Redesigning the stereospecificity of tyrosyl‐tRNA synthetase

d ‐Amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Unnatural amino acids have been introduced into proteins using engineered aminoacyl‐tRNA synthetases (aaRSs), and this strategy might be applicable to d ‐amino acids. Several aaRSs can aminoacylate their tRNA with a d ‐amino acid; of these, tyrosyl‐tRNA synthetase (TyrRS) has the weakest stereospecificity. We use computational protein design to suggest active site mutations in Escherichia coli TyrRS that could increase its d ‐Tyr binding further, relative to l ‐Tyr. The mutations selected all modify one or more sidechain charges in the Tyr binding pocket. We test their effect by probing the aminoacyl‐adenylation reaction through pyrophosphate exchange experiments. We also perform extensive alchemical free energy simulations to obtain l ‐Tyr/d ‐Tyr binding free energy differences. Agreement with experiment is good, validating the structural models and detailed thermodynamic predictions the simulations provide. The TyrRS stereospecificity proves hard to engineer through charge‐altering mutations in the first and second coordination shells of the Tyr ammonium group. Of six mutants tested, two are active towards d ‐Tyr; one of these has an inverted stereospecificity, with a large preference for d ‐Tyr. However, its activity is low. Evidently, the TyrRS stereospecificity is robust towards charge rearrangements near the ligand. Future design may have to consider more distant and/or electrically neutral target mutations, and possibly design for binding of the transition state, whose structure however can only be modeled. Proteins 2016; 84:240–253. © 2015 Wiley Periodicals, Inc.     

Discovery of keratin function and role in genetic diseases: the year that 1991 was

In 1991, a set of transgenic mouse studies took the fields of cell biology and dermatology by storm in providing the first credible evidence that keratin intermediate filaments play a unique and essential role in the structural and mechanical support in keratinocytes of the epidermis. Moreover, these studies intimated that mutations altering the primary structure and function of keratin filaments underlie genetic diseases typified by cellular fragility. This Retrospective on how these studies came to be is offered as a means to highlight the 25th anniversary of these discoveries.Although intermediate filaments (IFs) have been characterized at some level for a longer period of time (Oshima, 2007 ), they were officially discovered as such as recently as 1968 by Howard Holtzer and colleagues while studying the developing skeletal muscle (Ishikawa et al., 1968 ). The advent of gene cloning methods and monospecific antibody production in the late 1970s and throughout the 1980s led to an explosion of data and knowledge about IFs that established them as a large family of genes and proteins that are individually regulated in a tight and evolutionarily conserved tissue- and differentiation-specific manner. Researchers also uncovered some of the remarkable properties of IFs as purified elements in vitro and in living systems and recognized that they occur in the nucleus as well as in the cytoplasm. In spite of the fast pace of progress during that period, however, it was not possible to produce evidence that spoke unequivocally about the functional importance of IFs in cells and tissues, let alone their role in disease.Beginning in the mid- to late 1980s, pioneering experimentation along two distinct lines was underway in the laboratory of Elaine Fuchs, then at the University of Chicago. The eventual merger of these approaches yielded the first formal insight into IF function in vivo, as well as into their direct involvement in human disease. In an effort to define structure–function relationships with regard to the assembly and network formation properties of IFs, one such approach was the application of systematic deletion mutagenesis to keratin 14 (K14), a type I IF that is expressed with its type II partner keratin 5 (K5) in the progenitor basal layer of the epidermis and related complex epithelia. These studies demonstrated that deleting sequences from either end of the central α-helical rod domain of the K14 protein was deleterious for filament formation in a dominant manner both in transfected cells (Albers and Fuchs, 1987 , 1989 ; Figure 1) and the setting of IF polymerization assays involving purified proteins in vitro (e.g., Coulombe et al., 1990 ). The second key effort in the Fuchs lab in the late 1980s resulted in the demonstration that the proximal 2.5 kb and distal 700 base pairs corresponding respectively to the 5′ upstream and 3′ downstream regions of the cloned human K14 gene were sufficient to confer tissue-specific, that is, K14-like, regulation in transgenic mice in vivo (Vassar et al., 1989 ; Figure 1). This tour de force paved the way for the production of a human K14 gene promoter–based cassette (e.g., Saitou et al., 1995 ) that could reliably direct the expression of any open reading frame in a K14-like manner in transgenic mice. As an aside, this tool has had a profound effect on epithelial and skin biology research.Open in a separate windowFIGURE 1:Schematic representation of the strategy and outcome of the experiments that led to the discovery of keratin function and role in genetic disease. Original figures are reproduced to give a realistic account of the data. (A) Examples of a disrupted keratin filament network in cultured epithelial cells transfected with and expressing a dominantly acting K14 deletion mutant (arrows). (Reproduced from Albers and Fuchs, 1987 , with permission.) (B) Preferential expression of a substance P-epitope–tagged transgenic human K14 protein in the basal layer of tail skin epidermis in mouse, conveying the tissue- and differentiation-specific behavior of the transgene. (Reproduced from Vassar et al., 1989 , with permission.) (C) The two experimental approaches described in A and B were combined to assess the consequences of tissue-specific expression of dominantly acting K14 mutants in skin tissue in vivo. (D) Newborn mouse littermates. The mouse at the top is transgenic (Tg) and expresses a mutated form of K14 in the epidermis. It is showing severe skin blistering (arrows), particularly in its front paws, which are heavily used by mouse newborns to feed from their mother. The bottom mouse is a nontransgenic control showing no such blistering. (E, F) Hematoxylin-eosin–stained skin tissue sections showing the location of subepidermal cleavage within the epidermis of a K14 mutant–expressing transgenic mouse (opposing arrows in E). Cleavage occurs at the level of the basal layer, where the mutant keratin is expressed. Again, this is never seen in control wild-type (Wt) skin (F). Bar, 100 μm (E, F). (D–F are from Coulombe et al., 1991b , with permission.) (G) Leg skin in a patient suffering from the Dowling–Meara form of epidermolysis bullosa simplex. Characteristic of this severe variant of this disease, several skin blisters are often grouped in a herpetiform manner (Fine et al., 1991 ).Subsequent use of the human K14 promoter–based cassette to direct the expression of epitope-tagged and selected deletion mutants of K14 gave rise to transgenic mouse pups that exhibited extensive blistering of the skin preferentially at sites of frictional trauma (Coulombe et al., 1991b ; Vassar et al., 1991 ; Figure 1). Electron microscopy showed that skin blistering occurred secondary to a loss of the integrity of keratinocytes located in the basal layer of the epidermis, that is, the precise site of mutant K14 protein accumulation. Such blistering did not occur in transgenic mice expressing a full-length version of human K14 modified to carry only an epitope tag at the C-terminus at similar or higher levels (Coulombe et al., 1991b ; Vassar et al., 1991 ). In addition, the severity of skin blistering in mutant K14–expressing transgenic pups could be directly related to the extent to which the mutant protein had been shown to disrupt filament assembly in transfected cell assays and in IF reconstitution assays in vitro. For instance, tissue-specific expression of a K14 mutant that could severely disrupt 10-nm filament assembly was associated with whole-body skin blistering and the untimely death of mouse pups and, from a pathology perspective, with “tonofilament clumping” and a paucity of visible keratin IFs in transgenic basal keratinocytes. By comparison, expression of another K14 mutant with a less deleterious effect on 10-nm IF assembly was compatible with the survival of transgenic mouse pups and resulted in skin blistering largely limited to the front paws in newborn mice together with altered organization of keratin IFs in basal keratinocytes of transgenic epidermis in situ, albeit without tonofilament clumping. This initial set of mouse strains thus revealed the existence of a direct link between the so-called “genotype” (i.e., mutant K14 characteristics) and the skin phenotype (Coulombe et al., 1991b ; Vassar et al., 1991 ; Fuchs and Coulombe, 1992 ). Electrophoretic analyses of protein samples confirmed that the K14 mutant proteins acted dominantly to produce such spectacular phenotypes in transgenic mouse skin. Finally, blistering also occurred in the mutant K14–expressing transgenic mice in other stratified epithelia known both to express K14 and experience trauma, notably in the oral mucosa (Coulombe et al., 1991b ; Vassar et al., 1991 ).It is worth celebrating the 25th anniversary of these pioneering experiments for the following two reasons. First, the study of these mice provided the first formal demonstration that keratin IFs play a fundamentally important role in structural support in surface epithelia such as the epidermis and oral mucosa. Without proper IF support, epidermal keratinocytes are rendered fragile and cannot sustain trivial frictional stress (Coulombe et al., 1991b ; Fuchs and Coulombe, 1992 ). The second reason is the observation that the phenotype of these K14 mutant–expressing mice proved eerily similar to those of individuals afflicted with the disease epidermolysis bullosa simplex (EBS), a rare, dominantly inherited and debilitating skin condition in which the epidermis and oral mucosa undergo blistering after exposure to trivial mechanical trauma. As observed in the mouse model, tissue cleavage had been shown to result from the loss of integrity of keratinocytes located in the basal layer (Fine et al., 1991 ). Further, other researchers had previously reported on anomalies in the organization of keratin IFs in the basal epidermal keratinocytes of EBS patients (Anton-Lamprecht, 1983 ; Ito et al., 1991 ) or in cultures of epidermal keratinocytes established from EBS patients (Kitajima et al., 1989 ). The Fuchs laboratory thus teamed up with Amy Paller, a physician-scientist and pediatric dermatologist with deep expertise in genodermatoses, and mutations were soon discovered in the K14 gene of two independent and sporadic cases of a severe variant of the disease known as Dowling–Meara EBS (Coulombe et al., 1991a ; Figure 1). The two mutations were heterozygous missense alleles that affected the very same codon in K14 (Arg-125) and were correctly predicted at the time to correspond to a mutational hot spot in type I keratin genes. The mutations were shown to dominantly disrupt 10-nm IF assembly in vitro and/or in transfected keratinocytes in culture (Coulombe et al., 1991a ). Soon thereafter, a team led by Ervin Epstein at University of California, San Francisco (San Francisco, CA), reported on the use of classical linkage analysis to uncover a missense mutation in the K14 gene of a small pedigree with Koebner-type EBS, a less severe variant of the disease (Bonifas et al., 1991 ). The next year, Birgit Lane and colleagues (Lane et al., 1992 ) reported on the occurrence of mutations in keratin 5 (K5), the formal type II keratin assembly partner for K14 in vivo, in another instance of Dowling–Meara EBS.In the years since 1991, a role in structural support has been formally demonstrated for all classes of IFs (Coulombe et al., 2009 ), including the nuclear-localized lamins (e.g., Lammerding et al., 2004 ). Moreover, we now know of several hundred independent instances of mutations in either K5 or K14 in the setting of the EBS disease, with the vast majority of those consisting of dominantly acting missense alleles (Szeverenyi et al., 2008 ; Human Intermediate Filament Database, www.interfil.org, maintained at the Centre for Molecular Medicine and Bioinformatics Institute, Singapore). We also learned that, as anticipated, EBS largely represents a loss-of-function phenotype, since K14-null mice (Lloyd et al., 1995 ), K14-null individuals (Chan et al., 1994 ; Rugg et al., 1994 ), and K5-null mice (Peters et al., 2001 ) all exhibit an EBS-like skin-blistering phenotype (Coulombe et al., 2009 ). Mutations such as Arg125Cys in K14 markedly compromise the remarkable mechanical properties of keratin filaments (Ma et al., 2001 ), as well as the steady-state dynamics of keratin filaments in transfected keratinocytes in culture (Werner et al., 2004 ). Finally, mutations affecting the coding sequence of IF genes have been shown to underlie >100 diseases affecting the human population (Omary et al., 2004 ; Szeverenyi et al., 2008 ; www.interfil.org). Consistent with the exquisite tissue- and cell type–specific regulation of IF genes, these diseases collectively affect a myriad of tissues and organs and are relevant to nearly all branches of medicine. These observations attest to the importance and profound effect that the generation and characterization of mutant K14–expressing transgenic mice has had for cell biology, epithelial physiology, dermatology, and medicine.Many thoughts spring to mind when reminiscing about my involvement with this body of work. First, this effort was prescient of the power of team science and, in particular, of the potential effect of close collaborations involving biologists and physician-scientists. I learned a great deal and benefited immensely from working closely with many colleagues on this project, including Bob Vassar, Kathryn Albers, Linda Degenstein, Liz Hutton, Anthony Letai, Amy Paller, and, last but not least, my postdoctoral mentor and the laboratory head, Elaine Fuchs. Second, there is no substitute for elements such as innovation, hard work, perseverance, boldness, accountability, and great leadership. Elaine had the vision and created the exceptional circumstances necessary to make this set of discoveries possible, and, of equal importance, she was an integral part of the day-to-day progress and maturation of the entire project. Finally, as we all know, there is an intangible element of luck involved in discovery research. In this instance, a strong argument can be made that the studies highlighted here may not have had such a deep and defining effect had the effort been devoted to any IF other than the K5–K14 keratin pairing.What are some of the lingering issues regarding this specific topic that preoccupy us still, 25 years later? Two challenges loom particularly large. First, we have yet to achieve a satisfactory understanding of how mutations in keratin proteins can cause disease. This is due in part to the lack of an atomic-level understanding of the core structure of IFs (which has been a tough nut to crack; Lee et al., 2012 ), along with the reality that, for any relevant IF gene, there is a broad variety of disease-associated (mostly missense) mutations that pepper their primary structure (www.interfil.org). Second, we have yet to achieve success toward the treatment of EBS or any IF-based disorder. Disease characteristics such as low incidence, a dominantly inherited character, genetic heterogeneity (e.g., broad mutational landscape), and, in the case of EBS and related conditions, an intrinsically high rate of cell turnover within the main target tissue significantly add to the challenge of devising safe and effective therapeutic strategies (Coulombe et al., 2009 ). Although efforts are still underway to foster progress on these two challenging issues, the field as a whole has made significant progress in uncovering a plethora of noncanonical functions of keratin IFs (Hobbs et al., 2016 ) in addition to understanding their regulation, dynamics, and many remarkable properties.     

A novel fluorescent sensor based on electrosynthesized benzene sulfonic acid‐doped polypyrrole for determination of Pb(II) and Cu(II)

To develop conducting organic polymers (COPs) as luminescent sensors for determination of toxic heavy metals, a new benzene sulfonic acid‐doped polypyrrole (PPy‐BSA) thin film was electrochemically prepared by cyclic voltammetry (CV) on flexible indium tin oxide (ITO) electrode in aqueous solution. PPy‐BSA film was characterized by FTIR spectrometry, X‐ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). The optical properties of PPy‐BSA were investigated by ultraviolet (UV)‐visible absorption and fluorescence spectrometry in dimethylsulfoxide (DMSO) diluted solutions. PPy‐BSA fluorescence spectra were strongly quenched upon increasing copper(II) ion (Cu²⁺) and lead(II) ion (Pb²⁺) concentrations in aqueous medium, and linear Stern–Volmer relationships were obtained, which indicated the existence of a main dynamic fluorescence quenching mechanism. BSA‐PPy sensor showed a high sensitivity for detection of both metallic ions, Cu²⁺ and Pb²⁺, with very low limit of detection values of 3.1 and 18.0 nM, respectively. The proposed quenching‐fluorimetric sensor might be applied to the determination of traces of toxic heavy metallic ions in water samples.     

Chelate-assisted phytoextraction of lead using Fagopyrum esculentum: laboratory vs. field experiments

Abstract

The development of more sustainable remediation techniques has been receiving greater attention, as an alternative to soil excavation plan in urban gardens. An in situ phytoextraction experiment with buckwheat (Fagopyrum esculentum) was performed with a 5?mmol kg^?1 citric acid (CA) application. Joint experiments under laboratory conditions were conducted using various cultivars of F. esculentum in two soils with a Pb contamination of either geogenic or anthropogenic origin and various chelate concentrations. Results show that a minimum dose of 50?mmol kg^?1 of CA is required to lower soil pH and raise the concentration of mobile Pb–CaCl₂ for both soils. Consequently, Pb shoot uptake is increased from 6.3 to 8.9 times depending on soil type. Phytoextraction efficiency is found to be 1.3 to 2.0 times higher in the anthropogenic contaminated soil than in the soil with geogenic Pb. A scale effect has also been identified since Pb root accumulation under laboratory conditions was 2.4 times higher than in the field experiment. Despite an increase in the Pb extraction rate with CA, buckwheat appears to lack the efficiency needed to remove Pb in moderately contaminated soils. The calculated remediation period would last 166?years to remove the mobile Pb fraction.     

Unlocking biodiversity and conservation studies in high‐diversity environments using environmental DNA (eDNA): A test with Guianese freshwater fishes

Determining the species compositions of local assemblages is a prerequisite to understanding how anthropogenic disturbances affect biodiversity. However, biodiversity measurements often remain incomplete due to the limited efficiency of sampling methods. This is particularly true in freshwater tropical environments that host rich fish assemblages, for which assessments are uncertain and often rely on destructive methods. Developing an efficient and nondestructive method to assess biodiversity in tropical freshwaters is highly important. In this study, we tested the efficiency of environmental DNA (eDNA) metabarcoding to assess the fish diversity of 39 Guianese sites. We compared the diversity and composition of assemblages obtained using traditional and metabarcoding methods. More than 7,000 individual fish belonging to 203 Guianese fish species were collected by traditional sampling methods, and ~17 million reads were produced by metabarcoding, among which ~8 million reads were assigned to 148 fish taxonomic units, including 132 fish species. The two methods detected a similar number of species at each site, but the species identities partially matched. The assemblage compositions from the different drainage basins were better discriminated using metabarcoding, revealing that while traditional methods provide a more complete but spatially limited inventory of fish assemblages, metabarcoding provides a more partial but spatially extensive inventory. eDNA metabarcoding can therefore be used for rapid and large‐scale biodiversity assessments, while at a local scale, the two approaches are complementary and enable an understanding of realistic fish biodiversity.     

An evaluation of sequencing coverage and genotyping strategies to assess neutral and adaptive diversity

Whole genome sequences (WGS) greatly increase our ability to precisely infer population genetic parameters, demographic processes, and selection signatures. However, WGS may still be not affordable for a representative number of individuals/populations. In this context, our goal was to assess the efficiency of several SNP genotyping strategies by testing their ability to accurately estimate parameters describing neutral diversity and to detect signatures of selection. We analysed 110 WGS at 12× coverage for four different species, i.e., sheep, goats and their wild counterparts. From these data we generated 946 data sets corresponding to random panels of 1K to 5M variants, commercial SNP chips and exome capture, for sample sizes of five to 48 individuals. We also extracted low‐coverage genome resequencing of 1×, 2× and 5× by randomly subsampling reads from the 12× resequencing data. Globally, 5K to 10K random variants were enough for an accurate estimation of genome diversity. Conversely, commercial panels and exome capture displayed strong ascertainment biases. Besides the characterization of neutral diversity, the detection of the signature of selection and the accurate estimation of linkage disequilibrium (LD) required high‐density panels of at least 1M variants. Finally, genotype likelihoods increased the quality of variant calling from low coverage resequencing but proportions of incorrect genotypes remained substantial, especially for heterozygote sites. Whole genome resequencing coverage of at least 5× appeared to be necessary for accurate assessment of genomic variations. These results have implications for studies seeking to deploy low‐density SNP collections or genome scans across genetically diverse populations/species showing similar genetic characteristics and patterns of LD decay for a wide variety of purposes.     

Conjunctival melanoma copy number alterations and correlation with mutation status,tumor features,and clinical outcome

Relatively little is known about the genetic aberrations of conjunctival melanomas (CoM) and their correlation with clinical and histomorphological features as well as prognosis. The aim of this large collaborative multicenter study was to determine potential key biomarkers for metastatic risk and any druggable targets for high metastatic risk CoM. Using Affymetrix single nucleotide polymorphism genotyping arrays on 59 CoM, we detected frequent amplifications on chromosome (chr) 6p and deletions on 7q, and characterized mutation‐specific copy number alterations. Deletions on chr 10q11.21‐26.2, a region harboring the tumor suppressor genes, PDCD4, SUFU, NEURL1, PTEN, RASSF4, DMBT1, and C10orf90 and C10orf99, significantly correlated with metastasis (Fisher's exact, p ≤ 0.04), lymphatic invasion (Fisher's exact, p ≤ 0.02), increasing tumor thickness (Mann–Whitney, p ≤ 0.02), and BRAF mutation (Fisher's exact, p ≤ 0.05). This enhanced insight into CoM biology is a step toward identifying patients at risk of metastasis and potential therapeutic targets for systemic disease.     

Aquatic carbon fluxes dampen the overall variation of net ecosystem productivity in the Amazon basin: An analysis of the interannual variability in the boundless carbon cycle

The river–floodplain network plays an important role in the carbon (C) cycle of the Amazon basin, as it transports and processes a significant fraction of the C fixed by terrestrial vegetation, most of which evades as CO₂ from rivers and floodplains back to the atmosphere. There is empirical evidence that exceptionally dry or wet years have an impact on the net C balance in the Amazon. While seasonal and interannual variations in hydrology have a direct impact on the amounts of C transferred through the river–floodplain system, it is not known how far the variation of these fluxes affects the overall Amazon C balance. Here, we introduce a new wetland forcing file for the ORCHILEAK model, which improves the representation of floodplain dynamics and allows us to closely reproduce data‐driven estimates of net C exports through the river–floodplain network. Based on this new wetland forcing and two climate forcing datasets, we show that across the Amazon, the percentage of net primary productivity lost to the river–floodplain system is highly variable at the interannual timescale, and wet years fuel aquatic CO₂ evasion. However, at the same time overall net ecosystem productivity (NEP) and C sequestration are highest during wet years, partly due to reduced decomposition rates in water‐logged floodplain soils. It is years with the lowest discharge and floodplain inundation, often associated with El Nino events, that have the lowest NEP and the highest total (terrestrial plus aquatic) CO₂ emissions back to atmosphere. Furthermore, we find that aquatic C fluxes display greater variation than terrestrial C fluxes, and that this variation significantly dampens the interannual variability in NEP of the Amazon basin. These results call for a more integrative view of the C fluxes through the vegetation‐soil‐river‐floodplain continuum, which directly places aquatic C fluxes into the overall C budget of the Amazon basin.