Omega-3 Fatty Acids Activate Ciliary FFAR4 to Control Adipogenesis.

Adult mesenchymal stem cells, including preadipocytes, possess a cellular sensory organelle called the primary cilium. Ciliated preadipocytes abundantly populate perivascular compartments in fat and are activated by a high-fat diet. Here, we sought to understand whether preadipocytes use their cilia to sense and respond to external cues to remodel white adipose tissue. Abolishing preadipocyte cilia in mice severely impairs white adipose tissue expansion. We discover that TULP3-dependent ciliary localization of the omega-3 fatty acid receptor FFAR4/GPR120 promotes adipogenesis. FFAR4 agonists and ω-3 fatty acids, but not saturated fatty acids, trigger mitosis and adipogenesis by rapidly activating cAMP production inside cilia. Ciliary cAMP activates EPAC signaling, CTCF-dependent chromatin remodeling, and transcriptional activation of PPARγ and CEBPα to initiate adipogenesis. We propose that dietary ω-3 fatty acids selectively drive expansion of adipocyte numbers to produce new fat cells and store saturated fatty acids, enabling homeostasis of healthy fat tissue.

Metabolic compensation and circadian resilience in prokaryotic cyanobacteria.

For a biological oscillator to function as a circadian pacemaker that confers a fitness advantage, its timing functions must be stable in response to environmental and metabolic fluctuations. One such stability enhancer, temperature compensation, has long been a defining characteristic of these timekeepers. However, an accurate biological timekeeper must also resist changes in metabolism, and this review suggests that temperature compensation is actually a subset of a larger phenomenon, namely metabolic compensation, which maintains the frequency of circadian oscillators in response to a host of factors that impinge on metabolism and would otherwise destabilize these clocks. The circadian system of prokaryotic cyanobacteria is an illustrative model because it is composed of transcriptional and nontranscriptional oscillators that are coupled to promote resilience. Moreover, the cyanobacterial circadian program regulates gene activity and metabolic pathways, and it can be manipulated to improve the expression of bioproducts that have practical value.

Clocking out and letting go to unleash green biotech applications in a photosynthetic host.

Cyanobacteria are photosynthetic bacteria whose gene expression patterns are globally regulated by their circadian (daily) clocks. Due to their ability to use sunlight as their energy source, they are also attractive hosts for "green" production of pharmaceuticals, renewable fuels, and chemicals. However, despite the application of traditional genetic tools such as the identification of strong promoters to enhance the expression of heterologous genes, cyanobacteria have lagged behind other microorganisms such as Escherichia coli and yeast as economically efficient cell factories. The previous approaches have ignored large-scale constraints within cyanobacterial metabolic networks on transcription, predominantly the pervasive control of gene expression by the circadian (daily) clock. Here, we show that reprogramming gene expression by releasing circadian repressor elements in the transcriptional regulatory pathways coupled with inactivation of the central oscillating mechanism enables a dramatic enhancement of expression in cyanobacteria of heterologous genes encoding both catalytically active enzymes and polypeptides of biomedical significance.

Host circadian behaviors exert only weak selective pressure on the gut microbiome under stable conditions but are critical for recovery from antibiotic treatment.

The circadian rhythms of hosts dictate an approximately 24 h transformation in the environment experienced by their gut microbiome. The consequences of this cyclic environment on the intestinal microbiota are barely understood and are likely to have medical ramifications. Can daily rhythmicity in the gut act as a selective pressure that shapes the microbial community? Moreover, given that several bacterial species have been reported to exhibit circadian rhythms themselves, we test here whether a rhythmic environment is a selective pressure that favors clock-harboring bacteria that can anticipate and prepare for consistent daily changes in the environment. We observed that the daily rhythmicity of the mouse gut environment is a stabilizing influence that facilitates microbiotal recovery from antibiotic perturbation. The composition of the microbiome recovers to pretreatment conditions when exposed to consistent daily rhythmicity, whereas in hosts whose feeding and activity patterns are temporally disrupted, microbiotal recovery is incomplete and allows potentially unhealthy opportunists to exploit the temporal disarray. Unexpectedly, we found that in the absence of antibiotic perturbation, the gut microbiome is stable to rhythmic versus disrupted feeding and activity patterns. Comparison of our results with those of other studies reveals an intriguing correlation that a stable microbiome may be resilient to one perturbation alone (e.g., disruption of the daily timing of host behavior and feeding), but not to multiple perturbations in combination. However, after a perturbation of the stable microbiome, a regular daily pattern of host behavior/feeding appears to be essential for the microbiome to recover to the original steady state. Given the inconsistency of daily rhythms in modern human life (e.g., shiftwork, social jet-lag, irregular eating habits), these results emphasize the importance of consistent daily rhythmicity to optimal health not only directly to the host, but also indirectly by preserving the host's microbiome in the face of perturbations.

Accelerating strain phenotyping with desorption electrospray ionization-imaging mass spectrometry and untargeted analysis of intact microbial colonies.

Reading and writing DNA were once the rate-limiting step in synthetic biology workflows. This has been replaced by the search for the optimal target sequences to produce systems with desired properties. Directed evolution and screening mutant libraries are proven technologies for isolating strains with enhanced performance whenever specialized assays are available for rapidly detecting a phenotype of interest. Armed with technologies such as CRISPR-Cas9, these experiments are capable of generating libraries of up to 1010 genetic variants. At a rate of 102 samples per day, standard analytical methods for assessing metabolic phenotypes represent a major bottleneck to modern synthetic biology workflows. To address this issue, we have developed a desorption electrospray ionization-imaging mass spectrometry screening assay that directly samples microorganisms. This technology increases the throughput of metabolic measurements by reducing sample preparation and analyzing organisms in a multiplexed fashion. To further accelerate synthetic biology workflows, we utilized untargeted acquisitions and unsupervised analytics to assess multiple targets for future engineering strategies within a single acquisition. We demonstrate the utility of the developed method using Escherichia coli strains engineered to overproduce free fatty acids. We determined discrete metabolic phenotypes associated with each strain, which include the primary fatty acid product, secondary products, and additional metabolites outside the engineered product pathway. Furthermore, we measured changes in amino acid levels and membrane lipid composition, which affect cell viability. In sum, we present an analytical method to accelerate synthetic biology workflows through rapid, untargeted, and multiplexed metabolomic analyses.

Eating breakfast and avoiding late-evening snacking sustains lipid oxidation.

Circadian (daily) regulation of metabolic pathways implies that food may be metabolized differentially over the daily cycle. To test that hypothesis, we monitored the metabolism of older subjects in a whole-room respiratory chamber over two separate 56-h sessions in a random crossover design. In one session, one of the 3 daily meals was presented as breakfast, whereas in the other session, a nutritionally equivalent meal was presented as a late-evening snack. The duration of the overnight fast was the same for both sessions. Whereas the two sessions did not differ in overall energy expenditure, the respiratory exchange ratio (RER) was different during sleep between the two sessions. Unexpectedly, this difference in RER due to daily meal timing was not due to daily differences in physical activity, sleep disruption, or core body temperature (CBT). Rather, we found that the daily timing of nutrient availability coupled with daily/circadian control of metabolism drives a switch in substrate preference such that the late-evening Snack Session resulted in significantly lower lipid oxidation (LO) compared to the Breakfast Session. Therefore, the timing of meals during the day/night cycle affects how ingested food is oxidized or stored in humans, with important implications for optimal eating habits.

Targeted modification of the Per2 clock gene alters circadian function in mPer2luciferase (mPer2Luc) mice.

Modification of the Per2 clock gene in mPer2Luc reporter mice significantly alters circadian function. Behavioral period in constant dark is lengthened, and dissociates into two distinct components in constant light. Rhythms exhibit increased bimodality, enhanced phase resetting to light pulses, and altered entrainment to scheduled feeding. Mechanistic mathematical modelling predicts that enhanced protein interactions with the modified mPER2 C-terminus, combined with differential clock regulation among SCN subregions, can account for effects on circadian behavior via increased Per2 transcript and protein stability. PER2::LUC produces greater suppression of CLOCK:BMAL1 E-box activity than PER2. mPer2Luc carries a 72 bp deletion in exon 23 of Per2, and retains a neomycin resistance cassette that affects rhythm amplitude but not period. The results show that mPer2Luc acts as a circadian clock mutation illustrating a need for detailed assessment of potential impacts of c-terminal tags in genetically modified animal models.

Helium-flushed sheathed nickel tube reactor for continuous flow oxygen stable isotope compound-specific analysis.

RATIONALE: Compound-specific oxygen stable isotope analysis has been commercially available for about 20 years but the technique has been infrequently applied and practical operating knowledge is limited. Existing reactor designs and chemistry fail to fully exclude contaminating oxygen, are chromatographically flawed, and are generally fabricated from expensive platinum tubing. METHODS: As a new interface between the gas chromatograph and isotope ratio mass spectrometer, we used a nickel tube reactor (containing no platinum wires) in a ceramic sheath with countercurrent helium flushing, operated at 1250°C. We analyzed suites of four long-chain alcohols (LCAs), defining the least and most 18 O-enriched LCAs as internal standards. The other two LCAs, co-injected in varying amounts, were treated as unknowns and amplitude-corrected during data reduction. RESULTS: With this design, signals from non-oxygenated compounds are minimized with a hydrogen trickle, while excessive graphitization is counterproductive. A balance of hydrogen and graphite is required to maintain a proper, stable redox state in the reactor. With low extraneous oxygen, the exact hydrogen flow has little effect on final δ18 O data quality. Overall average δ18 O values precisions and errors for the unknowns were better than 0.4 ‰ for injections between 2 and 7 nmol per component. CONCLUSIONS: Our actively flushed sheath reduces oxygen infiltration to negligible levels and results in a chromatographic flow devoid of dead or poorly swept volumes. The improved design allows measurement of LCAs with accuracy and precision sufficient to enable investigating real processes, and may also have wider applications to other isotopes.

Systematic identification and elimination of flux bottlenecks in the aldehyde production pathway of Synechococcus elongatus PCC 7942.

Isotopically nonstationary metabolic flux analysis (INST-MFA) provides a versatile platform to quantitatively assess in vivo metabolic activities of autotrophic systems. By applying INST-MFA to recombinant aldehyde-producing cyanobacteria, we identified metabolic alterations that correlated with increased strain performance in order to guide rational metabolic engineering. We identified four reactions adjacent to the pyruvate node that varied significantly with increasing aldehyde production: pyruvate kinase (PK) and acetolactate synthase (ALS) fluxes were directly correlated with product formation, while pyruvate dehydrogenase (PDH) and phosphoenolpyruvate carboxylase (PPC) fluxes were inversely correlated. Overexpression of enzymes for PK or ALS did not result in further improvements to the previous best-performing strain, while downregulation of PDH expression (through antisense RNA expression) or PPC flux (through expression of the reverse reaction, phosphoenolpyruvate carboxykinase) provided significant improvements. These results illustrate the potential of INST-MFA to enable a systematic approach for iterative identification and removal of pathway bottlenecks in autotrophic host cells.

A bioluminescent reporter for the halophilic archaeon Haloferax volcanii.

Haloarchaea have evolved to thrive in hypersaline environments. Haloferax volcanii is of particular interest due to its genetic tractability; however, few in vivo reporters exist for halophiles. Haloarchaeal proteins evolved characteristics that promote proper folding and function at high salt concentrations, but many mesophilic reporter proteins lack these characteristics. Mesophilic proteins that acquire salt-stabilizing mutations, however, can lead to proper function in haloarchaea. Using laboratory-directed evolution, we developed and demonstrated an in vivo luciferase that functions in the hypersaline cytosol of H. volcanii.

Deletion of choline acetyltransferase in enteric neurons results in postnatal intestinal dysmotility and dysbiosis.

Acetylcholine (ACh)-synthesizing neurons are major components of the enteric nervous system (ENS). They release ACh and peptidergic neurotransmitters onto enteric neurons and muscle. However, pharmacological interrogation has proven inadequate to demonstrate an essential role for ACh. Our objective was to determine whether elimination of ACh synthesis during embryogenesis alters prenatal viability, intestinal function, the neurotransmitter complement, and the microbiome. Conditional deletion of choline acetyltransferase ( ChAT), the ACh synthetic enzyme, in neural crest-derived neurons ( ChAT-Null) was performed. Survival, ChAT activity, gut motility, and the microbiome were studied. ChAT was conditionally deleted in ENS neural crest-derived cells. Despite ChAT absence, mice were born live and survived the first 2 wk. They failed to gain significant weight in the third postnatal week, dying between postnatal d 18 and 30. Small intestinal transit of carmine red was 50% slower in ChAT-Nulls vs. WT and ChAT- Het. The colons of many neonatal ChAT-Null mice contained compacted feces, suggesting dysmotility. Microbiome analysis revealed dysbiosis in ChAT-Null mice. Developmental deletion of ChAT activity in enteric neurons results in proximal gastrointestinal tract dysmotility, critically diminished colonic transit, failure to thrive, intestinal dysbiosis, and death. ACh is necessary for sustained gut motility and survival of neonatal mice after weaning.-Johnson, C. D., Barlow-Anacker, A. J., Pierre, J. F., Touw, K., Erickson, C. S., Furness, J. B., Epstein, M. L., Gosain, A. Deletion of choline acetyltransferase in enteric neurons results in postnatal intestinal dysmotility and dysbiosis.

Non-optimal codon usage is a mechanism to achieve circadian clock conditionality.

Circadian rhythms are oscillations in biological processes that function as a key adaptation to the daily rhythms of most environments. In the model cyanobacterial circadian clock system, the core oscillator proteins are encoded by the gene cluster kaiABC. Genes with high expression and functional importance, such as the kai genes, are usually encoded by optimal codons, yet the codon-usage bias of the kaiBC genes is not optimized for translational efficiency. We discovered a relationship between codon usage and a general property of circadian rhythms called conditionality; namely, that endogenous rhythmicity is robustly expressed under some environmental conditions but not others. Despite the generality of circadian conditionality, however, its molecular basis is unknown for any system. Here we show that in the cyanobacterium Synechococcus elongate, non-optimal codon usage was selected as a post-transcriptional mechanism to switch between circadian and non-circadian regulation of gene expression as an adaptive response to environmental conditions. When the kaiBC sequence was experimentally optimized to enhance expression of the KaiB and KaiC proteins, intrinsic rhythmicity was enhanced at cool temperatures that are experienced by this organism in its natural habitat. However, fitness at those temperatures was highest in cells in which the endogenous rhythms were suppressed at cool temperatures as compared with cells exhibiting high-amplitude rhythmicity. These results indicate natural selection against circadian systems in cyanobacteria that are intrinsically robust at cool temperatures. Modulation of circadian amplitude is therefore crucial to its adaptive significance. Moreover, these results show the direct effects of codon usage on a complex phenotype and organismal fitness. Our work also challenges the long-standing view of directional selection towards optimal codons, and provides a key example of natural selection against optimal codons to achieve adaptive responses to environmental changes.

Evolution of KaiC-Dependent Timekeepers: A Proto-circadian Timing Mechanism Confers Adaptive Fitness in the Purple Bacterium Rhodopseudomonas palustris.

Circadian (daily) rhythms are a fundamental and ubiquitous property of eukaryotic organisms. However, cyanobacteria are the only prokaryotic group for which bona fide circadian properties have been persuasively documented, even though homologs of the cyanobacterial kaiABC central clock genes are distributed widely among Eubacteria and Archaea. We report the purple non-sulfur bacterium Rhodopseudomonas palustris (that harbors homologs of kaiB and kaiC) only poorly sustains rhythmicity in constant conditions-a defining characteristic of circadian rhythms. Moreover, the biochemical characteristics of the Rhodopseudomonas homolog of the KaiC protein in vivo and in vitro are different from those of cyanobacterial KaiC. Nevertheless, R. palustris cells exhibit adaptive kaiC-dependent growth enhancement in 24-h cyclic environments, but not under non-natural constant conditions. Therefore, our data indicate that Rhodopseudomonas does not have a classical circadian rhythm, but a novel timekeeping mechanism that does not sustain itself in constant conditions. These results question the adaptive value of self-sustained oscillatory capability for daily timekeepers and establish new criteria for circadian-like systems that are based on adaptive properties (i.e., fitness enhancement in rhythmic environments), rather than upon observations of persisting rhythms in constant conditions. We propose that the Rhodopseudomonas system is a "proto" circadian timekeeper, as in an ancestral system that is based on KaiC and KaiB proteins and includes some, but not necessarily all, of the canonical properties of circadian clocks. These data indicate reasonable intermediate steps by which bona fide circadian systems evolved in simple organisms.

Peroxiredoxins are conserved markers of circadian rhythms.

Cellular life emerged ∼3.7 billion years ago. With scant exception, terrestrial organisms have evolved under predictable daily cycles owing to the Earth's rotation. The advantage conferred on organisms that anticipate such environmental cycles has driven the evolution of endogenous circadian rhythms that tune internal physiology to external conditions. The molecular phylogeny of mechanisms driving these rhythms has been difficult to dissect because identified clock genes and proteins are not conserved across the domains of life: Bacteria, Archaea and Eukaryota. Here we show that oxidation-reduction cycles of peroxiredoxin proteins constitute a universal marker for circadian rhythms in all domains of life, by characterizing their oscillations in a variety of model organisms. Furthermore, we explore the interconnectivity between these metabolic cycles and transcription-translation feedback loops of the clockwork in each system. Our results suggest an intimate co-evolution of cellular timekeeping with redox homeostatic mechanisms after the Great Oxidation Event ∼2.5 billion years ago.

Assessment of Thrombelastography and Platelet Life Span in Ovines.

Ovines are a common animal model for the study of cardiovascular devices, where consideration of blood biocompatibility is an essential design criterion. In the ovine model, tools to assess blood biocompatibility are limited and continued investigation to identify and apply additional assays is merited. Toward this end, the thrombelastograph, clinically utilized to assess hemostasis, was used to characterize normal ovine parameters. In addition, platelet labeling with biotin was evaluated for its potential applicability to quantify ovine platelet life span. Mean ovine thrombelastograph values were reaction-time: 4.9 min, K-time: 2 min, angle: 64.1°, maximum amplitude: 68.6mm, actual clot strength: 11.9 kd/s, and coagulation index: 1.5. Reaction time was significantly shorter and maximum amplitude, actual clot strength, and coagulation index were all significantly higher when compared to normal human thrombelastograph values suggesting some hypercoagulability of sheep blood. Biotinylation and reinfusion of ovine platelets allowed temporal tracking of the labeled platelet cohort with flow cytometry. These data indicated a mean ovine platelet life span of 188h with a half-life of 84h. The collection of these parameters for normal ovines demonstrates the applicability of these techniques for subsequent studies where cardiovascular devices may be evaluated and provides an indication of normal ovine values for comparison purposes.

Isotopically nonstationary 13C flux analysis of cyanobacterial isobutyraldehyde production.

We applied isotopically nonstationary 13C metabolic flux analysis (INST-MFA) to compare the pathway fluxes of wild-type (WT) Synechococcus elongatus PCC 7942 to an engineered strain (SA590) that produces isobutyraldehyde (IBA). The flux maps revealed a potential bottleneck at the pyruvate kinase (PK) reaction step that was associated with diversion of flux into a three-step PK bypass pathway involving the enzymes PEP carboxylase (PEPC), malate dehydrogenase (MDH), and malic enzyme (ME). Overexpression of pk in SA590 led to a significant improvement in IBA specific productivity. Single-gene overexpression of the three enzymes in the proposed PK bypass pathway also led to improvements in IBA production, although to a lesser extent than pk overexpression. Combinatorial overexpression of two of the three genes in the proposed PK bypass pathway (mdh and me) led to improvements in specific productivity that were similar to those achieved by single-gene pk overexpression. Our work demonstrates how 13C flux analysis can be used to identify potential metabolic bottlenecks and novel metabolic routes, and how these findings can guide rational metabolic engineering of cyanobacteria for increased production of desired molecules.

Nicotine Metabolism in Young Adult Daily Menthol and Nonmenthol Smokers.

INTRODUCTION: Menthol cigarette smoking may increase the risk for tobacco smoke exposure and inhibit nicotine metabolism in the liver. Nicotine metabolism is primarily mediated by the enzyme CYP2A6 and the nicotine metabolite ratio (NMR = trans 3' hydroxycotinine/cotinine) is a phenotypic proxy for CYP2A6 activity. No studies have examined differences in this biomarker among young adult daily menthol and nonmenthol smokers. This study compares biomarkers of tobacco smoke exposure among young adult daily menthol and nonmenthol smokers. METHODS: Saliva cotinine and carbon monoxide were measured in a multiethnic sample of daily smokers aged 18-35 (n = 186). Nicotine, cotinine, the cotinine/cigarette per day ratio, trans 3' hydroxycotinine, the NMR, and expired carbon monoxide were compared. RESULTS: The geometric means for nicotine, cotinine, and the cotinine/cigarette per day ratio did not significantly differ between menthol and nonmenthol smokers. The NMR was significantly lower among menthol compared with nonmenthol smokers after adjusting for race/ethnicity, gender, body mass index, and cigarette smoked per day (0.19 vs. 0.24, P = .03). White menthol smokers had significantly higher cotinine/cigarettes per day ratio than white nonmenthol smokers in the adjusted model. White menthol smokers had a lower NMR in the unadjusted model (0.24 vs. 0.31, P = .05) and the differences remained marginally significant in the adjusted model (0.28 vs. 0.34, P = .06). We did not observe these differences in Native Hawaiians and Filipinos. CONCLUSIONS: Young adult daily menthol smokers have slower rates of nicotine metabolism than nonmenthol smokers. Studies are needed to determine the utility of this biomarker for smoking cessation treatment assignments.

Visible light alters yeast metabolic rhythms by inhibiting respiration.

Exposure of cells to visible light in nature or in fluorescence microscopy often is considered to be relatively innocuous. However, using the yeast respiratory oscillation (YRO) as a sensitive measurement of metabolism, we find that non-UV visible light has a significant impact on yeast metabolism. Blue/green wavelengths of visible light shorten the period and dampen the amplitude of the YRO, which is an ultradian rhythm of cell metabolism and transcription. The wavelengths of light that have the greatest effect coincide with the peak absorption regions of cytochromes. Moreover, treating yeast with the electron transport inhibitor sodium azide has similar effects on the YRO as visible light. Because impairment of respiration by light would change several state variables believed to play vital roles in the YRO (e.g., oxygen tension and ATP levels), we tested oxygen's role in YRO stability and found that externally induced oxygen depletion can reset the phase of the oscillation, demonstrating that respiratory capacity plays a role in the oscillation's period and phase. Light-induced damage to the cytochromes also produces reactive oxygen species that up-regulate the oxidative stress response gene TRX2 that is involved in pathways that enable sustained growth in bright visible light. Therefore, visible light can modulate cellular rhythmicity and metabolism through unexpectedly photosensitive pathways.

Polygenic control of Caenorhabditis elegans fat storage.

Tubby mice and individuals with Bardet-Biedl syndrome have defects in ciliated neuron function and obesity, suggesting an as-yet unknown metabolic signaling axis from ciliated neurons to fat storage tissues. Here we show coordinate regulation of Caenorhabditis elegans fat storage by orthologues of these genes acting in ciliated neurons and by a 3-ketoacyl-coA thiolase (encoded by kat-1) that acts in fat storage tissue. A genetic screen for markedly enhanced fat storage in tub-1 mutants led to the isolation only of kat-1 alleles, which impair fatty acid beta-oxidation. kat-1 acts in the intestine, the major C. elegans fat storage tissue, and is transcriptionally upregulated in animals with high fat storage. A genetic screen for synergistic increase in fat storage of a kat-1 mutant identified bbs-1. bbs-1 acts in 15 ciliated neurons that are poised to sense external and internal nutrient levels, supporting a model in which bbs-1 and tub-1 in ciliated neurons form part of an ancient, conserved neuroendocrine axis. This pathway also includes genes encoding intraflagellar transport proteins and cyclic nucleotide gated channels, demonstrating that C. elegans fat storage is under polygenic control.

The Effect of Isolated Hamstrings Fatigue on Landing and Cutting Mechanics.

The function of the hamstrings in protecting the ACL is not fully understood. The purpose of this study was to determine how landing knee mechanics were affected by hamstrings fatigue, analyzed with principal components analysis (PCA). Knee joint mechanics were collected during single-leg stride landings that were followed by lateral and vertical jumps. An isokinetic fatigue protocol was employed to reduce hamstrings strength by 75% at the cessation of the exercise protocol. On the landing test day, participants performed the stride landing maneuvers before and after the fatigue protocol. PCA was performed on the landing knee joint angle, moment, and power waveforms, and MANOVAs were conducted on the retained PCs of each waveform (P < .05). On the strength test day, hamstrings strength recovery was assessed with an identical fatigue protocol followed by strength assessment ~75 s after the cessation of exercise. Pre- and postexercise hamstrings strength on this day was assessed with a dependent t test (P < .05). The hamstrings strength remained significantly reduced by ~8% postexercise (75 s). For stride landings followed by vertical jumps, there were significantly reduced knee flexion angles, extensor moments, and energy absorption. This was indicative of a stiffer landing strategy postfatigue, which has been associated with increased ACL loading.