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Introduction: Non-invasive angiography with indocyanine green dye facilitates the assessment of flaps. Although ICG angiography has been successfully utilized in clinical settings for human beings, its application in experimental models exhibits certain limitations. This study aimed to address the encountered issues in angiography with different experimental models and introduce a novel modification to the ICG imaging of the McFarlane flap. Materials-methods: Rats were randomly divided into three groups: the first group received an epigastric flap (n = 4), the second group received a deep inferior epigastric perforator flap (n = 4), and the third group received a dorsal flap (n = 8). In the first group, sterile silicone background was placed under two flaps. In the second group, no background was used. In the third group, a sterile silicone or aluminum foil was placed under the flaps. Flap perfusions were assessed using fluorescent imaging after flap adaptations, at postoperative 30th minute and 3., 5. and 7. days. Necrosis calculations were performed using all images obtained from the digital camera and the fluorescent imaging. In the third group, the flow velocities were also calculated. All flaps were sent for histopathological examination. Results: Even with a silicone background, clear perfusion evaluation and determining the threshold value for predicting necrosis rates were challenging. Moreover, a portion of the flaps without background material survived as grafts. Using an aluminum foil background improved image quality by reducing reflection from interior organs. Conclusion: The use of an aluminum foil background is beneficial in non-invasive angiography for assessing flap perfusions accurately and predicting necrosis in experimental animal models.

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2-O-(α-d-glucopyranosyl)-sn-glycerol (2-αGG) has been applied in the food industry due to its numerous physiological benefits. The synthesis of 2-αGG can be achieved through a cascade catalytic reaction involving sucrose phosphorylase (SP) and 2-O-α-glucosylglycerol phosphorylase (GGP). However, the low substrate transfer rates between free enzymes have hindered the efficiency of 2-αGG synthesis. To address this issue, a novel technology was developed to prepare sequential multi-enzyme nanoflowers via chemical crosslinking and protein assembly, thus overcoming diffusion limitations. Specifically, spatially sequential co-immobilized enzymes, referred to as SP-GGP@Cap, were created through the targeted assembly of Bifidobacterium adolescentis SP and Marinobacter adhaerens GGP on Ca2+. This assembly was facilitated by the spontaneous protein reaction between SpyTag and SpyCatcher. Compared to free SP-GGP, SP-GGP@Cap demonstrated improved thermal and pH stability. Moreover, SP-GGP@Cap enhanced the biosynthesis of 2-αGG, achieving a relative concentration of 98 %. Additionally, it retained the ability to catalyze the substrate to yield 61 % relative concentration of 2-αGG even after ten cycles of recycling. This study presents a strategy for the spatially sequential co-immobilization of multiple enzymes in a confined environment and provides an exceptional biocatalyst for the potential industrial production of 2-αGG.

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OBJECTIVE: The objective of this study was to evaluate whether a systematic image assessment protocol using SPY Elite images (LifeCell Corp., US) of viable tissue at the periphery of the surgical field was associated with positive wound healing outcomes following mastectomy and breast reconstruction. METHOD: Patients undergoing mastectomy and subsequent breast reconstruction surgery at a single tertiary medical centre were included. SPY images were prospectively analysed using a systematic image assessment protocol, and an absolute value of mean fluorescence was calculated by measuring peripheral, in-situ tissue from each image. Patient medical records were retrospectively reviewed for demographics, surgical characteristics and postoperative outcomes. These variables were statistically tested for associations with mean fluorescence. RESULTS: A total of 63 patients were included in the final analysis. We found that objectively determined mean fluorescence values were not statistically significantly associated with postoperative complications. CONCLUSION: In this study, objectively measured mean fluorescence values representing breast tissue remaining after dissection showed little utility in the assessment of postoperative wound healing outcomes as they did not identify patients who would later have complications of wound healing. DECLARATION OF INTEREST: The authors have no conflicts of interest to declare.

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NHX5 and NHX6, Arabidopsis endosomal antiporters, play a vital role in facilitating ion and pH homeostasis in endosomal compartments. Studies have found that NHX5 and NHX6 are essential for protein trafficking, auxin homeostasis, and plant growth and development. Here, we report the role of NHX5 and NHX6 in brassinosteroid (BR) signaling. We found that hypocotyl growth was enhanced in nhx5 nhx6 under epibrassinolide (eBR) treatment. nhx5 nhx6 bri1 was insensitive to eBR treatment, indicating that NHX5 and NHX6 are downstream of the BRI1 receptor in BR signaling. Moreover, confocal observation with both hypocotyls and root tips showed that BRI1-YFP localization in the plasma membrane (PM) was reduced in nhx5 nhx6. Interestingly, brefeldin A (BFA) treatment showed that formation of the BFA bodies containing BRI1 and their disassembling were disrupted in nhx5 nhx6. Further genetic analysis showed that NHX5/NHX6 and SYP22 may act coordinately in BR signaling. NHX5 and NHX6 may regulate SYP22 function by modulating cellular K+ and pH homeostasis. Importantly, NHX5 and NHX6 colocalize and interact with SYP22, but do not interact with BRI1. In summary, our findings indicate that NHX5/NHX6/SYP22 complex is essential for the regulation of BRI1 recycling and PM localization. The H+-leak facilitated by NHX5 and NHX6 offers a means of controlling BR signaling in plants.

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There is an emerging strong demand for smart environmentally responsive protein-based biomaterials with improved adhesion properties, especially underwater adhesion for potential environmental and medical applications. Based on the fusion of elastin-like polypeptides (ELPs), SpyCatcher and SpyTag modules, biosynthetic barnacle-derived protein was genetically engineered and self-assembled with an enhanced adhesion ability and temperature response. The water resistance ability of the synthetic protein biopolymer with a network structure increased to 98.8 from 58.5% of the original Cp19k, and the nonaqueous adhesion strength enhanced to 1.26 from 0.68 MPa of Cp19k. The biopolymer showed an improved adhesion ability toward hydrophilic and hydrophobic surfaces as well as diatomite powders. The combination of functional module ELPs and SpyTag/SpyCatcher could endow the biosynthetic protein with temperature response, an insoluble form above 42 °C and a soluble form at 4 °C. The combinational advantages including temperature response and adhesion performance make the self-assembled protein an excellent candidate in surgical adhesion, underwater repair, and surface modification of various coatings. Distinct from the traditional approach of utilizing solely ELPs, the integration of short ELPs with Spy partners exhibited a synergistic enhancement in the temperature response. The synergistic effects of two functional modules provide a technical method and insight for designing smart self-assembled protein-based biopolymers.

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Reading spy fiction has imprinted its traces on the thinking patterns of the readers that encourage them to consider it as a real event in their life. This case study is about a 37 year old woman, referred by a senior clinical psychologist with complaints of having the feeling of being monitored through a device and hearing the voices of the people. The brief therapeutic plan was based on Cognitive Behavioural Therapy which focused on developing insight, identifying cognitive errors, and enhancing her socialisation skills. This case report holds its implications to drift the attention of the readers and clinicians toward the issue of consuming content that may potentially have an impact on its reader's mental health.

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PURPOSE: The study aims to investigate the complications and long-term outcomes associated with retropectoral DTI breast reconstruction with IDF utilizing the SPY-Elite laser angiographic system. MATERIAL AND METHOD: This retrospective study was conducted from June 2017 to January 2023. We examined 52 patients (85 breasts) treated with a direct-to-implant retropectoral dual plane approach with IDF implant coverage. Informed consent was duly obtained from every participant. Inclusion criteria dictated that patients should have medium to large breasts and a second or third degree of ptosis, as per the Regnault ptosis scale. During the intraoperative evaluation, the mastectomy flaps and IDF were assessed with the SPY-Elite laser angiographic system using near-infrared imaging. We recorded patient demographics, characteristic data, and complications. RESULTS: A total of 52 patients, aged 27 to 63, underwent 85 mastectomies using a direct-to-implant retropectoral approach with inferior dermal flap. The average age of the patients was 48, and their average body mass index was 30.8, with a range of 28 to 43. The distance from the nipple to the inframammary fold varied between 14 and 24 cm. The implants used had an average size of 275 cc, ranging from 250 to 650 cc. Textured anatomic implants with either moderate plus or high profile were used in all cases. The sternal notch to nipple distance for these patients ranged from 24 to 38 cm. During the evaluation using the SPY-Elite laser angiographic system, insufficient distal marginal perfusion was detected in five out of 85 inferior dermal flaps, measuring between 2 and 5 cm2. These areas were subsequently debrided, and the reconstructions were successfully completed, representing 5.8% of cases. No instances of necrosis related to IDF have been observed. There have been no failed assessments conducted by SPY ICG. In total, the complication rate was 15.2%, with minor complications occurring in 8.2% of the breasts (7 out of 85) and major ones in 7% (6 out of 85). The subjects were monitored for an average of 14 months, the duration ranging from 12 to 24 months. CONCLUSION: Inferior dermal flaps have considerable advantages, such as a natural autologous blood supply, a more realistic tissue thickness and texture, lower costs, and better tolerance to post-reconstruction radiation. Moreover, using the IDF technique and assessing the perfusion of IDF and mastectomy flaps through the SPY-Elite laser angiographic system appears to be a dependable, efficient way to achieve good cosmetic results in one operation, eliminating the need for additional surgeries. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine Ratings, please refer to Table of Contents or online Instructions to Authors www.springer.com/00266.

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Remote sensing data are important for assessing ecological change, but their value is often restricted by their limited temporal coverage. Major historical events that affected the environment, such as those associated with colonial history, World War II, or the Green Revolution are not captured by modern remote sensing. In the present article, we highlight the potential of globally available black-and-white satellite photographs to expand ecological and conservation assessments back to the 1960s and to illuminate ecological concepts such as shifting baselines, time-lag responses, and legacy effects. This historical satellite photography can be used to monitor ecosystem extent and structure, species' populations and habitats, and human pressures on the environment. Even though the data were declassified decades ago, their use in ecology and conservation remains limited. But recent advances in image processing and analysis can now unlock this research resource. We encourage the use of this opportunity to address important ecological and conservation questions.

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Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder caused by mutations in the tumor suppressor gene MEN1 and is characterized by parathyroid, pancreatic islet, and anterior pituitary tumors. Primary hyperparathyroidism is the most characteristic finding in MEN1, and intraoperative identification and accurate removal of the diseased parathyroid glands are vital since incomplete excision results in recurrence. This case report describes a 59-year-old woman who had pancreatic islet cell tumors and pituitary tumors and underwent selective transsphenoidal adenomectomy. Based on her medical history and examination, the diagnosis of primary hyperparathyroidism in MEN1 was made, and she underwent total parathyroidectomy with autotransplantation with SPY-Elite®ï¸ Fluorescence Imaging (Stryker Corp., Kalamazoo, MI). Intraoperative identification of the parathyroid glands using autofluorescence with real-time intrinsic near-infrared (NIR) imaging made it easier to detect all of the parathyroid hyperplasia. After the surgery, she had hypoparathyroidism and continued with her oral calcium and vitamin D supplementation to maintain normal calcium levels during follow-up. Herein, we would like to advocate that the use of parathyroid gland autofluorescence with real-time intrinsic NIR imaging may be useful for identifying parathyroid tumors in patients with primary hyperparathyroidism in MEN1.

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Streptococcus pyogenes, Group A Streptococcus (GAS), is a human pathogen that causes a spectrum of diseases from mild to severe, including GAS pharyngitis, a common acute respiratory disease in developed countries. Although wastewater-based epidemiology (WBE) has been extensively used to monitor viral pathogens such as severe acute respiratory syndrome coronavirus 2, its applicability to S. pyogenes remains unexplored. This study was conducted to investigate the feasibility of detecting and quantifying S. pyogenes in wastewater by quantitative polymerase chain reaction (qPCR) and evaluate the applicability of WBE for monitoring the prevalence of GAS pharyngitis. A total of 52 grab influent samples were collected from a wastewater treatment plant in Japan once a week between March 2023 and February 2024. The samples were centrifuged, followed by nucleic acid extraction and qPCR for the S. pyogenes-specific genes speB and spy1258. Of the 52 samples, 90 % and 81 % were positive for speB and spy1258 genes, respectively, indicating the feasibility of S. pyogenes for wastewater surveillance. However, the percentage of quantifiable samples for speB gene was significantly higher in winter than in spring and summer. Similarly, the concentrations of both genes in wastewater samples were significantly higher in winter (speB, 4.1 ± 0.27 log10 copies/L; spy1258, 4.1 ± 0.28 log10 copies/L; One-way ANOVA, p < 0.01) than in spring and summer. Higher concentrations and detection ratios of S. pyogenes genes were observed during increased GAS pharyngitis cases in the catchment. Significant moderate correlations were observed between target gene concentrations and reported GAS pharyngitis cases. This study enhances the understanding role of WBE in monitoring and managing infectious diseases within communities.

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Psyllids, members of the family Triozidae, represent a potential threat to the cultivation of solanaceous and apiaceous crops worldwide, mainly as vectors of the phloem-restricted bacterium 'Candidatus Liberibacter solanacearum' (Lso). The Lso haplotypes C, D and E are known to affect apiaceous crops, such as carrot and celery, in several European countries. In Italy, data on the incidence and natural spread of both Lso and psyllids have not been reported so far. In this study, the presence of the vectors was investigated in a main Italian district for carrot production, the "Altopiano del Fucino" area (Central Italy). Both occasional and regular surveys were carried out on a total of five carrot fields and one potato field in 2021 and 2022. Bactericera trigonica (Hodkinson), which is known to efficiently transmit Lso to carrots, was found to be well-established in the area. High levels of population density were recorded in the summer period (more than 100 adult specimens per trap caught every two weeks) and then sharply decreased after the carrot harvest, confirming the strict association of this psyllid species with crop availability. In 2022, 27.5% of the total tested psyllid samples resulted in being positive for Lso haplotypes D and E, the latter being prevalent. This survey revealed, for the first time in Italy, the presence of B. trigonica adults associated with Lso in carrot crops. Although this study was limited to a few fields located in one area, it provided important evidence of the risks for Lso outbreaks and prompted further research to assess the spread and incidence of the disease in apiaceous cultivations in Italy.

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Whole-cell biosensors have demonstrated promising capabilities in detecting target molecules. However, their limited selectivity and precision can be attributed to the broad substrate tolerance of natural proteins. In this study, we aim to enhance the performance of whole-cell biosensors by incorporating of logic AND gates. Specifically, we utilize the HrpR/S system, a widely employed hetero-regulation module from Pseudomonas syringae in synthetic biology, to construct an orthogonal AND gate in Escherichia coli. To accomplish this, we compare the HrpR/S system with self-associating split fluorescent proteins using the Spy Tag/Spy Catcher system. Our objective is to selectively activate a reporter gene in the presence of both IPTG and Hg(II) ions. Through systematic genetic engineering and evaluation of various biological parts under diverse working conditions, our research demonstrates the utility of self-associating split fluorescent proteins in developing high-performance whole-cell biosensors. This approach offers advantages such as engineering simplicity, reduced basal activity, and improved selectivity. Furthermore, the comparison with the HrpR/S system serves as a valuable control model, providing insights into the relative advantages and limitations of each approach. These findings present a systematic and adaptable strategy to overcome the substrate tolerance challenge faced by whole-cell biosensors.

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Purpose This study aims to assess and validate the role and cost-effectiveness of indocyanine green angiography (ICGA) in free flap surgery outcomes. A new intraoperative protocol of whole-body surface warming (WBSW) for all free flap surgeries during the strategic "microbreaks" is also described. Methods A retrospective analysis of 877 consecutive free flaps, performed over 12 years, is presented. The results of the ICGA group ( n = 438) were compared with the historical No-ICGA group ( n = 439), and statistical significance was calculated for three crucial flap-related adverse outcomes and cost-effectiveness. ICGA was also used as a tool to show the effect of WBSW on free flaps. Results ICGA showed a notably strong statistical significance in decreasing two outcome parameters, namely, partial flap loss and re-exploration rate. It was also cost-effective. ICGA also demonstrated the positive role of WBSW in increasing flap perfusion. Conclusions Our study shows that the usage of ICGA for intraoperative assessment of flap perfusion can significantly reduce the partial flap loss and re-exploration rate in free flap surgeries in a cost-effective manner. A new protocol of WBSW is also described and recommended to increase flap perfusion in all free flap surgeries.

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BACKGROUND: Mussel foot proteins (Mfps) are considered as remarkable materials due to their extraordinary adhesive capability. Recombinant expression is an ideal way to synthesis these proteins at large scale. However, secretory expression of Mfps into culture medium has not been achieved in a heterologous host. METHODS AND RESULTS: Here, to realize the secretion of Mfp3 and Mfp5 in Bacillus subtilis, signal peptide screening was first performed. Minimal Mfp3-6×His was targeted into the growth medium with AmyE signal peptide. We found that a small chaperone protein Spy was secreted efficiently in B. subtilis, and the fusion proteins Spy-Mfp3-6×His and Spy-Mfp5-6×His could also be delivered into growth medium well. The yield of Spy-Mfp3-6×His and Spy-Mfp5-6×His reached 255 and 119 mg L-1 at shake flask conditions, respectively. Mfp3-6×His and Mfp5-6×His were finally purified via TEV protease cleavage and NTA affinity chromatography. CONCLUSION: Mfp3-6×His and Mfp5-6×His could be efficiently secreted using a chaperone protein Spy as fusion tag in B. subtilis.

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Investor sentiment plays a crucial role in the stock market, and in recent years, numerous studies have aimed to predict future stock prices by analyzing market sentiment obtained from social media or news. This study investigates the use of investor sentiment from social media, with a focus on Stocktwits, a social media platform for investors. However, using investor sentiment on Stocktwits to predict stock price movements may be challenging due to a lack of user-initiated sentiment data and the limitations of existing sentiment analyzers, which may inaccurately classify neutral comments. To overcome these challenges, this study proposes an alternative approach using FinBERT, a pre-trained language model specifically designed to analyze the sentiment of financial text. This study proposes an ensemble support vector machine for improving the accuracy of stock price movement predictions. Then, it predicts the future movement of SPDR S&P 500 Index Exchange Traded Funds using the rolling window approach to prevent look-ahead bias. Through comparing various techniques for generating sentiment, our results show that using the FinBERT model for sentiment analysis yields the best results, with an F1-score that is 4-5% higher than other techniques. Additionally, the proposed ensemble support vector machine improves the accuracy of stock price movement predictions when compared to the original support vector machine in a series of experiments.

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PURPOSE: ROR1 and ROR2 are Type 1 tyrosine kinase-like orphan receptors for Wnt5a that are associated with breast cancer progression. Experimental agents targeting ROR1 and ROR2 are in clinical trials. This study evaluated whether expression levels of ROR1 or ROR2 correlated with one another or with clinical outcomes. METHODS: We interrogated the clinical significance of high-level gene expression of ROR1 and/or ROR2 in the annotated transcriptome dataset from 989 patients with high-risk early breast cancer enrolled in one of nine completed/graduated/experimental and control arms in the neoadjuvant I-SPY2 clinical trial (NCT01042379). RESULTS: High ROR1 or high ROR2 was associated with breast cancer subtypes. High ROR1 was more prevalent among hormone receptor-negative and human epidermal growth factor receptor 2-negative (HR-HER2-) tumors and high ROR2 was less prevalent in this subtype. Although not associated with pathologic complete response, high ROR1 or high ROR2 each was associated with event-free survival (EFS) in distinct subtypes. High ROR1 associated with a worse EFS in HR + HER2- patients with high post-treatment residual cancer burden (RCB-II/III) (HR 1.41, 95% CI = 1.11-1.80) but not in patients with minimal post-treatment disease (RCB-0/I) (HR 1.85, 95% CI = 0.74-4.61). High ROR2 associated with an increased risk of relapse in patients with HER2 + disease and RCB-0/I (HR 3.46, 95% CI = 1.33-9.020) but not RCB-II/III (HR 1.07, 95% CI = 0.69-1.64). CONCLUSION: High ROR1 or high ROR2 distinctly identified subsets of breast cancer patients with adverse outcomes. Further studies are warranted to determine if high ROR1 or high ROR2 may identify high-risk populations for studies of targeted therapies.

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Collective cell migration is crucial for a variety of pathophysiological processes including embryonic development, wound healing, carcinoma invasion, and sprouting angiogenesis. The behavior of leading and following cells during migration is highly dynamic and involves extensive cellular morphological changes mediated by the actin cytoskeleton. Imaging these rapid and dynamic changes over time requires expression of fluorescent proteins and/or live labeling with fluorescent probes, followed by acquiring series of image stacks at short intervals. This presents significant challenges related to dye cytotoxicity, signal loss, and in particular phototoxicity resulting from repeated irradiation, especially when using separate channels for multiple dyes and when imaging large z-stacks at short time intervals. In this chapter, we present methods for multicolor live-cell labeling of primary human endothelial cell populations, followed by multi-position time-lapse imaging in 2D and in 3D protein matrices. These approaches can be performed in combination with RNA interference to suppress the expression of specific proteins, as well as in mosaic assays using mixtures of differentially labeled cell populations. Finally, we present a protocol for long-term imaging at low laser intensity to minimize laser-induced cell damage, followed by post-imaging signal enhancement using artificial intelligence.

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We report a case of a middle-aged male patient with a diffuse large mixed infiltrating angiolipoma on the back with venous malformation that grew from the age of 3 years. We discussed the design of the surgical flap for this large lipoma, observed its blood supply using SPY, and screened for possible causative genes, FCGR2A, BMP5, MUC2, and KRTAP4-9. To date, no invasive lipomas of this size and duration have been reported.

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The platforms based on immobilization of transmembrane proteins have become an effective way to study drug-protein interaction and identify new leads for drug discovery. Herein, we exploited the protein superglue (i.e. SpyTag-SpyCatcher chemistry) for site-specific, oriented, and in-situ one-step beta2-adrenoceptor (ß2-AR) immobilization. SpyCatcher was used as a fusion tag at the C-terminal of ß2-AR and the macroporous silica gels were functionalized with the SpyTag peptide. Immobilization was realized by immersing the gels into the E.coli cell lysate containing ß2-AR-SpyCatcher. Characterization of the functionalized gels was performed by X-ray photoelectron spectroscopy and fluorescence microscopy. Adsorption energy distribution calculation, injection amount dependent analysis (IADA) and nonlinear chromatographic were used for receptor-ligand interaction analysis. The affinity rank order of four ligands to the receptor was tulobuterol> chlorprenaline> salbutamol> terbutaline, which showed highly consistent with data from the radioligand binding assay and the ß2-AR column prepared by HaloTag technology. Magnolol and honokiol were screened from Cortex Magnoliae Officinalis and proved to promote the expression of the receptor in human airway smooth muscle cells. Our work unraveled the great potential to generate good bioactivity of the immobilized ß2-AR through Spy toolbox. This technology can be extended to the immobilization of other functional proteins, providing a better alternative in the field of bioanalysis, biosensing, and separation science.

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OBJECTIVES: Highlight the use of fluorescent angiography in free flap reconstruction of the head and neck. Qualify how fluorescent angiography can be selectively added to management paradigms for head and neck free flap reconstruction. METHODS: Retrospective chart review of 993 free flaps completed from the time the SPY Elite® system first became available at our institution between September 2013, until August 2020. Cases that used the SPY Elite® system were grouped into three broad categories: evaluation during initial flap harvest while still attached to the donor site, evaluation after anastomosis in the head and neck area, and evaluation post-operatively for questionable flap viability. RESULTS: The SPY Elite® system was used in 64 cases. Forty flaps were evaluated intraoperatively during initial harvest and before anastomosis to the head and neck area. Of these, 20 had signs of poor perfusion of the entire skin paddle, 12 had large myogenous or skin flaps with questionable perfusion of the distal aspect, and 8 were evaluated for other reasons. In this group the use of SPY Elite® changed the management of the patient in 20 cases (50%). Ten flaps were evaluated intraoperatively after anastomosis to the head and neck to ascertain adequate flow to the entire flap. In this group management was changed in two (20%). Fourteen flaps were evaluated 3-5 days post operatively due to suspected failure of a component. In five cases (36%), the use of SPY Elite® determined management with either trimming or discarding the flap. CONCLUSION: Assessment of flap perfusion via fluorescent angiography during initial flap harvest or when flap compromise is suspected post-operatively can guide decision making in free flap reconstruction of the head and neck and can be added to existing planning and management paradigms. LEVEL OF EVIDENCE: 4 Laryngoscope, 133:1388-1393, 2023.

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