GLI1 is a transcriptional effector at the terminal end of the Hedgehog signaling (Hh) pathway and is tightly regulated during embryonic development and tissue patterning/differentiation. GLI1 has low-level expression in differentiated tissues, however, in certain cancers, aberrant activation of GLI1 has been linked to the promotion of numerous hallmarks of cancer, such as proliferation, survival, angiogenesis, metastasis, metabolic rewiring, and chemotherapeutic resistance. All of these are driven, in part, by GLI1’s role in regulating cell cycle, DNA replication and DNA damage repair processes. The consequences of GLI1 oncogenic activity, specifically the activity surrounding DNA damage repair proteins, such as NBS1, and cell cycle proteins, such as CDK1, can be linked to tumorigenesis and chemoresistance. Therefore, understanding the underlying mechanisms driving GLI1 dysregulation can provide prognostic and diagnostic biomarkers to identify a patient population that would derive therapeutic benefit from either direct inhibition of GLI1 or targeted therapy towards proteins downstream of GLI1 regulation.

Resistance to radiation and chemotherapy in colorectal cancer (CRC) patients contribute significantly to refractory disease and disease progression. Herein, we provide mechanistic rationale for acquired or inherent chemotherapeutic resistance to the anti-tumor effects of 5-fluorouracil (5-FU) that is linked to oncogenic GLI1 transcription activity and NBS1 overexpression. Patients with high levels of GLI1 also expressed high levels of NBS1. Non-canonical activation of GLI1 is driven through oncogenic pathways in CRC, like the BRAF^V600E mutation. GLI1 was identified as a novel regulator of NBS1 and discovered that by knocking down GLI1 levels in vitro, diminished NBS1 expression, increased DNA damage/apoptosis, and re-sensitization of 5-FU resistant cancer to treatment was observed. Furthermore, a novel GLI1 inhibitor, SRI-38832, which exhibited pharmacokinetic properties suitable for in vivo testing, was identified. GLI1 inhibition in a murine BRAF^V600E variant xenograft model of CRC resulted in the same down-regulation of NBS1 observed in vitro as well as significant reduction of tumor growth/burden. GLI1 inhibition could therefore be a therapeutic option for 5-FU resistant and BRAF^V600E variant CRC patients.

Over 90% of chronic wounds have biofilm infections, making the need for inhibiting reformation of biofilm post‐debridement paramount to support progression through the normal phases of wound healing. Herein, we describe a porcine wound model infected with methicillin‐resistant Staphylococcus aureus (MRSA) and examine the ability of an antimicrobial barrier composed of native type I collagen and polyhexamethylene biguanide (PCMP) to serve as a barrier to protect wounds and support progression through the innate wound healing cascade. Wounds were inoculated with MRSA and allowed to form a biofilm for 72 h, subjected to standard of care sharp debridement, then either left untreated or received PCMP for 5, 10, 15 or 20 days. Wounds were assessed for bioburden, wound closure and expression of genes related to wound healing. Wounds treated with PCMP exhibited statistically lower MRSA levels compared to untreated controls and achieved 90% closure by 2 weeks of treatment. Gene expression analysis demonstrated that by reducing bacterial load, wounds progressed through the innate wound healing cascade, while untreated wounds exhibited a dampening of the immune response. Additionally, for randomly assigned wounds, PCMP was not reapplied at dressing changes to assess the impact of inconsistent wound protection. At all timepoints, a resurgence in bioburden was observed following removal of PCMP if the wounds had not fully closed. This study highlights the value of PCMP as an antimicrobial barrier and the importance of protecting wounds through closure and resolution.

Genome-wide association studies (GWAS) and functional genomic analyses have implicated severalITGAM(CD11b) single-nucleotide polymorphisms (SNPs) in the development of SLE and other disorders.ITGAMencodes the α_Mchain of the β₂integrin Mac-1, a receptor that plays important roles in myeloid cell functions. TheITGAMSNP rs1143679, which results in an arginine to histidine change at amino acid position 77 of the CD11b protein, has been shown to reduce binding to several ligands and to alter Mac-1-mediated cellular response in vitro. Importantly, however, the potential contribution of this SNP variant to the initiation and/or progression of immune and inflammatory processes in vivo remains unexplored. Herein, we describe for the first time the generation and characterization of a mouse line expressing the 77His variant of CD11b. Surprisingly, we found that 77His did not significantly affect Mac-1-mediated leukocyte migration and activation as assessed using thioglycollate-induced peritonitis and LPS/TNF-α-induced dermal inflammation models. In contrast, expression of this variant did alter T cell immunity, as evidenced by significantly reduced proliferation of ovalbumin (OVA)-specific transgenic T cells in 77His mice immunized with OVA. Reduced antigen-specific T cell proliferation was also observed when either 77His splenic dendritic cells (DCs) or bone marrow-derived DCs were used as antigen-presenting cells (APCs). Although more work is necessary to determine how this alteration might influence the development of SLE or other diseases, these in vivo findings suggest that the 77His variant of CD11b can compromise the ability of DCs to induce antigen-driven T cell proliferation.

Placental-derived products have been used since the early 1900s for wound applications and have shown clinical utility in supporting wound healing. A hypothermically stored amniotic membrane (HSAM) was developed using a proprietary process to allow for the retention of the extracellular matrix (ECM), viable cells, and key proteins. To evaluate its utility, we characterized the HSAM and compared it to a native unprocessed amniotic membrane (uAM) and a dehydrated amniotic membrane (dAM), as well as assessing the functionality of the HSAM as a scaffold to promote cell growth. The HSAM, uAM, and dAM were compared using scanning electron microscopy (SEM), histology, and thickness. Scaffold durability was assessed in vitro using mechanical testing and a simulated wound fluid (SWF) model. The ability of the HSAM to act as a scaffold was evaluated using an in vitro attachment model. The HSAM showed similar structural characteristics compared to the uAM; however, the dAM was significantly more compact. There were no significant differences between the HSAM and the uAM following degradation in an SWF model. ECM- and placental-related proteins were shared between the HSAM and uAM, and the HSAM enhanced the attachment and proliferation of fibroblasts in vitro. The HSAM is substantially similar to the uAM by retaining key regulatory proteins, resisting degradation in SWF, and acting as a scaffold for cellular growth and invasion.

While acute wounds typically progress through the phases of wound healing, chronic wounds often stall in the inflammatory phase due to elevated levels of matrix metalloproteinases (MMPs) and proinflammatory cytokines. Dysregulated expression of MMPs can result in the breakdown of extracellular matrix (ECM) formed during the wound healing process, resulting in stalled wounds. Native collagen-based wound dressings offer a potential wound management option to sequester excess MMPs and support cellular interactions that allow wound progression through the natural healing process. Herein, we utilized commercially available ECM matrices, two derived from porcine small intestinal submucosa (PCMP, 2 layers; PCMP-XT, 5 layers) and one derived from propria submucosa (ovine forestomach matrix, OFM, 1 layer), to demonstrate the impact of processing methodologies (e.g., layering and crosslinking) on functional characteristics needed for the management of chronic wounds. Grafts were evaluated for structural composition using scanning electron microscopy and histology, ability to reduce MMPs using fluorometric assays, and durability in an in vitro degradation chronic wound model. Both intact (nondegraded) and partially degraded grafts were assessed for their ability to serve as a functional cell scaffold using primary human fibroblasts. Grafts differed in matrix substructure and composition. While all grafts demonstrated attenuation of MMP activity, PCMP and PCMP-XT showed larger reductions of MMP levels. OFM rapidly degraded in the in vitro degradation model (7days). The ability of PCMP and PCMP-XT to serve as scaffolds for cellular attachment was not impacted by degradation in vitro. Three ECM grafts with varying structural and functional characteristics exhibited differential durability when degraded in a simulated chronic wound model. Those that withstood rapid degradation maintained their ability to function as a scaffold to support attachment and proliferation of fibroblasts, a cell type important for wound healing.