of cataract-associated variants of human eye lens protein γS-crystallin reportedIncreasing deamidation of γS-crystallin decreases stability and affects aggregation propensityOverall fold of γS-crystallin maintained among deamidated and disulfide-bonded variantsDeamidated γS variants form disulfide bonds more rapidly than wild-type γSPotential functional advantage of disulfide bonding in the CXCXC motif proposed

A major challenge for science educators is teaching foundational concepts while introducing their students to current research. Here we describe an active learning module developed to teach protein structure fundamentals while supporting ongoing research in enzyme discovery. It can be readily implemented in both entry-level and upper-division college biochemistry or biophysics courses. Pre-activity lectures introduced fundamentals of protein secondary structure and provided context for the research projects, while a homework assignment familiarized students with 3D visualization of biomolecules using UCSF Chimera, a free protein structure viewer. The activity is an online survey in which students compare structure elements in papain, a well-characterized cysteine protease fromCarica papaya, to novel homologous proteases identified from the genomes of an extremophilic microbe (Halanaerobium praevalens) and two carnivorous plants (Drosera capensisandCephalotus follicularis). Students were then able to identify, with varying levels of accuracy, a number of structural features in cysteine proteases that could expedite the identification of novel or biochemically interesting cysteine proteases for experimental validation in a university laboratory. Student responses to a post-activity survey were largely positive and constructive, describing points in the activity that could be improved and indicating that the activity helped them learn about protein structure.

The emerging technique of mid-infrared optical coherence tomography (MIR-OCT) takes advantage of the reduced scattering of MIR light in various materials and devices, enabling tomographic imaging at deeper penetration depths. Because of challenges in MIR detection technology, the image acquisition time is, however, significantly longer than for tomographic imaging methods in the visible/near-infrared. Here we demonstrate an alternative approach to MIR tomography with high-speed imaging capabilities. Through femtosecond nondegenerate two-photon absorption of MIR light in a conventional Si-based CCD camera, we achieve wide-field, high-definition tomographic imaging with chemical selectivity of structured materials and biological samples in mere seconds.

The βγ-crystallin superfamily contains both β- and γ-crystallins of the vertebrate eye lens and the microbial calcium-binding proteins, all of which are characterized by a common double-Greek key domain structure. The vertebrate βγ-crystallins are long-lived structural proteins that refract light onto the retina. In contrast, the microbial βγ-crystallins bind calcium ions. The βγ-crystallin from the tunicate Ciona intestinalis (Ci-βγ) provides a potential link between these two functions. It binds calcium with high affinity and is found in a light-sensitive sensory organ that is highly enriched in metal ions. Thus, Ci-βγ is valuable for investigating the evolution of the βγ-crystallin fold away from calcium binding and toward stability in the apo form as part of the vertebrate lens. Here, we investigate the effect of Ca2+ and other divalent cations on the stability and aggregation propensity of Ci-βγ and human γS-crystallin (HγS). Beyond Ca2+, Ci-βγ is capable of coordinating Mg2+, Sr2+, Co2+, Mn2+, Ni2+, and Zn2+, although only Sr2+ is bound with comparable affinity to its preferred metal ion. The extent to which the tested divalent cations stabilize Ci-βγ structure correlates strongly with ionic radius. In contrast, none of the tested divalent cations improved the stability of HγS, and some of them induced aggregation. Zn2+, Ni2+, and Co2+ induce aggregation by interacting with cysteine residues, whereas Cu2+-mediated aggregation proceeds via a different binding site.

α-Crystallins are small heat-shock proteins that act as holdase chaperones. In humans, αA-crystallin is expressed only in the eye lens, while αB-crystallin is found in many tissues. α-Crystallins have a central domain flanked by flexible extensions and form dynamic, heterogeneous oligomers. Structural models show that both the C- and N-terminal extensions are important for controlling oligomerization through domain swapping. α-Crystallin prevents aggregation of damaged β- and γ-crystallins by binding to the client protein using a variety of binding modes. α-Crystallin chaperone activity can be compromised by mutation or posttranslational modifications, leading to protein aggregation and cataract. Because of their high solubility and their ability to form large, functional oligomers, α-crystallins are particularly amenable to structure determination by solid-state nuclear magnetic resonance (NMR) and solution NMR, as well as cryo-electron microscopy.

The hydroxyl radical is the primary reactive oxygen species produced by the radiolysis of water, and is a significant source of radiation damage to living organisms. Mobility of the hydroxyl radical at low temperatures and/or high pressures is hence a potentially important factor in determining the challenges facing psychrophilic and/or barophilic organisms in high-radiation environments (e.g., ice-interface or undersea environments in which radiative heating is a potential heat and energy source). Here, we estimate the diffusion coefficient for the hydroxyl radical in aqueous solution, using a hierarchical Bayesian model based on atomistic molecular dynamics trajectories in TIP4P/2005 water over a range of temperatures and pressures.

Amyloid fibril formation is central to the etiology of a wide range of serious human diseases, such as Alzheimer’s disease and prion diseases. Despite an ever growing collection of amyloid fibril structures found in the Protein Data Bank (PDB) and numerous clinical trials, therapeutic strategies remain elusive. One contributing factor to the lack of progress on this challenging problem is incomplete understanding of the mechanisms by which these locally ordered protein aggregates self-assemble in solution. Many current models of amyloid deposition diseases posit that the most toxic species are oligomers that form either along the pathway to forming fibrils or in competition with their formation, making it even more critical to understand the kinetics of fibrillization. A recently introduced topological model for aggregation based on network Hamiltonians is capable of recapitulating the entire process of amyloid fibril formation, beginning with thousands of free monomers and ending with kinetically accessible and thermodynamically stable amyloid fibril structures. The model can be parameterized to match the five topological classes encompassing all amyloid fibril structures so far discovered in the PDB. This paper introduces a set of network statistical and topological metrics for quantitative analysis and characterization of the fibrillization mechanisms predicted by the network Hamiltonian model. The results not only provide insight into different mechanisms leading to similar fibril structures, but also offer targets for future experimental exploration into the mechanisms by which fibrils form.

The SARS-CoV-2 main protease (Mpro) is essential to viral replication and cleaves highly specific substrate sequences, making it an obvious target for inhibitor design. However, as for any virus, SARS-CoV-2 is subject to constant selection pressure, with new Mpromutations arising over time. Identification and structural characterization of Mprovariants is thus critical for robust inhibitor design. Here we report sequence analysis, structure predictions, and molecular modeling for seventy-nine Mprovariants, constituting all clinically observed mutations in this protein as of April 29, 2020. Residue substitution is widely distributed, with some tendency toward larger and more hydrophobic residues. Modeling and protein structure network analysis suggest differences in cohesion and active site flexibility, revealing patterns in viral evolution that have relevance for drug discovery.

Divalent metal cations can play a role in protein aggregation diseases, including cataract. Here we compare the aggregation of human γS-crystallin, a key structural protein of the eye lens, via mutagenesis, ultraviolet light damage, and the addition of metal ions. All three aggregation pathways result in globular, amorphous-looking structures that do not elongate into fibers. We also investigate the molecular mechanism underlying copper(II)-induced aggregation. This work was motivated by the observation that zinc(II)-induced aggregation of γS-crystallin is driven by intermolecular bridging of solvent-accessible cysteine residues, while in contrast, copper(II)-induced aggregation of this protein is exacerbated by the removal of solvent-accessible cysteines via mutation. Here we find that copper(II)-induced aggregation results from a complex mechanism involving multiple interactions with the protein. The initial protein-metal interactions result in the reduction of Cu(II) to Cu(I) with concomitant oxidation of γS-crystallin. In addition to the intermolecular disulfides that represent a starting point for aggregation, intramolecular disulfides also occur in the cysteine loop, a region of the N-terminal domain that was previously found to mediate the early stages of cataract formation. This previously unobserved ability of γS-crystallin to transfer disulfides intramolecularly suggests that it may serve as an oxidation sink for the lens after glutathione levels have become depleted during aging. γS-Crystallin thus serves as the last line of defense against oxidation in the eye lens, a result that underscores the chemical functionality of this protein, which is generally considered to play a purely structural role.

Crystallins are transparent, refractive proteins that contribute to the focusing power of the vertebrate eye lens. These proteins are extremely soluble, resisting aggregation for decades, even under crowded conditions. Crystallins have evolved to avoid strong inter-protein interactions and have unusual hydration properties. Crystallin aggregation resulting from mutation, damage, or aging can lead to cataract, a disease state characterized by opacity of the lens. Different aggregation mechanisms can occur, following multiple pathways and leading to aggregates with varied morphologies. Studies of variant proteins found in individuals with childhood-onset cataract have provided insight into the molecular factors underlying crystallin stability and solubility. Modulation of exposed hydrophobic surface is critical, as is preventing specific intermolecular interactions that could provide nucleation sites for aggregation. Biophysical measurements and structural biology techniques are beginning to provide a detailed picture of how crystallins crowd into the lens, providing high refractivity while avoiding excessively tight binding that would lead to aggregation. Despite the central biological importance of refractivity, relatively few experimental measurements have been made for lens crystallins. Our work and that of others has shown that hydration is important to the high refractive index of crystallin proteins, as are interactions between pairs of aromatic residues and potentially other specific structural features. This Account describes our efforts to understand both the functional and disease states of vertebrate eye lens crystallins, particularly the γ-crystallins. We use a variety of biophysical techniques, notably NMR spectroscopy, to investigate crystallin stability and solubility. In the first section, we describe efforts to understand the relative stability and aggregation propensity of different γS-crystallin variants. The second section focuses on interactions of these proteins with the holdase chaperone αB-crystallin. The third, fourth, and fifth sections explore different modes of aggregation available to crystallin proteins, and the final section highlights the importance of refractive index and the sometimes conflicting demands of selection for refractivity and solubility.

Background The eye lens crystallins are highly soluble proteins that are required to last the lifespan of an organism due to low protein turnover in the lens. Crystallin aggregation leads to formation of light-scattering aggregates known as cataract. The G18V mutation of human γS-crystallin (γS-G18V), which is associated with childhood-onset cataract, causes structural changes throughout the N-terminal domain and increases aggregation propensity. The holdase chaperone protein αB-crystallin does not interact with wild-type γS-crystallin, but does bind its G18V variant. The specific molecular determinants of αB-crystallin binding to client proteins is incompletely charcterized. Here, a new variant of γS, γS-G18A, was created to test the limits of αB-crystallin selectivity. Methods Molecular dynamics simulations were used to investigate the structure and dynamics of γS-G18A. The overall fold of γS-G18A was assessed by circular dichroism (CD) spectroscopy and intrinsic tryptophan fluorescence. Its thermal unfolding temperature and aggregation propensity were characterized by CD and DLS, respectively. Solution-state NMR was used to characterize interactions between αB-crystallin and γS-G18A. Results γS-G18A exhibits minimal structural changes, but has compromised thermal stability relative to γS-WT. The placement of alanine, rather than valine, at this highly conserved glycine position produces minor changes in hydrophobic surface exposure. However, human αB-crystallin does not bind the G18A variant, in contrast to previous observations for γS-G18V, which aggregates at physiological temperature. Conclusions αB-crystallin is capable of distinguishing between aggregation-prone and function-preserving variants, and recognizing the transient unfolding or minor conformers that lead to aggregation in the disease-related variant. General significance Human αB-crystallin distinguishes between highly similar variants of a structural crystallin, binding the cataract-related γS-G18V variant, but not the function-preserving γS-G18A variant, which is monomeric at physiological temperature.

Static light scattering is a popular physical chemistry technique that enables calculation of physical attributes such as the radius of gyration and the second virial coefficient for a macromolecule (e.g., a polymer or a protein) in solution. The second virial coefficient is a physical quantity that characterizes the magnitude and sign of pairwise interactions between particles, and hence is related to aggregation propensity, a property of considerable scientific and practical interest. Estimating the second virial coefficient from experimental data is challenging due both to the degree of precision required and the complexity of the error structure involved. In contrast to conventional approaches based on heuristic ordinary least squares estimates, Bayesian inference for the second virial coefficient allows explicit modeling of error processes, incorporation of prior information, and the ability to directly test competing physical models. Here, we introduce a fully Bayesian model for static light scattering experiments on small-particle systems, with joint inference for concentration, index of refraction, oligomer size, and the second virial coefficient. We apply our proposed model to study the aggregation behavior of hen egg-white lysozyme and human γS-crystallin using in-house experimental data. Based on these observations, we also perform a simulation study on the primary drivers of uncertainty in this family of experiments, showing in particular the potential for improved monitoring and control of concentration to aid inference.

Coarse-graining is a powerful tool for extending the reach of dynamic models of proteins and other biological macromolecules. Topological coarse-graining, in which biomolecules or sets thereof are represented via graph structures, is a particularly useful way of obtaining highly compressed representations of molecular structures, and simulations operating via such representations can achieve substantial computational savings. A drawback of coarse-graining, however, is the loss of atomistic detail—an effect that is especially acute for topological representations such as protein structure networks (PSNs). Here, we introduce an approach based on a combination of machine learning and physically-guided refinement for inferring atomic coordinates from PSNs. This “neural upscaling” procedure exploits the constraints implied by PSNs on possible configurations, as well as differences in the likelihood of observing different configurations with the same PSN. Using a 1 μs atomistic molecular dynamics trajectory of Aβ1–40, we show that neural upscaling is able to effectively recapitulate detailed structural information for intrinsically disordered proteins, being particularly successful in recovering features such as transient secondary structure. These results suggest that scalable network-based models for protein structure and dynamics may be used in settings where atomistic detail is desired, with upscaling employed to impute atomic coordinates from PSNs.

We report the formation kinetics of trifluoromethane clathrate hydrate (CH) from less than 75 μm diameter ice particles and CHF3 gas. As previously observed for difluoromethane and propane hydrate formation, the initial stages of the reaction exhibit a strong negative correlation of the reaction rate with temperature, consistent with a negative activation energy of formation. The values obtained for trifluoromethane, ca. −6 kJ/mol (H2O), are similar to those for difluoromethane, even though the two molecules have different intermolecular interactions and sizes. The activation energy is lesser per mole of H2O, but greater per mole of guest molecule, than for propane hydrate, which has a different crystal structure. We propose a possible explanation for the negative activation barrier based on the stabilization of metastable structures at low temperature. A pronounced dependence of the formation kinetics on the gas flow rate into the cell is observed. At 253 K and a flow rate of 15 mmol/h, the stage II encl...

Background Carnivorous plants possess diverse sets of enzymes with novel functionalities applicable to biotechnology, proteomics, and bioanalytical research. Chitinases constitute an important class of such enzymes, with future applications including human-safe antifungal agents and pesticides. Here, we compare chitinases from the genome of the carnivorous plant Drosera capensis to those from related carnivorous plants and model organisms. Methods Using comparative modeling, in silico maturation, and molecular dynamics simulation, we produce models of the mature enzymes in aqueous solution. We utilize network analytic techniques to identify similarities and differences in chitinase topology. Results Here, we report molecular models and functional predictions from protein structure networks for eleven new chitinases from D. capensis, including a novel class IV chitinase with two active domains. This architecture has previously been observed in microorganisms but not in plants. We use a combination of comparative and de novo structure prediction followed by molecular dynamics simulation to produce models of the mature forms of these proteins in aqueous solution. Protein structure network analysis of these and other plant chitinases reveal characteristic features of the two major chitinase families. General significance This work demonstrates how computational techniques can facilitate quickly moving from raw sequence data to refined structural models and comparative analysis, and to select promising candidates for subsequent biochemical characterization. This capability is increasingly important given the large and growing body of data from high-throughput genome sequencing, which makes experimental characterization of every target impractical.

The tunicate (Ciona intestinalis) βγ-crystallin represents an intermediate case between the calcium-binding proteins ancestral to the vertebrate βγ-crystallin fold and the vertebrate structural crystallins. Unlike the structural βγ-crystallins in the vertebrate eye lens, this βγ-crystallin strongly binds Ca(2+). Furthermore, Ca(2+) binding greatly stabilizes the protein, an effect that has previously been observed in microbial βγ-crystallins but not in those of vertebrates. This relationship between binding and protein stabilization makes the tunicate βγ-crystallin an interesting model for studying the evolution of the human βγ-crystallin. We also compare and contrast the binding sites of tunicate βγ-crystallin with other βγ-crystallins in order to develop hypotheses as to the functional origin of the lack of the Ca(2+) binding sites in the human crystallins.

The βγ-crystallin superfamily contains both the β- and γ-crystallins of the vertebrate eye lens and the microbial calcium-binding proteins, all of which are characterized by a common double-Greek key domain structure. The vertebrate βγ-crystallins are long-lived structural proteins that refract light onto the retina. In contrast, the microbial βγ-crystallins bind calcium ions. The βγ-crystallin from the tunicate Ciona intestinalis (Ci-βγ) provides a potential link between these two functions: it binds calcium with high affinity and is found in a light-sensitive sensory organ that is highly enriched in metal ions. Thus Ci-βγ is valuable for investigating the evolution of the βγ-crystallin fold away from calcium binding and toward stability in the apo form as part of the vertebrate lens. Here we investigate the effect of Ca(2+) and other divalent cations on the stability and aggregation propensity of Ci-βγ and human γS-crystallin (H S). Beyond Ca(2+), Ci-βγ is capable of coordinating Mg(2+), Sr(2+), Co(2+), Mn(2+), Ni(2+), and Zn(2+), although only Sr(2+) is bound with comparable affinity to its preferred metalion. The extent to which the tested divalent cations stabilize Ci-βγ structure correlates strongly with ionic radius. In contrast, none of the tested divalent cations improved the stability of HγS, and some of them induced aggregation. Zn(2+), Ni(2+), and Co(2+) induce aggregation by interacting with cysteine residues, whereas Cu(2+)-mediated aggregation proceeds via a different binding site.

We describe a simplified method for improving the reproducibility of transceiver coil fabrication for nuclear magnetic resonance (NMR) through single-use templates made from 3D-printed polymer forms. The utility of dissolvable inserts for achieving performance enhanced resonators (DIAPERs) is tested herein by a comparison of RF homogeneity along the rotor axis for variable-pitch solenoids with different inter-turn spacing. Simulated B1 field profiles are compared to experimental homogeneity measurements, demonstrating the potential of this approach for making NMR coils quickly and reproducibly.

Amyloid fibrils are locally ordered protein aggregates that self-assemble under a variety of physiological and in vitro conditions. Their formation is of fundamental interest as a physical chemistry problem and plays a central role in Alzheimer's disease, Type II diabetes, and other human diseases. As the number of known amyloid fibril structures has grown, the need has arisen for a nomenclature for describing and classifying fibril types, as well as a theoretical description of the physics that gives rise to the self-assembly of these structures. Here, we introduce a systematic nomenclature and coarse-graining methodology for describing the topology of fibrils and other protein aggregates, along with a computational methodology for simulating protein aggregation. Both have mathematical underpinnings in graph theory and statistical mechanics and are consistent with available experimental data on the fibril structure and aggregation kinetics. Our graph representation of the fibril topology enables us to define a network Hamiltonian based on connectivity patterns among monomers rather than detailed intermolecular interactions, greatly speeding up the simulation of large ensembles. Our simulation strategy is capable of recapitulating the formation of all currently known amyloid fibril topologies found in the Protein Data Bank, as well as the formation kinetics of fibrils and oligomers.

Many advances in instrumentation and methodology have furthered the use of solid-state NMR as a technique for determining the structures and studying the dynamics of molecules involved in complex biological assemblies. Solid-state NMR does not require large crystals, has no inherent size limit, and with appropriate isotopic labeling schemes, supports solving one component of a complex assembly at a time. It is complementary to cryo-EM, in that it provides local, atomic-level detail that can be modeled into larger-scale structures. This review focuses on the development of high-field MAS instrumentation and methodology; including probe design, benchmarking strategies, labeling schemes, and experiments that enable the use of quadrupolar nuclei in biomolecular NMR. Current challenges facing solid-state NMR of biological assemblies and new directions in this dynamic research area are also discussed.