Determinants of Histone H3K4 Methylation Patterns.

Various factors differentially recognize trimethylated histone H3 lysine 4 (H3K4me3) near promoters, H3K4me2 just downstream, and promoter-distal H3K4me1 to modulate gene expression. This methylation "gradient" is thought to result from preferential binding of the H3K4 methyltransferase Set1/complex associated with Set1 (COMPASS) to promoter-proximal RNA polymerase II. However, other studies have suggested that location-specific cues allosterically activate Set1. Chromatin immunoprecipitation sequencing (ChIP-seq) experiments show that H3K4 methylation patterns on active genes are not universal or fixed and change in response to both transcription elongation rate and frequency as well as reduced COMPASS activity. Fusing Set1 to RNA polymerase II results in H3K4me2 throughout transcribed regions and similarly extended H3K4me3 on highly transcribed genes. Tethered Set1 still requires histone H2B ubiquitylation for activity. These results show that higher-level methylations reflect not only Set1/COMPASS recruitment but also multiple rounds of transcription. This model provides a simple explanation for non-canonical methylation patterns at some loci or in certain COMPASS mutants.

In Vivo Dynamic and Static Analysis of Lymphatic Dysfunction in Lymphedema Using Near-Infrared Fluorescence Indocyanine Green Lymphangiography.

BACKGROUND: Near-infrared fluorescence indocyanine green lymphangiography, a primary modality for detecting lymphedema, which is a disease due to lymphatic obstruction, enables real-time observations of lymphatics and reveals not only the spatial distribution of drainage (static analysis) but also information on the lymphatic contraction (dynamic analysis). METHODS: We have produced total lymphatic obstruction in the upper limbs of 18 Sprague-Dawley rats through the dissection of proximal (brachial and axillary) lymph nodes and 20-Gy radiation (dissection limbs). After the model formation for 1 week, 9 animal models were observed for 6 weeks using near-infrared fluorescence indocyanine green lymphangiography by injecting 6-µL ICG-BSA (indocyanine green-bovine serum albumin) solution of 20-µg/mL concentration. The drainage pattern and leakage of lymph fluid were evaluated and time-domain signals of lymphatic contraction were observed in the distal lymphatic vessels. The obtained signals were converted to frequency-domain spectrums using signal processing. RESULTS: The results of both static and dynamic analyses proved to be effective in accurately identifying the extent of lymphatic disruption in the dissection limbs. The static analysis showed abnormal drainage patterns and increased leakage of lymph fluid to the periphery of the vessels compared with the control (normal) limbs. Meanwhile, the waveforms were changed and the contractile signal frequency increased by 58% in the dynamic analysis. Specifically, our findings revealed that regular lymphatic contractions, observed at a frequency range of 0.08 to 0.13 Hz in the control limbs, were absent in the dissection limbs. The contractile regularity was not fully restored for the follow-up period, indicating a persistent lymphatic obstruction. CONCLUSIONS: The dynamic analysis could detect the abnormalities of lymphatic circulation by observing the characteristics of signals, and it provided additional evaluation indicators that cannot be provided by the static analysis. Our findings may be useful for the early detection of the circulation problem as a functional evaluation indicator of the lymphatic system.

Direct Analysis of Phosphorylation Sites on the Rpb1 C-Terminal Domain of RNA Polymerase II.

Dynamic interactions between RNA polymerase II and various mRNA-processing and chromatin-modifying enzymes are mediated by the changing phosphorylation pattern on the C-terminal domain (CTD) of polymerase subunit Rpb1 during different stages of transcription. Phosphorylations within the repetitive heptamer sequence (YSPTSPS) of CTD have primarily been defined using antibodies, but these do not distinguish different repeats or allow comparative quantitation. Using a CTD modified for mass spectrometry (msCTD), we show that Ser5-P and Ser2-P occur throughout the length of CTD and are far more abundant than other phosphorylation sites. msCTD extracted from cells mutated in several CTD kinases or phosphatases showed the expected changes in phosphorylation. Furthermore, msCTD associated with capping enzyme was enriched for Ser5-P while that bound to the transcription termination factor Rtt103 had higher levels of Ser2-P. These results suggest a relatively sparse and simple "CTD code."

Comparative Study of Small Vessel (under 0.8 mm) Anastomosed Free Flap and Larger Vessel (over 0.8 mm) Anastomosed Free Flap; Does Supermicrosurgery Provide Sufficient Blood Flow to the Free Flap?

BACKGROUND: This study aimed to quantify the blood flow of free flaps and compare the blood flow of small vessel (<0.8 mm) and larger vessel (>0.8 mm) anastomosed free flaps. METHODS: This retrospective study included patients treated successfully with a perforator free flap in the lower extremity between June 2015 and March 2017. A color duplex ultrasound system measured the flow volume through the pedicle by analyzing the mean flow peak velocity, flow volume, and flow volume per 100 g of the flap. RESULTS: A total of 69 patients were enrolled in this study. There was no statistical difference in peak velocity between the small vessel anastomosed free flap (25.2 ± 5.6) and larger vessel anastomosed free flap (26.5 ± 5.4). Flow volume (6.8 ± 4.2 vs. 6.3 ± 3.6) and flow volume/100 g (3.6 ± 3.9 vs. 6.2 ± 6.9) also did not show significant differences. CONCLUSION: Small vessel (<0.8 mm) free flaps showed similar flow velocity and flow volume to larger vessel (>0.8 mm) anastomosed free flaps. Blood flow to the small vessel anastomosed free flap was sufficient despite its small vessel size.

Identifying Functional Lymph Nodes in Lower Extremity Lymphedema Patients: The Role of High-frequency Ultrasound.

BACKGROUND: Lymph nodes may play a potential role in lymphedema surgery. Radiologic evaluation of nodes may reveal the status of pathologic conditions but with limited accuracy. This study is the first to evaluate the efficacy of ultrasound in detecting functioning nodes in lymphedema patients and presents a criterion for determining the functionality of the lymph nodes. METHODS: This retrospective study reviews 30 lower extremity lymphedema cases which were candidates for lymph node to vein anastomosis. Lymphoscintigraphy and magnetic resonant lymphangiography (MRL) imaging were compared with ultrasound features which were correlated to intraoperative indocyanine green (ICG) nodal uptake as an indication of functionality. RESULTS: Majority were International Society of Lymphology stage 2 late (50.0%) and stage 3 (26.7%). ICG positive uptake (functioning nodes) was noted in 22 (73.3%), while 8 patients (26.6%) had negative uptake (nonfunctioning). Ultrasound had significantly the highest specificity (100%) for identifying functional nodes followed by lymphoscintigraphy (55%) and MRL (36%; p = 0.002, p < 0.001, respectively). This was associated with 100% positive predictive value compared against lymphoscintigraphy (44%) and MRL (36%; p < 0.001 for both). The identified ultrasound imaging criteria for functioning lymph node were oval lymph node shape (Solbiati Index), morphology, vascularity pattern, and vascularity quantification. CONCLUSION: The use of ultrasound in nodal evaluation was proven effective in different pathologic conditions and demonstrated the best prediction for functionality of the lymph node based on the new evaluation criteria.

Effect of a portable light emitting diode device on wound healing in a rat model.

Light-emitting diode (LED) lights produce a variety of wavelengths that have demonstrable efficacy in therapeutic and aesthetic fields. However, a repetitive treatment regimen is required to produce treatment outcomes, which has created a need for portable LED devices. In this study, we aimed to develop a portable therapeutic LED device and investigate its healing effect on excisional wounds in a rat model. The 35 × 35 mm-sized LED device was used on a total of 30 rats with full-thickness wounds that were divided into two groups depending on radiation intensity (11.1 and 22.2 mW/cm2 group). LED irradiation was performed every 24 h for 30 min, over 14 days, in direct contact with the wound. Percentage wound closure was measured by photographic quantification and was assessed histologically using haematoxylin and eosin (H&E) and Masson's Trichrome staining, and immunohistochemistry for Vascular endothelial growth factor (VEGF) and CD31. Percentage wound closure was significantly higher in 22.2 mW/cm2 irradiated wounds than that in the control wounds on days 7 and 10. The area of collagen deposition was remarkably larger in 22.2 mW/cm2 irradiated wounds than that in the control, with more horizontally organized fibres. CD31 immunostaining confirmed a significant increase in the number of microvessels in 22.2 mW/cm2 irradiated wounds than that in the control wounds, although there was no difference in VEGF immunostaining. Our novel portable LED device accelerates wound healing in a rat model, raising the possibility that portable LED devices can combine convenience with accessibility to play an innovative role in wound dressing.

Maximizing the Versatility of Thin Flap from the Groin Area as a Workhorse Flap: The Selective Use of Superficial Circumflex Iliac Artery Perforator (SCIP) Free Flap and Superficial Inferior Epigastric Artery (SIEA) Free Flap with Precise Preoperative Planning.

BACKGROUND: This study aimed to evaluate the usefulness of the selective use of the superficial circumflex iliac artery perforator (SCIP) and the superficial inferior epigastric artery (SIEA) flap as a workhorse flap from the groin area with precise preoperative surgical planning. METHODS: A total of 79 free flap reconstructions were performed in the study period; 35 SCIP free flaps and 19 SIEA free flaps were performed in the study period. Detailed preoperative surgical planning was performed using computed tomography (CT) angiography and color Doppler ultrasound. Detailed anatomical information of the flaps and reconstructive outcomes were evaluated. RESULTS: Flap characteristics between SCIP free flaps and SIEA free flaps were similar. The average transverse distance of the perforator from anterior superior iliac spine was 15.91 cm in SCIP free flaps and 43.15 cm in SIEA free flaps. The overall flap success rate was 96.4%. Majority of the patients achieved satisfactory contour without debulking surgery. Donor site morbidity was minimal with one case of wound dehiscence. CONCLUSION: The selective use of the SCIP and SIEA free flap in groin area is a safe and useful technique. The surgical outcomes were reliable and similar between the SCIP and SIEA free flaps. Preoperative vascular planning using CT angiography and color Doppler ultrasound is essential for selecting the proper flap.

Planes for Perforator/Skin Flap Elevation-Definition, Classification, and Techniques.

BACKGROUND: Elevation in different layers achieving thin flaps are becoming relatively common practice for perforator flaps. Although postreconstruction debulking achieves pleasing aesthetic results and is widely practiced, customized approach during elevation to achieve the ideal thickness will increase efficiency while achieving the best possible aesthetic outcome. Multiple planes for elevation have been reported along with different techniques but it is quite confusing and may lack correspondence to the innate anatomy of the skin and subcutaneous tissue. METHODS: This article reviews the different planes of elevation and aims to clarify the definition and classification in accordance to anatomy and present the pros and cons of elevation based on the different layers and provide technical tips for elevation. RESULTS: Five different planes of elevation for perforator flaps are identified: subfascial, suprafacial, superthin, ultrathin, and subdermal (pure skin) layers based on experience, literature, and anatomy. CONCLUSION: These planes all have their unique properties and challenges. Understanding the benefits and limits along with the technical aspect will allow the surgeon to better apply the perforator flaps.

Evaluation of wound healing effects of micronized acellular dermal matrix in combination with negative pressure wound therapy: In vivo study.

Acellular dermal matrix (ADM) grafts can provide coverage for full-thickness skin defects and substitute for dermal defects. We tested the effectiveness of micronized ADM (mADM) as a dressing material, combined with negative pressure wound therapy (NPWT), for managing superficial wounds. We compared the wound healing effect of mADM in combination with NPWT with those of gelatin and mADM applied with a foam dressing. These therapeutic materials were applied to 36 cm2 excisional wounds in a porcine full-thickness skin defect model. Wound healing kinetics and new tissue formation were assessed 10 days after the initial treatment by measuring the wound area. Collagen deposition and neovascularization were histologically evaluated. Compared with the other two groups, mADM plus NPWT combination group had a significantly larger wound area at the baseline (P = .0040), but the smallest on the 7th day (P = .0093). In addition, collagen formation and neovascularization were more histologically promoted than in the other two groups. mADM showed better results than the gelatin group but less collagen and revascularization than the combination group, and there was no significant difference in wound area. Our results show that the combination of mADM and NPWT has a synergistic wound healing effect.

Using Duplex Ultrasound for Recipient Vessel Selection.

BACKGROUND: Among the many factors involved in reconstructive microsurgery, identifying a good recipient vessel is one of the key elements leading to a successful result. METHODS: Multiple modalities have been used to identify recipient vessels from simple palpation of axial arteries to hand-held Doppler, duplex ultrasound, computed tomography angiograms, and other advanced techniques. Although these various modalities bring their own unique advantages, using the duplex ultrasound can provide far superior and real-time information based on the anatomy and physiology of the recipient vessel. RESULTS: Duplex ultrasound is a valuable and powerful tool for reconstructive surgeons who are interested in performing microsurgery or supermicrosurgery. CONCLUSION: As we enter the era of individualized/customized reconstruction using superthin flaps, perforator-to-perforator anastomosis, and supermicrosurgery, understanding and becoming versatile with duplex ultrasound will be critical especially in choosing recipient vessels.

The Use of Color Duplex Ultrasound for Local Perforator Flaps in the Extremity.

BACKGROUND: The local flaps, especially perforator and keystone flaps, are used as first-line treatment option in reconstruction of small tomoderate-sized defect of the extremity. However, the high complication rate associated with these flaps may hinder this usage. METHODS: This article reviews the technical and clinical aspect of using color duplex ultrasound )CDU) in the preoperative, intraoperative, and postoperative period for propeller and keystone flaps. RESULTS: CDU allows the surgeon to understand the anatomical aspect of the perforator such as the location, point of penetration on the deep fascia, subcutaneous pathway )axiality) and physiological aspect such as velocity and flow volume. Understanding and utilizing this information will allow accurate preoperative design, intraoperative decision making, and postoperative monitoring, leading to better outcome. CONCLUSION: Carefully designed local perforator flaps based on anatomy and physiology using CDU will be a powerful armamentarium for reconstruction of the lower extremity.

Highly Multiplexed Kinase Profiling in Spleen with Targeted Mass Spectrometry Reveals Kinome Plasticity across Species.

Early attrition of drug candidates, including kinase inhibitors, often occurs due to issues that arise during preclinical safety and efficacy evaluation. This problem may be exacerbated by the fact that these studies might fail to consider the basic physiological differences that could exist between human patients and animal models. We report the development of a targeted mass spectrometry-based assay capable of monitoring >50 different kinases using peptides conserved in humans and the key preclinical species used in drug development (mouse, rat, dog, and cynomolgus monkey). These methods were then used to profile interspecies kinome variability in spleen with three of the current techniques used in targeted proteomics (MRM, PRM, and IS-PRM). IS-PRM provides the highest number of kinase identifications, and the results indicate that while this initial set of kinases exhibits high correlation between species for this tissue type, distinct species-specific differences do exist, especially within the cyclin-dependent kinase family. An initial screen in two species with the kinase inhibitor dasatinib in competition with the chemoproteomic kinase-binding probe XO44 demonstrated how the targeted methods can be further applied to study species-specific inhibitor occupancy profiles. Understanding such differences could help rationalize the findings of preclinical studies and have major implications for the selection of these animals as models in kinase drug development.

The C-terminal domain of Rpb1 functions on other RNA polymerase II subunits.

The C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II (RNApII), coordinates recruitment of RNA- and chromatin-modifying factors to transcription complexes. It is unclear whether the CTD communicates with the catalytic core region of Rpb1 and thus must be physically connected, or instead can function as an independent domain. To address this question, CTD was transferred to other RNApII subunits. Fusions to Rpb4 or Rpb6, two RNApII subunits located near the original position of CTD, support viability in a strain carrying a truncated Rpb1. In contrast, CTD fusion to Rpb9 on the other side of RNApII does not. Rpb4-CTD and Rpb6-CTD proteins are functional for phosphorylation and recruitment of various factors, albeit with some restrictions and minor defects. Normal CTD functions are not transferred to RNApI or RNApIII by Rbp6-CTD. These results show that, with some spatial constraints, CTD can function even when disconnected from Rpb1.

Importance of Vascularity and Selecting the Recipient Vessels of Lower Extremity Reconstruction.

BACKGROUND: Successful lower extremity reconstruction using free flaps begins by identifying a good recipient vessel and understanding the surrounding environment of the defect. METHODS: One should consider multiple factors when selecting the recipient vessel such as the status of the axial arteries, trying to preserve flow as much as possible, extent and severity of the defect, and ultimately what type of anastomosis will be ideal. RESULTS: Multiple factors of importance are reviewed and show the relevance in decision making and provide an algorithm. CONCLUSION: In addition to the multiple factors to be considered, the ultimate decision should be made on the table during surgery when the actual artery or vein is exposed and shows signs of good pulsation and flow.

Special Considerations for Diabetic Foot Reconstruction.

Successful reconstruction of diabetic ulcers using free flap surgery can prevent further amputation and promote limb salvage to maintain normal gait of the patient and increase the quality of life after surgery. To minimize the postoperative complication and days of stay in hospital, surgeons should thoroughly investigate the risk factors and underlying conditions, including the general condition and local factors.

Free Tissue Transfer after Open Transmetatarsal Amputation in Diabetic Patients.

BACKGROUND: Transmetatarsal amputation (TMA) preserves functional gait while avoiding the need for prosthesis. However, when primary closure is not possible after amputation, higher level amputation is recommended. We hypothesize that reconstruction of the amputation stump using free tissue transfer when closure is not possible can achieve similar benefits as primarily closed TMAs. METHODS: Twenty-eight TMAs with free flap reconstruction were retrospectively reviewed in 27 diabetic patients with a median age of 61.5 years from 2004 to 2018. The primary outcome was limb salvage rate, with additional evaluation of flap survival, ambulatory status, time until ambulation, and further amputation rate. In addition, subgroup analysis was performed based on the microanastomosis type. RESULTS: Flap survival was 93% (26 of 28 flaps) and limb salvage rate of 93% (25 of 27 limbs) was achieved. One patient underwent a second free flap reconstruction. In the two failed cases, higher level amputation was required. Thirteen flaps had partial loss or other complications which were salvaged with secondary intension or skin grafts. Median time until ambulation was 14 days following reconstruction (range: 9-20 days). Patients were followed-up for a median of 344 days (range: 142-594 days). Also, 88% of patients reported good ambulatory function, with a median ambulation score of 4 out of 5 at follow-up. There was no significant difference between the subgroups based on the microanastomosis type. CONCLUSION: TMA with free flap reconstruction is an effective method for diabetic limb salvage, yielding good functional outcomes and healing results.

Oncologic safety of propeller flap and free flap in reconstruction after soft tissue sarcoma resection.

BACKGROUND AND OBJECTIVES: The propeller flap is a reliable option for reconstruction after soft tissue sarcoma resection. However, some parts of the resection margin may move away from its original position during flap rotation and thus can be excluded from the clinical target volume of adjuvant radiotherapy. This study aimed to evaluate local recurrence after soft tissue sarcoma resection with propeller flap or free flap reconstruction. METHODS: Patients who underwent resection of soft tissue sarcoma followed by a free flap or propeller flap reconstruction and adjuvant radiotherapy at a single institution were retrospectively reviewed. RESULTS: The 1- and 3-year local control rates were 94.6% and 88.6% in the free flap group vs 90.6% and 87.5% in the propeller flap group, without statistical significance. There were no statistically significant differences in 5-year local recurrence-free survival (88.6% vs 87.5%) and disease-free survival (82.5% vs 74.8%) between the groups. CONCLUSIONS: Although there was no significant difference in local control and disease-free survival rates between propeller flap and free flap reconstruction after soft tissue sarcoma resection, a multidisciplinary approach is needed to obtain surgical information for determining the accurate clinical target volume of adjuvant radiotherapy and the area for meticulous follow-up postoperatively.

Transoral Endoscopic Thyroidectomy Vestibular Approach: A Single-institution Experience of the First 50 Cases.

Background. Transoral endoscopic thyroidectomy vestibular approach (TOETVA) has evolved from a novel procedure to a cosmetically appealing alternative to the traditional open thyroidectomy. TOETVA is limited to only high-volume centers with the hope to expand and demonstrate the safety, reproducibility, and application for the correct patient with thyroid disease. We present our experience with the first 50 TOETVA procedures performed at our institution. Methods. From September 2016 to June 2018, we performed 50 transoral endoscopic thyroidectomies via the vestibular approach for a variety of thyroid pathologies. Outcomes were analyzed from a prospectively maintained database. Patients were treated for a different range of thyroid pathologies. Results. A total of 12 (24%) patients underwent total thyroidectomy, whereas 38 (76%) patients underwent a subtotal thyroidectomy or thyroid lobectomy. The mean surgical time was 149 ± 40.0 (90-256) minutes for lobectomy and 217.6 ± 33.3 (175-276) minutes for total thyroidectomy. Of our 50 patients, 16 (32%) had postoperative transient lower lip numbness with an average time to recovery of 23.8 (.43-48) weeks, with 1 (2%) patient having persistent, but improving, lower lip numbness beyond the 6-month follow-up. There were 13 (26%) patients with transient chin numbness with an average time to recovery of 15.7 (2-48) weeks. Two (4%) patients had transient recurrent laryngeal nerve (RLN) injury with hoarseness, whereas 1 (2%) patient had permanent injury. Conclusion. The TOETVA is a safe and reproducible procedure. For selected patients, this technique is a viable alternative to conventional thyroidectomy.

Superficial Circumflex Iliac Artery Perforator Flap as a Workhorse Flap: Systematic Review and Meta-analysis.

BACKGROUND: There are various advantages and disadvantages attributed to superficial circumflex iliac artery perforator (SCIP) flap. The aim of this study is to evaluate the versatility and reliability of free SCIP flap by performing a systematic review and meta-analysis of the literature in terms of flap characteristics, pedicle types, and outcomes, including the different types of flap elevations. METHODS: PubMed, Embase OVID, and Cochrane CENTRAL were searched up to January 2019. All original articles and case reports published in English were included in the analysis. Anatomic descriptions, cadaveric studies, conference presentations, letter to the editors, local SCIP flaps, and review articles were excluded. RESULTS: A total of 36 articles including 907 SCIP flaps were available for the analysis. The most frequent causes of defects were tumors (38.2%) and lower extremities were the most common recipient areas (62.7%). The average flap dimension was 73.3 ± 23.0 cm2 with a pedicle length of 5.0 ± 0.6 cm. Vessel diameter average was 0.67 ± 0.12 mm. The average number of deep branch and superficial branch used per study was 14.4 ± 8.7 (18%) and 93.3 ± 75.0 (84%), respectively. Flap failure rate and complication rate were 2.7 and 4.2%, respectively. CONCLUSION: SCIP flaps have been shown to be versatile in various aspects of reconstruction. The attributed disadvantages such as having short pedicle and small vessel diameter do not seem to limit the variable usage of this flap. Therefore, SCIP flap should be considered a workhorse flap.

The Role of Duplex Ultrasound in Microsurgical Reconstruction: Review and Technical Considerations.

BACKGROUND: With the technological advances, microsurgery has evolved to the era of supermicrosurgery since its inception. With the popularization of these flaps, proficiency in using tools such as color Doppler ultrasound (CDU) has become crucial. Despite the increased interest, studies regarding the role of ultrasound in microsurgical reconstruction are scarce. Therefore, in this study, we discussed currently available imaging modalities for reconstructive surgeons, types of ultrasound, and the role of ultrasound in microsurgical reconstruction, and made recommendations for ultrasound use in reconstructive surgery. METHODS: We performed a computerized search of ultrasound in reconstructive surgery using the MEDLINE database. Data regarding ultrasound indications, usage, and outcomes were collected. RESULTS: Of the 115 articles identified, the majority was written on nonreconstructive ultrasound uses (51.4%). For the reconstructive uses, mapping of perforators was the most common usage (39.1%), followed by flap monitoring and volume measurement. In addition, we found that there are a limited number of literature on the role of CDU in reconstructive surgery, especially on its intraoperative and postoperative use. CONCLUSION: CDU is a valuable and powerful tool for any reconstructive surgeons who are interested in performing microsurgery or supermicrosurgery. As the demand of customized flaps using superthin flaps, perforator-to-perforator anastomosis, and supermicrosurgery increases, understanding and becoming versatile in CDU will be critical. We hope our experience with using ultrasound and refinements that we made are helpful for those who would like to include it as a part of their armamentarium.