Narrow Substrates Research Articles

How Enzyme Selectivity and Immobilization Affect Catalytic Yields in Lipase-Catalyzed Processes

Abstract: Herein, the influence of structural attributes, including the interactions of lipases with support systems, substrates, products/byproducts, and the media environment, on enzyme stability, selectivity and activity are discussed. Substrates/products, such as methanol, glycerol, phenolic acids and polyphenols, can inhibit lipase activity by influencing the mass flow of the reactants and products or by enzyme denaturation, which is also caused by extreme pH, high temperatures, and digestive action of most organic solvents. Immobilization techniques that involve chemical bonding between the functional groups of the support and the amino acids of the lipase maintain the enzyme’s active conformation via the formation of stable secondary structures. Functionalized metal nanoparticles and metal and covalent organic frameworks (COFs and MOFs) covalently bond to lipases, reducing the reliance of the active site conformation on hydrogen bonding and disulfide bonds. The crystallinity of COFand MOF-immobilized lipases allows them to be used in contrasting media environments and at high temperatures, which increases the reaction kinetics and improves the catalytic yield. On the other hand, inert support systems such as silica promote catalytic yields by minimizing protein leaching, which fairly maintains the amount of the preloaded lipase. The structure of substrates also plays a large role, whereas some lipases strictly prefer narrow substrates. In contrast, others, such as Candida species lipases, are liberal and allow substrates of varying bulkiness/steric hindrances.

Microhabitat and adhesive toepads shape gecko limb morphology.

Different substrates pose varied biomechanical challenges that select specific morphologies, such as long limbs for faster running and short limbs for balanced posture while climbing narrow substrates. We tested how gecko locomotion is affected by the microhabitat they occupy and by a key adaptation-adhesive toepads-through analyzing how those are related to limb morphology. We collected microhabitat and toepads data for over 90% of limbed gecko species, and limb measurements for 403 species from 83 of the 121 limbed gecko genera, which we then used in phylogenetic comparative analyses. Our data highlight the association of adhesive toepads with arboreality, but a phylogenetic analysis shows that this relationship is not significant, suggesting that these traits are phylogenetically constrained. Comparative analyses reveal that pad-bearing species possess shorter hindlimbs and feet, more even limb lengths, and lower crus: thigh ratios, than padless geckos, across microhabitats. Saxicolous geckos have the longest limbs and limb segments. This is probably influenced by selection for long strides, increased takeoff velocity, and static stability on inclined surfaces. Terrestrial geckos have more even hind- and forelimbs than arboreal geckos, unlike patterns found in other lizards. Our findings underline the difficulty to infer on microhabitat-morphology relationships from one taxon to another, given their differing ecologies and evolutionary pathways. We emphasize the importance of key innovation traits, such as adhesive toepads, in shaping limb morphology in geckos and, accordingly, their locomotion within their immediate environment.

Beakiation: how a novel parrot gait expands the locomotor repertoire of living birds.

Occupation of arboreal habitats poses myriad locomotor challenges, driving both anatomical and behavioural innovations across various tetrapod lineages. Here, we report and biomechanically assess a novel, beak-driven locomotor mode-'beakiation'-by which parrots advance along the underside of narrow arboreal substrates. Using high-speed videography and kinetic analyses, we describe the limb loading patterns and pendular mechanics of beakiation, and compare the biomechanical characteristics of this gait with other suspensory behaviours (namely, forelimb-driven brachiation and inverted quadrupedal walking). We report that the parrot beak experiences comparable force magnitudes (approx. 150% body weight in the normal plane; approx. 50% body weight in the fore-aft plane) to the forelimbs of brachiating primates. Parrot beakiation is also characterized by longer-than-expected pendular periods, similar to observations of gibbon brachiation. However, in terms of mechanical energy recovery, beakiation is typified by lower levels of energetic recovery than brachiating specialists: a product of its slower, more careful nature. The observation of this novel behaviour-which adds to a growing base of literature regarding beak-assisted locomotor strategies in birds-highlights the extraordinary behavioural plasticity of birds, the functional versatility of the avian beak, and the difficulties in reconstructing an animal's locomotor repertoire from morphological characteristics alone.

The Impact of Inflammation on the In Vivo Activity of the Renal Transporters OAT1/3 in Pregnant Women Diagnosed with Acute Pyelonephritis.

Inflammation can regulate hepatic drug metabolism enzymes and transporters. The impact of inflammation on renal drug transporters remains to be elucidated. We aimed to quantify the effect of inflammation (caused by acute pyelonephritis) on the in vivo activity of renal OAT1/3, using the probe drug furosemide. Pregnant women (second or third trimester) received a single oral dose of furosemide 40 mg during acute pyelonephritis (Phase 1; n = 7) and after its resolution (Phase 2; n = 7; by treatment with intravenous cefuroxime 750 mg TID for 3-7 days), separated by 10 to 14 days. The IL-6, IFN-γ, TNF-α, MCP-1, and C-reactive protein plasma concentrations were higher in Phase I vs. Phase II. The pregnant women had a lower geometric mean [CV%] furosemide CLsecretion (3.9 [43.4] vs. 6.7 [43.8] L/h) and formation clearance to the glucuronide (1.1 [85.9] vs. 2.3 [64.1] L/h) in Phase 1 vs. Phase 2. Inflammation reduced the in vivo activity of renal OAT1/3 (mediating furosemide CLsecretion) and UGT1A9/1A1 (mediating the formation of furosemide glucuronide) by approximately 40% and 54%, respectively, presumably by elevating the plasma cytokine concentrations. The dosing regimens of narrow therapeutic window OAT drug substrates may need to be adjusted during inflammatory conditions.

The role of sensory feedback from carpal sinus hairs in locomotor kinematics of rats (Rattus norvegicus, Rodentia) during walking on narrow substrates

Carpal sinus hairs on the forearms are assumed to have evolved within the stem lineage of Theria. The presence and similar position of these specialized tactile hairs in scansorial and terrestrial species as well as earlier studies on rats indicate a biological role in sensing substrate irregularities in high structured environments to ensure the dynamic stability of the body during locomotion. While these sensors were considered as one functional unit until so far, the present study deals with the biological role of the single tactile hairs of the trident, assuming a role in sensing substrate diameters and adapting limb coordination and body posture to different arboreal inclinations. To investigate the influence of each hair, we studied the locomotion of rats on poles of two different diameters whereby we selectively removed individual carpal sinus hairs. The rats walked at speeds ranging from 0.12 m/s to 0.58 m/s. Normal-light high-speed cameras and x-ray fluoroscopy visualized the hairs and body dynamics during locomotion. The time lag between first contact of the hairs to the branch until contact of the forepaw was 56–108 ms. Within this time window the pronation/supination of the paw and anterior body posture are adjusted to the substrate diameter. We presume that the most proximal sinus hair (located between the medial and lateral one) senses the paw-substrate distance through the increasing bend from its first branch-contact until the contact of the paw. The medial and the lateral hairs touch the pole sides and thereby, may collect information about the properties of the small-diameter substrate. The removal of single hairs from the group results in minor changes of kinematic parameters, but locomotor stability is seriously impaired when more than one hair is cut. The kinematic responses span from a more crouched body posture and higher forearm pronation to paw slipping, muscle tremor or complete refusal to walk on the narrow substrate.

In-situ benzoxazine-isocyanide chemistry (BIC)/sol-gel preparation and Pb(II) electrochemical probing investigation of modified polyamide/silica composite

The construction of polyamide (PA)/silica (PA/SiO2) composites was restricted by the tedious synthetic route, narrow substrates selection and harsh reaction condition of existing protocols. Herein, a series of novel PA/SiO2 composites were prepared via in-situ benzoxazine-isocyanide chemistry combined with a sol-gel process (BIC/sol-gel) under mild conditions (under 30 °C and ambient pressure). The influences of the amount of catalyst (octylphosphonic acid) and reaction time on the BIC/sol-gel process were explored. The chemical structure and other fundamental properties of a representative composite (PA-Si-100) were systematically characterized here. The downstream application of PA-Si-100 as Pb2+ electrochemical probe was further explored. The results showed that the introduction of PA-Si-100 composite significantly improved Pb2+ signaling. The interaction mechanism between PA-Si-100 and Pb2+ was investigated by X-ray photoelectron spectroscopy (XPS) and FT-IR analyses. The results indicated that amide, phenolic hydroxyl and Si-O segments acted as the primary binding sites for Pb2+, rendering the enhanced response signal of Pb2+ via “enrichment” effect.

The Stabilizing Function of the Tail During Arboreal Quadrupedalism.

Locomotion on the narrow and compliant supports of the arboreal environment is inherently precarious. Previous studies have identified a host of morphological and behavioral specializations in arboreal animals broadly thought to promote stability when on precarious substrates. Less well-studied is the role of the tail in maintaining balance. However, prior anatomical studies have found that arboreal taxa frequently have longer tails for their body size than their terrestrial counterparts, and prior laboratory studies of tail kinematics and the effects of tail reduction in focal taxa have broadly supported the hypothesis that the tail is functionally important for maintaining balance on narrow and mobile substrates. In this set of studies, we extend this work in two ways. First, we used a laboratory dataset on three-dimensional segmental kinematics and tail inertial properties in squirrel monkeys (Saimiri boliviensis) to investigate how tail angular momentum is modulated during steady-state locomotion on narrow supports. In the second study, we used a quantitative dataset on quadrupedal locomotion in wild platyrrhine monkeys to investigate how free-ranging arboreal animals adjust tail movements in response to substrate variation, focusing on kinematic measures validated in prior laboratory studies of tail mechanics (including the laboratory data presented). Our laboratory results show that S. boliviensis significantly increase average tail angular momentum magnitudes and amplitudes on narrow supports, and primarily regulate that momentum by adjusting the linear and angular velocity of the tail (rather than via changes in tail posture per se). We build on these findings in our second study by showing that wild platyrrhines responded to the precarity of narrow and mobile substrates by extending the tail and exaggerating tail displacements, providing ecological validity to the laboratory studies of tail mechanics presented here and elsewhere. In conclusion, our data support the hypothesis that the long and mobile tails of arboreal animals serve a biological role of enhancing stability when moving quadrupedally over narrow and mobile substrates. Tail angular momentum could be used to cancel out the angular momentum generated by other parts of the body during steady-state locomotion, thereby reducing whole-body angular momentum and promoting stability, and could also be used to mitigate the effects of destabilizing torques about the support should the animals encounter large, unexpected perturbations. Overall, these studies suggest that long and mobile tails should be considered among the fundamental suite of adaptations promoting safe and efficient arboreal locomotion.

Can Anyone Climb? The Skills of a Non-specialized Toad and its Bearing on the Evolution of New Niches

Several studies of arboreal anuran species show morphological specializations for clinging onto narrow substrates. However, little is known about these capacities in non-specialized anurans, which is crucial to understand the initial phases of adaptation to a new niche. To assess the functional requirements related to the evolution of arboreality in anurans we analyzed climbing performance, and correlated anatomical traits, in the terrestrial toad Rhinella arenarum, a species choose as a proxy for the ancestral condition regarding the evolution of this specialized niche. We studied the impact of a substrate of wooden rods with different diameters, arrangements, and slopes on locomotion, grasping, and climbing with a comparative framework. Animals were confronted with climbing tests, video recording their behaviors. Preserved specimens were dissected to assess limb myology, osteology, and tendons’ characteristics. Our results show that how terrestrial toad R. arenarum climbs is different from those displayed by specialized tree frogs. Animals flexed their fingers and toes, grasping the substrate displaying hookings and partial graspings. The palm was scarcely involved in the grip, as in specialized anurans. These actions were performed although flexor and extensor muscles of the digits are highly conserved and generalized. Further, we formally assess the evolutionary history of ecological and anatomical traits related to climbing among Rhinella species to improving the comprehension of the relation between morphofunctional patterns and behavioral climbing skills. Our experiments revealed that this terrestrial toad possesses unexpected climbing capacities, suggesting a way in which evolution of new niches could have developed in the evolution of anurans.

Tail Length Evolution in Deer Mice: Linking Morphology, Behavior, and Function.

Determining how variation in morphology affects animal performance (and ultimately fitness) is key to understanding the complete process of evolutionary adaptation. Long tails have evolved many times in arboreal and semi-arboreal rodents; in deer mice, long tails have evolved repeatedly in populations occupying forested habitat even within a single species (Peromyscus maniculatus). Here, we use a combination of functional modeling, laboratory studies, and museum records to test hypotheses about the function of tail-length variation in deer mice. First, we use computational models, informed by museum records documenting natural variation in tail length, to test whether differences in tail morphology between forest and prairie subspecies can influence performance in behavioral contexts relevant for tail use. We find that the deer- mouse tail plays little role in statically adjusting center of mass or in correcting body pitch and yaw, but rather it can affect body roll during arboreal locomotion. In this context, we find that even intraspecific tail-length variation could result in substantial differences in how much body rotation results from equivalent tail motions (i.e., tail effectiveness), but the relationship between commonly-used metrics of tail-length variation and effectiveness is non-linear. We further test whether caudal vertebra length, number, and shape are associated with differences in how much the tail can bend to curve around narrow substrates (i.e., tail curvature) and find that, as predicted, the shape of the caudal vertebrae is associated with intervertebral bending angle across taxa. However, although forest and prairie mice typically differ in both the length and number of caudal vertebrae, we do not find evidence that this pattern is the result of a functional trade-off related to tail curvature. Together, these results highlight how even simple models can both generate and exclude hypotheses about the functional consequences of trait variation for organismal-level performance.

Adhesion and Running Speed of a Tropical Arboreal Ant (Cephalotes atratus) on Rough, Narrow, and Inclined Substrates.

Arboreal ants must navigate variably sized and inclined linear structures across a range of substrate roughness when foraging tens of meters above the ground. To achieve this, arboreal ants use specialized adhesive pads and claws to maintain effective attachment to canopy substrates. Here, we explored the effect of substrate structure, including small and large-scale substrate roughness, substrate diameter, and substrate orientation (inclination), on adhesion and running speed of workers of one common, intermediately-sized, arboreal ant species. Normal (orthogonal) and shear (parallel) adhesive performance varied on sandpaper and natural leaf substrates, particularly at small size scales, but running speed on these substrates remained relatively constant. Running speed also varied minimally when running up and down inclined substrates, except when the substrate was positioned completely vertical. On vertical surfaces, ants ran significantly faster down than up. Ant running speed was slower on relatively narrow substrates. The results of this study show that variation in the physical properties of tree surfaces differentially affects arboreal ant adhesive and locomotor performance. Specifically, locomotor performance was much more robust to surface roughness than was adhesive performance. The results provide a basis for understanding how performance correlates of functional morphology contribute to determining local ant distributions and foraging decisions in the tropical rainforest canopy.

Ontogeny of locomotion in mouse lemurs: Implications for primate evolution

The environment of juvenile primates is very challenging. They have to forage and move on the same substrates as adults do and escape the same predators, despite their immature state. In this study, we explore the developmental strategies that may provide effective locomotor abilities early in life. This could provide new insights into the selective pressures acting on juvenile primates and into evolution of primate locomotion. We conducted an ontogenetic study of 36 arboreal gray mouse lemurs from birth to adulthood (6 months of age). The investigated parameters were, for both limbs, (1) grasping behavior during locomotion (i.e., grip postures), (2) grasping performance (i.e., pull strength), and (3) motor coordination (i.e., rotarod test). Our results show that 8-day-old babies are able to climb substrates of various slopes and diameters outside of their nest. Although juveniles cannot successfully complete a motor coordination test before 30 days of age, young individuals display relative pull strengths that are very high or even on par with adults, guaranteeing stability on narrow substrates. These powerful grasps highlight the importance of the grasping function for these juveniles that are not carried and move independently on arboreal substrates shortly after their first week of life. Moreover, the pedal grasping provides a secure grasp on all substrates across ontogeny; however, manual secure grasps decrease during development, being highly used only shortly after birth on vertical and narrow substrates. These results first suggest different functional roles of the hands and feet, with the hind limbs ensuring body balance on the substrates, freeing the upper limbs for manipulation. They further show vertical and narrow branches to be especially challenging, requiring strong grasps, which suggests that they may drive the evolution of strong grasping abilities in primates.

Quantifying koala locomotion strategies: implications for the evolution of arborealism in marsupials.

The morphology and locomotor performance of a species can determine their inherent fitness within a habitat type. Koalas have an unusual morphology for marsupials, with several key adaptations suggested to increase stability in arboreal environments. We quantified the kinematics of their movement over ground and along narrow arboreal trackways to determine the extent to which their locomotion resembled that of primates, occupying similar niches, or basal marsupials from which they evolved. On the ground, the locomotion of koalas resembled a combination of marsupial behaviours and primate-like mechanics. For example, their fastest strides were bounding type gaits with a top speed of 2.78 m s-1 (mean 1.20 m s-1), resembling marsupials, while the relatively longer stride length was reflective of primate locomotion. Speed was increased using equal modification of stride length and frequency. On narrow substrates, koalas took longer but slower strides (mean 0.42 m s-1), adopting diagonally coupled gaits including both lateral and diagonal sequence gaits, the latter being a strategy distinctive among arboreal primates. The use of diagonally coupled gaits in the arboreal environment is likely only possible because of the unique gripping hand morphology of both the fore and hind feet of koalas. These results suggest that during ground locomotion, they use marsupial-like strategies but alternate to primate-like strategies when moving amongst branches, maximising stability in these environments. The locomotion strategies of koalas provide key insights into an independent evolutionary branch for an arboreal specialist, highlighting how locomotor strategies can convergently evolve between distant lineages.

Effects of substrate and phylogeny on quadrupedal gait in free-ranging platyrrhines.

Primate diagonal sequence (DS) gaits are often argued to be an adaptation for moving and foraging in the fine-branch niche; however, existing data have come predominantly from laboratory studies that are limited in taxonomic breadth and fail to account for the structural and ecological variation of natural substrates. We test the extent to which substrate diameter and orientation influence gait sequence type and limb phase in free-ranging primates, as well as how phylogenetic relatedness might condition response patterns. We filmed quadrupedal locomotion in 11 platyrrhine species at field sites in Ecuador and Costa Rica and measured the diameter and orientation of locomotor substrates using remote sensors. We quantified limb phase values and classified strides by gait sequence type (N = 988 strides). Our results show that most of the species in our sample consistently used DS gaits, regardless of substrate diameter or orientation; however, all taxa also used asymmetrical and/or lateral sequence gaits. By incorporating phylogenetic eigenvectors into our models, we found significant differences in gait sequence patterns and limb phase values among the major platyrrhine clades, suggesting that phylogeny may be a better predictor of gait than substrate diameter or orientation. Our field data generally corroborate locomotor patterns from laboratory studies but capture additional aspects of gait variability and flexibility in response to the complexity of natural environments. Overall, our results suggest that DS gaits are not exclusively tailored to narrow or oblique substrates but are used on arboreal substrates in general.

Functional Characterization of Novel CYP2C9 Variants Found in an Alaska Native Population

Coding‐region variants of CYP2C9 can dramatically influence the pharmacokinetics and drug response of therapeutic agents, an important clinical consideration for commonly used CYP2C9 narrow therapeutic index substrates, including (S)‐warfarin and phenytoin. In the Yup'ik Alaska Native people, novel CYP2C9 protein variants M1L, N218I, and P279T are expressed with higher frequencies than the well‐characterized CYP2C9*2 and CYP2C9*3 alleles. This study's objective was to determine the relative expression of these CYP2C9 variants in HepG2 cells and to characterize the function of the purified reconstituted enzymes expressed in E. coli, towards several CYP2C9 drug substrates.While mRNA expression of the CYP2C9 M1L, N218I, and P279T gene variants and the reference (wildtype) protein in HepG2 cells were similar, the protein was undetectable for the M1L variant. This is expected, due to disruption of the start codon in CYP2C9 M1L. His‐tagged CYP2C9 reference protein and N218I and P279T variants expressed well in E. coli and were highly purified after affinity chromatography. Following reconstitution with cytochrome P450 reductase and cytochrome b5, the N218I and P279T variants metabolized (S)‐warfarin, but not (R)‐warfarin, to the principal 7‐ and 6‐hydroxy metabolites formed by the reference enzyme. Kinetic studies of (S)‐warfarin metabolism demonstrate that the N218I variant has reduced function, with less than 40% of the catalytic efficiency of reference enzyme, while the P279T variant had similar catalytic efficiency to that of reference CYP2C9. Similar, although not identical concentration–rate profiles were obtained for phenytoin, flurbiprofen and naproxen metabolism.The potential of CYP2C9 M1L, N218I, and perhaps even P279T alleles to alter the pharmacokinetics of drugs metabolized by CYP2C9 may put carriers at risk of exacerbated therapeutic effects from drugs that rely predominantly on CYP2C9 for their metabolic clearance. Further clinical implications for the Alaska Native people include phenotypic misclassification from currently available pharmacogenetic tests, which typically screen only for the common CYP2C9*2 (R144C) and CYP2C9*3 (I359T) variants.Support or Funding InformationP01 GM116691, T32 GM 007750This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Moving in complex environments: a biomechanical analysis of locomotion on inclined and narrow substrates.

Characterisation of an organism's performance in different habitats provides insight into the conditions that allow it to survive and reproduce. In recent years, the northern quoll (Dasyurus hallucatus) - a medium-sized semi-arboreal marsupial native to northern Australia - has undergone significant population declines within open forest, woodland and riparian habitats, but less so in rocky areas. To help understand this decline, we quantified the biomechanical performance of wild northern quolls as they ran up inclined narrow (13 mm pole) and inclined wide (90 mm platform) substrates. We predicted that quolls may possess biomechanical adaptations to increase stability on narrow surfaces, which are more common in rocky habitats. Our results showed that quolls have some biomechanical characteristics consistent with a stability advantage on narrow surfaces. This includes the coupled use of limb pairs, as indicated via a decrease in footfall time, and an ability to produce corrective torques to counteract the toppling moments commonly encountered during gait on narrow surfaces. However, speed was constrained on narrow surfaces, and quolls did not adopt diagonal sequence gaits, unlike true arboreal specialists such as primates. In comparison with key predators, such as cats and dogs, northern quolls appear inferior in terrestrial environments but have a stability advantage at higher speeds on narrow supports. This may partially explain the heterogeneous declines in northern quoll populations among various habitats on mainland Australia.

Locomotor kinematics of tree squirrels (Sciurus carolinensis) in free‐ranging and laboratory environments: Implications for primate locomotion and evolution

The grasping capabilities and gait kinematics characteristic of primates are often argued to be adaptations for safely moving on small terminal branches. The goal of this study was to identify whether Eastern gray squirrels (Sciurus carolinensis)-arboreal rodents that frequently move and forage on small branches, lack primate-like grasping and gait patterns, and arguably represent extant analogs of a stem primate ancestor-adjust gait kinematics to narrow and nonhorizontal branches. We studied locomotor kinematics of free-ranging and laboratory-housed squirrels moving over various substrates. We used high-speed video to film (a) a population of free-ranging squirrels moving on natural substrates and (b) laboratory-housed squirrels moving on horizontal poles. Substrates were coded as small, medium, or large relative to squirrel trunk diameter, and as inclined, declined, or horizontal. Free-ranging squirrels used more gallops and half-bounds on small- and medium-sized substrates, and more high-impact bounds, with reduced limb-lead durations, on declined substrates. Laboratory squirrels moved at higher speeds than free-ranging squirrels and responded to decreasing diameter by using more gallops and half-bounds, lowering speed, and-controlling for speed-increasing mean duty factor, mean number of supporting limbs, and relative forelimb lead duration. Our inability to detect substantial diameter or orientation-related gait adjustments in the wild may be due to a limited accounting of confounding influences (e.g., substrate compliance). Ultimately, studies assessing stability measures (e.g., center of mass fluctuations and peak vertical force) are required to assess whether primates' enhanced grasping and gait patterns engender performance advantages on narrow or oblique substrates.

Characterization of the In Vivo Catalytic Efficiency of CYP2C9 M1L, a Novel and Common Variant in the Yup'ik Alaska Native Population

The Yup'ik Alaska Native people are under‐represented in genetic research, but have unique pharmacogene variation that may critically impact their response to drug therapy. Full gene resequencing of CYP2C9 in the Yup'ik population identified novel, relatively common CYP2C9 variants, including CYP2C9M1L (M1L), a coding region single nucleotide polymorphism hypothesized to confer a poor metabolism activity phenotype by disrupting the start codon. M1L is present at a minor allele frequency of 7.1% in the Yup'ik, placing the population at risk for adverse drug interactions with narrow therapeutic index CYP2C9 substrates such as (S)‐warfarin, phenytoin, and tolbutamide. This study's objective was to characterize the catalytic efficiency of MIL in vivo by evaluating the pharmacokinetic behavior of naproxen, a probe for CYP2C9 activity, in genotyped Yup'ik participants. Targeted genotyping was conducted to identify heterozygous and homozygous M1L individuals from 906 representative members of the Yup'ik population. CYP2C9 mediates the O‐demethylation of naproxen to its 6‐O‐desmethyl metabolite, which is glucuronidated or sulfonated to secondary metabolites, all of which are excreted into the urine. Thus, a change in the urinary metabolite to parent ratio is indicative of a change in CYP2C9 intrinsic formation clearance to 6‐O‐desmethyl naproxen. We developed and validated a novel LC/MS method for simultaneous quantification of (S)‐naproxen, (S)‐6‐O‐desmethyl naproxen, and (S)‐naproxen acyl glucuronide in human urine. Preliminary analysis of one‐half of the final study population revealed an average ratio of 6‐O‐desmethyl naproxen to unchanged naproxen of 18.0 ± 7.7 (n = 7), 10.1 ± 4.5 (n = 7), and 15.6 (n = 2) for the M1L reference, heterozygote, and homozygote variant groups, respectively. ANOVA analysis was borderline significant (p = 0.06) and pairwise comparison of the homozygous reference and heterozygous groups (p = 0.06) suggested reduced activity for the M1L variant. The effect of the novel M1L variant and its potential to alter the pharmacokinetics of drugs metabolized by CYP2C9 have clinical implications, including phenotypic misclassification from currently available pharmacogenetic tests, in the Alaska Native population.Support or Funding InformationP01 GM116691, T32 GM 007750This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In vitro evaluation of the inhibition and induction potential of olaparib, a potent poly(ADP-ribose) polymerase inhibitor, on cytochrome P450

1. In vitro studies were conducted to evaluate potential inhibitory and inductive effects of the poly(ADP-ribose) polymerase (PARP) inhibitor, olaparib, on cytochrome P450 (CYP) enzymes. Inhibitory effects were determined in human liver microsomes (HLM); inductive effects were evaluated in cultured human hepatocytes.2. Olaparib did not inhibit CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2D6 or CYP2E1 and caused slight inhibition of CYP2C9, CYP2C19 and CYP3A4/5 in HLM up to a concentration of 100 μM. However, olaparib (17–500 μM) inhibited CYP3A4/5 with an IC50 of 119 μM. In time-dependent CYP inhibition assays, olaparib (10 μM) had no effect against CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1 and a minor effect against CYP3A4/5. In a further study, olaparib (2–200 μM) functioned as a time-dependent inhibitor of CYP3A4/5 (KI, 72.2 μM and Kinact, 0.0675 min−1). Assessment of the CYP induction potential of olaparib (0.061–44 μM) showed minor concentration-related increases in CYP1A2 and more marked increases in CYP2B6 and CYP3A4 mRNA, compared with positive control activity; however, no significant change in CYP3A4/5 enzyme activity was observed.3. Clinically significant drug–drug interactions due to olaparib inhibition or induction of hepatic or intestinal CYP3A4/5 cannot be excluded. It is recommended that olaparib is given with caution with narrow therapeutic range or sensitive CYP3A substrates, and that prescribers are aware that olaparib may reduce exposure to substrates of CYP2B6.

Arboreal gaits in three sympatric rodents Apodemus agrarius, Apodemus flavicollis (Rodentia, Muridae) and Myodes glareolus (Rodentia, Cricetidae)

Vertical stratification of the arboreal habitat allows the coexistence of several species in a given area, because the complex arboreal strata can be used in different ways by arboreal and scansorial mammals. The present report experimentally investigated the gait metrics on different arboreal substrates, of three sympatric rodents living in a deciduous forest in Poznań, Poland. Arboreal locomotion was compared between the burrowing striped field mouse, Apodemus agrarius, the scansorial bank vole, Myodes glareolus, and the more arboreal yellow-necked mouse, Apodemus flavicollis. We filmed two wild-caught individuals from each species walking on four different substrate diameters (2mm, 5mm, 10mm, 25mm) and three different inclinations (45° descending, horizontal, 45° ascending) at 240fps and collected a set of gait parameters from a total of 273 complete cycles. Our results did not demonstrate clear relationships between arboreal locomotion and the ecology of the three species. Only A. flavicollis exhibited locomotor features partly associated with arboreal competence, including lower velocity and diagonality on narrow substrates and asymmetrical gaits on wider ones. On the other hand, the two Apodemus species, despite their different ecologies, shared a few locomotor similarities, such as velocity regulation primarily by stride frequency, and similar effects of substrate size and inclination on diagonality, duty factor, and duty factor index indicating the possibility of a phylogenetic signal. Because the selected gait parameters provided limited insight into the ability of small mammals to move competently through an arboreal habitat, these findings indicate that the relationship between behaviour and ecology is complex.

Precision dicing of hard materials with abrasive blade

The present work shows the experiments on dicing of high alumina substrates using abrasive blades. The technology used by modern electronic components is based on narrow and thin ceramic and single crystal substrates. One of the techniques used to obtain these products is to dice a larger substrate using an abrasive blade to generate low damages and high productivity called dicing process. Samples with high alumina (99.8 %) were diced with diamond abrasive blades in a creep feed process. The process parameters were changed as cuts 1 to 3 mm deep were made. The feed speed varied from 1 to 19 mm/s and the blade rotation varied from 10,000 to 30,000 rpm. The blade wearing as well as the linearity and the quality of dicing were analyzed. Higher blade rotation increases the blade stiffness and on these experiments a more stable cutting process was achieved at 30,000 rpm. The removal material mechanism was also explored comparing the chipping between silicon and the high alumina specimens.