MTGR1 is required to maintain small intestinal stem cell populations.

Undifferentiated intestinal stem cells (ISCs) are crucial for maintaining homeostasis and resolving injury. Lgr5+ cells in the crypt base constantly divide, pushing daughter cells upward along the crypt axis where they differentiate into specialized cell types. Coordinated execution of complex transcriptional programs is necessary to allow for the maintenance of undifferentiated stem cells while permitting differentiation of the wide array of intestinal cells necessary for homeostasis. Previously, members of the myeloid translocation gene (MTG) family have been identified as transcriptional co-repressors that regulate stem cell maintenance and differentiation programs in multiple organ systems, including the intestine. One MTG family member, myeloid translocation gene related 1 (MTGR1), has been recognized as a crucial regulator of secretory cell differentiation and response to injury. However, whether MTGR1 contributes to the function of ISCs has not yet been examined. Here, using Mtgr1-/- mice, we have assessed the effects of MTGR1 loss specifically in ISC biology. Interestingly, loss of MTGR1 increased the total number of cells expressing Lgr5, the canonical marker of cycling ISCs, suggesting higher overall stem cell numbers. However, expanded transcriptomic and functional analyses revealed deficiencies in Mtgr1-null ISCs, including deregulated ISC-associated transcriptional programs. Ex vivo, intestinal organoids established from Mtgr1-null mice were unable to survive and expand due to aberrant differentiation and loss of stem and proliferative cells. Together, these results indicate that the role of MTGR1 in intestinal differentiation is likely stem cell intrinsic and identify a novel role for MTGR1 in maintaining ISC function.

Tobramycin and Vancomycin in an In Vitro Model of Anterior Cruciate Ligament Allograft Decontamination.

BACKGROUND: Approximately 100,000 anterior cruciate ligament (ACL) reconstructions (ACLRs) occur annually in the United States, and postoperative surgical-site infection is a relatively rare but devastating complication, often leading to graft failure or septic arthritis of the knee, necessitating repeat surgery. Wrapping allografts in vancomycin-soaked gauze has been adopted as a common sterilization technique in the operating room to reduce surgical-site infection; however, identifying effective alternatives to vancomycin has not been extensively pursued. HYPOTHESIS: Tobramycin would be as effective as vancomycin in reducing the concentrations of Staphylococcus epidermidis bacteria on tendon allografts. STUDY DESIGN: Controlled laboratory study. METHODS: S. epidermidis strain ATCC 12228 was inoculated onto the human cadaveric gracilis tendon. The tendons were wrapped in sterile gauze saturated with tobramycin or vancomycin at various experimental concentrations. Bacteria remaining on the tendon were dislodged, serially diluted, and plated for colony counting. Statistical analysis was performed utilizing 2-way analysis of variance testing. Results were considered statistically significant when P < .05. RESULTS: Vancomycin (P = .0001) and tobramycin (P < .0001) reduced bacterial concentration. Tobramycin was found to produce a statistically significant reduction in bacterial concentration at concentrations as low as 0.1 mg/mL (P < .0001 and P = .01 at 10 and 20 minutes), while vancomycin produced a statistically significant reduction at a concentration as low as 2.5 mg/mL (P < .0001 at both 10 and 20 minutes). CONCLUSION: This study demonstrates that tobramycin is as effective as vancomycin in bacterial concentration reduction but can achieve this reduction level at lower doses. Further studies clarifying the biomechanical and cytotoxic effects of tobramycin on tendon tissue are indicated to solidify its use as a clinical alternative to vancomycin in ACLR. CLINICAL RELEVANCE: These results will begin establishing tobramycin as an alternative to vancomycin in ACL graft decontamination. Because of relatively frequent shortages of vancomycin, establishing tobramycin as an alternative agent is a useful option for the orthopaedic surgeon.

MTGR1 is required to maintain small intestinal stem cell populations.

Undifferentiated intestinal stem cells (ISCs), particularly those marked by Lgr5, are crucial for maintaining homeostasis and resolving injury. Lgr5+ cells in the crypt base constantly divide, pushing daughter cells upward along the crypt axis, where they differentiate into a variety of specialized cell types. This process requires coordinated execution of complex transcriptional programs, which allow for the maintenance of undifferentiated stem cells while permitting differentiation of the wide array of intestinal cells necessary for homeostasis. Thus, disrupting these programs may negatively impact homeostasis and response to injury. Previously, members of the myeloid translocation gene (MTG) family have been identified as transcriptional co-repressors that regulate stem cell maintenance and differentiation programs in multiple organ systems, including the intestine. One MTG family member, myeloid translocation gene related 1 (MTGR1), has been recognized as a crucial regulator of secretory cell differentiation and response to injury. However, whether MTGR1 contributes to the function of ISCs has not yet been examined. Here, using Mtgr1-/- mice, we have assessed the effects of MTGR1 loss on ISC biology and differentiation programs. Interestingly, loss of MTGR1 increased the total number of cells expressing Lgr5, the canonical marker of cycling ISCs, suggesting higher overall stem cell numbers. However, expanded transcriptomic analyses revealed MTGR1 loss may instead promote stem cell differentiation into transit-amplifying cells at the expense of cycling ISC populations. Furthermore, ex vivo intestinal organoids established from Mtgr1 null were found nearly completely unable to survive and expand, likely due to aberrant ISC differentiation, suggesting that Mtgr1 null ISCs were functionally deficient as compared to WT ISCs. Together, these results identify a novel role for MTGR1 in ISC function and suggest that MTGR1 is required to maintain the undifferentiated state.

Influence of Staphylococcus epidermidis on Collagen Crimp Patterns of Soft Tissue Allograft.

BACKGROUND: Postoperative infections, commonly from Staphylococcus epidermidis, may result in anterior cruciate ligament graft failure and necessitate revision surgery. In biomechanical studies, S. epidermidis has been shown to establish biofilms on tendons and reduce graft strength. PURPOSE/HYPOTHESIS: The goal of this study was to determine the effect of bacterial bioburden on the collagen structure of tendon. It was hypothesized that an increase in S. epidermidis biofilm would compromise tendon crimp, a pattern necessary for mechanical integrity, of soft tissue allografts. STUDY DESIGN: Controlled laboratory study. METHODS: Cultures of S. epidermidis were used to inoculate tibialis anterior cadaveric tendons. Conditions assessed included 5 × 105 colony-forming units or concentrated spent media from culture (no living bacteria). Incubation times of 30 minutes, 3 hours, 6 hours, and 24 hours were utilized. Second-harmonic generation imaging allowed for visualization of collagen autofluorescence. Crimp lengths were determined using ImageJ and compared based on incubation time. RESULTS: Incubation time positively correlated with increasing S. epidermidis bioburden. Both fine and coarse crimp patterns lengthened with increasing incubation time. Significant coarse crimp changes were observed after only 30-minute incubations (P < .029), whereas significant fine crimp lengthening occurred after 6 hours (P < .0001). No changes in crimp length were identified after incubation in media lacking living bacteria. CONCLUSION: The results of this study demonstrate that exposure to S. epidermidis negatively affects collagen crimp structure. Structural alterations at the collagen fiber level occur within 30 minutes of exposure to media containing S. epidermidis. CLINICAL RELEVANCE: Our study highlights the need for antimicrobial precautions to prevent graft colonization and maximize graft mechanical strength.

MTG16 regulates colonic epithelial differentiation, colitis, and tumorigenesis by repressing E protein transcription factors.

Aberrant epithelial differentiation and regeneration contribute to colon pathologies, including inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). Myeloid translocation gene 16 (MTG16, also known as CBFA2T3) is a transcriptional corepressor expressed in the colonic epithelium. MTG16 deficiency in mice exacerbates colitis and increases tumor burden in CAC, though the underlying mechanisms remain unclear. Here, we identified MTG16 as a central mediator of epithelial differentiation, promoting goblet and restraining enteroendocrine cell development in homeostasis and enabling regeneration following dextran sulfate sodium-induced (DSS-induced) colitis. Transcriptomic analyses implicated increased Ephrussi box-binding transcription factor (E protein) activity in MTG16-deficient colon crypts. Using a mouse model with a point mutation that attenuates MTG16:E protein interactions (Mtg16P209T), we showed that MTG16 exerts control over colonic epithelial differentiation and regeneration by repressing E protein-mediated transcription. Mimicking murine colitis, MTG16 expression was increased in biopsies from patients with active IBD compared with unaffected controls. Finally, uncoupling MTG16:E protein interactions partially phenocopied the enhanced tumorigenicity of Mtg16-/- colon in the azoxymethane/DSS-induced model of CAC, indicating that MTG16 protects from tumorigenesis through additional mechanisms. Collectively, our results demonstrate that MTG16, via its repression of E protein targets, is a key regulator of cell fate decisions during colon homeostasis, colitis, and cancer.