A distinct Fusobacterium nucleatum clade dominates the colorectal cancer niche.

Fusobacterium nucleatum (Fn), a bacterium present in the human oral cavity and rarely found in the lower gastrointestinal tract of healthy individuals1, is enriched in human colorectal cancer (CRC) tumours2-5. High intratumoural Fn loads are associated with recurrence, metastases and poorer patient prognosis5-8. Here, to delineate Fn genetic factors facilitating tumour colonization, we generated closed genomes for 135 Fn strains; 80 oral strains from individuals without cancer and 55 unique cancer strains cultured from tumours from 51 patients with CRC. Pangenomic analyses identified 483 CRC-enriched genetic factors. Tumour-isolated strains predominantly belong to Fn subspecies animalis (Fna). However, genomic analyses reveal that Fna, considered a single subspecies, is instead composed of two distinct clades (Fna C1 and Fna C2). Of these, only Fna C2 dominates the CRC tumour niche. Inter-Fna analyses identified 195 Fna C2-associated genetic factors consistent with increased metabolic potential and colonization of the gastrointestinal tract. In support of this, Fna C2-treated mice had an increased number of intestinal adenomas and altered metabolites. Microbiome analysis of human tumour tissue from 116 patients with CRC demonstrated Fna C2 enrichment. Comparison of 62 paired specimens showed that only Fna C2 is tumour enriched compared to normal adjacent tissue. This was further supported by metagenomic analysis of stool samples from 627 patients with CRC and 619 healthy individuals. Collectively, our results identify the Fna clade bifurcation, show that specifically Fna C2 drives the reported Fn enrichment in human CRC and reveal the genetic underpinnings of pathoadaptation of Fna C2 to the CRC niche.

Effect of the intratumoral microbiota on spatial and cellular heterogeneity in cancer.

The tumour-associated microbiota is an intrinsic component of the tumour microenvironment across human cancer types1,2. Intratumoral host-microbiota studies have so far largely relied on bulk tissue analysis1-3, which obscures the spatial distribution and localized effect of the microbiota within tumours. Here, by applying in situ spatial-profiling technologies4 and single-cell RNA sequencing5 to oral squamous cell carcinoma and colorectal cancer, we reveal spatial, cellular and molecular host-microbe interactions. We adapted 10x Visium spatial transcriptomics to determine the identity and in situ location of intratumoral microbial communities within patient tissues. Using GeoMx digital spatial profiling6, we show that bacterial communities populate microniches that are less vascularized, highly immuno­suppressive and associated with malignant cells with lower levels of Ki-67 as compared to bacteria-negative tumour regions. We developed a single-cell RNA-sequencing method that we name INVADEseq (invasion-adhesion-directed expression sequencing) and, by applying this to patient tumours, identify cell-associated bacteria and the host cells with which they interact, as well as uncovering alterations in transcriptional pathways that are involved in inflammation, metastasis, cell dormancy and DNA repair. Through functional studies, we show that cancer cells that are infected with bacteria invade their surrounding environment as single cells and recruit myeloid cells to bacterial regions. Collectively, our data reveal that the distribution of the microbiota within a tumour is not random; instead, it is highly organized in microniches with immune and epithelial cell functions that promote cancer progression.

Systematic evasion of the restriction-modification barrier in bacteria.

Bacteria that are recalcitrant to genetic manipulation using modern in vitro techniques are termed genetically intractable. Genetic intractability is a fundamental barrier to progress that hinders basic, synthetic, and translational microbiology research and development beyond a few model organisms. The most common underlying causes of genetic intractability are restriction-modification (RM) systems, ubiquitous defense mechanisms against xenogeneic DNA that hinder the use of genetic approaches in the vast majority of bacteria and exhibit strain-level variation. Here, we describe a systematic approach to overcome RM systems. Our approach was inspired by a simple hypothesis: if a synthetic piece of DNA lacks the highly specific target recognition motifs for a host's RM systems, then it is invisible to these systems and will not be degraded during artificial transformation. Accordingly, in this process, we determine the genome and methylome of an individual bacterial strain and use this information to define the bacterium's RM target motifs. We then synonymously eliminate RM targets from the nucleotide sequence of a genetic tool in silico, synthesize an RM-silent "SyngenicDNA" tool, and propagate the tool as minicircle plasmids, termed SyMPL (SyngenicDNA Minicircle Plasmid) tools, before transformation. In a proof-of-principle of our approach, we demonstrate a profound improvement (five orders of magnitude) in the transformation of a clinically relevant USA300 strain of Staphylococcus aureus This stealth-by-engineering SyngenicDNA approach is effective, flexible, and we expect in future applications could enable microbial genetics free of the restraints of restriction-modification barriers.

Modulation of infection-mediated migration of neutrophils and CXCR2 trafficking by osteopontin.

Osteopontin (OPN) is a pro-inflammatory protein that paradoxically protects against inflammation and bone destruction in a mouse model of endodontic infection. Here we have tested the hypothesis that this effect of OPN is mediated by effects on migration of innate immune cells to the site of infection. Using the air pouch as a model of endodontic infection in mice, we showed that neutrophil accumulation at the site of infection with a mixture of endodontic pathogens is significantly reduced in OPN-deficient mice. Reduced neutrophil accumulation in the absence of OPN was accompanied by an increase in bacterial load. OPN-deficiency did not affect neutrophil survival, CXCR2 ligand expression, or the production of inflammatory cytokines in the air pouch. In vitro, OPN enhanced neutrophil migration to CXCL1, whereas in vivo, inhibition of CXCR2 suppressed cellular infiltration in air pouches of infected wild-type mice by > 50%, but had no effect in OPN-deficient mice. OPN increased cell surface expression of CXCR2 on bone marrow neutrophils in an integrin-αv -dependent manner, and suppressed the internalization of CXCR2 in the absence of ligand. Together, these results support a model where the protective effect of OPN results from enhanced initial neutrophil accumulation at sites of infection resulting in optimal bacterial killing. We describe a novel mechanism for this effect of OPN: integrin-αv -dependent suppression of CXCR2 internalization in neutrophils, which increases the ability of these cells to migrate to sites of infection in response to CXCR2 ligands.

Pathogenic bacterial species associated with endodontic infection evade innate immune control by disabling neutrophils.

Endodontic infections, in which oral bacteria access the tooth pulp chamber, are common and do not resolve once established. To investigate the effects of these infections on the innate immune response, we established a mouse subcutaneous chamber model, where a mixture of four oral pathogens commonly associated with these infections (endodontic pathogens [EP]), i.e., Fusobacterium nucleatum, Streptococcus intermedius, Parvimonas micra, and Prevotella intermedia, was inoculated into subcutaneously implanted titanium chambers. Cells that infiltrated the chamber after these infections were primarily neutrophils; however, these neutrophils were unable to control the infection. Infection with a nonpathogenic oral bacterial species, Streptococcus mitis, resulted in well-controlled infection, with bacterial numbers reduced by 4 to 5 log units after 7 days. Propidium iodide (PI) staining of the chamber neutrophils identified three distinct populations: neutrophils from EP-infected chambers were intermediate in PI staining, while cells in chambers from mice infected with S. mitis were PI positive (apoptotic) or negative (live). Strikingly, neutrophils from EP-infected chambers were severely impaired in their ability to phagocytose and to generate reactive oxygen species in vitro after removal from the chamber compared to cells from S. mitis-infected chambers. The mechanism of neutrophil impairment was necrotic cell death as determined by morphological analyses. P. intermedia alone could induce a similar neutrophil phenotype. We conclude that the endodontic pathogens, particularly P. intermedia, can efficiently disable and kill infiltrating neutrophils, allowing these infections to become established. These results can help explain the persistence of endodontic infections and demonstrate a new virulence mechanism associated with P. intermedia.

Context, engagement, and the (multiple) functions of negativity bias.

Hibbing and colleagues argue that political attitudes may be rooted in individual differences in negativity bias. Here, we highlight the complex, conditional nature of the relationship between negativity bias and ideology by arguing that the political impact of negativity bias should vary as a function of (1) issue domain and (2) political engagement.

Bacteria-derived L-lactate fuels cervical cancer chemoradiotherapy resistance.

Accumulating studies have demonstrated the presence of viable and metabolically active bacterial communities within a range of solid tumor types. However, the precise mechanisms by which these microbes modulate their infected tumor niches or impact patient responses to cancer treatments remain to be elucidated. Recently, Colbert et al. revealed that L-lactate produced by intratumoral Lactobacillus iners reprograms metabolic capabilities of cervical tumors to support chemoradiotherapy resistance. This finding has implications for many solid cancer types.

Quadriceps composition and function influence downhill gait biomechanics >1 year following anterior cruciate ligament reconstruction.

BACKGROUND: Quadriceps dysfunction is common following anterior cruciate ligament reconstruction and contributes to aberrant gait biomechanics. Changes in quadriceps composition also occur in these patients including greater concentrations of non-contractile tissue. The purpose of this study was to evaluate associations between quadriceps composition, function, and gait biomechanics in individuals with anterior cruciate ligament reconstruction. METHODS: Forty-eight volunteers with anterior cruciate ligament reconstruction completed gait biomechanics and quadriceps function and composition assessments. Gait biomechanics were sampled during downhill walking (-10° slope) on an instrumented treadmill. Quadriceps function (peak torque and rate of torque development) was assessed via maximal isometric contractions, while composition was evaluated via ultrasound echo intensity. FINDINGS: Greater quadriceps peak torque was associated with a greater peak knee extension moment (r = 0.365, p = 0.015). Greater vastus lateralis echo intensity (i.e. poorer muscle quality) was associated with less knee flexion displacement (r = -0.316, p = 0.032). Greater echo intensity of the vastus lateralis (r = -0.298, p = 0.044) and rectus femoris (r = -0.322, p = 0.029) was associated with a more abducted knee angle at heel strike. Quadriceps peak torque explained 11-16% of the variance in echo intensity. INTERPRETATION: Both quadriceps function and composition influence aberrant gait biomechanics following anterior cruciate ligament reconstruction. Quadriceps composition appears to provide insight into quadriceps dysfunction independent of muscle strength, as they associated with different gait biomechanics outcomes and shared minimal variance. Future research is necessary to determine the influence of changes in quadriceps composition on joint health outcomes.

Longitudinal Changes in Quadriceps Morphology over the First 3 Months after Anterior Cruciate Ligament Reconstruction.

PURPOSE: Neuromuscular deficits and atrophy after anterior cruciate ligament reconstruction (ACLR) may be accompanied by changes in muscle composition and poor quadriceps muscle quality (QMQ). Quadriceps atrophy occurs after ACLR but improves within the first three postoperative months, yet this hypertrophy could be attributable to increases in noncontractile tissue (i.e., poor QMQ). The purposes of this study were to evaluate changes in QMQ after ACLR and to determine if changes in QMQ and cross-sectional area (CSA) occur in parallel or independently. METHODS: A longitudinal prospective cohort design was implemented to evaluate QMQ and CSA in 20 individuals with ACLR and 12 healthy controls. Participants completed three testing sessions (baseline/presurgery, 1 month, and 3 months) during which ultrasound images were obtained from the vastus lateralis (VL) and rectus femoris (RF). QMQ was calculated as the echo intensity (EI) of each image, with high EI representing poorer QMQ. Anatomical CSA was also obtained from each image. RESULTS: RF and VL EI were greater at 1 and 3 months in the ACLR limb compared with baseline and the contralateral limb and did not change between 1 and 3 months. VL and RF CSA in the ACLR limb were smaller at 1 and 3 months compared with the contralateral limb and controls (VL only) but increased from 1 to 3 months. Changes in QMQ and CSA were not correlated. CONCLUSIONS: QMQ declines within the first month after ACLR and does not improve by 3 months although hypertrophy occurs, suggesting that these morphological characteristics change independently after ACLR. Poorer QMQ represents greater concentration of noncontractile tissues within the muscle and potentially contributes to chronic quadriceps dysfunction observed after ACLR.

Immediate Effects of Walking With a Knee Brace After Anterior Cruciate Ligament Reconstruction: A Biomechanical, Biochemical, and Structural Approach.

CONTEXT: Individuals who undergo anterior cruciate ligament reconstruction (ACLR) are at higher risk of posttraumatic osteoarthritis. Altered joint tissue loading caused by aberrant gait biomechanics leads to deleterious changes in joint health linked to the onset of posttraumatic osteoarthritis. Knee braces have been used to modify joint tissue loading in individuals with joint injury, yet the effects of walking with a brace after ACLR on biomechanical, biochemical, and structural cartilage outcomes are unknown. OBJECTIVE: To compare biomechanical, biochemical, and structural outcomes between braced and nonbraced walking in individuals with ACLR. DESIGN: Crossover study. SETTING: Research laboratory. PATIENTS OR OTHER PARTICIPANTS: A total of 34 individuals with unilateral ACLR (18 females, 16 males; time since ACLR = 50.1 ± 36.8 months). INTERVENTION(S): Gait biomechanics were assessed during braced and unbraced conditions on separate days. MAIN OUTCOME MEASURE(S): Vertical ground reaction force, knee-flexion angle, and internal knee-extension moment waveforms were evaluated throughout the stance phase and compared between conditions. Percentage changes in serum cartilage oligomeric matrix protein (%ΔCOMP) and femoral cartilage cross-sectional area (%ΔCSA) measured via ultrasound were calculated after a 3000-step walking protocol. RESULTS: Braced walking increased the knee-flexion angle (largest difference = 3.56°; Cohen d effect size = 1.72) and knee-extension moment (largest difference = -0.48% body weight × height; Cohen d effect size = -1.14) compared with nonbraced walking but did not influence vertical ground reaction force. Whereas no difference (P = .20) in %ΔCOMP existed between the braced and nonbraced conditions in the entire cohort (n = 30 with complete blood data), a larger increase (P = .04) in %ΔCOMP was seen during nonbraced than braced walking in individuals who demonstrated increased COMP during nonbraced walking. No difference (P = .86) in %ΔCSA was present between the braced and nonbraced conditions. CONCLUSIONS: Braced walking may improve sagittal-plane gait biomechanics and %ΔCOMP in a subset of individuals who demonstrate a typical increased COMP response to load (ie, increase in COMP) after nonbraced walking.

Expanding the rumen Prevotella collection: The description of Prevotella communis, sp. nov. of ovine origin.

27 strains representing eight new Prevotella species were isolated from rumen of a single sheep in eight weeks interval. One of the putative species encompassing the highest number of isolated strains which also exhibited some genetic variability in preliminary data, was then selected for description of a novel species. We examined six strains in genomic and phenotypic detail, two of which may actually be the same strain isolated nearly three weeks apart. Other strains formed clearly diverged intraspecies lineages as evidenced by core genome phylogeny and phenotypic differences. Strains of the proposed new Prevotella species are strictly saccharolytic as is usual for rumen Prevotella, and use plant cell-wall xylans and pectins for growth. However, the range of cell-wall polysaccharides utilised for growth is rather limited compared to rumen generalists such as Prevotella bryantii or Prevotella ruminicola and this extends also to the inability to utilise starch, which is unexpected for the members of the genus Prevotella. Based on the data obtained, we propose Prevotella communis sp. nov. to accommodate strain E1-9T as well as other strains with the similar properties. The proposed species is widespread: two other strains were previously isolated from sheep in Japan and is also common in metagenomic data of cattle and sheep rumen samples from Scotland and New Zealand. It was also found in a collection of metagenome-assembled genomes originating from cattle in Scotland. Thus, it is a ubiquitous bacterium of domesticated ruminants specialising in degradation of a somewhat restricted set of plant cell wall components.

Enhanced transformation efficiency in Treponema denticola enabled by SyngenicDNA-based plasmids lacking restriction-modification target motifs.

Oral spirochetes are among a small group of keystone pathogens contributing to dysregulation of tissue homeostatic processes that leads to breakdown of the tissue and bone supporting the teeth in periodontal disease. Additionally, our group has recently demonstrated that Treponema are among the dominant microbial genera detected intracellularly in tumor specimens from patients with oral squamous cell carcinoma. While over 60 species and phylotypes of oral Treponema have been detected, T. denticola is one of the few that can be grown in culture and the only one in which genetic manipulation is regularly performed. Thus, T. denticola is a key model organism for studying spirochete metabolic processes, interactions with other microbes, and host cell and tissue responses relevant to oral diseases, as well as venereal and nonvenereal treponematoses whose agents lack workable genetic systems. We previously demonstrated improved transformation efficiency using an Escherichia coli-T. denticola shuttle plasmid and its utility for expression in T. denticola of an exogenous fluorescent protein that is active under anaerobic conditions. Here, we expand on this work by characterizing T. denticola Type I and Type II restriction-modification (R-M) systems and designing a high-efficiency R-M-silent "SyngenicDNA" shuttle plasmid resistant to all T. denticola ATCC 35405 R-M systems. Resequencing of the ATCC 33520 genome revealed an additional Type I R-M system consistent with the relatively low transformation efficiency of the shuttle plasmid in this strain. Using SyngenicDNA approaches, we optimized shuttle plasmid transformation efficiency in T. denticola and used it to complement a defined T. denticola ΔfhbB mutant strain. We further report the first high-efficiency transposon mutagenesis of T. denticola using an R-M-silent, codon-optimized, himarC9 transposase-based plasmid. Thus, use of SyngenicDNA-based strategies and tools can enable further mechanistic examinations of T. denticola physiology and behavior.

INVADEseq to identify cell-adherent or invasive bacteria and the associated host transcriptome at single-cell-level resolution.

Single-cell RNA sequencing (scRNAseq) technologies have been beneficial in revealing and describing cellular heterogeneity within mammalian tissues, including solid tumors. However, many of these techniques apply poly(A) selection of RNA, and thus have primarily focused on determining the gene signatures of eukaryotic cellular components of the tumor microenvironment. Microbiome analysis has revealed the presence of microbial ecosystems, including bacteria and fungi, within human tumor tissues from major cancer types. Imaging data have revealed that intratumoral bacteria may be located within epithelial and immune cell types. However, as bacterial RNA typically lacks a poly(A) tail, standard scRNAseq approaches have limited ability to capture this microbial component of the tumor microenvironment. To overcome this, we describe the invasion-adhesion-directed expression sequencing (INVADEseq) approach, whereby we adapt 10x Genomics 5' scRNAseq protocol by introducing a primer that targets a conserved region of the bacterial 16S ribosomal RNA gene in addition to the standard primer for eukaryotic poly(A) RNA selection. This 'add-on' approach enables the generation of eukaryotic and bacterial DNA libraries at eukaryotic single-cell level resolution, utilizing the 10x barcode to identify single cells with intracellular bacteria. The INVADEseq method takes 30 h to complete, including tissue processing, sequencing and computational analysis. As an output, INVADEseq has shown to be a reliable tool in human cancer cell lines and patient tumor specimens by detecting the proportion of human cells that harbor bacteria and the identities of human cells and intracellular bacteria, along with identifying host transcriptional programs that are modulated on the basis of associated bacteria.

Fusobacterium sphaericum sp. nov. , isolated from a human colon tumor, is prevalent in various human body sites and induces IL-8 secretion from colorectal cancer cells.

Cancerous tissue is a largely unexplored microbial niche that provides a unique environment for the colonization and growth of specific bacterial communities, and with it, the opportunity to identify novel bacterial species. Here, we report distinct features of a novel Fusobacterium species, F. sphaericum sp. nov. ( Fs ), isolated from primary colon adenocarcinoma tissue. We acquire the complete, closed genome of this organism and phylogenetically confirm its classification into the Fusobacterium genus. Phenotypic and genomic analysis of Fs reveal that this novel organism is of coccoid shape, rare for Fusobacterium members, and has species-distinct gene content. Fs displays a metabolic profile and antibiotic resistance repertoire consistent with other Fusobacterium species. In vitro, Fs has adherent and immunomodulatory capabilities, as it intimately associates with human colon cancer epithelial cells and promotes IL-8 secretion. Analysis of the prevalence and abundance of Fs in ∼1,750 human metagenomic samples shows that it is a moderately prevalent member of the human oral cavity and stool. Intriguingly, analysis of ∼1,270 specimens from patients with colorectal cancer demonstrate that Fs is significantly enriched in colonic and tumor tissue as compared to mucosa or feces. Our study sheds light on a novel bacterial species that is prevalent within the human intestinal microbiota and whose role in human health and disease requires further investigation.

Negativity bias, personality and political ideology.

Research suggests that right-wing ideology is associated with negativity bias: a tendency to pay more attention and give more weight to negative versus positive stimuli. This work typically relies on either self-reported traits related to negativity bias in large, often-representative, samples or physiological and behavioural indicators of negativity bias in small convenience samples. We extend this literature and examine the relationship of negativity bias to political ideology using five distinct behavioural measures of negativity bias in four national samples of US residents with a total analytical sample size of about 4,000 respondents. We also examine the association of these behavioural measures to four of the most common self-report measures of personality in the literature on ideology. Across a wide range of tests, we find no consistent evidence for a relationship of negativity bias to either ideology or self-reported personality.

Complete Genome Sequence of Morganella morganii CTX51T, Isolated from a Human Cecal Adenocarcinoma.

We report the complete genome sequence of Morganella morganii CTX51T, a strain isolated from the resected tumor of a patient with cecal colorectal adenocarcinoma of the cecum. The genome comprises a circular chromosome of 4.19 Mbp, with an overall GC content of 50.4% and one circular plasmid of 8.48 kbp.

Complete Genome Sequence of Clostridium cadaveris IFB3C5, Isolated from a Human Colonic Adenocarcinoma.

We report the complete genome sequence of Clostridium cadaveris IFB3C5, a strain isolated from the resected tumor of a treatment naive colorectal cancer patient. This genome is comprised of a singular chromosome of approximately 3.63 Mbp in length, contains two plasmids, and has an overall mean GC content of 31.7%.

The cancer chemotherapeutic 5-fluorouracil is a potent Fusobacterium nucleatum inhibitor and its activity is modified by intratumoral microbiota.

Fusobacterium nucleatum (Fn) is a dominant bacterial species in colorectal cancer (CRC) tissue that is associated with cancer progression and poorer patient prognosis. Following a small-molecule inhibitor screen of 1,846 bioactive compounds against a Fn CRC isolate, we find that 15% of inhibitors are antineoplastic agents including fluoropyrimidines. Validation of these findings reveals that 5-fluorouracil (5-FU), a first-line CRC chemotherapeutic, is a potent inhibitor of Fn CRC isolates. We also identify members of the intratumoral microbiota, including Escherichia coli, that are resistant to 5-FU. Further, CRC E. coli isolates can modify 5-FU and relieve 5-FU toxicity toward otherwise-sensitive Fn and human CRC epithelial cells. Lastly, we demonstrate that ex vivo patient CRC tumor microbiota undergo community disruption after 5-FU exposure and have the potential to deplete 5-FU levels, reducing local drug efficacy. Together, these observations argue for further investigation into the role of the CRC intratumoral microbiota in patient response to chemotherapy.

A bacterial pan-genome makes gene essentiality strain-dependent and evolvable.

Many bacterial species are represented by a pan-genome, whose genetic repertoire far outstrips that of any single bacterial genome. Here we investigate how a bacterial pan-genome might influence gene essentiality and whether essential genes that are initially critical for the survival of an organism can evolve to become non-essential. By using Transposon insertion sequencing (Tn-seq), whole-genome sequencing and RNA-seq on a set of 36 clinical Streptococcus pneumoniae strains representative of >68% of the species' pan-genome, we identify a species-wide 'essentialome' that can be subdivided into universal, core strain-specific and accessory essential genes. By employing 'forced-evolution experiments', we show that specific genetic changes allow bacteria to bypass essentiality. Moreover, by untangling several genetic mechanisms, we show that gene essentiality can be highly influenced by and/or be dependent on: (1) the composition of the accessory genome, (2) the accumulation of toxic intermediates, (3) functional redundancy, (4) efficient recycling of critical metabolites and (5) pathway rewiring. While this functional characterization underscores the evolvability potential of many essential genes, we also show that genes with differential essentiality remain important antimicrobial drug target candidates, as their inactivation almost always has a severe fitness cost in vivo.

Fewer daily steps are associated with greater cartilage oligomeric matrix protein response to loading post-ACL reconstruction.

Aberrant joint loading contributes to the development of posttraumatic knee osteoarthritis (PTOA) following anterior cruciate ligament reconstruction (ACLR); yet little is known about the association between joint loading due to daily walking and cartilage health post-ACLR. Accelerometer-based measures of daily steps and cadence (i.e., rate of steps/min) provide information regarding daily walking in a real-world setting. The purpose of this study was to determine the association between changes in serum cartilage oligomeric matrix protein (COMP; %∆COMP), a mechanosensitive biomarker that is associated with osteoarthritis progression, following a standardized walking protocol and daily walking in individuals with ACLR and uninjured controls. Daily walking was assessed over 7 days using an accelerometer worn on the right hip in 31 individuals with ACLR and 21 controls and quantified as mean steps/day and time spent in ≥100 steps/min. Serum COMP was measured before and following a 3000-step walking protocol at a preferred speed. %∆COMP was calculated as a change in COMP relative to the prewalking value. Linear regressions were used to examine associations between daily walking and %∆COMP after adjusting for preferred speed. Fewer daily steps (ΔR2 = 0.18, p = 0.02) and fewer minutes spent in ≥100 steps/min (ΔR2 = 0.16, p = 0.03) were associated with greater %∆COMP following walking in individuals with ACLR; no statistically significant associations existed in controls (daily steps: ΔR2 = 0.03, p = 0.47; time ≥100 steps/min: ΔR2 < 0.01, p = 0.81). Clinical significance: Individuals with ACLR who engage in less daily walking undergo greater %ΔCOMP, which may represent greater cartilage degradation or turnover in response to walking.