OBJECTIVE:The National Institute of Diabetes and Digestive and Kidney Diseases Diabetic Foot Consortium tested the hypothesis that compromised restoration of the skin barrier function of closed diabetic foot ulcers (DFUs), as measured by high transepidermal water loss (TEWL), is associated with an increased risk of DFU recurrence. RESEARCH DESIGN AND METHODS:This was a multicenter noninterventional study measuring TEWL in 418 adult participants with diabetes and a recently healed DFU. TEWL was measured at the center of the closed wound and at an anatomically similar reference area on the contralateral foot within 2 weeks of wound closure (visit 1); measurements were repeated at a wound closure confirmation visit 2 weeks later (visit 2). Participants were observed for up to 16 weeks to assess for wound recurrence. Participant self-reported and clinician assessments of DFU wound recurrence were recorded. RESULTS:DFU recurrence by week 16 occurred in 21.5% of participants. Mean TEWL at the center of the healed DFU at visit 1 was higher for those with recurrence compared with those without (P = 0.006). Among participants with high TEWL (>30.05 g · m-2 · h-1), 35% reported wound recurrence by 16 weeks versus 17% of those with low TEWL. The odds ratio for recurrence for participants with high TEWL was 2.66 (P < 0.001). Self-reported wound recurrence was highly concordant with clinician assessment of wound recurrence. CONCLUSIONS:Compromised wound healing mechanisms culminating in wound closure associated with defective skin barrier function is associated with increased risk of DFU recurrence. Measurement of TEWL has value as a predictor of functional wound healing and could affect clinical practice, leading to better outcomes.

Objective: Wound closure is skin reepithelialization confirmed at two consecutive clinical visits 2 weeks apart. Our objective was to identify participant characteristics, including transepidermal water loss (TEWL), associated with complete wound closure of diabetic foot ulcers (DFUs) and reopening of a DFU within 2 weeks after initial closure in the National Institute of Diabetes and Digestive and Kidney Diseases-sponsored Diabetic Foot Consortium TEWL prospective observational cohort study of wound recurrence. At the site of wound closure, TEWL measures restoration of skin barrier function and functional wound closure. Approach: Four hundred and sixty-six eligible participants had physician-assessed wound closure at baseline. Of which, 418 (90%) had confirmed closure 2 weeks later and remained in the study, whereas 29 had their DFU reopen 2 weeks later and were not eligible for follow-up (i.e., screen failures). We compared baseline characteristics of 418 enrolled and 29 screen fail individuals using Wilcoxon rank sum and Fisher's exact tests p value for continuous and categorical outcomes, respectively. Results: There were no statistically significant differences in demographics, including age, sex, race, education, employment status, social support, or dressing change requirements between groups. The failure to maintain closure group had longer median duration of index DFU before initial closure (25.8 vs. 14 weeks, p = 0.003), higher frequency of prior total contact casting use (37% vs. 14%, p = 0.003), and a higher median initial TEWL measurement at the healed ulcer midpoint (27.1 vs. 21.0 g/m2/h, p = 0.006). Innovation: TEWL measurement at the site of wound closure can assess functional capacity of the skin in conjunction with current standards of wound closure end point in DFU and has significant potential to add quantitative measurement to assist in clinical assessment of healing wounds. Conclusion: Individuals with DFU who did not maintain wound closure had higher TEWL values at baseline, longer DFU wound duration, and more prior off-loading use. These findings are clinically relevant as a higher TEWL measurement demonstrates incomplete functional wound closure, supporting the use of TEWL to identify a healed DFU.

Uncontrolled fibrosis via excess deposition of extracellular matrix (ECM) is a hallmark of hypertrophic scars and keloids. A decellularized ECM biomaterial from porcine small intestinal submucosa (SIS; Biodesign or BioD, Cook Biotech, Inc.) is widely used in clinical applications for tissue repair. The objective of the current study was to test the effects of BioD scaffolds, as compared with collagen constructs, on normal human skin (nFB) and keloid fibroblasts (kFBs). Immortalized human dermal fibroblasts (hFBs) and human keloid fibroblasts (hKFs) were utilized for all experiments. Cells were cultured either on BioD membranes or on collagen gel (used as a control). To investigate pro-fibrotic signaling pathways, real-time quantitative PCR (qPCR), ELISA, and gene knockdown studies were conducted on cultured cells. ECM gene expression array revealed that BioD significantly attenuated (p < 0.05) the expression of thrombospondin-1 and fibronectin-1, two drivers of fibrosis in nFB as well as kFB. BioD-repressed thrombospondin-1 and fibronectin-1 gene expression manifested as significant downregulation (n = 5–6; p < 0.05) of both proteins in nFB and kFB. The levels of latent transforming-growth factor (LAP-TGFβ-1) were markedly reduced (n = 5; p < 0.05) in both nFB and kFB cultured on BioD, but not the other constructs. Knockdown of FN1 using siRNA significantly attenuated (n = 5, p < 0.05) pro-fibrotic responses, including expression of Col1A1 and the levels of LAP-TGFβ-1 in nFB, suggesting that downregulation of FN1 by BioD is one of the primary underlying mechanisms of attenuated pro-fibrotic responses in keloid fibroblasts. This study reports that a decellularized ECM scaffold may significantly attenuate pro-fibrotic responses in both normal and keloid fibroblasts via TSP1 and FN1-dependent mechanisms.

Lymphedema is chronic limb swelling resulting from lymphatic dysfunction. There is no cure for the disease. Clinically, a preventive surgical approach called immediate lymphatic reconstruction (ILR) has gained traction. Experimental gene-based therapeutic approaches (e.g., using viral vectors) have had limited translational applicability. Tissue nanotransfection (TNT) technology uses a direct, transcutaneous nonviral vector, gene delivery using a chip with nanochannel poration in response to a rapid (<100 ms) focused electric field. The purpose of this study was to experimentally prevent lymphedema using focal delivery of a specific gene Prox1 (a master regulator of lymphangiogenesis). TNT was applied to the previously optimized lymphedematous mice tail (day 0) directly at the surgical site with genetic cargo loaded into the TNT reservoir: group I (sham) was given pCMV6 (expression vector backbone alone) and group II was treated with pCMV6-Prox1. Group II mice had decreased tail volume (47.8%) compared to sham and greater lymphatic clearance on lymphangiography. Immunohistochemistry showed greater lymphatic vessel density and RNA sequencing exhibited reduced inflammatory markers in group II compared to group I. Prox1 prophylactically delivered using TNT to the surgical site on the day of injury decreased the manifestations of lymphedema in the murine tail model compared to control.

Background: Lymphedema is chronic limb swelling resulting from lymphatic dysfunction. It affects an estimated five million Americans. There is no cure for this disease. Assessing lymphatic growth is essential in developing novel therapeutics. Intravital microscopy (IVM) is a powerful imaging tool for investigating various biological processes in live animals. Tissue nanotransfection technology (TNT) facilitates a direct, transcutaneous nonviral vector gene delivery using a chip with nanochannel poration in a rapid (<100 ms) focused electric field. TNT was used in this study to deliver the genetic cargo in the murine tail lymphedema to assess the lymphangiogenesis. The purpose of this study is to experimentally evaluate the applicability of IVM to visualize and quantify lymphatics in the live mice model. Methods and Results: The murine tail model of lymphedema was utilized. TNT was applied to the murine tail (day 0) directly at the surgical site with genetic cargo loaded into the TNT reservoir: TNTpCMV6 group receives pCMV6 (expression vector backbone alone) (n = 6); TNTProx1 group receives pCMV6-Prox1 (n = 6). Lymphatic vessels (fluorescein isothiocyanate [FITC]-dextran stained) and lymphatic branch points (indicating lymphangiogenesis) were analyzed with the confocal/multiphoton microscope. The experimental group TNTProx1 exhibited reduced postsurgical tail lymphedema and increased lymphatic distribution compared to TNTpCMV6 group. More lymphatic branching points (>3-fold) were observed at the TNT site in TNTProx1 group. Conclusions: This study demonstrates a novel, powerful imaging tool for investigating lymphatic vessels in live murine tail model of lymphedema. IVM can be utilized for functional assessment of lymphatics and visualization of lymphangiogenesis following gene-based therapy.

Introduction and Objective: It’s been more than 25 years since the FDA approved becaplermin gel for diabetic wound healing. The FDA definition for wound closure is complete re-epithelialization confirmed at two consecutive clinical visits two weeks apart. Our objective was to identify participant characteristics, including TEWL, associated with complete wound closure of diabetic foot ulcers (DFU) and re-opening of a DFU within 2 weeks after initial closure in the NIDDK-sponsored Diabetic Foot Consortium TEWL prospective observational cohort study. TEWL measures skin barrier function and functional wound closure by objectively determining restoration of barrier function at the wound-site. Methods: Among 466 eligible participants who had physician-assessed wound closure at visit 1, 418 (90%) remained eligible and had confirmed closure 2 weeks later. 29 individuals screen failed because of DFU re-opening. We compared the baseline characteristics of the 418 enrolled and 29 screen fail individuals using Wilcoxon rank sum and Fisher’s exact test p-value for continuous and categorical outcomes, respectively. Results: There were no differences in demographics including age, sex, race, education, employment status, or social support for transportation and dressing change requirements (p>0.1) between groups. However, the screen failure group had a longer duration of the index DFU prior to initial closure (46.6 vs 27.7 weeks, p=0.003), higher frequency of prior total contact casting (TCC) use for DFU offloading (37% vs. 14%, p=0.003), and a higher initial TEWL measurement at healed ulcer midpoint (31.9 vs. 23.5 g/m2/h, p=0.006). Conclusion: Individuals who did not maintain wound closure had higher TEWL values at baseline, longer DFU wound duration, and more prior offloading use. These findings are clinically relevant as a higher TEWL measurement suggests complete wound closure may not have occurred, supporting the use of TEWL to identify a healed DFU at the point of care. Disclosure B.M. Schmidt: None. C. Spino: Other Relationship; Astellas Pharma Inc., Endo International, JW Pharmaceutical. Consultant; Certa Therapeutics. S. Roy: None. G. Gordillo: Consultant; Biocomposites LLC. Stock/Shareholder; Med Compliance IQ. C.M. Holmes: None. C.K. Sen: Consultant; SouthWest Technologies. Board Member; VisopalExo. Consultant; Vomaris Inc. R. Busui: Board Member; American Diabetes Association. Consultant; Procter & Gamble, AstraZeneca, Averitas Pharma, Inc., Bayer Inc., Lexicon Pharmaceuticals, Inc., Nevro Corp., Ono Pharmaceutical Co., Ltd., Novo Nordisk, Roche Diagnostics. Advisory Panel; ADA/ACC Diabetes by Heart Program. Funding BMS is supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) grant K23DK131261 and U01DK119083. CAS is supported by U24DK122927CMH is supported by U01DK119083CKS is supported by U01DK119099RPB was supported by R01DK107956, R01DK11672, U01DK119083 and the JDRF Center of Excellence at University of Michigan.

Background Microsurgical techniques have a steep learning curve. We adapted validated surgical approaches to develop a novel, competency-based microsurgical simulation curriculum called Fundamentals of Microsurgery (FMS). The purpose of this study is to present our experience with FMS and quantify the effect of the curriculum on resident performance in the operating room.Methods Trainees underwent the FMS curriculum requiring task progression: (1) rubber band transfer, (2) coupler tine grasping, (3) glove laceration repair, (4) synthetic vessel anastomosis, and (5) vessel anastomosis in a deep cavity. Resident anastomoses were also evaluated in the operative room with the Stanford Microsurgery and Resident Training (SMaRT) tool to evaluate technical performance. The National Aeronautics and Space Administration Task Load Index (NASA-TLX) and Short-Form Spielberger State-Trait Anxiety Inventory (STAI-6) quantified learner anxiety and workload.Results A total of 62 anastomoses were performed by residents in the operating room during patient care. Higher FMS task completion showed an increased mean SMaRT score ( p = 0.05), and a lower mean STAI-6 score (performance anxiety) ( p = 0.03). Regression analysis demonstrated residents with higher SMaRT score had lower NASA-TLX score (mental workload) ( p < 0.01) and STAI-6 scores ( p < 0.01).Conclusion A novel microsurgical simulation program FMS was implemented. We found progression of trainees through the program translated to better technique (higher SMaRT scores) in the operating room and lower performance anxiety on STAI-6 surveys. This suggests that the FMS curriculum improves proficiency in basic microsurgical skills, reduces trainee mental workload, anxiety, and improves intraoperative clinical proficiency.

Introduction Between 5% and 20% of all combat-related casualties are attributed to burn wounds. A decrease in the mortality rate of burns by about 36% can be achieved with early treatment, but this is contingent upon accurate characterization of the burn. Precise burn injury classification is recognized as a crucial aspect of the medical artificial intelligence (AI) field. An autonomous AI system designed to analyze multiple characteristics of burns using modalities including ultrasound and RGB images is described. Materials and Methods A two-part dataset is created for the training and validation of the AI: in vivo B-mode ultrasound scans collected from porcine subjects (10,085 frames), and RGB images manually collected from web sources (338 images). The framework in use leverages an explanation system to corroborate and integrate burn expert's knowledge, suggesting new features and ensuring the validity of the model. Through the utilization of this framework, it is discovered that B-mode ultrasound classifiers can be enhanced by supplying textural features. More specifically, it is confirmed that statistical texture features extracted from ultrasound frames can increase the accuracy of the burn depth classifier. Results The system, with all included features selected using explainable AI, is capable of classifying burn depth with accuracy and F1 average above 80%. Additionally, the segmentation module has been found capable of segmenting with a mean global accuracy greater than 84%, and a mean intersection-over-union score over 0.74. Conclusions This work demonstrates the feasibility of accurate and automated burn characterization for AI and indicates that these systems can be improved with additional features when a human expert is combined with explainable AI. This is demonstrated on real data (human for segmentation and porcine for depth classification) and establishes the groundwork for further deep-learning thrusts in the area of burn analysis.

BACKGROUND: In the United States (US), about 1.25 million people are treated each year for burns, and 40,000 are hospitalized for the treatment of these injuries resulting in high medical costs, approximately $7.9 billion per year. Early assessment of burn depth considered a predictor of pathological scarring that occurs in 30%-91% of burn injuries, and prioritizing burns that require surgical intervention is a critical task. However, it continues to be an open clinical challenge. We sought to develop a high accuracy automated system, that relies on multimodal Harmonic B-mode ultrasound (HUSD B-mode) and Tissue Elastography imaging (TEI), to classify burn pathology using novel techniques based on machine learning and artificial intelligence (AI). METHODS: Burn wounds of different degrees (superficial, partial, and full thickness; n=2 each per pig; size 2“x2”) were created on the dorsum of female domestic pigs (70-80lbs) (n=6 pigs) using a standardized burner. Burn wounds were treated with the same dressing. Progression of burn wounds was followed by non-invasive imaging using digital photographs, HUSD B-mode, and TEI videos at day 0 - postburn, and on days 3, 7, 14, 21, 28, 35 and 42 postburn. Burn depth was validated by histopathological analysis and results were compared with US-acquired data at different time points. State-of-the-art deep learning methods to analyze images and videos such as convolutional neural networks (CNNs) were employed. These features were used to train task-specific networks. In the case of depth classification, the classifier was further enhanced using traditional computer vision features. RESULTS: Burns of different degrees were successfully created on all the pigs. HUSD B- mode and TEI showed characteristic biomechanical and biological response patterns unique to the different degrees of burn which was validated by H&E staining. Histological pattern graded the burn injury from superficial involving only epidermal layer to the full thickness burn involving all skin and subcutaneous layers. Data labelling, segmentation and augmentation was done and fed into the AI system. Our system was able to classify burn wounds with a mean accuracy greater than 90%. In burn segmentation, our system achieved a mean global accuracy greater than 0.87. Further, we calculated a mean intersection over union (IoU) score of ~0.8. These scores represent a statistically significant improvement over our baseline segmentation model. Critically, this part of our system presented a clear and human-readable masks to understand the surface of burn wounds, allowing a high degree of explainability often required to interpret AI-produced results. CONCLUSIONS: This work presented elements of an autonomous AI system to analyze and predict burn depth via texture-based image processing algorithms using multiple common medical modalities.

Background: Lymphedema is chronic limb swelling resulting from lymphatic dysfunction. It affects an estimated 5 million Americans. There is no cure for this disease. Experimental gene-based therapeutic approaches (e.g., using viral vectors) have had limited translational applicability. Tissue nanotransfection technology (TNT) utilizes a direct, transcutaneous non-viral vector, gene delivery using a chip with nanochannel poration in a rapid (<100ms) focused electric field. This platform technology has been used for various applications in tissue reprogramming. The ability to deliver genetic cargo at a focal, non-global site would have practical clinical potential in lymphedema treatment. The purpose of this study is to experimentally evaluate the applicability of TNT for lymphedema. Methods: The murine tail model of lymphedema was utilized. A 3 mm full thickness skin excision and lymphatic vessel disruption was performed 20 mm from the base of the tail in twelve C57BL/6 mice. TNT was applied to the murine tail (day 0) directly at the surgical site with genetic cargo loaded into the TNT reservoir: Group I (control) was given pCMV6 (expression vector backbone alone) (n=6); Group II had pCMV6-Prox1 (n=6). TNT was applied with square wave pulse electric stimulation (10x10ms pulses, 250 V, 10 mA). The efficiency of gene delivery was assessed through qRT-PCR using primers with SYBR Green fluorescence quantification and immunostaining with anti-Prox1 antibody. Results: The experimental Group II exhibited four-fold increased expression of Prox1 using qRT-PCR compared to control Group I at the site of TNT treatment (P<0.05). Increased expression of Prox1 was also observed with immunohistochemistry 3 days post-TNT application. Intensity quantification of immunohistochemistry revealed greater expression of Prox1 in Group II when compared to Group I (P<0.05). Conclusion: This study demonstrates a novel, focal nanotechnology approach for local genetic cargo delivery in murine tail lymphedema model without the use of viral vectors for transfection. TNT can rapidly and effectively be applied for potentially therapeutic delivery of factors locally at the site of lymphedema.

Extracellular vesicles (EVs) are nanovesicles released by all eu-karyotic cells. This work reports the first nanoscale fluorescent visualization of tumor-originating vesicles bearing an angio-genic microRNA (miR)-126 cargo. In a validated experimental model of lethal murine vascular neoplasm, tumor-originating EV delivered its miR-126 cargo to tumor-associated macro-phages (TAMs). Such delivery resulted in an angiogenic (LYVE+) change of state in TAM that supported tumor formation. Study of the trafficking of tumor-originating fluo-rescently tagged EV revealed colocalization with TAM demon-strating uptake by these cells. Ex vivo treatment of macro-phages with tumor-derived EVs led to gain of tumorigenicity in these isolated cells. Single-cell RNA sequencing of macro-phages revealed that EV-borne miR-126 characterized the angiogenic change of state. Unique gene expression signatures of specific macrophage clusters responsive to miR-126-en-riched tumor-derived EVs were revealed. Topical tissue nano-transfection (TNT) delivery of an oligonucleotide comprising an anti-miR against miR-126 resulted in significant knock-down of miR-126 in the tumor tissue. miR-126 knockdown resulted in complete involution of the tumor and improved survival rate of tumor-affected mice. This work identifies a novel tumorigenic mechanism that relies on tumorigenic state change of TAM caused by tumor-originating EV-borne angio-miR. This disease process can be effectively targeted by topical TNT of superficial tumors.

PURPOSE: We have previously shown that urinary microRNA126 (miR126) levels are a biomarker for hemangioma growth based on a prospective longitudinal study of children with hemangiomas and healthy age matched controls. We sought to determine the significance of miR126 on the regulation of hemangioma growth using a validated mouse model. METHODS: qPCR measurement of miR126 levels was done in EOMA cells and non-tumor forming endothelial cells. 6 weeks old female 129P/3 mice received a subcutaneous injection of 5x106 EOMA cells. 4 days post injection tumors were visible and topical administration of control antagomiR (n=8) or miR126 antagomiR (n=15) delivered transcutaneously using tissue nanotransfection electroporation technique. Treatments were given twice weekly RESULTS: EOMA cells had 8,000-fold increase in miR126 levels compared to non-tumor forming endothelial cells. Control mice had significantly larger tumors with 100% mortality by 17 days post EOMA cell injection. miR126 antagomiR treated mice were significantly different from controls with decreased mortality (40%), smaller tumor size, prolonged survival with the last mouse death at 29 days post-injection, complete tumor regression by 6 weeks (n=9) and no observed recurrence at 90 days. CONCLUSION: These are the first results to demonstrate complete regression of hemangioendothelioma in response to miR26 inhibition. Elevation of miR126 levels in human hemangioma and murine hemangioendothelioma indicate shared mechanisms of growth. These findings demonstrate that miR126 is necessary for growth of both tumor types and represents a valid therapeutic target to treat hemangiomas.

PURPOSE: The spread of biofilms on medical implants represents one of the principal triggers of persistent and chronic infections in clinical settings. Nearly 300,000 women annually have breast implant surgery in the United States, for reasons including post-mastectomy breast reconstruction, revision of prior augmentation/ reconstruction, cosmetic augmentation, and gender affirmation. There has been increased identification of patients experiencing a constellation of symptoms related to their implants termed as breast implant illness (BII). In this work, we report that bacterial biofilm associated with breast implant, metabolize fatty acid oleic acid present in the breast tissue milieu to oxylipins, one such oxylipin identified from this study is (E)-10-hydroxy-8-octadecenoic acid (10-HOME). We hypothesize that immunomodulatory effects of oxylipin 10-HOME produced by biofilm present on the implant could be a possible etiology for BII pathogenesis. METHOD: Implants, peri-prosthetic tissues and blood was collected from BII subjects (n=46) and two control groups, group I, (non-BII, n=34) patients with breast implants, no BII symptoms. Group II (normal tissue, n = 20), patients without an implant, whose breast tissue was removed due to surgical procedures. A questionnaire developed based on epidemiological studies on BII screened for the commonly reported symptoms associated with BII. Predictive variables included age, diabetes status, co-morbidities, type (smooth/textured) and duration of implant. Scanning electron microscopy (SEM), 16S rRNA (genomic) next generation sequencing (NGS) were used for bacterial biofilm identification. 10-HOME was quantitated through targeted and untargeted lipidomic analyses using LC-MS-MS. RNA-Seq analysis was performed on peri-prosthetic breast tissues. Flow cytometry and mass cytometry (CyToF) were conducted to investigate the role of immune cells. RESULTS: Bacterial biofilm was detected through SEM and 16SrRNA NGS. Bivariate analysis using cross-tabulation was performed between presence of biofilm and the study groups. Using the two-sample test of proportions with z-tests, Staphylococcus epidermidis colonization was observed to be higher in the BII group (73.33%) compared to non-BII group (16.67%, p=0.018) and the normal group (10%, p=0.036). The BII group was 2.4 times more likely to have S. epidermidis colonization compared to the non-BII group (Odds Ratio=2.4). Similarly, when comparing with normal group, the BII group was 3.4 times more likely to have S. epidermidis. Elevated levels of 10-HOME in BII compared to non-BII samples, (p<0.0001) were observed through mass spectrometry. Positive correlation was observed between bacterial abundance and concentration of 10-HOME in BII subjects (R2=0.88). RNA-Seq analysis on peri-prosthetic tissue and flow/ mass cytometry analyses from peripheral blood derived lymphocytes showed increased abundance of CD4+ Th1 cells. Th1 cells have been reported to be activated in auto-immune diseases. No significant difference was observed in the abundance of other Th subtypes (Th2, Th9 and Th22). Oxylipin 10-HOME polarized CD4+ naïve T cells to Th1 subtype in vitro. CONCLUSION: This study investigated the biofilm hypothesis of BII through a biofilm derived immunogenic metabolite. Through a systematic cause-effect based studies, the work shows activation of Th1 cells in presence of 10-HOME. The study provides the first evidence of a possible etiology of BII mediated via bacterial biofilm derived 10-HOME.

An extreme chronic wound tissue microenvironment causes epigenetic gene silencing. An unbiased whole-genome methylome was studied in the wound-edge tissue of patients with chronic wounds. A total of 4,689 differentially methylated regions (DMRs) were identified in chronic wound-edge skin compared with unwounded human skin. Hypermethylation was more frequently observed (3,661 DMRs) in the chronic wound-edge tissue compared with hypomethylation (1,028 DMRs). Twenty-six hypermethylated DMRs were involved in epithelial-mesenchymal transition (EMT). Bisulfite sequencing validated hypermethylation of a predicted specific upstream regulator TP53. RNA-Seq analysis was performed to qualify findings from methylome analysis. Analysis of the downregulated genes identified the TP53 signaling pathway as being significantly silenced. Direct comparison of hypermethylation and downregulated genes identified 4 genes, ADAM17, NOTCH, TWIST1, and SMURF1, that functionally represent the EMT pathway. Single-cell RNA-Seq studies revealed that these effects on gene expression were limited to the keratinocyte cell compartment. Experimental murine studies established that tissue ischemia potently induces wound-edge gene methylation and that 5′-azacytidine, inhibitor of methylation, improved wound closure. To specifically address the significance of TP53 methylation, keratinocyte-specific editing of TP53 methylation at the wound edge was achieved by a tissue nanotransfection-based CRISPR/dCas9 approach. This work identified that reversal of methylation-dependent keratinocyte gene silencing represents a productive therapeutic strategy to improve wound closure.

Therapeutic VEGF replenishment has met with limited success for the management of critical limb threatening ischemia (CLTI). To improve outcomes of VEGF therapy we applied single-cell RNA sequencing technology to study the endothelial cells of the human diabetic skin. Single-cell suspensions were generated from the human skin followed by cDNA preparation using Chromium Next GEM Single-cell 3' Kit v3.1. Using appropriate quality control measures, 36,487 cells were chosen for downstream analysis. scRNA-seq studies identified that although VEGF signaling was not significantly altered in diabetic vs non-diabetic skin, phospholipase-C-Gamma-2 (PLCg2) was down-regulated. The significance of PLCg2 in VEGF mediated increase in endothelial cell metabolism and function was assessed in cultured human microvascular endothelial cells. In these cells, VEGF enhanced mitochondrial function as indicated by elevation in oxygen consumption rate and extracellular acidification rate. VEGF-dependent increase in cell metabolism was blunted in response to PLCg2 inhibition. Follow-up rescue studies therefore focused on understanding the significance of VEGF therapy in presence or absence of endothelial PLCg2 in type-1 (streptozotocin-injected) and type-2 (db/db) diabetic ischemic tissue. Non-viral topical tissue nanotransfection (TNT) delivery of CDH5 promoter driven PLCg2-ORF promoted the rescue of hind-limb ischemia in diabetic mice. Improvement of blood flow was also associated with higher abundance of VWF+/CD31+ and VWF+/SMA+ immunohistochemical staining. TNT-based gene delivery was not associated with tissue edema, a commonly noted complication associated with pro-angiogenic gene therapies. Taken together our study demonstrates that TNT mediated delivery of endothelial PLCg2, as part of combination gene therapy, is effective in diabetic ischemic limb rescue.

Approximately 1.25 million people in the United States are treated each year for burn injuries. Precise burn injury classification is an important aspect of the medical AI field. In this work, we propose an explainable human-in-the-loop framework for improving burn ultrasound classification models. Our framework leverages an explanation system based on the LIME classification explainer to corroborate and integrate a burn expert's knowledge -- suggesting new features and ensuring the validity of the model. Using this framework, we discover that B-mode ultrasound classifiers can be enhanced by supplying textural features. More specifically, we confirm that texture features based on the Gray Level Co-occurance Matrix (GLCM) of ultrasound frames can increase the accuracy of transfer learned burn depth classifiers. We test our hypothesis on real data from porcine subjects. We show improvements in the accuracy of burn depth classification -- from ~88% to ~94% -- once modified according to our framework.

LEARNING OBJECTIVES:After studying this article, the participant should be able to: 1. Understand the basics of biofilm infection and be able to distinguish between planktonic and biofilm modes of growth. 2. Have a working knowledge of conventional and emerging antibiofilm therapies and their modes of action as they pertain to wound care. 3. Understand the challenges associated with testing and marketing antibiofilm strategies and the context within which these strategies may have effective value.SUMMARY:The Centers for Disease Control and Prevention estimate for human infectious diseases caused by bacteria with a biofilm phenotype is 65 percent and the National Institutes of Health estimate is closer to 80 percent. Biofilms are hostile microbial aggregates because, within their polymeric matrix cocoons, they are protected from antimicrobial therapy and attack from host defenses. Biofilm-infected wounds, even when closed, show functional deficits such as deficient extracellular matrix and impaired barrier function, which are likely to cause wound recidivism. The management of invasive wound infection often includes systemic antimicrobial therapy in combination with débridement of wounds to a healthy tissue bed as determined by the surgeon who has no way of visualizing the biofilm. The exceedingly high incidence of false-negative cultures for bacteria in a biofilm state leads to missed diagnoses of wound infection. The use of topical and parenteral antimicrobial therapy without wound débridement have had limited impact on decreasing biofilm infection, which remains a major problem in wound care. Current claims to manage wound biofilm infection rest on limited early-stage data. In most cases, such data originate from limited experimental systems that lack host immune defense. In making decisions on the choice of commercial products to manage wound biofilm infection, it is important to critically appreciate the mechanism of action and significance of the relevant experimental system. In this work, the authors critically review different categories of antibiofilm products, with emphasis on their strengths and limitations as evident from the published literature.

Objective: This work addressing complexities in wound infection, seeks to test the reliance of bacterial pathogen Pseudomonas aeruginosa (PA) on host skin lipids to form biofilm with pathological consequences. Background: PA biofilm causes wound chronicity. Both CDC as well as NIH recognizes biofilm infection as a threat leading to wound chronicity. Chronic wounds on lower extremities often lead to surgical limb amputation. Methods: An established preclinical porcine chronic wound biofilm model, infected with PA or Pseudomonas aeruginosa ceramidase mutant (PA∆Cer), was used. Results: We observed that bacteria drew resource from host lipids to induce PA ceramidase expression by three orders of magnitude. PA utilized product of host ceramide catabolism to augment transcription of PA ceramidase. Biofilm formation was more robust in PA compared to PA∆Cer. Downstream products of such metabolism such as sphingosine and sphingosine-1-phosphate were both directly implicated in the induction of ceramidase and inhibition of peroxisome proliferator-activated receptor (PPAR)δ, respectively. PA biofilm, in a ceram-idastin-sensitive manner, also silenced PPARδ via induction of miR-106b. Low PPARδ limited ABCA12 expression resulting in disruption of skin lipid homeostasis. Barrier function of the wound-site was thus compromised. Conclusions: This work demonstrates that microbial pathogens must co-opt host skin lipids to unleash biofilm pathogenicity. Anti-biofilm strategies must not necessarily always target the microbe and targeting host lipids at risk of infection could be productive. This work may be viewed as a first step, laying fundamental mechanistic groundwork, toward a paradigm change in biofilm management.

The visiting professor is an important tradition in plastic and reconstructive surgery. Innovative techniques and developing frontiers in our field are disseminated in cross-institutional learning opportunities such as visiting professorships. Residents can network with the visiting professor, which can enhance career opportunities. During the peak of coronavirus disease of 2019 (COVID-19) social distancing restrictions, seven virtual visiting professor lectures were performed during educational conferences at our institution over a 3-week period. The lectures followed a standard format to capture aspects of a true visiting professor over 1 hour: (1) introduction of virtual visiting professor by faculty (3 minutes); senior/chief residents introduce themselves (7 minutes); interactive life lessons from the virtual visiting professor (10 minutes); and lecture and questions (40 minutes). Visiting professors and residents were surveyed after the lectures. (See Figure, Supplemental Digital Content 1, which shows the survey administered to visiting professors immediately after virtual lecture, https://links.lww.com/PRS/E626. See Figure, Supplemental Digital Content 2, which shows the survey administered to residents immediately after virtual lecture, https://links.lww.com/PRS/E627.) All visiting professors found the virtual lecturer program convenient. Before the COVID-19 pandemic, one professor (14.3 percent) was in favor of teleconferencing for lectures, two of seven (28.6 percent) were against teleconferencing for lectures, and four of seven (57.1 percent) were neutral. Following relaxing of COVID-19 social distancing, six of seven visiting professors (85.7 percent) would like to continue virtual visiting professorships (Tables 1 and 2). Table 1. - Virtual Visiting Professor Survey Responses Visiting Professor Responses Total 7 Approximately how many lectures do you give per year? 11.3 How many lectures have you ever given while visiting other institutions? 4.9 How many years have you been in practice? 6.4 yr How convenient do you find virtual visiting professor lectures? Not convenient 0% Neutral 0% Somewhat convenient 0% Convenient 14.3% Very convenient 85.7% Is a virtual visiting professorship something you would like to continue after COVID-19? Yes 85.7% No 14.3% How similar did your virtual visiting professor lecture allow you to connect with residents at the visiting institution compared to an in-person visit? In-person is far superior 42.9% In-person is somewhat better 57.1% Which manner do you think provides a larger connection to the residents? In-person lecture 85.7% Virtual lecture 14.3% Equal 0% How likely are you to give visiting professor lectures at additional institutions if a virtual option is offered? I would give fewer lectures 0% I would give the same amount 0% I would probably give more lectures 71.4% I would definitely give more lectures 28.6% How many previous Webinars or teleconferences have you given before the Covid-19 pandemic? 0 28.6% 1–5 57.1% 6–10 14.3% >10 0% Before the Covid-19 pandemic, what was your opinion on teleconferencing for educational lectures? In favor of teleconferencing 14.3% Neutral 57.1% Not in favor of teleconferencing 28.6% How many residency programs would you prefer to have at each visiting professor lecture? 1 42.9% 2–5 42.9% 6–10 0% >10 14.3% On average, how many cases did you do per week before Covid-19? 10.3 On average, how many cases do you do per week during Covid-19? 2.8 Table 2. - Resident Survey Responses Resident Responses Distribution Integrated 1 2 Integrated 2 2 Integrated 3 2 Integrated 4/independent 1 4 Integrated 5/independent 2 4 Integrated 6/independent 3 3 How educational do you find the visiting professors? Not educational 0% Neutral 0% Somewhat educational 5.9% Educational 29.4% Very educational 64.7% What is your preferred learning method? Reading 29.4% Online lecture/video 52.9% In-person lecture 17.6% Do you prefer in-person or virtual visiting professor? In-person 35.3% Virtual 29.4% No preference 35.3% Based on the lectures you received, how much did you learn? Nothing 0% Minimal 0% Moderate educational amount 64.7% Large educational amount 35.3% How educational do you find the virtual visiting professor lecture compared to previous in-person visiting professor lecture? Far less educational 5.9% Somewhat less educational 5.9% Same educational 41.2% Somewhat more educational 47.1% Far more educational 0% What characteristics of virtual conferencing do you agree with? Worse image quality 5.9% Better image quality 52.9% Harder to hear speaker 17.6% Better to hear speaker 47.1% More distractions 23.5% Less distractions 64.7% How important do you think a face-to-face meeting with a visiting professor is for your career aspirations? Not important at all 0% Neutral 35.3% Somewhat important 29.4% Important 23.5% Very important 11.8% Do you plan to pursue a fellowship? Yes 64.7% No 35.3% When surveyed on their experiences, six of 17 residents (35.3 percent) preferred in-person visiting professors and five of 17 (29.4 percent) preferred virtual visiting professors. Residents found the virtual visiting professor more educational [eight of 17 (47.1 percent)], whereas seven of 17 (41.2 percent) found the virtual visiting professors to have the same educational value as in-person, and two of 17 (11.8 percent) found the virtual visiting professors to be less educational. Residents thought face-to-face meetings with a visiting professor were more important for career aspirations, and only six of 17 (35.3 percent) and five of 17 (29.4 percent) found it to be somewhat important. The mean cost of each visiting professor event during the 2018 academic year was $1780 ± $238. The cost of an average virtual visiting professor was $15 per visit (gift card) (p < 0.0001). This study revealed that both visiting professors and residents found our virtual visiting professor program maintained its educational value and elements of personal connection. Visiting professors found them convenient and worth continuing after travel restoration after COVID-19. The benefits of a virtual professorship are clear. A visiting professor may be able to give a visiting lecture, establish a national reputation in their area of expertise, and share their educational approach with learners at another program without having to travel or sacrifice their routine clinical or research opportunities. There is a financial burden to the institution with an in-person visiting professor. The hosting plastic surgery program typically pays airfare, hotel, meals, and an honorarium. The visiting professor sacrifices clinical and research opportunities including missed relative value unit earning potential. A routine virtual visiting professor represents a potential paradigm shift. It may not replace all in-person visiting professors entirely given some of the advantages an in-person experience offers, but the virtual visiting professor offers a comparable experience at a lower institutional cost. These results suggest that institutions should consider implementing a virtual visiting professor program to augment traditional visiting professor lectures even after social and travel normalization after the COVID-19 pandemic. Facilitating virtual personal interactions between the professor and the residents during the program is necessary. DISCLOSURE The authors have no financial disclosures to report.