Stomatal conductance, not biochemistry, drives low temperature acclimation of photosynthesis in Populus balsamifera, regardless of nitrogen availability.

Low-temperature thermal acclimation may require adjustments to N and water use to sustain photosynthesis because of slow enzyme functioning and high water viscosity. However, understanding of photosynthetic acclimation to temperatures below 11 °C is limited. We acclimated Populus balsamifera to 6 °C and 10 °C (6A and 10A, respectively) and provided the trees with either high or low N fertilizer. We measured net CO2 assimilation (Anet ), stomatal conductance (gs ), maximum rates of Rubisco carboxylation (Vcmax ), electron transport (Jmax ) and dark respiration (Rd ) at leaf temperatures of 2, 6, 10, 14 and 18 °C, along with leaf N concentrations. The 10A trees had higher Anet than the 6A trees at warmer leaf temperatures, which was correlated with higher gs in the 10A trees. The instantaneous temperature responses of Vcmax , Jmax and Rd were similar for trees from both acclimation temperatures. While soil N availability increased leaf N concentrations, this had no effect on acclimation of photosynthesis or respiration. Our results indicate that acclimation below 11 °C occurred primarily through changes in stomatal conductance, not photosynthetic biochemistry, and was unaffected by short-term N supply. Thermal acclimation of stomatal conductance should therefore be a priority for future carbon cycle model development.

A constraint on historic growth in global photosynthesis due to increasing CO2.

The global terrestrial carbon sink is increasing1-3, offsetting roughly a third of anthropogenic CO2 released into the atmosphere each decade1, and thus serving to slow4 the growth of atmospheric CO2. It has been suggested that a CO2-induced long-term increase in global photosynthesis, a process known as CO2 fertilization, is responsible for a large proportion of the current terrestrial carbon sink4-7. The estimated magnitude of the historic increase in photosynthesis as result of increasing atmospheric CO2 concentrations, however, differs by an order of magnitude between long-term proxies and terrestrial biosphere models7-13. Here we quantify the historic effect of CO2 on global photosynthesis by identifying an emergent constraint14-16 that combines terrestrial biosphere models with global carbon budget estimates. Our analysis suggests that CO2 fertilization increased global annual photosynthesis by 11.85 ± 1.4%, or 13.98 ± 1.63 petagrams carbon (mean ± 95% confidence interval) between 1981 and 2020. Our results help resolve conflicting estimates of the historic sensitivity of global photosynthesis to CO2, and highlight the large impact anthropogenic emissions have had on ecosystems worldwide.

Synthesis and characterization of injectable bioadhesive hydrogels for nucleus pulposus replacement and repair of the damaged intervertebral disc.

Bioadhesive polymers are natural or synthetic materials that can be used for soft tissue repair. The aim of this investigation was to develop an injectable, bioadhesive hydrogel with the potential to serve as a synthetic replacement for the nucleus pulposus of the intervertebral disc or as an annulus closure material. Branched copolymers of poly(N-isopropylacrylamide) (PNIPAAm) and poly(ethylene glycol) (PEG) were blended with poly(ethylene imine) (PEI). This three component injectable system can form a precipitated gel at physiological temperature due to the phase transition of PNIPAAm. The injection of glutaraldehyde into the gel core will adhere the implant to the surrounding tissues. (1)H NMR results indicated the successful physical incorporation of PEI into the PNIPAAm-PEG network by blending. In addition, the covalent crosslinking between the amine functionalities on the PEI and the aldehyde functionalities on the glutaraldehyde was verified using FTIR difference spectroscopy. Mechanical characterization of these blends showed a significant increase (p < 0.05) in compressive modulus following glutaraldehyde injection. The in vitro bioadhesive force studies with porcine skin showed a significant increase (p < 0.05) in the mean maximum force of detachment for PNIPAAm-PEG/PEI gels when glutaraldehyde was injected into the gel core. The results of this study indicate that the reactivity between amines and aldehyde functionalities can be exploited to impart bioadhesive properties to PNIPAAm-PEG/PEI copolymers.

Evaluation of novel injectable hydrogels for nucleus pulposus replacement.

Branched copolymers composed of poly(N-isopropylacrylamide) (PNIPAAm) and poly(ethylene glycol) (PEG) are being investigated as an in situ forming replacement for the nucleus pulposus of the intervertebral disc. A family of copolymers was synthesized by varying the molecular weight of the PEG blocks and molar ratio of NIPAAm monomer units to PEG branches. Gel swelling, dissolution, and compressive mechanical properties were characterized over 90 days and stress relaxation behavior over 30 days immersion in vitro. It was found that the NIPAAm to PEG molar ratio did not affect the equilibrium swelling and compressive mechanical properties. However, gel elasticity exhibited a dependency on both the PEG block molecular weight and content. The equilibrium gel water content increased and compressive modulus decreased with increasing PEG block size. While all of the branched copolymers showed significant increases in stress relaxation time constant compared to the homopolymer (p < 0.05), the high PEG content PNIPAAm-PEG (4600 and 8000 g/mol) exhibited the maximum elasticity. Because of its high water content, requisite stiffness and high elastic response, PNIPAAm-PEG (4600 g/mol) will be further evaluated as a candidate material for nucleus pulposus replacement.

Why are translationally sub-optimal synonymous codons used in Escherichia coli?

Natural selection favors certain synonymous codons which aid translation in Escherichia coli, yet codons not favored by translational selection persist. We use the frequency distributions of synonymous polymorphisms to test three hypotheses for the existence of translationally sub-optimal codons: (1) selection is a relatively weak force, so there is a balance between mutation, selection, and drift; (2) at some sites there is no selection on codon usage, so some synonymous sites are unaffected by translational selection; and (3) translationally sub-optimal codons are favored by alternative selection pressures at certain synonymous sites. We find that when all the data is considered, model 1 is supported and both models 2 and 3 are rejected as sole explanations for the existence of translationally sub-optimal codons. However, we find evidence in favor of both models 2 and 3 when the data is partitioned between groups of amino acids and between regions of the genes. Thus, all three mechanisms appear to contribute to the existence of translationally sub-optimal codons in E. coli.

A test of amino acid reversibility.

In studies of molecular evolution, the assumption that protein evolution is reversible has often been made, but rarely tested. Here we use a large set of orthologous murid protein coding sequences to perform a simple test of reversibility, and find no evidence to reject the assumption of reversibility in protein evolution.

Synonymous codon bias is not caused by mutation bias in G+C-rich genes in humans.

It is has been suggested that synonymous codon bias is a consequence of mutation bias in mammals. We tested this hypothesis in humans using single-nucleotide polymorphism data. We found a pattern of polymorphism which was inconsistent with the mutation bias hypothesis in G+C-rich genes. However, the data were consistent with the action of natural selection or biased gene conversion. Similar patterns of polymorphism were also observed in noncoding DNA, suggesting that natural selection or biased gene conversion may affect large tracts of the human genome.

In-depth morphological changes and embrittlement near the wear surface of UHMWPE inserts from uncemented hip systems.

Polyethylene hip cups were examined with optical and electron microscopy after permanganic etching, a technique that allows in-depth examination from the articulating surface downward. In addition to wear features present on the surface, novel defects were revealed in implants after retrieval from the body but not in as-manufactured controls. They were incipient cracks that indicated the existence of an embrittled layer extending 10 microm or more into the implant from the wear surface after exposure to the body environment. The lengths of the cracks, which were perpendicular to the tensile stresses responsible for their formation, were mostly more or less parallel to the wear surface. The embrittlement and cracking revealed are probably major contributors to the wear of polyethylene implants in the body. Poor particle consolidation may be a contributory factor, but it was not observed to be the primary cause of implant wear within the body.

The evolutionary dynamics of male-killers and their hosts.

Male-killing bacteria are cytoplasmic sex-ratio distorters that are transmitted vertically through females of their insect hosts. The killing of male hosts by their bacteria is thought to be an adaptive bacterial trait because it augments the fitness of female hosts carrying clonal relatives of those bacteria. Here we attempt to explain observations of multiple male-killers in natural host populations. First we show that such male-killer polymorphism cannot be explained by a classical model of male-killing. We then show that more complicated models incorporating the evolution of resistance in hosts can explain male-killer polymorphism. However, this is only likely if resistance genes are very costly. We also consider the long-term evolutionary dynamics of male-killers, and show that evolution towards progressively more 'efficient' male-killers can be thwarted by the appearance of host resistance. The presence of a resistance gene can allow a less efficient male-killer to outcompete its rival and hence reverse the trend towards more efficient transmission and reduced metabolic load on the host.

The evolution of haplodiploidy under inbreeding.

Although haplodiploid organisms tend to be inbred, previous models of the evolution of haplodiploidy have assumed outbred populations. Here a model for the evolution of haplodiploidy is developed which incorporates sib mating, deleterious mutations generated by mutation, and fitness differences between haploids and diploids. Simulations of the model allow an assessment of the effect of inbreeding on the deleterious mutation and maternal transmission theories for the evolution of haplodiploidy. As expected from intuitive arguments, inbreeding favours haplodiploidy under the deleterious mutation hypothesis but disfavours haplodiploidy under the maternal transmission hypothesis. It appears that the effect of inbreeding is greater on the maternal transmission theory, and thus inbreeding may restrict the evolution of haplodiploidy.

The effect of tandem substitutions on the correlation between synonymous and nonsynonymous rates in rodents.

Nonsynonymous substitutions in DNA cause amino acid substitutions while synonymous substitutions in DNA leave amino acids unchanged. The cause of the correlation between the substitution rates at nonsynonymous (K(A)) and synonymous (K(S)) sites in mammals is a contentious issue, and one that impacts on many aspects of molecular evolution. Here we use a large set of orthologous mammalian genes to investigate the causes of the K(A)-K(S) correlation in rodents. The strength of the K(A)-K(S) correlation exceeds the neutral theory expectation when substitution rates are estimated using algorithmic methods, but not when substitution rates are estimated by maximum likelihood. Irrespective of this methodological uncertainty the strength of the K(A)-K(S) correlation appears mostly due to tandem substitutions, an excess of which is generated by substitutional nonindependence. Doublet mutations cannot explain the excess of tandem synonymous-nonsynonymous substitutions, and substitution patterns indicate that selection on silent sites is the likely cause. We find no evidence for selection on codon usage. The nature of the relationship between synonymous divergence and base composition is unclear because we find a significant correlation if we use maximum-likelihood methods but not if we use algorithmic methods. Finally, we find that K(S) is reduced at the start of genes, which suggests that selection for RNA structure may affect silent sites in mammalian protein-coding genes.

On morphology of consolidated UHMWPE resin in hip cups.

The microstructure of replacement hip cups made from ultra-high molecular weight polyethylene (UHMWPE) has been revealed by permanganic etching to bring out the fine details. Specimens are examined by optical microscopy according to the Nomarski technique, and by scanning and transmission electron microscopy. In all cups the original reactor particles are visible. Optical microscopy is most productive in revealing variations not only in the texture of different individual particles, but also in the degree of consolidation of a particle with its neighbours. Both these factors differ according to the material and process. Electron microscopy reveals the existence of pockets of lower molecular weight material which has been expelled from the particles by the original sintering process.

Vertebrate genome evolution: a slow shuffle or a big bang?

In vertebrates it is often found that if one considers a group of genes clustered on a certain chromosome, then the homologues of those genes often form another cluster on a different chromosome. There are four explanations, not necessarily mutually exclusive, to explain how such homologous clusters appeared. Homologous clusters are expected at a low probability even if genes are distributed at random. The duplication of a subset of the genome might create homologous clusters, as would a duplication of the entire genome. Alternatively, it may be adaptive for certain combinations of genes to cluster, although clearly the genes must have duplicated prior to rearrangement into clusters. Molecular phylogenetics provides a means to examine the origins of homologous clusters, although it is difficult to discriminate between the different explanations using current data. However, with more extensive sequencing and mapping of vertebrate genomes, especially those of the early diverging chordates, it should soon become possible to resolve the origins of homologous clusters.

Do essential genes evolve slowly?

Approximately two thirds of all knockouts of individual mouse genes give rise to viable fertile mice. These genes have thus been termed 'non-essential' in contrast to 'essential' genes, the knockouts of which result in death or infertility. Although non-essential genes are likely to be under selection that favours sequence conservation [1], it is predicted that they are less subject to such stabilising selection than essential genes, and hence evolve faster [2]. We have addressed this issue by analysing the molecular evolution of 108 non-essential and 67 essential genes that have been sequenced in both mouse and rat. On preliminary analysis, the non-essential genes appeared to be faster evolving than the essential ones. We found, however, that the non-essential class contains a disproportionate number of immune-system genes that may be under directional selection (that is, selection favouring change) because of host-parasite coevolution. After correction for this bias, we found that the rate at which genes evolve does not correlate with the severity of the knockout phenotype. This was corroborated by the finding that, whereas neuron-specific genes have significantly lower rates of change than other genes, essential and non-essential neuronal genes have comparable rates of evolution. Our findings most probably reflect strong selection acting against even very subtle deleterious phenotypes, and indicate that the putative involvement of directional selection in host-parasite coevolution and gene expression within the nervous system explains much more of the variance in rates of gene evolution than does the knockout phenotype.

The causes of synonymous rate variation in the rodent genome. Can substitution rates be used to estimate the sex bias in mutation rate?

Miyata et al. have suggested that the male-to-female mutation rate ratio (alpha) can be estimated by comparing the neutral substitution rates of X-linked (X), Y-linked (Y), and autosomal (A) genes. Rodent silent site X/A comparisons provide very different estimates from X/Y comparisons. We examine three explanations for this discrepancy: (1) statistical biases and artifacts, (2) nonneutral evolution, and (3) differences in mutation rate per germline replication. By estimating errors and using a variety of methodologies, we tentatively reject explanation 1. Our analyses of patterns of codon usage, synonymous rates, and nonsynonymous rates suggest that silent sites in rodents are evolving neutrally, and we can therefore reject explanation 2. We find both base composition and methylation differences between the different sets of chromosomes, a result consistent with explanation 3, but these differences do not appear to explain the observed discrepancies in estimates of alpha. Our finding of significantly low synonymous substitution rates in genomically imprinted genes suggests a link between hemizygous expression and an adaptive reduction in the mutation rate, which is consistent with explanation 3. Therefore our results provide circumstantial evidence in favor of the hypothesis that the discrepancies in estimates of alpha are due to differences in the mutation rate per germline replication between different parts of the genome. This explanation violates a critical assumption of the method of Miyata et al., and hence we suggest that estimates of alpha, obtained using this method, need to be treated with caution.

Molecular evolution of an imprinted gene: repeatability of patterns of evolution within the mammalian insulin-like growth factor type II receptor.

The repeatability of patterns of variation in Ka/Ks and Ks is expected if such patterns are the result of deterministic forces. We have contrasted the molecular evolution of the mammalian insulin-like growth factor type II receptor (Igf2r) in the mouse-rat comparison with that in the human-cow comparison. In so doing, we investigate explanations for both the evolution of genomic imprinting and for Ks variation (and hence putatively for mutation rate evolution). Previous analysis of Igf2r, in the mouse-rat comparison, found Ka/Ks patterns that were suggested to be contrary to those expected under the conflict theory of imprinting. We find that Ka/Ks variation is repeatable and hence confirm these patterns. However, we also find that the molecular evolution of Igf2r signal sequences suggests that positive selection, and hence conflict, may be affecting this region. The variation in Ks across Igf2r is also repeatable. To the best of our knowledge this is the first demonstration of such repeatability. We consider three explanations for the variation in Ks across the gene: (1) that it is the result of mutational biases, (2) that it is the result of selection on the mutation rate, and (3) that it is the product of selection on codon usage. Explanations 2 and 3 predict a Ka-Ks correlation, which is not found. Explanation 3 also predicts a negative correlation between codon bias and Ks, which is also not found. However, in support of explanation 1 we do find that in rodents the rate of silent C --> T mutations at CpG sites does covary with Ks, suggesting that methylation-induced mutational patterns can explain some of the variation in Ks. We find evidence to suggest that this CpG effect is due to both variation in CpG density, and to variation in the frequency with which CpGs mutate. Interestingly, however, a GC4 analysis shows no covariance with Ks, suggesting that to eliminate methyl-associated effects CpG rates themselves must be analyzed. These results suggest that, in contrast to previous studies of intragenic variation, Ks patterns are not simply caused by the same forces responsible for Ka/Ks correlations.