First draft reference genome and annotation of the alternative oil species Physaria fendleri.

In the wake of increasing demand for renewable energy sources, plant-based sources including alternative oilseeds have come to the forefront of interest. Hydroxy fatty acids (HFAs), produced in a few oilseed species, are important chemical feedstocks for industrial applications. An integrated approach was taken to assemble the first draft genome of the alternative HFA producer Physaria fendleri (n = 6), an outcrossing species with high heterozygosity. Both de novo transcriptome assemblies and genome assemblies were produced with public and generated sequencing reads. Resulting intermediate assemblies were then scaffolded and patched with multiple data sources, followed by super-scaffolding onto a masked genome of Camelina laxa (n = 6). Despite a current lack of available resources for the physical mapping of genomic scaffolds of Physaria fendleri, topography of the genome with respect to repeat and gene content was preserved at the scaffold level and not significantly lost via super-scaffolding. Read representation, gene and genome completion statistics, and annotation results illustrated the creation of a functional draft genome and a tool for future research on alternative oil species.

Investigating skeletal muscle biomarkers for the early detection of Australian myotoxic snake envenoming: an animal model pilot study.

INTRODUCTION: Myotoxicity is an important toxidrome that can occur with envenoming from multiple Australian snake types. Early antivenom administration is an important strategy to reduce the incidence and severity of myotoxicity. The current gold standard biomarker, serum creatine kinase activity, does not rise early enough to facilitate early antivenom administration. Several other skeletal muscle biomarkers have shown promise in other animal models and scenarios. The aim of this study was to examine the predictive values of six skeletal muscle biomarkers in a rat model of Australian snake myotoxicity. METHODS: Sprague-Dawley rats were anaesthetised and administered either Pseudechis porphyriacus (red-bellied black snake) or Notechis scutatus (tiger snake) venom, or normal saline via intramuscular injection. Blood samples were collected. Assays were performed for serum creatine kinase skeletal muscle troponin-I concentration, skeletal muscle troponin-C concentration, myoglobin activity, skeletal muscle myosin light chain-1 concentration, and creatine kinase-MM activity. Serum markers were plotted against time, with comparison of area under the concentration (or activity)-time curve. The predictive values of six skeletal muscle biomarkers were examined using receiver operating characteristic curves. RESULTS: There was no difference in area under the serum creatine kinase activity-time curve between venom and control groups. Serum creatine kinase-MM activity rose early in the venom treated rats, which had a significantly greater area under the serum activity-time curve. No difference in area under the serum concentration-time curve was demonstrated for the other biomarkers. Creatine kinase-MM activity had a superior predictive values than creatine kinase activity at 0-4 hours and 0-10 hours after venom administration, as indicated by area under the receiver operating characteristic curves (95 per cent confidence intervals) of 0.91 (0.78-1.00) and 0.88 (0.73-1.00) versus 0.79 (0.63-0.95) and 0.66 (0.51-0.80). DISCUSSION: The limitations of serum creatine kinase activity in early detection of myotoxicity were demonstrated in this rat model. CONCLUSION: Serum creatine kinase-MM activity was superior for early detection of Australian myotoxic snake envenoming.

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.

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.

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.

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.

Training Load and Current Soreness Predict Future Delayed Onset Muscle Soreness in Collegiate Female Soccer Athletes.

Background: Delayed onset muscles soreness (DOMS) is an indication of muscle stress and trauma that develops from excessive musculoskeletal loads. Musculoskeletal loads can be measured with wearable devices, but there is limited research on specific training load metrics that most correlate with DOMS after activity. Purpose: To determine the predictive capabilities of training load variables on the development of lower extremity DOMS in female collegiate soccer athletes throughout an entire season. Study Design: Prospective Cohort. Methods: Twenty-seven collegiate female soccer athletes reported their lower extremity DOMS each day prior to all soccer activity. Participants wore Polar heart rate and global positioning monitors to capture training load measures. Pearson correlation coefficients were used to assess the relationships between the training load variables and change in DOMS when collapsed across dates. Separate linear mixed models were performed with the following day's DOMS as the outcome variable, training load and the current day's DOMS as predictor variables, and participants serving as random intercepts. Results: All training load variables significantly predicted change in DOMS, with number of decelerations (ρ=0.72, p <0.001), minutes spent at greater than 80% of maximum heart rate (HRmax) (ρ=0.71 , p <0.001), and distance (ρ=0.70 , p <0.001) best correlating with change in DOMS. Linear mixed models revealed a significant interaction of all training load and current day's DOMS on the following day's DOMS (p<0.001), but number of decelerations, HRmax, and total number of accelerations demonstrated the highest coefficient of determination (R2 marginal=33.2% - 29.2% , R2 conditional= 46.9% - 44.8%). Conclusions: Training load variables paired with the current day's DOMS significantly predict lower extremity DOMS in the future, with number of decelerations, accelerations, and HRmax best predicting future DOMS. Although this demonstrates that training load variables predict lower extremity DOMS, future research should incorporate objective measures of strength or jump kinetics to identify if similar relationships exist. Level of Evidence: Level 3.

Phase Separation as a Driver of Stem Cell Organization and Function during Development.

A properly organized subcellular composition is essential to cell function. The canonical organizing principle within eukaryotic cells involves membrane-bound organelles; yet, such structures do not fully explain cellular complexity. Furthermore, discrete non-membrane-bound structures have been known for over a century. Liquid-liquid phase separation (LLPS) has emerged as a ubiquitous mode of cellular organization without the need for formal lipid membranes, with an ever-expanding and diverse list of cellular functions that appear to be regulated by this process. In comparison to traditional organelles, LLPS can occur across wider spatial and temporal scales and involves more distinct protein and RNA complexes. In this review, we discuss the impacts of LLPS on the organization of stem cells and their function during development. Specifically, the roles of LLPS in developmental signaling pathways, chromatin organization, and gene expression will be detailed, as well as its impacts on essential processes of asymmetric cell division. We will also discuss how the dynamic and regulated nature of LLPS may afford stem cells an adaptable mode of organization throughout the developmental time to control cell fate. Finally, we will discuss how aberrant LLPS in these processes may contribute to developmental defects and disease.

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.

Drosophila Adducin facilitates phase separation and function of a conserved spindle orientation complex.

Asymmetric cell division (ACD) allows stem cells to generate differentiating progeny while simultaneously maintaining their own pluripotent state. ACD involves coupling mitotic spindle orientation with cortical polarity cues to direct unequal segregation of cell fate determinants. In Drosophila neural stem cells (neuroblasts; NBs), spindles orient along an apical-basal polarity axis through a conserved complex of Partner of Inscuteable (Pins; human LGN) and Mushroom body defect (Mud; human NuMA). While many details of its function are well known, the molecular mechanics that drive assembly of the cortical Pins/Mud complex remain unclear, particularly with respect to the mutually exclusive Pins complex formed with the apical scaffold protein Inscuteable (Insc). Here we identify Hu li tai shao (Hts; human Adducin) as a direct Mud-binding protein, using an aldolase fold within its head domain (HtsHEAD) to bind a short Mud coiled-coil domain (MudCC) that is adjacent to the Pins-binding domain (MudPBD). Hts is expressed throughout the larval central brain and apically polarizes in mitotic NBs where it is required for Mud-dependent spindle orientation. In vitro analyses reveal that Pins undergoes liquid-liquid phase separation with Mud, but not with Insc, suggesting a potential molecular basis for differential assembly mechanics between these two competing apical protein complexes. Furthermore, we find that Hts binds an intact Pins/Mud complex, reduces the concentration threshold for its phase separation, and alters the liquid-like property of the resulting phase separated droplets. Domain mapping and mutational analyses implicate critical roles for both multivalent interactions (via MudCC oligomerization) and protein disorder (via an intrinsically disordered region in Hts; HtsIDR) in phase separation of the Hts/Mud/Pins complex. Our study identifies a new component of the spindle positioning machinery in NBs and suggests that phase separation of specific protein complexes might regulate ordered assembly within the apical domain to ensure proper signaling output.

Modeling a variant of unknown significance in the Drosophila ortholog of the human cardiogenic gene NKX2.5.

Sequencing of human genome samples has unearthed genetic variants for which functional testing is necessary to validate their clinical significance. We used the Drosophila system to analyze a variant of unknown significance in the human congenital heart disease gene NKX2.5 (also known as NKX2-5). We generated an R321N allele of the NKX2.5 ortholog tinman (tin) to model a human K158N variant and tested its function in vitro and in vivo. The R321N Tin isoform bound poorly to DNA in vitro and was deficient in activating a Tin-dependent enhancer in tissue culture. Mutant Tin also showed a significantly reduced interaction with a Drosophila T-box cardiac factor named Dorsocross1. We generated a tinR321N allele using CRISPR/Cas9, for which homozygotes were viable and had normal heart specification, but showed defects in the differentiation of the adult heart that were exacerbated by further loss of tin function. We propose that the human K158N variant is pathogenic through causing a deficiency in DNA binding and a reduced ability to interact with a cardiac co-factor, and that cardiac defects might arise later in development or adult life.

Using Drosophila to model a variant of unknown significance in the human cardiogenic gene Nkx2.5.

Sequencing of human genome samples has unearthed genetic variants for which functional testing is necessary to validate their clinical significance. We used the Drosophila system to analyze a variant of unknown significance in the human congenital heart disease gene, Nkx2 . 5 . We generated an R321N allele of the Nkx2 . 5 ortholog tinman ( tin ) to model a human K158N variant and tested its function in vitro and in vivo. The R321N Tin isoform bound poorly to DNA in vitro and was deficient in activating a Tin-dependent enhancer in tissue culture. Mutant Tin also showed a significantly reduced interaction with a Drosophila Tbox cardiac factor named Dorsocross1. We generated a tin R321N allele using CRISPR/Cas9, for which homozygotes were viable and had normal heart specification, but showed defects in the differentiation of the adult heart that were exacerbated by further loss of tin function. We conclude that the human K158N mutation is likely pathogenic through causing both a deficiency in DNA binding and a reduced ability to interact with a cardiac cofactor, and that cardiac defects might arise later in development or adult life.

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.

Optimising mechanical separation of anaerobic digestate for total solids and nutrient removal.

Mechanical separation of anaerobic digestate has been identified as a method to reduce pollution risk to waterways by partitioning phosphorus in the solid fraction and reducing its application to land. Separators have adjustable parameters which affect separation efficiency, and hence the degree of phosphorous partitioning, but information on how these parameters affect separation performance is limited in the literature. Two well known technologies were investigated, decanter centrifuge and screw press, to determine the most efficient method of separation. Counterweight load and the use of an oscillator were adjusted for the screw press, while bowl speed, auger differential speed, feed rate and polymer addition were modified for the decanter centrifuge. Separation efficiency was determined for total solids, phosphorus, nitrogen, potassium, and carbon, and the total solids content of resulting fractions was measured. The decanter centrifuge had higher separation efficiency for phosphorus in all cases, ranging from 51% to 71.5%, while the screw press had a phosphorus separation efficiency ranging from 8.5% to 10.9% for digestate of ∼5% solids (slurry/grass silage mix). Separation by decanter centrifuge partitioned up to 56% of nitrogen in the solid fraction leaving a reduced nitrogen content in the liquid fraction available for land spreading; this nitrogen would most likely need to be replaced by chemical fertiliser which would add to the cost of the system. The decanter centrifuge is better suited to cases where phosphorus recovery is the most important factor, while the screw press could be advantageous in cases where cost is a limiting factor.

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.

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.

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.

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.