How Nature discovered genetic coding is a largely ignored question, yet the answer is key to explaining the transition from biochemical building blocks to life. Other, related puzzles also fall inside the aegis enclosing the codes themselves. The peptide bond is unstable with respect to hydrolysis. So, it requires some form of chemical free energy to drive it. Amino acid activation and acyl-transfer are also slow and call for catalysis. All living things must thus also convert free energy and synchronize cellular chemistry. Most importantly, functional proteins occupy only small, isolated regions of sequence space. Nature evolved heritable symbolic data processing to seek out and use those sequences. That system has three parts: a memory of how amino acids behave in solution and inside proteins, a set of code-keys to access that memory, and a scoring function. The code-keys themselves are the genes for cognate pairs of tRNA and aminoacyl-tRNA synthetases, AARS. I outline the surprising links between all these questions and the structural duality of the base-pairing that holds genes together and how those links arise from the experiments.

The folding of certain proteins (e.g., enzymes) into perfectly defined 3D conformations via multi-orthogonal interactions is critical to their function. Concerning synthetic polymers chains, the “folding” of individual polymer chains at high dilution via intra-chain interactions leads to so-called single-chain nanoparticles (SCNPs). This review article describes the advances carried out in recent years in the folding of single polymer chains into discrete SCNPs via multi-orthogonal interactions using different reactive chemical species where intra-chain bonding only occurs between groups of the same species. First, we summarize results from computer simulations of multi-orthogonally folded SCNPs. Next, we comprehensively review multi-orthogonally folded SCNPs synthesized via either non-covalent bonds or covalent interactions. Finally, we conclude by summarizing recent research about multi-orthogonally folded SCNPs prepared through both reversible (dynamic) and permanent bonds.

“Clickase” single-chain nanoparticles (Ck-SCNPs) are folded, enzyme-mimetic unimolecular polymeric nano-objects containing copper (Cu) ions able to catalyze the azide-alkyne Huisgen cycloaddition reaction in water and/or selected organic solvents, often in the presence of a reductant. Herein, we investigate the effect of morphology on catalytic activity of Ck-SCNPs synthesized by means of two different routes. An amphiphilic random copolymer composed of oligo(ethylene glycol) methyl ether methyl methacrylate (OEGMA) and 2-acetoacetoxy ethyl methacrylate (AEMA) units was used as precursor of these Ck-SCNPs. Folding was promoted through metal complexation between Cu(II) ions and beta-ketoester-containing AEMA moieties. The first route resulted in Ck-SCNPs1 containing Cu ions homogeneously distributed within each nanoparticle, whereas the second one promoted intra-chain clustering of Cu ions inside Ck-SCNPs2. A model fluorogenic “click” reaction between 9-(azidomethyl)anthracene and phenylacetylene, which was catalyzed either by Ck-SCNPs1 or Ck-SCNPs2, was used to unravel the effect of morphology on catalytic activity. This work paves the way to improve the catalytic activity of metallo-folded SCNPs through control of the intra-chain distribution of catalytic sites.

The potential to affect gene expression via G-quadruplex stabilization has been extended to all domains of life, including viruses. Here, we investigate the polymorphism and structures of G-quadruplexes of the human papillomavirus type 52 with UV, CD and NMR spectroscopy and gel electrophoresis. We show that oligonucleotide with five G-tracts folds into several structures and that naturally occurring single nucleotide polymorphisms (SNPs) have profound effects on the structural polymorphism in the context of G-quadruplex forming propensity, conformational heterogeneity and folding stability. With help of SNP analysis, we were able to select one of the predominant forms, formed by G-rich sequence d(G3TAG3CAG4ACACAG3T). This oligonucleotide termed HPV52(1-4) adopts a three G-quartet snap back (3+1) type scaffold with four syn guanine residues, two edgewise loops spanning the same groove, a no-residue V loop and a propeller type loop. The first guanine residue is incorporated in the central G-quartet and all four-guanine residues from G4 stretch are included in the three quartet G-quadruplex core. Modification studies identified several structural elements that are important for stabilization of the described G-quadruplex fold. Our results expand set of G-rich targets in viral genomes and address the fundamental questions regarding folding of G-rich sequences.

Intrinsically disordered proteins mediate crucial biological functions through their interactions with other proteins. Mutual synergistic folding (MSF) occurs when all interacting proteins are disordered, folding into a stable structure in the course of the complex formation. In these cases, the folding and binding processes occur in parallel, lending the resulting structures uniquely heterogeneous features. Currently there are no dedicated classification approaches that would take into account the particular biological and biophysical properties of MSF complexes. Here we present a scalable clustering-based classification scheme, built on redundancy-filtered features that describe the sequence and structure properties of the complexes, and the role of the interaction, which is directly responsible for structure formation. Using this approach, we define six major types of MSF complexes, corresponding to biologically meaningful groups. Hence, the presented method also shows that differences in binding strength, subcellular localization, and regulation are encoded in the sequence and structural properties of proteins. While current structure classification methods can also handle complex structures, we show that the developed scheme is fundamentally different, and since it takes into account defining features of MSF complexes, it serves as a better representation of structures arising through this specific interaction mode.

Succinic semialdehyde dehydrogenase (SSADH) is a mitochondrial enzyme involved in the catabolism of the neurotransmitter γ-amino butyric acid. Pathogenic variants in the gene encoding this enzyme cause SSADH deficiency, a developmental disease manifesting as hypotonia, autism and epilepsy. SSADH deficiency patients usually have family-specific gene variants. Here, we describe a family exhibiting four different SSADH variants, Val90Ala, Cys93Phe, and His180Tyr/Asn255Asp (a double variant). We provide a structural and functional characterization of these variants and show that Cys93Phe and Asn255Asp are pathogenic variants that affect the stability of the SSADH protein. Due to impairment of the cofactor NAD+ binding, these variants show a highly reduced enzyme activity. However, Val90Ala and His180Tyr exhibit normal activity and expression. The His180Tyr/Asn255Asp variant exhibits a highly reduced activity as a recombinant species, is inactive and shows a very low expression in eukaryotic cells. A treatment with substances that support protein folding by either increasing chaperone protein expression or by chemical means was not capable of increasing the expression of the pathogenic variants of the SSADH deficiency patient. However, stabilization of the folding of pathogenic SSADH variants by other substances may provide a treatment option for this disease.

We show that Universe inflation occurs when Einstein's gravity couples to nonlinear electromagnetic fields with cosmic stochastic magnetic fields background. Nonlinear electrodynamics with two parameters is used. The strength of magnetic fields to have the Universe inflation is obtained. It is shown that singularities of the energy density and pressure are absent for any scale factors. At large scale factors one arrives at equation of state for ultra-relativistic case. It is demonstrated that the curvature invariants do not have singularities. By computing the deceleration parameter we show that graceful exit takes place with reasonable e-folding number. The duration of Universe inflation as a function of model parameters is obtained. The classical stability and causality are analyzed by calculating the speed of the sound.

Development of methods and algorithms to predict the effect of mutations on protein stability, protein-protein, and protein-DNA/RNA binding is necessitated by the needs of protein engineering and for understanding the molecular mechanism of disease-causing variants. The vast majority of the leading methods are either methods with adjustable parameters or machine learning algorithms, both requiring a database of experimentally measured folding and binding free energy changes. These databases are collections of experimental data taken from scientific investigations typically aimed at probing the role of particular residue on the above-mentioned thermodynamics characteristics, i.e., the mutations are not introduced at random and do not necessarily represent mutations originating from single nucleotide variant (SNV). Thus, the reported performance of the leading algorithms assessed on these databases or other limited cases, may not be applicable for predicting the effect of SNVs seen in the human population. Indeed, we demonstrate that the SNVs and non-SNVs are not equally presented in the corresponding databases and the distribution of the free energy changes are not the same. Furthermore, the Pearson correlation coefficients (PCCs) obtained on cases involving SNVs are less impressive than for non-SNVs, indicating that caution should be used in applying them to reveal the effect of human SNVs. All methods are found to underestimate the energy changes by roughly a factor of 2.

From experimental studies of protein folding, it is now clear that there are two types of folding behavior, i.e., two-state folding and non-two-state folding, and understanding the relationships between these apparently different folding behaviors is essential for fully elucidating the molecular mechanisms of protein folding. This article describes how the presence of the two types of folding behavior has been confirmed experimentally, and discusses the relationships between the two-state and the non-two-state folding reactions, on the basis of available data on the correlations of the folding rate constant with various structure-based properties, which are determined primarily by the backbone topology of proteins. Finally, a two-stage hierarchical model is proposed as a general mechanism of protein folding. In this model, protein folding occurs in a hierarchical manner, reflecting the hierarchy of the native three-dimensional structure, as embodied in the case of non-two-state folding with an accumulation of the molten globule state as a folding intermediate. The two-state folding is thus merely a simplified version of the hierarchical folding caused either by an alteration in the rate-limiting step of folding or by destabilization of the intermediate.

The conformational preferences of the ester group have the potential to facilitate the large amplitude folding of long alkyl chains in the gas phase. They are monitored by Raman spectroscopy in supersonic jet expansions for the model system methyl butanoate, after establishing a quantitative relationship to quantum-chemical predictions for methyl methanoate. This requires a careful analysis of experimental details, and a simulation of the rovibrational contours for near-symmetric top molecules. The technique is shown to be complementary to microwave spectroscopy in quantifying coexisting conformations. It confirms that a C-O-C(=O)-C-C chain segment can be collapsed into a single all-trans conformation by collisional cooling, whereas alkyl chain isomerism beyond this five-membered chain largely survives the jet expansion. This sets the stage for the investigation of linear alkyl alkanoates in terms of dispersion-induced stretched-chain to hairpin transitions by Raman spectroscopy.

The global pandemic caused by the new SARS-COV-2 coronavirus makes it necessary to search for drugs for its control. Within of this research it has been known that the ivermectin drug, a FDA-approved drugs which is formulated as an 80:20 mixture of ivermectin B1a and B1b and used commonly for parasitic infections, has an inhibitory effect on viruses, includes SARS-COV-2 at in vitro level. In the particular case of SARS-COV-2 its mechanism of action remains elusive and controversial. Interestingly, the energy of interaction of ivermectin with any of the proteins the SARS-CoV-2 and the possible structural alterations at the protein level that this drug can cause have not been reported. In this sense, we carried out a bioinformatics study with docking strategies and molecular dynamics to predict the binding and disturbance induced by ivermectin in proteins associated with SARS-CoV-2. We use DockThor and Molegro docking scores. The DockThor server and myPresto software were used to build complexes and dynamics studies, respectively. The results obtained suggested that ivermectin is capable of docking with the 3CL protease and the HR2 domain, and may promote structural changes in these proteins by inducing unfolding/folding. Specifically, ivermectin brings protease to a significantly more deployed conformational state and the HR2 domain to a more compact state compared to the native state. Finally, it is shown that B1a and B1b macrocyclic lactones have a behavior different from to each target protein. These results suggest a possible inhibitory effect against SARS-CoV-2 due to a synergistic role of this drug to spontaneously bind to two important proteins involve in the proliferation of this virus. However, more studies are required on this possible mechanism of action.

The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b protein’s host targets is the RNA silencing factor Argonaute 1 (AGO1), which it binds to and inhibits. In Arabidopsis thaliana, if 2b-induced inhibition of AGO1 is too efficient it induces reinforcement of antiviral silencing by AGO2, and triggers increased resistance against aphids, CMV’s insect vectors. These effects would be deleterious to CMV replication and transmission, respectively, but are moderated by the CMV 1a protein by sequestering sufficient 2b protein molecules into P-bodies to prevent excessive inhibition of AGO1. Mutant 2b protein variants were generated and red and green fluorescent protein fusions used to investigate subcellular colocalization with AGO1 and the 1a protein, and the effects of mutations on complex formation with the 1a protein and AGO1 were investigated using bimolecular fluorescence complementation and co-immunoprecipitation assays. Although we found that residues 56-60 influenced the 2b protein’s interactions with the 1a protein and AGO1, it appears unlikely that any single residue or sequence domain is solely responsible. In silico predictions of intrinsic disorder within the 2b protein secondary structure were supported by circular dichroism (CD) but not by nuclear magnetic resonance (NMR) spectroscopy. Intrinsic disorder provides a plausible model to explain the 2b protein’s ability to interact with AGO1, the 1a protein and other factors. However, the reasons for the conflicting conclusions provided by CD and NMR must first be resolved.

Proteomic analysis revealed the preservation of many proteins in the “Heslington brain” (which is at least 2,600-year-old brain tissue uncovered within the skull excavated in 2008 from a pit in Heslington, Yorkshire, England). Five of these proteins (“main proteins”), heavy, medium, and light neurofilament proteins (NFH, NFM, and NFL), glial fibrillary acidic protein (GFAP), and myelin basic (MBP) protein are engaged in the formation non-amyloid protein aggregates, such as intermediate filaments and myelin sheath. We used a wide spectrum of bioinformatics tools to evaluate the prevalence of functional disorder in several related sets of proteins, such as “main proteins” and their 44 interactors, as well as all other protein identified in the Heslington brain. These analyses revealed that all five “main proteins”, half of their interactors and almost one third of the Heslington brain proteins are expected to be mostly disordered. Furthermore, most of the remaining proteins are expected to contain sizable disordered regions. This is in contrary the expected substantial (if not complete) elimination of the disordered proteins from the Heslington brain. Therefore, it seems that the intrinsic disorder of NFH, NFM, NFL, GFAP, and MBP, their interactors and many other proteins might play a crucial role in preserving the Heslington brain by forming tightly folded brain protein aggregates, in which different parts are glued together via the disorder-to-order transitions.

Heat shock proteins (HSPs) are a family of molecular chaperones that regulate essential protein refolding and triage decisions to maintaining protein homeostasis. Numerous co-chaperone proteins directly interact and modify the function of HSPs, and these interactions impact the outcome of protein triage, impacting everything from structural proteins to cell signaling mediators. The chaperone/co-chaperone machinery protects against various stressors to ensuring cellular function in the face of stress. However, coding mutations, expression changes, and post-translational modifications of the chaperone/co-chaperone machinery can alter the cellular stress response. Importantly, these dysfunctions appear to contribute to numerous human diseases. Therapeutic targeting of chaperones is an attractive but challenging approach due to the vast functions of HSPs, likely contributing to the off-target effects of these therapies. Current efforts focus on targeting co-chaperones to develop precise treatments for numerous diseases caused by defects in protein quality control. This review focuses on the recent developments regarding selected HSP70/HSP90 co-chaperones, focusing on cardioprotection, neuroprotection, and cancer. We also discuss therapeutic approaches that highlight both the utility and challenges of targeting co-chaperones.

Myelin protein zero (P0), a type I transmembrane protein, is the most abundant protein in peripheral nervous system (PNS) myelin – the lipid-rich, periodic structure that concentrically encloses long axonal segments. Schwann cells, the myelinating glia of the PNS, express P0 throughout their development until the formation of mature myelin. In the intramyelinic compartment, the immunoglobulin-like domain of P0 bridges apposing membranes together via homophilic adhesion, forming a dense, macroscopic ultrastructure known as the intraperiod line. The C-terminal tail of P0 adheres apposing membranes together in the narrow cytoplasmic compartment of compact myelin, much like myelin basic protein (MBP). In mouse models, the absence of P0, unlike that of MBP or P2, severely disturbs the formation of myelin. Therefore, P0 is the executive molecule of PNS myelin maturation. How and when is P0 trafficked and modified to enable myelin compaction, and how disease mutations that give rise to incurable peripheral neuropathies alter the function of P0, are currently open questions. The potential mechanisms of P0 function in myelination are discussed, providing a foundation for the understanding of mature myelin development and how it derails in peripheral neuropathies.

Cardiac myosin-binding protein C (MyBPC) is a thick-filament associated regulatory protein in the sarcomere. It regulates the sensitive contractile system of the myocardium by acting as a mechanical tether, sensitizing the thin filament or modulating myosin motor activity. Mutations in the MYBPC3 gene are a frequent cause for the development of hypertrophic cardiomyopathy, the most frequent cardiac disorder. Recently, the monoallelic double mutation MYBPC3Δ25bp/D389V has been discovered as a subset of the common MYBPC3Δ25bp variant in South Asia. MYBPC3Δ25bp/D389V carriers exhibit hyperdynamic features, which are considered an early finding for the development of hypertrophic cardiomyopathy. Using correlation-guided molecular dynamics simulations sampling, we show that the D389V mutation shifts the conformational distribution of the C2 domain of MyBPC. We further applied biochemical approaches to probe the effects of the D389V mutation on structure, thermostability and protein-protein interactions of MyBPC C2. The melting temperature (Tm) of MyBPC C2 D389V is decreased by 4 to 7 °C compared to wild type while the interaction of the C0-C2 domains with myosin and actin remains unchanged. Additionally, we utilized steered molecular dynamics (SMD) simulations to investigate the altered unfolding pathway of MyBPC C2 D389V. Based on our data, we propose a pathomechanism for the development of HCM in MYBPC3Δ25bp and MYBPC3Δ25bp/D389V carriers.

Myelin ensheathes selected axonal segments within the nervous system, resulting primarily in nerve impulse acceleration, as well as mechanical and trophic support for neurons. In the central and peripheral nervous systems, various proteins that contribute to the formation and stability of myelin are present, which also harbour pathophysiological roles in myelin disease. Many myelin proteins share common attributes, including small size, high hydrophobicity, multifunctionality, longevity, and intrinsic disorder. With recent advances in protein biophysical characterization and bioinformatics, it has become evident that intrinsically disordered proteins (IDPs) are abundant in myelin, and their flexible nature enables multifunctionality. Here, we review known myelin IDPs, their conservation, molecular characteristics and functions, and their disease relevance, along with open questions and speculations. We place emphasis on classifying the molecular details of IDPs in myelin and correlate these with their various functions, including susceptibility to post-translational modifications, function in protein-protein and protein-membrane interactions, as well as their role as extended entropic chains. We discuss how myelin pathology can relate to IDPs and which molecular factors are potentially involved.

The structural transitions RNAs undergo during trafficking are not well-understood. Here, we used the well-developed yeast Ty1 retrotransposon to provide the first structural model of genome (g) RNA in the nucleus from a retrovirus-like transposon. Through a detailed comparison of nuclear Ty1 gRNA structure with those established in the cytoplasm, virus-like particles (VLPs), and synthesized in vitro, we detected Ty1 gRNA structural alternations that occur during retrotransposition. Full-length Ty1 gRNA serves as the mRNA for Gag and Gag-Pol proteins and as the genome that is reverse transcribed within VLPs. We show that about 60% of base pairs predicted for the nuclear Ty1 gRNA appear in the cytoplasm, and active translation does not account for such structural differences. Most of the shared base pairs are represented by short-range interactions, while the long-distance pairings seem unique for each compartment. Highly structured motifs tend to be preserved after nuclear export of Ty1 gRNA. In addition, our study highlights the important role of Ty1 Gag in mediating critical RNA:RNA interactions required for retrotransposition.

Structural information of biological macromolecules is crucial and necessary to deliver predictions about the effects of mutations—whether polymorphic or deleterious (i.e., disease causing), wherein, thermodynamic parameters, namely, folding and binding free energies potentially serve as effective biomarkers. It may be emphasized that the effect of a mutation depends on various factors, including the type of protein (globular, membrane or intrinsically disordered protein) and the structural context to which it occurs. Such information may positively aid drug-design. Furthermore, due to the intrinsic plasticity of proteins, even mutations involving radical change of the structural and physico-chemical properties of the amino acids (native vs. mutant) can still have minimal effects of protein thermodynamics. However, if a mutation causes significant perturbation of either folding or binding free energies, it is quite likely to be deleterious. Mitigating such effects is a promising alternative to the traditional approaches of designing inhibitors. This can be done by structure-based in silico screening of small molecules for which binding to the dysfunctional protein restores its wild type thermodynamics. In this review we emphasize on the effects of mutations on two important biophysical characteristics, stability and binding affinity, and how structures can be used for structure-based drug design to mitigate the effects of disease-causing variants on the above biophysical characteristics.

Homochirality is an obvious feature of life on Earth. On the other hand, extraterrestrial samples contain largely racemic compounds. The same is true for any common organic synthesis. Therefore, it has been a perplexing puzzle for decades how these racemates could have formed enantiomerically enriched fractions as a basis for the origin of homochiral life forms. Numerous hypotheses have been put forward as to how preferentially homochiral molecules could have formed and accumulated on Earth. In this article, it is shown that homochirality of the abiotic organic pool at the time of formation of the first self-replicating molecules is not necessary and not even probable. It is proposed to abandon the notion of a molecular ensemble and to focus on the level of individual molecules. Although the formation of the first self-replicating, most likely homochiral molecule is a seemingly improbable event, on a closer look, it is almost inevitable that homochiral molecules have formed simply on a statistical basis. In this case, the non-selective leap to homochirality would be one of the first steps in chemical evolution directly out of a racemic “ocean”. Moreover, most studies focus on the chirality of the primordial monomers with respect to an asymmetric carbon atom. However, any polymer with a minimal size that allows folding to a secondary structure, would spontaneously lead to asymmetric higher structures (conformations). Most of the functions of these polymers would be influenced by this inherently asymmetric folding. To summarize, simple and universal mechanisms may have led to homochiral self-replicating systems in the context of chemical evolution. A homochiral monomer pool is deemed unnecessary and probably never existed on primordial Earth.

Plants offer a simpler and cheaper alternative to mammalian animal models for the study of Endoplasmic Reticulum glycoprotein folding Quality Control (ERQC). In particular, the Arabidopsis thaliana (At) innate immune response to bacterial peptides provides an easy means of assaying ERQC function in vivo. A number of mutants that are useful to study ERQC in planta have been described in the literature, but only for a subset of these mutants the innate immune response to bacterial elicitors has been measured beyond monitoring plant weight and some physio-pathological parameters related to the plant immune response. In order to probe deeper into the role of ERQC in the plant immune response, we monitored expression levels of the PHI-1 and RET-OX genes in the At ER α-Glu II rsw3 and the At UGGT uggt1-1 mutant plants, in response to bacterial peptides elf18 and flg22. The elf18 response was impaired in the rsw3 but not completely abrogated in the uggt1-1 mutant plants, raising the possibility that the latter enzyme is partly dispensable for ERF signalling. In the rsw3 mutant, seedling growth was impaired only by concomitant application of the At ER α-Glu II NB-DNJ inhibitor at concentrations above 500 nM, suggesting residual activity in this mutant. The study highlights the need for extending plant innate immune response studies to assays sampling EFR signalling at the molecular level.

Ribosome assembly is one of the most fundamental processes in gene expression and has served as a playground to investigate the molecular mechanisms of how protein-RNA complexes (RNPs) assemble. The bacterial ribosome is composed of around 50 ribosomal proteins several of which are co-transcriptionally assembled on a ~4,500 nucleotides long pre-rRNA transcript that is further processed and modified during transcription, the entire process taking around 2 minutes in vivo and assisted by dozens of assembly factors. How this complex molecular process works so efficiently to produce an active ribosome has been investigated over decades and has resulted in the development of a plethora of novel approaches that can also be used to study the assembly of other RNPs. Here we review biochemical, structural and biophysical methods that have been developed and integrated to provide a detailed and quantitative understanding of this complex and intricate molecular process of assembly. We also discuss emerging cutting-edge approaches that could be used in the future to study how transcription, rRNA processing, cellular factors and the native cellular environment shape ribosome assembly and RNP assembly at large.

There are no theorems (proven theories) in the biological sciences. We propose that the 3 31 nt minihelix tRNA evolution theorem be universally accepted as one. The 3 31 nt minihelix theorem completely describes evolution of type I and type II tRNAs from ordered precursors (RNA repeats and inverted repeats). Despite diversification of tRNAome sequences, statistical tests overwhelmingly support the theorem. Furthermore, the theorem relates the dominant pathway for the origin of life on Earth, specifically, how tRNAomes and the genetic code may have coevolved. Alternate models for tRNA evolution (i.e., 2 minihelix, convergent and accretion models) are falsified. In the context of the pre-life world, tRNA was a molecule that via mutation could modify anticodon sequences and teach itself to code. We relate the clearest history to date of the chemical evolution of life on Earth. From analysis of tRNA evolution, ribozyme-mediated RNA ligation was a primary driving force in evolution of complexity during the pre-life to life transition on Earth.

Transition between the unfolded and native states of the ordered globular proteins is accompanied by accumulation of several intermediates, such as pre-molten globule, wet molten globule, and dry molten globule. Structurally equivalent conformations can serve as native functional states of intrinsically disordered proteins. This overview captures the characteristics and importance of these molten globules in both structured and intrinsically disordered proteins. It also discusses examples of engineered molten globules. The formation of these intermediates under the conditions of macromolecular crowding and their interactions with nanomaterials are also reviewed.