Fibroblast-specific upregulation of Flightless I impairs wound healing.

The cytoskeletal protein Flightless (Flii) is a negative regulator of wound healing. Upregulation of Flii is associated with impaired migration, proliferation and adhesion of both fibroblasts and keratinocytes. Importantly, Flii translocates from the cytoplasm to the nucleus in response to wounding in fibroblasts but not keratinocytes. This cell-specific nuclear translocation of Flii suggests that Flii may directly regulate gene expression in fibroblasts, providing one potential mechanism of action for Flii in the wound healing response. To determine whether the tissue-specific upregulation of Flii in fibroblasts was important for the observed inhibitory effects of Flii on wound healing, an inducible fibroblast-specific Flii overexpressing mouse model was generated. The inducible ROSA26 system allowed the overexpression of Flii in a temporal and tissue-specific manner in response to tamoxifen treatment. Wound healing in the inducible mice was impaired, with wounds at day 7 postwounding significantly larger than those from non-inducible controls. There was also reduced collagen maturation, increased myofibroblast infiltration and elevated inflammation. The impaired healing response was similar in magnitude to that observed in mice with non-tissue-specific upregulation of Flii suggesting that fibroblast-derived Flii may have an important role in the wound healing response.

Flightless I over-expression impairs skin barrier development, function and recovery following skin blistering.

Development of an intact epidermis is critical for maintaining the integrity of the skin. Patients with epidermolysis bullosa (EB) experience multiple erosions, which breach the epidermal barrier and lead to increased microbial colocalization of wounds, infections and sepsis. The cytoskeletal protein Flightless I (Flii) is a known regulator of both development and wound healing. Using Flii(+/-), WT and Flii(Tg/Tg) mice, we investigated the effect of altering Flii levels in embryos and adult mice on the development of the epidermal barrier and, consequently, how this affects the integrity of the skin in EB. Flii over-expression resulted in delayed formation of the epidermal barrier in embryos and decreased expression of tight junction (TJ) proteins Claudin-1 and ZO-2. Increased intercellular space and transepidermal water loss was observed in Flii(Tg)(/Tg) adult mouse skin, while Flii(Tg/Tg) keratinocytes showed altered TJ protein localization and reduced transepithelial resistance. Flii is increased in the blistered skin of patients with EB, and over-expression of Flii in experimental EBA showed impaired Claudin-1 and -4 TJ protein expression and delayed recovery of functional barrier post-blistering. Immunoprecipitation confirmed Flii associated with TJ proteins and in vivo actin assays showed that the effect of Flii on actin polymerization underpinned the impaired barrier function observed in Flii(Tg/Tg) mice. These results therefore demonstrate an important role for Flii in the development and regulation of the epidermal barrier, which may contribute to the impaired healing and skin fragility of EB patients.

Comparing the Mental Health of Healthcare Students: Mental Health Shame and Self-compassion in Counselling, Occupational Therapy, Nursing and Social Work Students.

Poor mental health of healthcare students is a cause for concern in many universities. Though previous research has identified mental health shame and self-compassion as critical in this student group, how these variables differ across different healthcare disciplines remains to be evaluated. Healthcare students (n = 344; counselling, occupational therapy, social work and nursing) completed measures regarding these variables. MANOVA and regression analyses were performed. (1) Counselling and nursing students were more depressed than occupational therapy students; (2) nursing students were more anxious than occupational therapy and social work students; (3) occupational therapy students had more positive attitudes towards mental health than the others; and (4) nursing students worried about their own reputation associated with their family more than counselling students. Self-compassion was the strongest predictor of mental health in all groups; however, the effect sizes varied: largest in nursing and smallest in social work students. Findings will help inform effective interventions for students in each healthcare discipline.

Flii neutralizing antibodies improve wound healing in porcine preclinical studies.

Wound healing is an important area of widely unmet medical need, with millions of procedures carried out worldwide which could potentially benefit from a product to improve the wound repair process. Our studies investigating the actin-remodeling protein Flightless I (Flii) show it to be an important regulator of wound healing. Flii-deficient mice have enhanced wound healing in comparison to Flii overexpressing mice which have impaired wound healing. For the first time, we show that a Flightless I neutralizing monoclonal antibody (FnAb) therapy is effective in a large animal model of wound repair. Porcine 5 cm incisional and 6.25 cm(2) excisional wounds were treated with FnAb at the time of wounding and for two subsequent days. The wounds were dressed in Tegaderm dressings and left to heal by secondary intention for 7 and 35 days, respectively. At the relevant end points, the wounds were excised and processed for histological analysis. Parameters of wound area, collagen deposition, and scar appearance were analyzed. The results show that treatment with FnAb accelerates reepithelialization and improves the macroscopic appearance of early scars. FnAbs have the potential to enhance wound repair and reduce scar formation.

The Feasibility and Impact of Practising Online Forest Bathing to Improve Anxiety, Rumination, Social Connection and Long-COVID Symptoms: A Pilot Study.

BACKGROUND: Long-COVID affects over 144 million people globally. In the absence of treatments, there is a need to establish the efficacy of therapies that improve patient outcomes. Forest bathing has been demonstrated to improve physical and mental outcomes but there is no evidence in Long-COVID patients. Accordingly, this pilot study sought to determine the feasibility and effectiveness of online forest bathing in adults with Long-COVID. METHODS: Feasibility was assessed by monitoring retention rates and participant feedback. In a waitlist controlled, repeated measures design, 22 Long-COVID patients completed weekly online surveys during a four-week waitlist control period, before engaging in four weekly online forest bathing sessions, completing post-intervention surveys following each session. RESULTS: In terms of retention, 27% did not provide post-intervention data, reasons for non-adherence were: feeling too ill, having medical appointments, or having career responsibilities. Compared with the waitlist control period, there were statistically significant improvements in Anxiety (49% decrease), Rumination (48% decrease), Social Connection (78% increase), and Long-COVID symptoms (22% decrease). Written qualitative comments indicated that participants experienced feelings of calm and joy, felt more connected socially and with nature, and experienced a break from the pain and rumination surrounding their illness. CONCLUSIONS: Online Forest bathing resulted in significant improvements in well-being and symptom severity and could be considered an accessible and inexpensive adjunct therapy for Long-COVID patients. Where people have limited access to in-person nature, virtual nature may offer an alternative to improve health and well-being outcomes.

In vitro analysis of the effect of Flightless I on murine tenocyte cellular functions.

BACKGROUND: Healing of tendons after injury involves the proliferation of tenocytes and the production of extracellular matrix; however, their capacity to heal is limited by poor cell density and limited growth factor activity. Flightless I (Flii) has previously been identified as an important regulator of cellular proliferation and migration, and the purpose of this study was to evaluate the effect of differential Flii gene expression on tenocyte function in vitro. METHODS: The role of Flii on tenocyte proliferation, migration, and contraction was assessed using established assays. Tenocytes from Flii+/-, wild-type, and Flii overexpressing mice were obtained and the effect of differential Flii expression on migration, proliferation, contraction, and collagen synthesis determined in vitro. Statistical differences were determined using unpaired Student's t test and statistical outliers were identified using the Grubbs' test. RESULTS: Flii overexpressing tenocytes showed significantly improved migration and proliferation as well as increased collagen I secretion. Explanted tendons from Flii overexpressing mice also showed significantly elevated tenocyte outgrowth compared to Flii+/- mice. In contrast to its role in dermal wound repair, Flii positively affects cellular processes in tendons. CONCLUSIONS: These findings suggest that Flii could be a novel target for modulating tenocyte activity and improving tendon repair. This could have significant clinical implications as novel therapeutic targets for improved healing of tendon injuries are urgently needed.

Increasing the level of cytoskeletal protein Flightless I reduces adhesion formation in a murine digital flexor tendon model.

BACKGROUND: Surgical repair of tendons is common, but function is often limited due to the formation of flexor tendon adhesions which reduce the mobility and use of the affected digit and hand. The severity of adhesion formation is dependent on numerous cellular processes many of which involve the actin cytoskeleton. Flightless I (Flii) is a highly conserved cytoskeletal protein, which has previously been identified as a potential target for improved healing of tendon injuries. Using human in vitro cell studies in conjunction with a murine model of partial laceration of the digital flexor tendon, we investigated the effect of modulating Flii levels on tenocyte function and formation of adhesions. METHODS: Human tenocyte proliferation and migration was determined using WST-1 and scratch wound assays following Flii knockdown by siRNA in vitro. Additionally, mice with normal and increased levels of Flii were subjected to a partial laceration of the digital flexor tendon in conjunction with a full tenotomy to immobilise the paw. Resulting adhesions were assessed using histology and immunohistochemistry for collagen I, III, TGF-ß1and -ß3 RESULTS: Flii knockdown significantly reduced human tenocyte proliferation and migration in vitro. Increasing the expression of Flii significantly reduced digital tendon adhesion formation in vivo which was confirmed through significantly smaller adhesion scores based on collagen fibre orientation, thickness, proximity to other fibres and crimping. Reduced adhesion formation was accompanied with significantly decreased deposition of type I collagen and increased expression of TGF-ß1 in vivo. CONCLUSIONS: These findings suggest that increasing the level of Flii in an injured tendon may be beneficial for decreasing tendon adhesion formation.

Cytoskeletal protein Flightless I inhibits apoptosis, enhances tumor cell invasion and promotes cutaneous squamous cell carcinoma progression.

Flightless I (Flii) is an actin remodeling protein that affects cellular processes including adhesion, proliferation and migration. In order to determine the role of Flii during carcinogenesis, squamous cell carcinomas (SCCs) were induced in Flii heterozygous (Flii+/-), wild-type and Flii overexpressing (FliiTg/Tg) mice by intradermal injection of 3-methylcholanthrene (MCA). Flii levels were further assessed in biopsies from human SCCs and the human SCC cell line (MET-1) was used to determine the effect of Flii on cellular invasion. Flii was highly expressed in human SCC biopsies particularly by the invading cells at the tumor edge. FliiTg/Tg mice developed large, aggressive SCCs in response to MCA. In contrast Flii+/- mice had significantly smaller tumors that were less invasive. Intradermal injection of Flii neutralizing antibodies during SCC initiation and progression significantly reduced the size of the tumors and, in vitro, decreased cellular sphere formation and invasion. Analysis of the tumors from the Flii overexpressing mice showed reduced caspase I and annexin V expression suggesting Flii may negatively regulate apoptosis within these tumors. These studies therefore suggest that Flii enhances SCC tumor progression by decreasing apoptosis and enhancing tumor cell invasion. Targeting Flii may be a potential strategy for reducing the severity of SCCs.