Bacteroides ovatus alleviates dysbiotic microbiota-induced graft-versus-host disease.

Acute lower gastrointestinal GVHD (aLGI-GVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation. Although the intestinal microbiota is associated with the incidence of aLGI-GVHD, how the intestinal microbiota impacts treatment responses in aLGI-GVHD has not been thoroughly studied. In a cohort of patients with aLGI-GVHD (n = 37), we found that non-response to standard therapy with corticosteroids was associated with prior treatment with carbapenem antibiotics and a disrupted fecal microbiome characterized by reduced abundances of Bacteroides ovatus. In a murine GVHD model aggravated by carbapenem antibiotics, introducing B. ovatus reduced GVHD severity and improved survival. These beneficial effects of Bacteroides ovatus were linked to its ability to metabolize dietary polysaccharides into monosaccharides, which suppressed the mucus-degrading capabilities of colonic mucus degraders such as Bacteroides thetaiotaomicron and Akkermansia muciniphila, thus reducing GVHD-related mortality. Collectively, these findings reveal the importance of microbiota in aLGI-GVHD and therapeutic potential of B. ovatus.

Continued development of a tool to measure intramuscular fat of Australian lamb carcases in commercial situations.

Despite the importance of intramuscular fat (IMF) to eating quality, as yet no methodology has been widely adopted by the whole of industry in Australia to measure it routinely. Thus, a study was conducted to investigate the potential for a Near Infra-Red (NIR) device to predict the IMF content of the loin from spectra collected on the topside which is externally located on a hanging carcase and therefore easily accessible. To this end, NIR spectra were collected from topsides (m. semimembranosus) of 258 lamb carcases over 5 data collections and a sample of muscle was collected from the loin and the topside for IMF determination using a wet chemistry method. Subsequent Partial Least Square (PLS) models suggested the ability to predict the absolute IMF content of loins was poor (R2 = 0.28, RMSE = 1.26), yet there was a moderate ability to predict the IMF content of the topside (R2 = 0.56, RMSE = 0.82). Partial Least Square Discrimination Analysis (PLS-DA) models to classify cuts based on the IMF eating quality threshold of 4.5% yielded better predictive outcomes with accuracies of 66.7% and 76.7% for loin and topside respectively. However, further research to assess the relationship between the IMF of the loin and topside and reduce the impact of differences in overall absorbance between data collections will improve predictive outcomes.

Validation of Raman spectroscopic models to verify the origin of Australian beef grown under different production systems.

Verification of beef production systems and authentication of origin is becoming increasingly important as consumers base purchase decisions on a greater number of perceived values including the healthiness and environmental impact of products. Previously Raman spectroscopy has been explored as a tool to classify carcases from grass and grain fed cattle. Thus, the aim of the current study was to validate Partial Least Squares Discriminant Analysis (PLS-DA) models created using independent samples from carcases sampled from northern and southern Australian production systems in 2019, 2020 and 2021. Validation of the robustness of discrimination models was undertaken using spectral measures of fat from 585 carcases which were measured in 2022 using a Raman handheld device with a sample excised for fatty acid analysis. PLS-DA models were constructed and then employed to classify samples as either grass or grain fed in a two-class model. Overall, predictions were high with accuracies of up to 95.7% however, variation in the predictive ability was noted with models created for southern cattle yielding an accuracy of 73.2%. While some variation in fatty acids and therefore models can be attributed to differences in genetics, management and diet, the impact of duration of feeding is currently unknown and thus further work is warranted.

Vaginal microbial dynamics and pathogen colonization in a humanized microbiota mouse model.

Vaginal microbial composition is associated with differential risk of urogenital infection. Although Lactobacillus spp. are thought to confer protection against infection, the lack of in vivo models resembling the human vaginal microbiota remains a prominent barrier to mechanistic discovery. Using 16S rRNA amplicon sequencing of C57BL/6J female mice, we found that vaginal microbial composition varies within and between colonies across three vivaria. Noting vaginal microbial plasticity in conventional mice, we assessed the vaginal microbiome of humanized microbiota mice (HMbmice). Like the community structure in conventional mice, HMbmice vaginal microbiota clustered into community state types but, uniquely, HMbmice communities were frequently dominated by Lactobacillus or Enterobacteriaceae. Compared to conventional mice, HMbmice were less susceptible to uterine ascension by urogenital pathobionts group B Streptococcus (GBS) and Prevotella bivia. Although Escherichia and Lactobacillus both correlated with the absence of uterine GBS, vaginal pre-inoculation with exogenous HMbmouse-derived E. coli, but not Ligilactobacillus murinus, reduced vaginal GBS burden. Overall, HMbmice serve as a useful model to elucidate the role of endogenous microbes in conferring protection against urogenital pathogens.

Site-specific pathophysiology in a neonatal mouse model of gastroparesis.

BACKGROUND: Early-life events impact maturation of the gut microbiome, enteric nervous system, and gastrointestinal motility. We examined three regions of gastric tissue to determine how maternal separation and gut microbes influence the structure and motor function of specific regions of the neonatal mouse stomach. METHODS: Germ-free and conventionally housed C57BL/6J mouse pups underwent timed maternal separation (TmSep) or nursed uninterrupted (controls) until 14 days of life. We assessed gastric emptying by quantifying the progression of gavaged fluorescein isothiocyanate (FITC)-dextran. With isolated rings of forestomach, corpus, and antrum, we measured tone and contractility by force transduction, gastric wall thickness by light microscopy, and myenteric plexus neurochemistry by whole-mount immunostaining. KEY RESULTS: Regional gastric sampling revealed site-specific differences in contractile patterns and myenteric plexus structure. In neonatal mice, TmSep prolonged gastric emptying. In the forestomach, TmSep increased contractile responses to carbachol, decreased muscularis externa and mucosa thickness, and increased the relative proportion of myenteric plexus nNOS+ neurons. Germ-free conditions did not appreciably alter the structure or function of the neonatal mouse stomach and did not impact the changes caused by TmSep. CONCLUSIONS AND INFERENCES: A regional sampling approach facilitates site-specific investigations of murine gastric motor physiology and histology to identify site-specific alterations that may impact gastrointestinal function. Delayed gastric emptying in TmSep is associated with a thinner muscle wall, exaggerated cholinergic contractile responses, and increased proportions of inhibitory myenteric plexus nNOS+ neurons in the forestomach. Gut microbes do not profoundly affect the development of the neonatal mouse stomach or the gastric pathophysiology that results from TmSep.

Internal endpoint temperature (level of cooking doneness) effects on the fatty acid and mineral profiles of grilled lamb m. longissimus lumborum.

This study compared the fatty acid and mineral concentrations of lamb meat that was prepared to different levels of cooking doneness. Ten m. longissimus lumborum were each sectioned into 4 slices that were randomly assigned to be uncooked or grilled to an internal end-point temperature of 60 °C (rare), 71 °C (medium), or 77 °C (well done). It was found that cooking loss increased as the level of cooking doneness increased. The proportion of most major fatty acids were not altered by cooking. However, when adjusted for cooking loss (i.e., mg/135 g serve of lamb as-is equivalent prepared to each level of cooking doneness), the concentration of most major fatty acids, including C16:0, C18:0, C18:1n-9, and many conjugated linoleic acids, were lowered after grilling to any level of cooking doneness and compared to the uncooked samples. The omega-6 to omega-3 ratio was lowest for the uncooked samples and highest for those prepared to a well done level of cooking doneness. Conversely, the concentration of health claimable omega-3 fatty acids in the uncooked meat was retained upon cooking and across all the different internal endpoint temperatures. Calcium, magnesium, phosphorous, potassium, and sodium were reduced with preparation of lamb meat to any level of cooking doneness, compared with uncooked meat. Zinc, iron, and selenium were retained within the cooked samples. These findings show that consumer preference for a level of cooking doneness will have only minor effects on the concentration of minerals and fatty acids in lamb meat.

Common Variable Immunodeficiency Patient Fecal Microbiota Transplant Recapitulates Gut Dysbiosis.

Purpose: Patients with non-infectious complications have worse clinical outcomes in common variable immunodeficiency (CVID) than those with infections-only. Non-infectious complications are associated with gut microbiome aberrations, but there are no reductionist animal models that emulate CVID. Our aim in this study was to uncover potential microbiome roles in the development of non-infectious complications in CVID. Methods: We examined fecal whole genome shotgun sequencing from patients CVID, and non-infectious complications, infections-only, and their household controls. We also performed Fecal Microbiota transplant from CVID patients to Germ-Free Mice. Results: We found potentially pathogenic microbes Streptococcus parasanguinis and Erysipelatoclostridium ramosum were enriched in gut microbiomes of CVID patients with non-infectious complications. In contrast, Fusicatenibacter saccharivorans and Anaerostipes hadrus, known to suppress inflammation and promote healthy metabolism, were enriched in gut microbiomes of infections-only CVID patients. Fecal microbiota transplant from non-infectious complications, infections-only, and their household controls into germ-free mice revealed gut dysbiosis patterns in recipients from CVID patients with non-infectious complications, but not infections-only CVID, or household controls recipients. Conclusion: Our findings provide a proof of concept that fecal microbiota transplant from CVID patients with non-infectious complications to Germ-Free mice recapitulates microbiome alterations observed in the donors.

Preliminary investigation into the prediction of indicators of beef spoilage using Raman and Fourier transform infrared spectroscopy.

The objective of this study was to assess the potential to predict the microbial beef spoilage indicators by Raman and Fourier transform infrared (FT-IR) spectroscopies. Vacuum skin packaged (VSP) beef steaks were stored at 0 °C, 4 °C, 8 °C and under a dynamic temperature condition (0 °C âˆ¼ 4 °C âˆ¼ 8 °C, for 36 d). Total viable count (TVC) and total volatile basic nitrogen (TVB-N) were obtained during the storage period along with spectroscopic data. The Raman and FTIR spectra were baseline corrected, pre-processed using Savitzky-Golay smoothing and normalized. Subsequently partial least squares regression (PLSR) models of TVC and TVB-N were developed and evaluated. The root mean squared error (RMSE) ranged from 0.81 to1.59 (log CFU/g or mg/100 g) and the determination coefficient (R2) from 0.54 to 0.75. The performance of PLSR model based on data fusion (combination of Raman and FT-IR data) is better than that based on Raman spectra and similar to that of FT-IR. Overall, Raman spectroscopy, FT-IR spectroscopy, and a combination of both exhibited a potential for the prediction of the beef spoilage.

Bacteroides ovatus alleviates dysbiotic microbiota-induced intestinal graft-versus-host disease.

Acute gastrointestinal intestinal GVHD (aGI-GVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation, and the intestinal microbiota is known to impact on its severity. However, an association between treatment response of aGI-GVHD and the intestinal microbiota has not been well-studied. In a cohort of patients with aGI-GVHD (n=37), we found that non-response to standard therapy with corticosteroids was associated with prior treatment with carbapenem antibiotics and loss of Bacteroides ovatus from the microbiome. In a mouse model of carbapenem-aggravated GVHD, introducing Bacteroides ovatus reduced severity of GVHD and improved survival. Bacteroides ovatus reduced degradation of colonic mucus by another intestinal commensal, Bacteroides thetaiotaomicron, via its ability to metabolize dietary polysaccharides into monosaccharides, which then inhibit mucus degradation by Bacteroides thetaiotaomicron and reduce GVHD-related mortality.

Diet-derived metabolites and mucus link the gut microbiome to fever after cytotoxic cancer treatment.

Not all patients with cancer and severe neutropenia develop fever, and the fecal microbiome may play a role. In a single-center study of patients undergoing hematopoietic cell transplant (n = 119), the fecal microbiome was characterized at onset of severe neutropenia. A total of 63 patients (53%) developed a subsequent fever, and their fecal microbiome displayed increased relative abundances of Akkermansia muciniphila, a species of mucin-degrading bacteria (P = 0.006, corrected for multiple comparisons). Two therapies that induce neutropenia, irradiation and melphalan, similarly expanded A. muciniphila and additionally thinned the colonic mucus layer in mice. Caloric restriction of unirradiated mice also expanded A. muciniphila and thinned the colonic mucus layer. Antibiotic treatment to eradicate A. muciniphila before caloric restriction preserved colonic mucus, whereas A. muciniphila reintroduction restored mucus thinning. Caloric restriction of unirradiated mice raised colonic luminal pH and reduced acetate, propionate, and butyrate. Culturing A. muciniphila in vitro with propionate reduced utilization of mucin as well as of fucose. Treating irradiated mice with an antibiotic targeting A. muciniphila or propionate preserved the mucus layer, suppressed translocation of flagellin, reduced inflammatory cytokines in the colon, and improved thermoregulation. These results suggest that diet, metabolites, and colonic mucus link the microbiome to neutropenic fever and may guide future microbiome-based preventive strategies.

There is no relationship between lamb particle size and consumer scores for tenderness, flavour, juiciness, overall liking or quality rank.

With the aim to define an objective threshold for consumer satisfaction, this study investigated the relationship between lamb particle size data and consumer scores for tenderness, juiciness, flavour and overall liking (sensorial properties). Data were sourced from the longissimus lumborum muscles of 273 Australian Merino lambs, these being aged for 5-d and then analysed for particle size and sensorial properties - the latter using untrained consumer sensory panels. Pearson's correlation and principal component analyses identified no significant relationship between particle size and consumer sensory scores. Linear regression models found the sensorial properties of lamb could not be predicted using particle size, indicating no univariate relationship. Further, a backwards stepwise regression analysis found there to be no multivariate or univariate relationship between the sensorial properties of lamb and its particle size. These findings demonstrate that there is little value in defining a particle size threshold for consumer satisfaction based on the sensorial properties of lamb.

Assessing chemometric models developed using Raman spectroscopy and fatty acid data for Northern and Southern Australian beef production systems.

A total of 960 beef carcases from northern and southern Australian production regions were assessed by examining the subcutaneous fat. Carcases from four different production systems within each region were assessed, by Raman spectroscopy and the fatty acid composition determined to develop models that best classified the various production systems. As a result, 12 Partial Least Square Discriminant Analysis models were developed. A two-class model based on fatty acid composition was able to correctly classify 99% of grass and grain fed animals. The best Raman spectroscopic model correctly classified 94% of grass vs grain carcases produced in the northern region. For the southern production region, the models had the following classification accuracies; southern long-term grain fed (98%), southern short-term grain fed (95%), southern grass (96%), southern grass supplemented (97%), and the southern model classified grass vs grain (97%). Raman spectroscopy is considered a useful rapid method for classification of beef carcases based upon production system.

The effect of perennial and annual wheat forages, fed with or without lucerne, on the fatty acid profile and oxidative status of lamb meat.

The current study investigated the fatty acid profile and oxidative status of the meat from lambs that were fed a novel perennial wheat or a conventional annual wheat, either as a cereal monoculture or lucerne biculture. Twelve lambs were assigned to each of the four dietary treatments (48 lambs in total) and held within individual pens for the duration of the 28 day feeding study. Lambs were slaughtered and the longissimus lumborum (LL) and semimembranosus (SM) muscles analysed. The independent effect of wheat type on fatty acid concentrations was negligible. The concentration of long-chain saturated and omega-6 polyunsaturated fatty acids was higher when lucerne was included in the diet. Only monounsaturated fatty acids were affected by the interaction between wheat type and lucerne. The three-way interaction between wheat type, lucerne and muscle was only significant for the concentration of C12:0 and anteiso-C15:0. The concentration of thiobarbituric reactive substances and vitamin E was higher in meat from lambs fed a lucerne biculture, compared with those fed a cereal monoculture. Furthermore, and independent to dietary treatment, higher concentration of omega-3, omega-6 and other health claimable fatty acids were found in the SM, compared with the LL. This information will support industry adoption of novel perennial wheat polycultures and help producers to utilise it as a dual-purpose crop for the production of grain and/or sheep meat.

P62 accumulates through neuroanatomical circuits in response to tauopathy propagation.

In Alzheimer's disease and related tauopathies, trans-synaptic transfer and accumulation of pathological tau from donor to recipient neurons is thought to contribute to disease progression, but the underlying mechanisms are poorly understood. Using complementary in vivo and in vitro models, we examined the relationship between these two processes and neuronal clearance. Accumulation of p62 (a marker of defective protein clearance) correlated with pathological tau accumulation in two mouse models of tauopathy spread; Entorhinal Cortex-tau (EC-Tau) mice where tau pathology progresses in time from EC to other brain regions, and PS19 mice injected with tau seeds. In both models and in several brain regions, p62 colocalized with human tau in a pathological conformation (MC1 antibody). In EC-Tau mice, p62 accumulated before overt tau pathology had developed and was associated with the presence of aggregation-competent tau seeds identified using a FRET-based assay. Furthermore, p62 accumulated in the cytoplasm of neurons in the dentate gyrus of EC-Tau mice prior to the appearance of MC1 positive tauopathy. However, MC1 positive tau was shown to be present at the synapse and to colocalize with p62 as shown by immuno electron microscopy. In vitro, p62 colocalized with tau inclusions in two primary cortical neuron models of tau pathology. In a three-chamber microfluidic device containing neurons overexpressing fluorescent tau, seeding of tau in the donor chamber led to tau pathology spread and p62 accumulation in both the donor and the recipient chamber. Overall, these data are in accordance with the hypothesis that the accumulation and trans-synaptic spread of pathological tau disrupts clearance mechanisms, preceding the appearance of obvious tau aggregation. A vicious cycle of tau accumulation and clearance deficit would be expected to feed-forward and exacerbate disease progression across neuronal circuits in human tauopathies.

3' UTR-truncated HMGA2 overexpression induces non-malignant in vivo expansion of hematopoietic stem cells in non-human primates.

Vector-mediated mutagenesis remains a major safety concern for many gene therapy clinical protocols. Indeed, lentiviral-based gene therapy treatments of hematologic disease can result in oligoclonal blood reconstitution in the transduced cell graft. Specifically, clonal expansion of hematopoietic stem cells (HSCs) highly expressing HMGA2, a chromatin architectural factor found in many human cancers, is reported in patients undergoing gene therapy for hematologic diseases, raising concerns about the safety of these integrations. Here, we show for the first time in vivo multilineage and multiclonal expansion of non-human primate HSCs expressing a 3' UTR-truncated version of HMGA2 without evidence of any hematologic malignancy >7 years post-transplantation, which is significantly longer than most non-human gene therapy pre-clinical studies. This expansion is accompanied by an increase in HSC survival, cell cycle activation of downstream progenitors, and changes in gene expression led by the upregulation of IGF2BP2, a mRNA binding regulator of survival and proliferation. Thus, we conclude that prolonged ectopic expression of HMGA2 in hematopoietic progenitors is not sufficient to drive hematologic malignancy and is not an acute safety concern in lentiviral-based gene therapy clinical protocols.

The quality and mineral composition of the longissimus lumborum and semimembranosus muscles from lambs fed perennial or annual wheat forage with or without lucerne.

This study compared the effect of a novel perennial wheat forage and a conventional annual wheat forage, fed to lambs with or without lucerne, on the quality and mineral profile of the longissimus lumborum and semimembranosus muscles. This involved 48 Poll Dorset × Merino ewe lambs, 12 per treatment, managed for 28 days within individual pens. Consumer sensory panel scores showed 'good eating quality' for all samples irrespective of diet, a conclusion supported by the shear force, cooking loss, particle size, and intramuscular fat results. Drip loss was affected by a significant interaction between diet and muscle. The ultimate pH of meat from lambs fed annual wheat was highest. Comparable colour stability and purge results suggested that diet did not impact on lamb retail-potential. Sodium, sulphur and zinc concentrations were higher in the meat from lambs fed annual wheat, compared to perennial wheat. Overall, these outcomes support the use of perennial wheat as a forage for lamb production.

PAC1 receptor-mediated clearance of tau in postsynaptic compartments attenuates tau pathology in mouse brain.

Accumulation of pathological tau in synapses has been identified as an early event in Alzheimer's disease (AD) and correlates with cognitive decline in patients with AD. Tau is a cytosolic axonal protein, but under disease conditions, tau accumulates in postsynaptic compartments and presynaptic terminals, due to missorting within neurons, transsynaptic transfer between neurons, or a failure of clearance pathways. Using subcellular fractionation of brain tissue from rTg4510 tau transgenic mice with tauopathy and human postmortem brain tissue from patients with AD, we found accumulation of seed-competent tau predominantly in postsynaptic compartments. Tau-mediated toxicity in postsynaptic compartments was exacerbated by impaired proteasome activity detected by measuring lysine-48 polyubiquitination of proteins targeted for proteasomal degradation. To combat the accumulation of tau and proteasome impairment in the postsynaptic compartments of rTg4510 mouse brain, we stimulated the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1R) with its ligand PACAP administered intracerebroventricularly to rTg4510 mice. We observed enhanced synaptic proteasome activity and reduced total tau in postsynaptic compartments in mouse brain after PACAP treatment. The clearance of tau from postsynaptic compartments correlated with attenuated tauopathy and improved cognitive performance of rTg4510 transgenic mice on two behavioral tests. These results suggest that activating PAC1R could prevent accumulation of aggregate-prone tau and indicate a potential therapeutic approach for AD and other tauopathies.

Partial least squares and machine learning for the prediction of intramuscular fat content of lamb loin.

Given the paucity of lamb carcase grading tools, there is a distinct need for the development of rapid, non-destructive grading tools for Australian lamb carcases, particularly fat content given its importance to meat and eating quality. The aim of the current study was to determine the potential for Near Infrared (NIR) spectroscopy to predict IMF using Partial Least Squares (PLS) and machine learning analysis methods. As such, 299 lamb loins were measured using a NIR fibre optic device, a sample was excised for Soxhlet determination of IMF content and prediction models were created using either PLS or machine learning analyses methods. IMF prediction model outcomes were similar between analysis methods with an R2 = 0.6 and RMSE = 0.84 and R2 = 0.65 and RMSE = 0.72, respectively. This study highlighted that spectra from one slaughter varied greatly from the two succeeding slaughters and wavelengths selected between studies are not consistent.

Music Perception and Speech-in-Noise Skills of Typical Hearing and Cochlear Implant Listeners.

Purpose Adult cochlear implant (CI) users rate music as one of the most important auditory stimuli, second to speech perception. However, few studies simultaneously examine music perception and speech-in-noise perception in adult CI recipients. This study explores the effect of auditory status on music perception and speech-in-noise perception recognition in noise as well as the relationship among music engagement, music perception, and speech-in-noise perception. Method Participants include 10 adults with typical hearing (TH) and 10 adults with long-term CI use. All participants completed the Music-Related Quality of Life Questionnaire, which assesses subjective music experiences and their importance; the Pitch Direction Discrimination, Familiar Melody Recognition, and Timbre Recognition subtests of the Clinical Assessment of Music Perception for Cochlear Implants; the Unfamiliar Melody Recognition subtest of the Profile of Music Perception Skills; and the Bamford-Kowal-Bench Speech-in-Noise Test. Results The TH group significantly outperformed the CI group for speech-in-noise perception and on all four music perception tasks. The CI group exhibited not only significantly poorer mean scores but also greater variability in performance compared to the TH group. Only Familiar Melody Recognition and Unfamiliar Melody Recognition subtests significantly correlated with speech-in-noise scores. Conclusions Patients and professionals should not assume speech perception and music perception in adult CI users derive from the same auditory or cognitive foundations. The lack of significant relationships among music engagement, music perception, and speech-in-noise perception scores in adult CI users suggests this population enjoys music despite poor and variable performance in discrete music tasks.

Identification of pannexin 1-regulated genes, interactome, and pathways in rhabdomyosarcoma and its tumor inhibitory interaction with AHNAK.

Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, is an aggressive cancer with a poor prognosis. Despite current management, the 5-year survival rate for patients with metastatic RMS is ∼30%; underscoring the need to develop better treatment strategies. We have recently reported that pannexin 1 (PANX1) levels are downregulated in RMS and that restoring its expression inhibits RMS progression. Here, we have surveyed and characterized the molecular changes induced by PANX1 re-expression in RMS. We cataloged transcriptomic changes in this context by RNA sequencing. At the protein level, we unveiled PANX1 interactors using BioID, complemented by co-immunoprecipitation coupled to high-performance liquid chromatography/electrospray ionization tandem mass spectrometry performed in PANX1-enriched fractions. Using these data, we generated searchable public databases for the PANX1 interactome and changes to the RMS transcriptome occurring when PANX1 expression is restored. STRING network analyses revealed a PANX1 interactome involving plasma membrane and cytoskeleton-associated proteins including the previously undescribed interactor AHNAK. Indeed, AHNAK knockdown abrogated the PANX1-mediated reduction in RMS cell viability and migration. Using these unbiased approaches, we bring insight to the mechanisms by which PANX1 inhibits RMS progression, identifying the cell migration protein AHNAK as a key modifier of PANX1-mediated changes in RMS malignant properties.