Noncanonical mono(ADP-ribosyl)ation of zinc finger SZF proteins counteracts ubiquitination for protein homeostasis in plant immunity.

Protein ADP-ribosylation is a reversible post-translational modification that transfers ADP-ribose from NAD+ onto acceptor proteins. Poly(ADP-ribosyl)ation (PARylation), catalyzed by poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolases (PARGs), which remove the modification, regulates diverse cellular processes. However, the chemistry and physiological functions of mono(ADP-ribosyl)ation (MARylation) remain elusive. Here, we report that Arabidopsis zinc finger proteins SZF1 and SZF2, key regulators of immune gene expression, are MARylated by the noncanonical ADP-ribosyltransferase SRO2. Immune elicitation promotes MARylation of SZF1/SZF2 via dissociation from PARG1, which has an unconventional activity in hydrolyzing both poly(ADP-ribose) and mono(ADP-ribose) from acceptor proteins. MARylation antagonizes polyubiquitination of SZF1 mediated by the SH3 domain-containing proteins SH3P1/SH3P2, thereby stabilizing SZF1 proteins. Our study uncovers a noncanonical ADP-ribosyltransferase mediating MARylation of immune regulators and underpins the molecular mechanism of maintaining protein homeostasis by the counter-regulation of ADP-ribosylation and polyubiquitination to ensure proper immune responses.

Perfluorooctanesulfonic Acid Alters the Plant's Phosphate Transport Gene Network and Exhibits Antagonistic Effects on the Phosphate Uptake.

Per- and polyfluoroalkyl substances (PFASs), with significant health risks to humans and wildlife, bioaccumulate in plants. However, the mechanisms underlying plant uptake remain poorly understood. This study deployed transcriptomic analysis coupled with genetic and physiological studies using Arabidopsis to investigate how plants respond to perfluorooctanesulfonic acid (PFOS), a long-chain PFAS. We observed increased expressions of genes involved in plant uptake and transport of phosphorus, an essential plant nutrient, suggesting intertwined uptake and transport processes of phosphorus and PFOS. Furthermore, PFOS-altered response differed from the phosphorus deficiency response, disrupting phosphorus metabolism to increase phosphate transporter (PHT) transcript. Interestingly, pht1;2 and pht1;8 mutants showed reduced sensitivity to PFOS compared to that of the wild type, implying an important role of phosphate transporters in PFOS sensing. Furthermore, PFOS accumulated less in the shoots of the pht1;8 mutant, indicating the involvement of PHT1;8 protein in translocating PFOS from roots to shoots. Supplementing phosphate improved plant's tolerance to PFOS and reduced PFOS uptake, suggesting that manipulating the phosphate source in PFOS-contaminated soils may be a promising strategy for minimizing PFOS uptake by edible crops or promoting PFOS uptake during phytoremediation. This study highlighted the critical role of phosphate sensing and transport system in the uptake and translocation of PFOS in plants.

Classification of Alzheimer's Progression Using fMRI Data.

In the last three decades, the development of functional magnetic resonance imaging (fMRI) has significantly contributed to the understanding of the brain, functional brain mapping, and resting-state brain networks. Given the recent successes of deep learning in various fields, we propose a 3D-CNN-LSTM classification model to diagnose health conditions with the following classes: condition normal (CN), early mild cognitive impairment (EMCI), late mild cognitive impairment (LMCI), and Alzheimer's disease (AD). The proposed method employs spatial and temporal feature extractors, wherein the former utilizes a U-Net architecture to extract spatial features, and the latter utilizes long short-term memory (LSTM) to extract temporal features. Prior to feature extraction, we performed four-step pre-processing to remove noise from the fMRI data. In the comparative experiments, we trained each of the three models by adjusting the time dimension. The network exhibited an average accuracy of 96.4% when using five-fold cross-validation. These results show that the proposed method has high potential for identifying the progression of Alzheimer's by analyzing 4D fMRI data.

Arabidopsis sirtuins and poly(ADP-ribose) polymerases regulate gene expression in the day but do not affect circadian rhythms.

Nicotinamide-adenine dinucleotide (NAD) is involved in redox homeostasis and acts as a substrate for NADases, including poly(ADP-ribose) polymerases (PARPs) that add poly(ADP-ribose) polymers to proteins and DNA, and sirtuins that deacetylate proteins. Nicotinamide, a by-product of NADases increases circadian period in both plants and animals. In mammals, the effect of nicotinamide on circadian period might be mediated by the PARPs and sirtuins because they directly bind to core circadian oscillator genes. We have investigated whether PARPs and sirtuins contribute to the regulation of the circadian oscillator in Arabidopsis. We found no evidence that PARPs and sirtuins regulate the circadian oscillator of Arabidopsis or are involved in the response to nicotinamide. RNA-seq analysis indicated that PARPs regulate the expression of only a few genes, including FLOWERING LOCUS C. However, we found profound effects of reduced sirtuin 1 expression on gene expression during the day but not at night, and an embryo lethal phenotype in knockouts. Our results demonstrate that PARPs and sirtuins are not associated with NAD regulation of the circadian oscillator and that sirtuin 1 is associated with daytime regulation of gene expression.

Angiotensin-Converting Enzyme Inhibitor, Captopril, Improves Scar Healing in Hypertensive Rats.

Pathological cutaneous scars, with aberrant extracellular matrix accumulation, have multiple origins. Antihypertensive medications, such as calcium channel blockers, have been used to treat pathological scars. However, a relationship between angiotensin-converting enzyme (ACE) inhibitors, pathological scars, and blood pressure (BP) has never been reported. Here, we aimed to compare the differences in scar development and the effects of the administration of systemic ACE inhibitor on scar tissue in a normotensive rat, the Wistar Kyoto rat (WKY), a hypertensive rat, and the spontaneously hypertensive rat (SHR). Using an 8-mm punch, we created two full-thickness skin defects in a total of 32 rats (16 WKY and 16 SHR) to obtain a total of 64 wounds. We established control WKY (n = 16), captopril-treated WKY (n = 16), control SHR (n = 16), and captopril-treated SHR (n = 16) groups and started captopril (100 mg/g per day) treatment on day 21 in the appropriate groups. The BP of all groups was measured at 0, 3, and 5 weeks. The scar area was measured by histopathological examination, and scarring was expressed in terms of scar area and fibroblast and capillary counts. The expression of heat shock protein (HSP) 47, type I and III collagens, alpha-smooth muscle actin (α-SMA), Ki67, and vascular endothelial growth factor (VEGF) was investigated using immunohistochemistry. The scar area and fibroblast count were significantly higher in control SHR than in control WKY. The scar area, fibroblast count, and capillary count were significantly smaller in captopril-treated SHR than in control SHR. Immunostaining for α-SMA, Ki67, and VEGF also showed a noticeable decrease in scarring in the treated SHR compared with that in control SHR. Thus, BP affects scar development in a rat model, and an ACE inhibitor is more effective at reducing scars in hypertensive rats than in normotensive rats.

Restoration of the inferomedial orbital strut using a standardized three-dimensional printing implant.

The inferomedial orbital strut (IOS) is the thin bony junction of the orbital medial wall and floor. Its fracture is common and leads to serious complications, including enophthalmos, globe dystopia and diplopia. However, anatomical restoration of the IOS is challenging owing to reduced structural support; sound anatomical background and accurate implants are therefore essential. The aim of the present study was to incorporate data from cadaveric orbit anatomy into three-dimensional (3D) printing technology and to reconstruct the complex orbital fracture elaborately. After averaging the data from computed tomography (CT) images of 100 adult cadavers, the dimensions of the IOS were extracted, and a tangent sphere was created using a computer-aided design program. The curves were compared with the CT data of 10 adult patients from the simulation test. Based on these data, a standardized 3D implant, 1.15 mm thick, was designed using polycaprolactone. The implant was placed in five patients with complex orbital fractures. The radius of the sphere in contact with the orbit, measuring 33.54 mm, was confirmed to be appropriate. A comparison between the normal side volume (V0) and the postoperative volume (Vpost ) showed that they were statistically similar. Furthermore, a comparison between V0 and the preoperative volume (Vpre ), and Vpost compared with Vpre also showed a statistically significant difference (P < 0.05). On follow-up, the preoperative ocular symptoms were resolved. The orbital data obtained from 100 cadavers provided standardized orbital anatomy, and 3D printed implants were created. The implants were anatomically accurate with regard to the orbital cavity and adequately covered the simulation model. The implant also showed satisfactory results when applied clinically in actual patients.

Trochanteric area reconstruction with free flap using perforators as recipients: An alternative and effective option.

BACKGROUND: Reconstructing soft tissue defect on the trochanteric area is challenging. Due to the significant complications associated with regional flap, free tissue transfer is an appropriate option. However, this area has poor recipient vessels. Therefore, we present perforators as suitable recipient vessels to facilitate the use of free flap coverage for the successful reconstruction of defects in the trochanteric area. PATIENTS AND METHODS: From 2013 to 2017, 10 patients underwent free flap reconstruction for soft tissue defects of the trochanteric area. After preoperative computed tomography or magnetic resonance imaging images confirmed the enhanced perforating artery, the skin site was identified by Doppler. If the vessel was confirmed as reliable, the operation was performed in the same manner as for other free flaps. RESULTS: All of the flaps survived, and the perforators selected for surgery included four superficial circumflex iliac artery perforators, four tensor fasciae latae artery perforators, and two inferior gluteal artery perforators. The average diameter of the recipient artery was 0.97 mm and that of the vein was 0.94 mm. One case exhibited arterial insufficiency caused by compression of hematoma; however, complete flap survival was achieved in this case with revised surgery. CONCLUSION: Reconstructing soft tissue defects in the trochanteric area is limited in recipient vessels. However, using a perforator vessel as a recipient facilitates the reconstruction by free flap coverage. This method leads to acceptable flap survival and sufficient padding, with reduced morbidity and collateral injury.

Physics-Based Capacitance Model of Drift Region in Laterally Diffused Metal-Oxide Semiconductor and Its Implementation with BSIM4.

A physical capacitance model is presented for the drift region in a laterally diffused metal-oxide semiconductor (LDMOS). It is derived as a surface-potential-based model of nodal charge for the drift region. The model is combined with BSIM4, which is for the intrinsic MOSFET region of LDMOS, and it is validated with TCAD and measurement data. The model accurately predicts the capacitance of each node for the entire bias region.

Identifying Preoperative Factors Associated with the Volume Discrepancy in Patients Undergoing Breast Reconstruction with the Extended Latissimus Dorsi Musculocutaneous Flap Coverage.

BACKGROUND: The latissimus dorsi (LD) flap is a versatile option for breast reconstruction. However, the indications are limited because of volume discrepancy between the breast and the flap. We conducted this study to identify preoperative factors associated with the volume discrepancy in patients undergoing breast reconstruction with the extended LD flap. METHODS: A retrospective study was performed in 69 patients (69 breasts) who underwent breast reconstruction with the extended LD flap between March 2015 and March 2018. We evaluated age, body weight, height, preoperative body mass index (BMI), postoperative BMI, breast skin defect size, breast volume, flap volume, and volume discrepancy [breast volume - flap volume]. RESULTS: Mean age, height, body weight, preoperative BMI, postoperative BMI, skin defect size, breast volume, flap volume, and volume discrepancy were 45.6 ± 7.1, 157.8 ± 0.1, 59 ± 8.1, 23.7 ± 3.2, 23.5 ± 3.3, 16.5 ± 9.3, 252.2 ± 107.1, 229.4 ± 95.6, and 32.6 ± 31.4, respectively. Spearman's rank correlation coefficients indicated significant positive linear correlations between volume discrepancy and preoperative BMI (correlation coefficient = 0.267, P = 0.027), volume discrepancy and breast volume (correlation coefficient = 0.472, P < 0.001), and between volume discrepancy and skin defect size (correlation coefficient = 0.609, P < 0.001). Stepwise multiple regression analysis yielded the following formula: predicted log volume discrepancy (ml) = 1.2891 + 0.0639 × skin defect size + 0.0025 × breast volume (R2 = 0.421). CONCLUSION: Skin defect size and breast volume were preoperative factors associated with volume discrepancy in patients who have undergone breast reconstruction with the extended LD flap. Considering these factors, we can predict the lack of volume and plan any necessary secondary procedures. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Chiral differentiation of d- and l-alanine by permethylated ß-cyclodextrin: IRMPD spectroscopy and DFT methods.

The gaseous chiral differentiation of alanine by permethylated ß-cyclodextrin was studied using IRMPD spectroscopy and density functional theory calculations. The protonated non-covalent complexes of permethylated ß-cyclodextrin and d- or l-alanine were mass-selected and investigated by IR laser pulses in the wavelength region of 2650-3800 cm-1. The remarkably different features of the IRMPD spectra for d- and l-alanine are described, and their origin is elucidated by quantum chemical calculations. We show that the differentiation of the experimentally observed spectral features is the result of different local interactions of d- and l-alanine with permethylated ß-cyclodextrin. We also assign the extremely high-frequency (>3700 cm-1) bands in the observed spectra to the stretch motions of completely isolated alanine -OH groups.

Can a Morel-Lavallée lesion be misdiagnosed as a mass like lesion?

The Morel-Lavallée lesion (MLL) is a post-traumatic closed soft tissue degloving injury. Common complaints of MLL patients are a haematoma or fluid collection on the trunk or the lower extremity. However, the authors introduce unique cases of MLL that present an atypical appearance. The fluid collection was not apparent, and the capsule formation was not detected on preoperative image study. The main complaint of patients was the uncomfortable mass-like lesion that was regarded as a simple benign lump. The purpose of this case study is to introduce the atypical cases of MLL and to help other physicians make accurate diagnosis based on trial and error of our cases.

AtMyb7, a subgroup 4 R2R3 Myb, negatively regulates ABA-induced inhibition of seed germination by blocking the expression of the bZIP transcription factor ABI5.

Various Myb proteins have been shown to play crucial roles in plants, including primary and secondary metabolism, determination of cell fate and identity, regulation of development and involvement in responses to biotic and abiotic stresses. The 126 R2R3 Myb proteins (with two Myb repeats) have been found in Arabidopsis; however, the functions of most of these proteins remain to be fully elucidated. In the present study, we characterized the function of AtMyb7 using molecular biological and genetic analyses. We used qRT-PCR to determine the levels of stress-response gene transcripts in wild-type and atmyb7 plants. We showed that Arabidopsis AtMyb7 plays a critical role in seed germination. Under abscisic acid (ABA) and high-salt stress conditions, atmyb7 plants showed a lower germination rate than did wild-type plants. Furthermore, AtMyb7 promoter:GUS seeds exhibited different expression patterns in response to variations in the seed imbibition period. AtMyb7 negatively controls the expression of the gene encoding bZIP transcription factor, ABI5, which is a key transcription factor in ABA signalling and serves as a crucial regulator of germination inhibition in Arabidopsis.

The Role of Adipokines in Tumor Progression and Its Association with Obesity.

Obesity is a well-established risk factor for various malignancies and emerging evidence suggests that adipokines play a pivotal role in linking excess adiposity to tumorigenesis. Adipokines are bioactive molecules secreted by adipose tissue and their altered expression in obesity contributes to a pro-inflammatory, pro-angiogenic, and growth-promoting microenvironment conducive to tumorigenesis. Leptin, a key adipokine, activates survival and proliferative signaling pathways whereas adiponectin exhibits tumor-suppressive effects by inducing apoptosis and cell cycle arrest. Visfatin has also been documented to promote tumor growth, angiogenesis, migration, and invasion. Moreover, emerging studies suggest that adipokines, such as resistin, apelin, and chemerin, which are overexpressed in obesity, may also possess oncogenic functions. Despite advancements in our understanding of the roles of individual adipokines in cancer, the intricate interplay and crosstalk between adipokines, tumor cells, and the tumor microenvironment remain complex and multifaceted. This review highlights the evolving knowledge of how adipokines contribute to obesity-related tumorigenesis, shedding light on the potential of targeting adipokine signaling pathways as a novel therapeutic approach for obesity-associated cancers. Further research on the specific mechanisms and interactions between adipokines and tumor cells is crucial for a comprehensive understanding of obesity-associated cancer pathogenesis.

Effects of a 2-Week Kinect-Based Mixed-Reality Exercise Program on Prediabetes: A Pilot Trial during COVID-19.

Background: Pre-diabetes can develop into type 2 diabetes mellitus, but can prevented by regular exercise. However, the outcomes when combining unsupervised Kinect-based mixed-reality (KMR) exercise with continuous glucose monitoring (CGM) remain unclear. Therefore, this single-arm pilot trial examined changes in blood glucose (BG) concentrations over 672 hours (4 weeks), including a 2-week period of KMR exercise and CGM in individuals with pre-diabetes. Methods: This was a pre-and post-treatment case-control study with nine participants. General questionnaires were administered and body composition, fasting BG concentrations, and 2-hour oral glucose tolerance test (2-OGTT) results were measured pre-and post-treatment. Weekly average glucose concentrations, hyperglycemia rate, hypoglycemia rate, average glucose concentration over time, amount of physical activity, amount of food intake, and pre- and postprandial BG (immediately and 30, 60, 90, and 120 minutes after lunch) were measured over 4 weeks (pre-test, exercise, and post -test weeks). Glucose concentrations were measured before exercising, between sets, and 30 and 60 minutes after exercise during the 2 weeks of unsupervised exercise (3 days/week). Results: In all participants, body mass index (27.16±2.92 kg/m2), fasting BG (108.00±7.19 mg/dL), 2-OGTT (162.56±18.12 mg/dL), hyperglycemia rate (P=0.040), and 90-minute postprandial BG (P=0.035) were significantly reduced during the 2 exercise weeks, and the 2-OGTT result (P=0.044) and diastolic blood pressure (DBP) (P=0.046) were significantly reduced at the post -test as compared with the pre-test. Conclusion: This study found that 2 weeks of unsupervised KMR exercise reduced 2-OGTT, DBP, hyperglycemia rate, and 90-minute postprandial BG concentration. We believed this effect could be identified more clearly in studies involving a larger number of participants and longer durations of exercise.

Methods for Improving the Germination of Rhodotypos scandens (Thunb.) Makino Seeds through Endocarp Removal.

Rhodotypos scandens (Thunb.) Makino is known to have a seed dispersal that is thick and stony (endocarp + seeds) and has potential as a landscaping tree seed. In several Rosaceae species, seeds are covered with a hard endocarp, making the internal seeds water-impermeable and germination difficult. Here, we analyzed the morphoanatomical traits and germination properties of R. scandens seeds. To identify ideal seed propagation conditions, we immersed R. scandens seeds in sulfuric acid for varying durations and subjected them to phytohormone (gibberellic acid A3 and fluridone) and a cold stratification (CS) (5 °C) treatment after endocarp removal (ER). The R. scandens stony seeds did not increase in mass by ≥25.0%. Following ER, the seed mass increased by ≥50.0% with water absorption when compared to the initial dry mass. Seed surfaces showed damage and cracks through scarification after 1 h of immersion in sulfuric acid, failing to germinate. A combination of ER, phytohormone treatment, and CS improved seed germination compared to ER alone (26.0 ± 5.3%). Overall, R. scandens seeds showed a dispersal with a hard endocarp from the parent plant, and a pre-treatment with ER, phytohormones, and CS was required for effective seed propagation.

A novel Arabidopsis MYB-like transcription factor, MYBH, regulates hypocotyl elongation by enhancing auxin accumulation.

Critical responses to developmental or environmental stimuli are mediated by different transcription factors, including members of the ERF, bZIP, MYB, MYC, and WRKY families. Of these, MYB genes play roles in many developmental processes. The overexpression of one MYB gene, MYBH, significantly increased hypocotyl elongation in Arabidopsis thaliana plants grown in the light, and the expression of this gene increased markedly in the dark. The MYBH protein contains a conserved motif, R/KLFGV, which was implicated in transcriptional repression. Interestingly, the gibberellin biosynthesis inhibitor paclobutrazol blocked the increase in hypocotyl elongation in seedlings that overexpressed MYBH. Moreover, the function of MYBH was dependent on phytochrome-interacting factor (PIF) proteins. Taken together, these results suggest that hypocotyl elongation is regulated by a delicate and efficient mechanism in which MYBH expression is triggered by challenging environmental conditions such as darkness, leading to an increase in PIF accumulation and subsequent enhanced auxin biosynthesis. These results indicate that MYBH is one of the molecular components that regulate hypocotyl elongation in response to darkness.

Loss of all three calreticulins, CRT1, CRT2 and CRT3, causes enhanced sensitivity to water stress in Arabidopsis.

KEY MESSAGE: The calreticulin triple knockout mutant shows growth defects in response to abiotic stress. The endoplasmic reticulum (ER) is an essential organelle that is responsible for the folding and maturation of proteins. During ER stress, unfolded protein aggregates accumulate in the cell, leading to the unfolded protein response (UPR). The UPR up-regulates the expression of ER-stress-responsive genes encoding calreticulin (CRT), an ER-localized Ca2+-binding protein. To understand the function of plant CRTs, we generated a triple knockout mutant, t123, which lacks CRT1, CRT2 and CRT3 and examined the roles of calreticulins in abiotic stress tolerance. A triple knockout mutant increased sensitivity to water stress which implies that calreticulins are involved in the Arabidopsis response to water stress. We identified that the cyclophilin AtCYP21-2, which is located in the ER, was specifically enhanced in the t123 mutants. Seed germination of the atcyp21-1 mutant was retarded by water stress. Taken together, these results suggest that regulatory proteins that serve to protect plants from water stress are folded properly in part with the help of calreticulins. The AtCYP21-2 may also participate in this protein-folding process in association with calreticulins.

Inhibition of histone deacetylation alters Arabidopsis root growth in response to auxin via PIN1 degradation.

KEY MESSAGE: Our results showed the histone deacetylase inhibitors (HDIs) control root development in Arabidopsis via regulation of PIN1 degradation. Epigenetic regulation plays a crucial role in the expression of many genes in response to exogenous or endogenous signals in plants as well as other organisms. One of epigenetic mechanisms is modifications of histone, such as acetylation and deacetylation, are catalyzed by histone acetyltransferase (HAT) and histone deacetylase (HDAC), respectively. The Arabidopsis HDACs, HDA6, and HDA19, were reported to function in physiological processes, including embryo development, abiotic stress response, and flowering. In this study, we demonstrated that histone deacetylase inhibitors (HDIs) inhibit primary root elongation and lateral root emergence. In response to HDIs treatment, the PIN1 protein was almost abolished in the root tip. However, the PIN1 gene did not show decreased expression in the presence of HDIs, whereas IAA genes exhibited increases in transcript levels. In contrast, we observed a stable level of gene expression of stress markers (KIN1 and COR15A) and a cell division marker (CYCB1). Taken together, these results suggest that epigenetic regulation may control auxin-mediated root development through the 26S proteasome-mediated degradation of PIN1 protein.

TTG1-mediated flavonols biosynthesis alleviates root growth inhibition in response to ABA.

KEY MESSAGE: Our results demonstrate that the flavonoids biosynthetic pathway can be effectively manipulated to confer enhanced plant root growth under water-stress conditions. Abscisic acid (ABA) is one of most important phytohormones. It functions in various processes during the plant lifecycle. Previous studies indicate that ABA has a negative effect on root growth and branching. Auxin is another key plant growth regulator that plays an essential role in plant growth and development. In contrast to ABA, auxin is a positive regulator of root growth and development at low concentrations. This study was performed to help understand whether flavonoids can suppress the effect of ABA on lateral root growth. The recessive TRANSPARENT TESTA GLABRA 1 (ttg1) mutant was characterized on ABA and sucrose treatments. It was determined that auxin mobilization could be altered by modifying flavonoids biosynthesis, which resulted in alterations of root architecture in response to ABA treatment. Moreover, transgenic TTG1-overexpression (TTG1-OX) seedlings exhibited enhanced root length and lateral root number compared to wild-type seedlings grown under normal or stress conditions. Genetic manipulation of the flavonoids biosynthetic pathway could therefore be employed successfully for the improvement of plant root systems by overcoming the inhibition of ABA and some abiotic stresses.

Safe and effective thrombolysis in free flap salvage: Intra-arterial urokinase infusion.

Despite the high success rate in reconstruction using free tissue transfer, flap failure is often caused by microvascular thrombosis. In a small percentage of cases with complete flap loss, a salvage procedure is performed. In the present study, the effectiveness of intra-arterial urokinase infusion through the free flap tissue was investigated to develop a protocol to prevent thrombotic failure. The retrospective study evaluated the medical records of patients who underwent salvage procedure with intra-arterial urokinase infusion after reconstruction with free flap transfer between January 2013 and July 2019. Thrombolysis with urokinase infusion was administered as salvage treatment for patients who experienced flap compromise more than 24 hours after free flap surgery. Because of an external venous drainage through the resected vein, 100,000 IU of urokinase was infused into the arterial pedicle only into the flap circulation. A total of 16 patients was included in the present study. The mean time to re-exploration was 45.4 hours (range: 24-88 hours), and the mean quantity of infused urokinase was 69,688 IU (range: 30,000-100,000 IU). 5 cases presented with both arterial and venous thrombosis, while 10 cases had only venous thrombosis and 1 case had only arterial thrombosis; in a study of 16 patients undergoing flap surgery, 11 flaps were found to have survived completely, while 2 flaps experienced transient partial necrosis and 3 were lost despite salvage efforts. In other word, 81.3% (13 of 16) of flaps survived. Systemic complications, including gastrointestinal bleeding, hematemesis, and hemorrhagic stroke, were not observed. The free flap can be effectively and safely salvaged without systemic hemorrhagic complications using high-dose intra-arterial urokinase infusion within a short period of time without systemic circulation, even in delayed salvage cases. Urokinase infusion results in successful salvage and low rate of fat necrosis.