A novel vector element providing multicopy vector integration in Arxula adeninivorans

An Arxula adeninivorans vector element has been identified that provides multicopy integration in an atrp1 host strain. The element consists of the ATRP1 selection marker fused to a newly generated truncated ALEU2 promoter of 53 bp. In the described example eight copies of an amyA expression vector encoding heterologous alpha-amylase from Bacillus amyloliquefaciens are integrated in the genome of the recombinant strain instead of a single copy observed when using the ATRP1 element with the complete promoter. The high copy number results in strains of superior productivity for a secreted recombinant alpha-amylase. The vector design enables the integration of a small vector fragment that consists of yeast DNA only providing high transformation frequencies and a high mitotic stability.     

Front Cover Image,Volume 116, Number 11, November 2019

CRISPR/Cas9-guided cytidine deaminase enables C:G to T:A base editing in bacterial genome without introduction of lethal double-stranded DNA break, supplement of foreign DNA template, or dependence on inefficient homologous recombination. However, limited by genome-targeting scope, editing window, and base transition capability, the application of base editing in metabolic engineering has not been explored. Herein, four Cas9 variants accepting different protospacer adjacent motif (PAM) sequences were used to increase the genome-targeting scope of bacterial base editing. After a comprehensive evaluation, we demonstrated that PAM requirement of bacterial base editing can be relaxed from NGG to NG using the Cas9 variants, providing 3.9-fold more target loci for gene inactivation in Corynebacterium glutamicum. Truncated or extended guide RNAs were employed to expand the canonical 5-bp editing window to 7-bp. Bacterial adenine base editing was also achieved with Cas9 fused to adenosine deaminase. With these updates, base editing can serve as an enabling tool for fast metabolic engineering. To demonstrate its potential, base editing was used to deregulate feedback inhibition of aspartokinase via amino acid substitution for lysine overproduction. Finally, a user-friendly online tool named gBIG was provided for designing guide RNAs for base editing-mediated inactivation of given genes in any given sequenced genome ( www.ibiodesign.net/gBIG ).     

Energy requirements of the red kangaroo (<Emphasis Type="Italic">Macropus rufus</Emphasis>): impacts of age,growth and body size in a large desert-dwelling herbivore.

Generally, young growing mammals have resting metabolic rates (RMRs) that are proportionally greater than those of adult animals. This is seen in the red kangaroo (Macropus rufus), a large (>20 kg) herbivorous marsupial common to arid and semi-arid inland Australia. Juvenile red kangaroos have RMRs 1.5–1.6 times those expected for adult marsupials of an equivalent body mass. When fed high-quality chopped lucerne hay, young-at-foot (YAF) kangaroos, which have permanently left the mother's pouch but are still sucking, and recently weaned red kangaroos had digestible energy intakes of 641±27 kJ kg^–0.75 day^–1 and 677±26 kJ kg^–0.75 day^–1, respectively, significantly higher than the 385±37 kJ kg^–0.75 day^–1 ingested by mature, non-lactating females. However, YAF and weaned red kangaroos had maintenance energy requirements (MERs) that were not significantly higher than those of mature, non-lactating females, the values ranging between 384 kJ kg^–0.75 day^–1 and 390 kJ kg^–0.75 day^–1 digestible energy. Importantly, the MER of mature female red kangaroos was 84% of that previously reported for similarly sized, but still growing, male red kangaroos. Growth was the main factor affecting the proportionally higher energy requirements of the juvenile red kangaroos relative to non-reproductive mature females. On a good quality diet, juvenile red kangaroos from permanent pouch exit until shortly after weaning (ca. 220–400 days) had average growth rates of 55 g body mass day^–1. At this level of growth, juveniles had total daily digestible energy requirements (i.e. MER plus growth energy requirements) that were 1.7–1.8 times the MER of mature, non-reproductive females. Our data suggest that the proportionally higher RMR of juvenile red kangaroos is largely explained by the additional energy needed for growth. Energy contents of the tissue gained by the YAF and weaned red kangaroos during growth were estimated to be 5.3 kJ g^–1, within the range found for most young growing mammals.Abbreviations BMR basal metabolic rate - DEI digestible energy intake - MER maintenance energy requirement - MER_g maintenance plus growth energy requirement - PPE permanent pouch exit - RMR resting metabolic rate - YAF young-at-foot Communicated by I.D. Hume     

Soil nutrient budgets following projected corn stover harvest for biofuel production in the conterminous United States

Increasing demand for food and biofuel feedstocks may substantially affect soil nutrient budgets, especially in the United States where there is great potential for corn (Zea mays L) stover as a biofuel feedstock. This study was designed to evaluate impacts of projected stover harvest scenarios on budgets of soil nitrogen (N), phosphorus (P), and potassium (K) currently and in the future across the conterminous United States. The required and removed N, P, and K amounts under each scenario were estimated on the basis of both their average contents in grain and stover and from an empirical model. Our analyses indicate a small depletion of soil N (?4 ± 35 kg ha^?1) and K (?6 ± 36 kg ha^?1) and a moderate surplus of P (37 ± 21 kg ha^?1) currently on the national average, but with a noticeable variation from state to state. After harvesting both grain and projected stover, the deficits of soil N, P, and K were estimated at 114–127, 26–27, and 36–53 kg ha^?1 yr^?1, respectively, in 2006–2010; 131–173, 29–32, and 41–96 kg ha^?1 yr^?1, respectively, in 2020; and 161–207, 35–39, and 51–111 kg ha^?1 yr^?1, respectively, in 2050. This study indicates that the harvestable stover amount derived from the minimum stover requirement for maintaining soil organic carbon level scenarios under current fertilization rates can be sustainable for soil nutrient supply and corn production at present, but the deficit of P and K at the national scale would become larger in the future.     

Leaf photoacclimatory responses of the tropical seagrass Thalassia testudinum under mesocosm conditions: a mechanistic scaling-up study

Here, the leaf photoacclimatory plasticity and efficiency of the tropical seagrass Thalassia testudinum were examined. Mesocosms were used to compare the variability induced by three light conditions, two leaf sections and the variability observed at the collection site. The study revealed an efficient photosynthetic light use at low irradiances, but limited photoacclimatory plasticity to increase maximum photosynthetic rates (P(max)) and saturation (E(k)) and compensation (E(c)) irradiances under high light irradiance. A strong, positive and linear association between the percentage of daylight hours above saturation and the relative maximum photochemical efficiency (F(V)/F(M)) reduction observed between basal and apical leaf sections was also found. The results indicate that T. testudinum leaves have a shade-adapted physiology. However, the large amount of heterotrophic biomass that this seagrass maintains may considerably increase plant respiratory demands and their minimum quantum requirements for growth (MQR). Although the MQR still needs to be quantified, it is hypothesized that the ecological success of this climax species in the oligotrophic and highly illuminated waters of the Caribbean may rely on the ability of the canopy to regulate the optimal leaf light environment and the morphological plasticity of the whole plant to enhance total leaf area and to reduce carbon respiratory losses.     

Increased heat requirement for leaf flushing in temperate woody species over 1980–2012: effects of chilling,precipitation and insolation

Recent studies have revealed large unexplained variation in heat requirement‐based phenology models, resulting in large uncertainty when predicting ecosystem carbon and water balance responses to climate variability. Improving our understanding of the heat requirement for spring phenology is thus urgently needed. In this study, we estimated the species‐specific heat requirement for leaf flushing of 13 temperate woody species using long‐term phenological observations from Europe and North America. The species were defined as early and late flushing species according to the mean date of leaf flushing across all sites. Partial correlation analyses were applied to determine the temporal correlations between heat requirement and chilling accumulation, precipitation and insolation sum during dormancy. We found that the heat requirement for leaf flushing increased by almost 50% over the study period 1980–2012, with an average of 30 heat units per decade. This temporal increase in heat requirement was observed in all species, but was much larger for late than for early flushing species. Consistent with previous studies, we found that the heat requirement negatively correlates with chilling accumulation. Interestingly, after removing the variation induced by chilling accumulation, a predominantly positive partial correlation exists between heat requirement and precipitation sum, and a predominantly negative correlation between heat requirement and insolation sum. This suggests that besides the well‐known effect of chilling, the heat requirement for leaf flushing is also influenced by precipitation and insolation sum during dormancy. However, we hypothesize that the observed precipitation and insolation effects might be artefacts attributable to the inappropriate use of air temperature in the heat requirement quantification. Rather than air temperature, meristem temperature is probably the prominent driver of the leaf flushing process, but these data are not available. Further experimental research is thus needed to verify whether insolation and precipitation sums directly affect the heat requirement for leaf flushing.     

Design and synthesis of tricyclic tetrahydroquinolines as a new series of nonsteroidal selective androgen receptor modulators (SARMs)

Some tricyclic tetrahydroquinolines (THQs) were found to have the potential of a new series of nonsteroidal selective androgen receptor modulators (SARMs). Compound 5b was first designed and synthesized under our hypothesis based on a four-point pharmacophoric requirement of the 3-carbonyl, 18-methyl, 17-hydroxyl, and 13-quaternary carbon groups of dihydrotestosterone (DHT). It was revealed that this compound exhibits not only a strong androgen receptor (AR) agonistic activity (EC₅₀ = 9.2 nM) but also the highest selectivity in binding affinity to AR among the steroid hormone receptors. Furthermore, this compound showed a weak virilizing effect with retention of the desired anabolic effect as compared with DHT in vivo.     

Dynamics of energy utilization in male and female turkeys during growth

Determining energy utilization in growing animals enables to adjust the nutritional constraints to nutrient requirements while maximizing the ratio between lean retention and fat retention to improve feed efficiency. In turkey production, the important sexual dimorphism and differences between strains may contribute to differences in basal energy metabolism and the partitioning of energy retention between protein and lipid. The objective of this study was to determine the dynamics of energy utilization in males and females of a heavy strain of turkeys fed ad libitum from 1 to 23 weeks of age. Heat production (HP) was determined by indirect calorimetry and retained energy (RE) was calculated as the difference between metabolizable energy (ME) intake and HP. The RE as protein was determined by a nitrogen balance, while the remaining RE was assumed to be lipid. A modeling procedure allowed partitioning HP between fasting HP (FHP), activity-related HP and thermic effect of feeding. A multiple regression analysis was used to estimate the maintenance energy expenditure (ME(m)) and the energy efficiencies of protein and lipid retention (k(p) and k(f), respectively). Results were expressed either per day or per kg BW(0.75) per day. In comparison with females, males consumed more feed (440 v. 368 g/day), grew faster (163 v. 147 g/day) and retained more protein (38 v. 28 g/day) during the experimental period. Expressed per kg BW(0.75) per day, ME intake decreased linearly with increasing age and was not affected by gender. Similarly, RE as protein decreased with increasing age and tended to be greater in males than in females, whereas RE as lipid increased with increasing age and was lower in males than in females. In addition, HP decreased with increasing age and was greater in males than in females, because of greater activity-related HP and FHP (47% and 9% greater in males compared with females). The FHP averaged 417 kJ/(kg BW)(0.75) per day during the first 3 weeks of age and decreased to 317 and 277 kJ/(kg BW)(0.75) per day in males and females, respectively, from 20 weeks of age onwards. Similar to FHP, ME(m) was lower in females than in males ((586 to 12 × BW) and (586 to 5 × BW) kJ/(kg BW)(0.75) per day, respectively) and the k(p) and k(f) were estimated at 0.63 and 0.87, respectively. This study shows that the partitioning of RE and HP differs between genders in growing turkeys, which likely results in differences in nutrient requirements.     

Comparisons of germination traits of alpine plants between fellfield and snowbed habitats

We examined the seed-germination responses of 27 alpine species with reference to habitat type (fellfield and snowbed), temperature (five regimes), and light requirement. About 70% of species showed >40% germination at warm temperatures without cold stratification. However, a moist-chilling treatment markedly improved the germination percentages in most species, especially under cool conditions. Thus, cold stratification effectively reduced the temperature requirement for germination. Patterns of germination response within species were not consistent between the fellfield and snowbed habitats for species inhabiting both habitats. For interspecific comparisons, there were no significant differences in germination responses to the temperature regimes and the cold stratification between the fellfield and snowbed species. Also, germination speed and the length of germinating period did not differ between fellfield and snowbed species. Most species (86%) showed a requirement for light for germination without cold stratification. Although the extent of the light requirement was reduced after cold stratification in some species, the light requirement of most small-seeded species remained. The combination of cold stratification and the light requirement is a major factor determining the seedling emergence and formation of seed banks in alpine plants. However, habitat-specific patterns of germination traits were less clear, suggesting similar germination traits in fellfield and snowbed plants, at least under controlled conditions in the laboratory.     

Effect of Screening Out Implausible Energy Intake Reports on Relationships between Diet and BMI

Objective: We present an updated method for identifying physiologically implausible dietary reports by comparing reported energy intake (rEI) with predicted energy requirements (pER), and we examine the impact of excluding these reports. Research Methods and Procedures: Adult data from the Continuing Survey of Food Intakes by Individuals 1994 to 1996 were used. pER was calculated from the dietary reference intake equations. Within‐subject variations and errors in rEI [coefficient of variation (CV) ～ 23%] over 2 days (d), pER (CV ～ 11%), and measured total energy expenditure (mTEE; doubly labeled water, CV ～ 8.2%) were propagated, where ±1 SD = . Thus, a report was identified as implausible if rEI was not within 78% to 122% of pER. Multiple cut‐offs between ±1 and ±2 SD were tested. Results: %rEI/pER = 81% in the total sample (n = 6499) and progressively increased to 95% in the ±1 SD sample (n = 2685). The ±1 to 1.4 SD samples yielded rEI‐weight associations closest to the theoretical relationship (mTEE to weight). Weak or spurious diet—BMI associations were present in the total sample; ±1 to 1.4 SD samples showed the strongest set of associations and provided the maximum n while maintaining biological plausibility. Discussion: Our methodology can be applied to different data sets to evaluate the impact of implausible rEIs on health outcomes. Implausible rEIs reduce the overall validity of a sample, and not excluding them may lead to inappropriate conclusions about potential dietary causes of health outcomes such as obesity.