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Recent advances in polymerase engineering have enabled the replication of xenonucleic acid (XNA) polymers with backbone structures distinct from those found in nature. By introducing a selective amplification step into the replication cycle, functional XNA molecules have been isolated by in vitro selection with binding and catalytic activity. Despite these successes, coding and decoding genetic information in XNA polymers remains limited by the fidelity and catalytic efficiency of engineered XNA polymerases. In particular, the process of reverse transcribing XNA back into DNA for amplification by PCR has been problematic. Here, we show that Geobacillus stearothermophilus (Bst) DNA polymerase I functions as an efficient and faithful threose nucleic acid (TNA)-dependent DNA polymerase. Bst DNA polymerase generates ∼twofold more cDNA with threefold fewer mutations than Superscript II (SSII), which was previously the best TNA reverse transcriptase. Notably, Bst also functions under standard magnesium-dependent conditions, whereas SSII requires manganese ions to relax the enzyme's substrate specificity. We further demonstrate that Bst DNA polymerase can support the in vitro selection of TNA aptamers by evolving a TNA aptamer to human α-thrombin.

The ability to synthesize and propagate genetic information encoded in the framework of xeno-nucleic acid (XNA) polymers would inform a wide range of topics from the origins of life to synthetic biology. While directed evolution has produced examples of engineered polymerases that can accept XNA substrates, these enzymes function with reduced activity relative to their natural counterparts. Here, we describe a biochemical strategy that enables the discovery of engineered polymerases with improved activity for a given unnatural polymerase function. Our approach involves identifying specificity determining residues (SDRs) that control polymerase activity, screening mutations at SDR positions in a model polymerase scaffold, and assaying key gain-of-function mutations in orthologous protein architectures. By transferring beneficial mutations between homologous protein structures, we show that new polymerases can be identified that function with superior activity relative to their starting donor scaffold. This concept, which we call scaffold sampling, was used to generate engineered DNA polymerases that can faithfully synthesize RNA and TNA (threose nucleic acid), respectively, on a DNA template with high primer-extension efficiency and low template sequence bias. We suggest that the ability to combine phenotypes from different donor and recipient scaffolds provides a new paradigm in polymerase engineering where natural structural diversity can be used to refine the catalytic activity of synthetic enzymes.

Polymerases that synthesize artificial genetic polymers hold great promise for advancing future applications in synthetic biology. However, engineering natural polymerases to replicate unnatural genetic polymers is a challenging problem. Here we present droplet-based optical polymerase sorting (DrOPS) as a general strategy for expanding polymerase function that employs an optical sensor to monitor polymerase activity inside the microenvironment of a uniform synthetic compartment generated by microfluidics. We validated this approach by performing a complete cycle of encapsulation, sorting and recovery on a doped library and observed an enrichment of ∼1,200-fold for a model engineered polymerase. We then applied our method to evolve a manganese-independent α-L-threofuranosyl nucleic acid (TNA) polymerase that functions with >99% template-copying fidelity. Based on our findings, we suggest that DrOPS is a versatile tool that could be used to evolve any polymerase function, where optical detection can be achieved by Watson-Crick base pairing.

Synthetic genetics is a subdiscipline of synthetic biology that aims to develop artificial genetic polymers (also referred to as xeno-nucleic acids or XNAs) that can replicate in vitro and eventually in model cellular organisms. This field of science combines organic chemistry with polymerase engineering to create alternative forms of DNA that can store genetic information and evolve in response to external stimuli. Practitioners of synthetic genetics postulate that XNA could be used to safeguard synthetic biology organisms by storing genetic information in orthogonal chromosomes. XNA polymers are also under active investigation as a source of nuclease resistant affinity reagents (aptamers) and catalysts (xenozymes) with practical applications in disease diagnosis and treatment. In this review, we provide a structural perspective on known antiparallel duplex structures in which at least one strand of the Watson-Crick duplex is composed entirely of XNA. Currently, only a handful of XNA structures have been archived in the Protein Data Bank as compared to the more than 100 000 structures that are now available. Given the growing interest in xenobiology projects, we chose to compare the structural features of XNA polymers and discuss their potential to access new regions of nucleic acid fold space.

Length-mass relationships, while often overlooked, form the basis of many fisheries science applications. Fisheriesindependent research surveys compile large databases of biological data that could hold a wealth of information regarding species' life history, which, for many, are data deficient and infrequently sampled. A flexible model using permutations of a broken stick and variance shift was applied to length-mass relationships to evaluate changes in the mean or variability of mass-at-length using data from deep-sea fishes and focusing on particularly poorly known deep-sea chondrichthyans. Changes in body shape and (or) in the scale of variability around mean mass-at-length were estimated for most species (94% of data sets examined). Such changes seemed likely to be correlated with biological factors, such as the onset of reproduction; 70% of length estimates for a variance shift correlated with the expected length-at-maturity. The model presented here could be applied to any fish where length and mass data are available, providing a way to estimate, validate, and investigate biological factors in species where macroscopic evaluations are unavailable or difficult to estimate. Les relations longueur-poids, bien que souvent negligees, forment la base de nombreuses applications en sciences halieutiques. Les releves de recherche independants des peches compilent d'imposantes bases de donnees biologiques qui pourraient contenir un tresor d'information concernant les cycles biologiques d'especes dont, pour bon nombre, les donnees sont insuffisantes et l'echantillonnage n'est pas frequent. Un modele souple reposant sur des permutations d'un baton rompu et d'un changement de variance a ete applique a des relations longueur-poids pour evaluer les variations de la moyenne ou de la variabilite du poids en fonction de la longueur en utilisant des donnees sur des poissons de grands fonds et en mettant l'accent sur des chondrichthyens de grands fonds particulierement meconnus. Les variations de la forme du corps ou de l'ampleur de la variabilite autour de la moyenne du poids en fonction de la longueur ont ete estimees pour la plupart des especes (94% des ensembles de donnees examines). Il semble probable que de telles variations soient correlees a des facteurs biologiques, comme le debut de la reproduction; 70% des estimations de la longueur pour un changement de variance etaient correlees a la longueur a la maturite attendue. Le modele presente pourrait etre applique a n'importe quel poisson pour lequel des donnees de longueur et de poids sont disponibles, offrant ainsi un moyen d'estimer, de valider et d'etudier des facteurs biologiques chez des especes pour lesquelles des evaluations macroscopiques sont indisponibles ou difficiles a estimer. [Traduit par la Redaction], Introduction The relationship between length and mass, while often overlooked, is an important component of fisheries population models (Froese 2006). The relationship between length (L) and mass (W) is conventionally [...]

Forage comprises the majority of beef cattle diets globally, and its quality can vary. Supplementation presents a significant opportunity to improve the efficiency of production. Supplementation has been shown to increase ruminally available nitrogen, improve forage utilization, and reduce livestock methane (CH4) emissions. However, corresponding effects of supplementation on gene expression of rumen microbiota, in particular metabolic pathways involved in methanogenesis, have yet to be studied. This study aimed to evaluate the effect of supplement composition before and after feeding on rumen archaeal and bacterial gene expression in steers consuming a lowquality forage diet. Ruminal samples were collected from five ruminally cannulated Angus steers [body weight (BW) = 375 ± 45 kg] used in a 4 × 4 Latin square fed a basal diet of King Ranch Bluestem Hay (3.5% CP, 73% NDF), and one of four isonitrogenous (130 mg N/ kg BW) supplements. Steers provided the two most divergent supplements (2% starch, 43% CP; 56% starch, 21% CP) were used for metatranscriptomic analysis. Rumen fluid samples were collected via rumen cannula 0 and 4 h post-feeding. Total RNA was extracted and sequenced. Differentially expressed genes (DEGs) were identified and functionally annotated. Pathway enrichment analysis was completed to determine enriched pathways (broad metabolic pathways) and modules (specific metabolic reactions). Only main effects are presented in this abstract. Archaeal hydrogenotrophic and methylotrophic methanogenesis reactions and tryptophan biosynthesis reactions were upregulated in the 2% starch supplement (P < 0.05). The 2% starch supplement resulted in fewer bacterial total upregulated carbohydrate-active enzymes compared with the 56% starch supplement (957 vs. 1,695 DEGs, respectively; P < 0.05), and an increased proportion of cellulose and hemicellulose-specific enzymes (35 vs. 29%, respectively; P < 0.05). The 56% starch supplement resulted in upregulation of the bacterial 3-Hydroxypropionate bi-cycle reaction (P < 0.05). Collection time exerted a larger effect than supplement composition on both archaeal and bacterial gene expression, with 83% and 63% of DEGs associated with time, respectively (P < 0.05). Archaeal hydrogenotrophic methanogenesis and carbon metabolism were upregulated 4 h post-feeding. In addition, 1,704 bacterial carbohydrate-active enzymes were upregulated 4 h post-feeding, as compared with 975 upregulated at 0 h (P < 0.05). However, these differences in DEGs did not correlate with significantly enriched pathways of bacterial carbohydrate metabolism. These findings demonstrate improvements to low-quality forage utilization, through CP and starch supplementation, can be correlated to relevant changes in gene expression of rumen microbiota. Furthermore, they demonstrate that supplementation influences the expression of methanogenesis reactions, suggesting an opportunity to affect CH4 production. Additional studies quantifying forage utilization, rumen microbiota gene expression and CH4 production are needed to further optimize supplementation strategies for improved efficiency of beef production whilst maintaining or reducing its carbon footprint. [ABSTRACT FROM AUTHOR]

Life-span estimates for orange roughy (Hoplostethus adanticus) range from ~20 years to well over 100 years. In this study, an improved lead-radium dating technique provided independent age estimates from sagittal otoliths. This technique used the known properties of radioactivity for lead-210 and radium-226 to determine the validity of fish age estimates. An improvement to lead-radium dating using mass spectrometry allowed the use of smaller samples than previously possible; therefore, an application was made to otolith cores, the first few years of otolith growth. This approach circumvented the use of whole otoliths and alleviated many of the assumptions that were necessary in previous lead-radium dating applications. Hence, it was possible to critically evaluate lead-radium dating as a tool in fish age validation. The measurement of lead-radium ratios for a series of age groups that consisted of otolith cores, grouped based on growth-zone counts from thin sections, showed a high degree of correlation to the expected lead-radium in-growth curve. This finding provided support for age estimation procedures using thin otolith sectioning. As independent estimates of age, the results indicated that fish in the oldest age group were at least 93 years old, providing robust support for a centenarian life span. Les estimations de la longevite de l'hoplostethe orange (Hoplostethus atlanticus) varient de ~20 ans a nettement plus d'un siecle. Dans notre etude, une methode amelioree de determination de l'age basee sur le plomb et le radium fournit des estimations de l'age independantes a partir des otolithes sagittaux. La technique utilise les proprietes connues de radioactivite du plomb-210 et du radium-226 pour determiner la validite des estimations de l'age chez les poissons. Une amelioration de la determination de l'age basee sur le plomb et le radium par l'utilisation de la spectrometrie de masse permet l'analyse d'echantillons plus petits qu'il n'etait possible anterieurement; c'est pourquoi nous l'avons appliquee aux noyaux des otolithes qui representent les premieres annees de croissance des otolithes. Cette methodologie evite l'utilisation d'otolithes entiers et attenue plusieurs des presuppositions anterieurement necessaires pour la determination de l'age a l'aide du plomb-radium. Il est ainsi possible d'evaluer de maniere critique la determination de l'age par le plombradium comme outil de validation de l'age chez les poissons. Les mesures des rapports plomb-radium pour une serie de classes d'age representees par des noyaux d'otolithes regroupes d'apres le nombre de zones de croissance sur des coupes minces, presentent une forte correlation avec la courbe attendue de recrutement plomb-radium. Cette observation vient valider les procedures d'estimation de l'age basees sur les coupes minces d'otolithes. En tant qu'estimations independantes de l'age, nos resultats indiquent que les poissons appartenant au groupe le plus age ont au moins 93 ans, ce qui appuie solidement l'hypothese d'une longevite d'un siecle. [Traduit par la Redaction], Introduction Orange roughy (Hoplostethus atlanticus, family Trachichthyidae) is widely distributed at depths of 600-1400 m in most temperate oceans and is a common target species for deepwater trawl fisheries around [...]

Sharks (class Chondrichthyes, subclass Elasmobranchii) typically have a long lifespan, slow growth rate and low fecundity, leading to low productivity and hence relatively high vulnerability to fishing. Information for managing fisheries catches of elasmobranchs is often lacking, because elasmobranchs are usually non‐target species, often with low abundance. It is more feasible to develop management reference points for elasmobranchs based on their life‐history information than through traditional stock assessments. The natural mortality rate (M) is the leading life‐history parameter (LHP) required by many methods for developing reference points and is itself often derived indirectly from other LHPs. In this paper, we evaluate nine M estimators, using 15 shark stocks in the Western and Central Pacific Ocean as examples. We then compare four methods for developing fishing mortality reference points Fmsy, including empirical modelling, the Euler‐Lotka equation, an M‐productivity‐based method and the spawning potential rate converted method. Our analyses show that popular M estimators developed mainly from teleost data resulted in large deviations from the average and were not suitable for elasmobranchs. All four methods for estimating Fmsy performed similarly. However, the empirical method is very simple and cost‐effective and tended to produce smaller deviations from the average than the other three methods. Nonetheless, it is recommended that multiple methods should be used to minimize possible bias and reflect uncertainty, if the required LHPs are available. [ABSTRACT FROM AUTHOR]

A literature review on longevity of New Zealand deep-sea fishes provided life history parameter estimates for 17 bony and five cartilaginous species. Preliminary age estimates for a further nine deep-sea bony fishes were made from otolith zone counts and included results from a pilot analysis of radiocarbon age estimates from the otoliths of three of the species. Radiocarbon age estimates corroborated the otolith zone count age estimates made for white rattail (Trachyrincus aphyodes) and smallhead cod (Lepidion microcephalus) but were substantially lower for the third species, smallscale brown slickhead (Alepocephalus antipodianus) which suggested that it was not exposed to elevated (near-surface) levels of 14C in early life. Validation of otolith zone age estimates is difficult for deep-sea species but the radiocarbon analysis provided a practical method to corroborate age estimates for some bony fishes. Of the 31 fish in this study (known and new age estimates), five of the fishery target species and 10 of the bycatch were long-lived (50 years or more). The hypothesis that all New Zealand deep-sea species are long-lived was not supported, but the chance of a species being long-lived seem to be higher in deeper water. [ABSTRACT FROM AUTHOR]

Feeding habits of six deep‐sea demersal trawl‐caught macrourids on Chatham Rise, New Zealand, were examined from stomach contents during the austral summer. Three species were predominantly benthic foragers: smallbanded rattail Coelorinchus parvifasciatus on small epifaunal crustaceans, twosaddle rattail Coelorinchus biclinozonalis on epifaunal decapods and humpback rattail Coryphaenoides dossenus on benthic fishes and epifaunal decapods. Three species were predominantly benthopelagic foragers: banded rattail Coelorinchus fasciatus on hyperiid and gammarid amphipods and calanoid copepods, blackspot rattail Lucigadus nigromaculatus on small epifaunal crustaceans and suprabenthic mysids and Mahia rattail Coelorinchus matamua on epifaunal decapods and calanoid copepods. The most important predictors of diet variability were identified using distance‐based linear models and included areal predictors in C. parvifasciatus, L. nigromaculatus and C. dossenus, fish size in C. dossenus, C. biclinozonalis and C. matamua, sample year in C. biclinozonalis and C. fasciatus and depth in C. matamua. Results are compared with previously published data for four other macrourid species from the same study area. The 10 grenadier species comprise benthic, benthopelagic and mesopelagic foraging guilds. This study brings the number of grenadier species for which diet on Chatham Rise has been described in detail to 12. [ABSTRACT FROM AUTHOR]