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Throughout molecular evolution, organisms create assorted chemicals in response to varying ecological niches. Catalytic landscapes underlie metabolic evolution, wherein mutational steps alter the biosynthetic properties of enzymes. Here we report the first systematic quantitative characterization of the catalytic landscape underlying the evolution of sesquiterpene chemical diversity. On the basis of our previous discovery of a set of nine naturally occurring amino acid substitutions that functionally interconverted orthologous sesquiterpene synthases from Nicotiana tabacum and Hyoscyamus muticus , we created a library of all possible residue combinations (2 9 = 512) in the N. tabacum enzyme. The product spectra of 418 active enzymes revealed a rugged landscape where several minimal combinations of the nine mutations encode convergent solutions to the interconversions of parental activities. Quantitative comparisons indicated context dependence for mutational effects—epistasis—in product specificity and promiscuity. These results provide a measure of the mutational accessibility of phenotypic variability in a diverging lineage of terpene synthases.

A procedure was developed for the rapid analysis of titanium dioxide (TiO2) concentrations in feed and fecal samples. Samples were digested in concentrated H2SO4 for 2 h, followed by addition of 30% H2O2, and absorbance was measured at 410 nm. Standards were prepared by spiking blanks with increasing amounts of TiO2, resulting in a linear standard curve. Complete analysis using this procedure can typically be accomplished within 4.5 h. This procedure was compared to a previously published dry-ash procedure for the analysis of TiO2 in bovine fecal samples. Three sources of OM devoid of TiO2 (a forage sample, a bovine fecal sample without Cr2O3, and a bovine fecal sample containing Cr2O3) were spiked with graded amounts (0, 2, 4, 6, 8, or 10 mg) of TiO2. With our procedure, TiO2 recoveries averaged 96.7, 97.5, and 98.5%, for the three OM sources, respectively, vs. 74.3, 83.8, and 53.1% for the same samples analyzed using the dry-ash method. These results suggest that our procedure is a rapid and accurate alternative to dry-ash procedures for the determination of TiO2.

Supplementing ruminant animal diets with fat has been investigated as a means to influence a variety of physiological processes or to alter fatty acid composition of food products derived from ruminant animals. Several digestion experiments have been conducted with beef cattle and sheep to elucidate the effects of supplemental fat on utilization of other dietary components. Negative associative effects are not likely to be observed in ruminants consuming forage-based diets with supplemental fat at <=2% of DMI. Inclusion of supplemental fat at <=3% of DM is recommended to obtain the most benefit from the energy contained within the fat and other dietary components in high-forage diets. For ruminants fed high-concentrate diets, supplementing fat at 6% of diet DM is expected to have minimal impacts on utilization of other dietary components. Although there is greater potential to supply the ruminant animal with unsaturated fatty acids from dietary origin if fat is added to high-concentrate diets, incomplete ruminal biohydrogenation of C18 unsaturated fatty acids results in an increase in duodenal flow of 18:1 trans fatty acids regardless of basal diet consumed by the animal. The biohydrogenation intermediate 18:1 trans-11 (trans-vaccenic acid) is the likely precursor to cis-9, trans-11 CLA because the magnitude of increase in CLA content in tissues or milk of ruminants fed fat is much greater than the increase in CLA presented to the small intestine of ruminants fed fat supplements. Duodenal flow of trans-vaccenic acid is also substantially greater than CLA. Increasing unsaturated fatty acids status of ruminants imparts physiological responses that are separate than the energy value of supplemental fat. Manipulating maternal diet to improve unsaturated fatty acid status of the neonate has practical benefits for animals experiencing stress due to exposure to cold environments or conditions which mount an immune response. Supplementing fat to provide an additional 16 to 18 g/d of 18:2n-6 to the small intestine of beef cows for the first 60 to 90 d of lactation will have negative impacts on reproduction and may impair immune function of the suckling calf. Consequences of the suckling animal increasing its intake of unsaturated fatty acids because of manipulation of maternal diet warrants further investigation.

RationaleAcetazolamide and atomoxetine-plus-oxybutynin (‘AtoOxy’) can improve obstructive sleep apnoea (OSA) by stabilising ventilatory control and improving dilator muscle responsiveness respectively. Given the different pathophysiological mechanisms targeted by each intervention, we tested whether AtoOxy-plus-acetazolamide would be more efficacious than AtoOxy alone.MethodsIn a multicentre randomised crossover trial, 19 patients with moderate-to-severe OSA received AtoOxy (80/5 mg), acetazolamide (500 mg), combined AtoOxy-plus-acetazolamide or placebo at bedtime for three nights (half doses on first night) with a 4-day washout between conditions. Outcomes were assessed at baseline and night 3 of each treatment period. Mixed model analysis compared the reduction in Apnoea-Hypopnoea Index (AHI) from baseline between AtoOxy-plus-acetazolamide and AtoOxy (primary outcome). Secondary outcomes included hypoxic burden and arousal index.ResultsAlthough AtoOxy lowered AHI by 49 (33, 62)%baseline (estimate (95% CI)) vs placebo, and acetazolamide lowered AHI by+34 (14, 50)%baseline vs placebo, AtoOxy-plus-acetazolamide was not superior to AtoOxy alone (difference: −2 (−18, 11)%baseline, primary outcome p=0.8). Likewise, the hypoxic burden was lowered with AtoOxy (+58 (37, 71)%baseline) and acetazolamide (+37 (5, 58)%baseline), but no added benefit versus AtoOxy occurred when combined (difference: −13 (−5, 39)%baseline). Arousal index was also modestly reduced with each intervention (11%baseline–16%baseline). Mechanistic analyses revealed that similar traits (ie, higher baseline compensation, lower loop gain) were associated with both AtoOxy and acetazolamide efficacy.ConclusionsWhile AtoOxy halved AHI, and acetazolamide lowered AHI by a third, the combination of these leading experimental interventions provided no greater efficacy than AtoOxy alone. Failure of acetazolamide to further increase efficacy suggests overlapping physiological mechanisms.Trial registration number NCT03892772.

In this volume, the emergence of English in Saipan is being examined in the complex context of its colonial past. The focus lies on the influence of the American era on the linguistic outcomes in Saipan. Sociolinguistic interviews with indigenous Chamorros and Saipan Carolinians were analyzed using qualitative and quantitative methods.

This study utilized maternal undernutrition from early to midgestation in the ewe to determine the impact(s) of intrauterine growth restriction on postpartum growth of male offspring and the potential mechanisms involved. Multiparous ewes were fed 50% (nutrient-restricted) or 100% (control-fed) of their nutrient requirements (NRC, 1985) between d 28 and 78 of gestation, and then all ewes were fed 100% of the NRC requirements from d 79 through lambing. Male lambs born to nutrient-restricted (n = 9) and control-fed (n = 9) ewes exhibited similar BW (5.8 vs. 6.0 ± 0.3 kg) and crown-rump lengths (53.8 vs. 55.4 ± 1.0 cm) at birth. At 63 and 250 d of postnatal age, wether lambs were subjected to a glucose tolerance test, in which a bolus of glucose was administered i.v. to evaluate changes in glucose and insulin concentrations. After i.v. glucose administration at 63 d of age, lambs from nutrient-restricted ewes exhibited a greater area under the curve for glucose (AUCg; 6,281 vs. 5,242 ± 429; P < 0.05) and insulin (AUCi; 21.0 vs. 8.6 ± 1.9; P < 0.001) than lambs from control-fed ewes. After glucose administration at 250 d of age, lambs from nutrient-restricted ewes had greater AUCg (7,147 vs. 5,823 ± 361; P < 0.01) but a lower AUCi (6.4 vs. 10.2 ± 1.9; P = 0.05) than lambs from control-fed ewes. Lambs from nutrient-restricted ewes were heavier (26.6 vs. 21.8 ± 2.3 kg; P < 0.05) and had more backfat (0.30 vs. 0.21 ± 0.03 cm, P < 0.05) by 4 mo of age than the lambs from control-fed ewes. At slaughter at 280 d of age, lambs from nutrient-restricted ewes remained heavier than lambs from control-fed ewes, had greater (P < 0.05) amounts of kidney and pelvic-area adipose tissue, and tended (P < 0.10) to have reduced LM and semitendinosus muscle weights as a percentage of HCW. These data demonstrate that a bout of maternal undernutrition during early to midgestation in sheep increased BW and fat deposition during adolescence and dysregulated glucose uptake in the absence of any change in birth weight.

Fetal intrauterine growth restriction (IUGR) is known to negatively affect offspring health postnatally. This study evaluated the impacts of early gestational undernutrition followed by realimentation on bovine fetal and placental growth. Thirty multiparous beef cows bred to a single sire and gestating female fetuses were fed to meet NRC recommendations (control; n = 15) or fed below NRC recommendations (68.1% of NEm and 86.7% of MP recommendations; nutrient restricted, NR; n = 15) from d 30 to 125 of gestation. On d 125 of gestation, 10 control and 10 NR cows were necropsied. The remaining 5 NR cows were realimented to achieve similar BW and BCS with the remaining 5 control cows by d 190 of gestation; both groups were necropsied at d 245 of gestation. Fetal weight at d 125 of gestation was 948 ± 14 g (n = 10) for control cows; however, fetal weights of NR cows fell into 2 distinct groups: NR non-IUGR cows had fetal weights similar to control cows (974 ± 20 g, n = 6), whereas fetal weights of NR IUGR cows were reduced (773 ± 23 g, n = 4; P < 0.01). Fetal brain weight as a percentage of fetal weight was increased (approximately 11%; P < 0.01) in the NR IUGR fetuses compared with fetuses from the other 2 groups, which were similar. Fetal heart weight as a percentage of fetal weight also tended to be increased (approximately 10%; P = 0.08) in NR IUGR fetuses compared with control fetuses. Nutrient-restricted IUGR cows exhibited reduced (P < 0.01) cotyledonary weights compared with NR non-IUGR and control cows, which were similar (192 ± 27 vs. 309 ± 22, and 337 ± 17 g, respectively). Total placentome surface area also tended to be reduced (P = 0.07) in NR IUGR cows compared with NR non-IUGR and control cows, which again were similar (685.0 ± 45.6 vs. 828.7 ± 37.2 and 790.7 ± 28.9 mm², respectively). On d 245 of gestation, fetal weights and caruncle weight were similar for NR and control cows; cotyledonary weights, however, were reduced in NR vs. control cows (1,430 ± 133 vs. 2,137 ± 133 g, P < 0.01). Decreased fetal growth in NR IUGR cows on d 125 of gestation was associated with decreased cotyledonary weights and reduced placentomal surface areas. The return of NR cows to a BW and BCS similar to that of control cows through realimentation beginning on d 126 resulted in similar fetal weights of NR and control cows by d 245 of gestation. Thus, a bout of fetal IUGR may go undetected if cows undernourished during early gestation receive feed supplementation in the second half of gestation to assure normal birth weight.

Two experiments were conducted to compare ruminal, physiological, and performance responses of forage-fed cattle consuming grain-based supplements without (NF) or with the inclusion (10%; DM basis) of a rumen-protected PUFA (PF) or SFA source (SF). Supplements were offered and consumed at 0.6% of BW/animal daily (DM basis). In Exp. 1, DMI and ruminal in situ forage degradability were evaluated in 3 Angus x Hereford cows fitted with ruminal cannulas and allocated to a 3 x 3 Latin square design. Within each experimental period, hay was offered in amounts to ensure ad libitum access from d 1 to 13, DMI was recorded from d 8 to 13, and cows were limited to receive 90% of their average hay DMI (d 1 to 13) from d 14 to 21. On d 16, polyester bags containing 4 g of ground hay (DM basis) were incubated within the rumen of each cow for 0, 4, 8, 12, 24, 36, 48, 72, and 96 h. Hay and total DMI were reduced (P < 0.05) in cows receiving PF compared with cows receiving SF and NF. No treatment effects were detected (P > 0.48) for ruminal disappearance rate and effective ruminal degradability of hay DM and NDF. In Exp. 2, preconditioning DMI, ADG, carcass traits, and plasma concentrations of cortisol, fatty acids, acute-phase proteins, and proinflammatory cytokines were assessed in 72 Angus x Hereford steers receiving supplement treatments during a 28-d preconditioning period. All steers were transported to a commercial growing lot after preconditioning (d 1) and were later moved to an adjacent commercial finishing yard (d 144), where they remained until slaughter. No treatment effects were detected (P ≥ 0.52) for preconditioning ADG and G:F, but DMI tended (P = 0.09) to be reduced in steers receiving PF compared with those receiving NF and SF. Plasma PUFA concentrations were greater in steers receiving PF compared with those receiving NF and SF (P = 0.01). After transportation, concentration of tumor necrosis factor-α increased for steers receiving NF, did not change for steers receiving SF, but decreased for steers receiving PF (treatment x day interaction, P < 0.01). Steers fed PF had greater (P = 0.02) ADG compared with those fed NF during the growing phase. Carcass yield grade and marbling were greater (P < 0.05) for steers fed PF compared with those fed NF. In conclusion, PUFA supplementation did not affect ruminal forage degradability but did impair DMI in beef cows. Further, PUFA supplementation to steers during preconditioning reduced plasma concentrations of tumor necrosis factor-α after transportation, and benefited growing lot ADG and carcass marbling.

Angus x Gelbvieh cows with 2 to 3 previous pregnancies were used to evaluate effects of maternal nutrient restriction on offspring adipose tissue morphology at standard production endpoints. At 45 d after AI to a single sire, pregnancy was confirmed and cows randomly allotted into groups and fed a control (Con, 100% of NRC recommendations), nutrient-restricted (NR, 70% of Con diet), or nutrient-restricted + protein-supplemented (NRP, 70% of Con + essential AA supply to the small intestine equal to Con) diet. At d 185 of gestation, cows were commingled and received the Con diet thereafter. Bull calves were castrated at 2 mo of age. Calves were weaned at 210 d, backgrounded for 28 d, and then placed in the feedlot for 195 d. Steers and heifers were slaughtered at an average 12th-rib fat thickness of 7.6 mm. Adipose tissue from selected depots was collected for adipocyte size analysis. There was no significant difference in BW or BCS between Con, NRP, and NR cows at d 45 of gestation, which averaged 489.7 ± 17.7 kg and 5.35 ± 0.13, respectively. At d 185 of gestation, Con and NRP groups had similar BW (566.1 ± 14.8 and 550.2 ± 14.8 kg) and BCS (6.34 ± 0.27 and 5.59 ± 0.27), but NR cows exhibited reduced (P < 0.05) BW (517.9 ± 14.8 kg) and BCS (4.81 ± 0.27). Among offspring (steers and heifers) at slaughter, there were no significant differences in BW or organ weights among treatment groups. Yield grade was reduced (P < 0.05) and semitendinosus weight/HCW tended (P = 0.09) to be reduced in NR offspring compared with Con and NRP offspring. Average adipocyte diameter was increased (P < 0.05) in subcutaneous, mesenteric, and omental adipose tissue and tended (P = 0.09) to increase in perirenal adipose tissue in NR compared with Con offspring with NRP offspring adipocyte diameter being either intermediate or similar to Con calves. The adipocyte size alterations observed in NR offspring were confirmed by DNA concentration of the adipose tissue depots. There also was an increased mRNA expression (P < 0.05) of fatty acid transporter 1 in subcutaneous adipose tissue from NR offspring compared with Con and NRP offspring. Nutritional restriction during early and mid gestation increased or tended to increase (P < 0.09) adipocyte diameter in all adipose tissue depots in finished steer and heifer calves.

Therapies for obstructive sleep apnea (OSA) could be administered on the basis of a patient's own phenotypic causes (\"traits\") if a clinically applicable approach were available. Here we aimed to provide a means to quantify two key contributors to OSA-pharyngeal collapsibility and compensatory muscle responsiveness-that is applicable to diagnostic polysomnography. Based on physiological definitions, pharyngeal collapsibility determines the ventilation at normal (eupneic) ventilatory drive during sleep, and pharyngeal compensation determines the rise in ventilation accompanying a rising ventilatory drive. Thus, measuring ventilation and ventilatory drive (e.g., during spontaneous cyclic events) should reveal a patient's phenotypic traits without specialized intervention. We demonstrate this concept in patients with OSA (N = 29), using a novel automated noninvasive method to estimate ventilatory drive (polysomnographic method) and using \"gold standard\" ventilatory drive (intraesophageal diaphragm EMG) for comparison. Specialized physiological measurements using continuous positive airway pressure manipulation were employed for further comparison. The validity of nasal pressure as a ventilation surrogate was also tested (N = 11). Polysomnography-derived collapsibility and compensation estimates correlated favorably with those quantified using gold standard ventilatory drive (R = 0.83, P < 0.0001; and R = 0.76, P < 0.0001; respectively) and using continuous positive airway pressure manipulation (R = 0.67, P < 0.0001; and R = 0.64, P < 0.001; respectively). Polysomnographic estimates effectively stratified patients into high versus low subgroups (accuracy, 69-86% vs. ventilatory drive measures; P < 0.05). Traits were near-identical using nasal pressure versus pneumotach (N = 11, R ≥ 0.98, both traits; P < 0.001). Phenotypes of pharyngeal dysfunction in OSA are evident from spontaneous changes in ventilation and ventilatory drive during sleep, enabling noninvasive phenotyping in the clinic. Our approach may facilitate precision therapeutic interventions for OSA.