Lyoprotectant Constituents Suited for Lyophilization and Reconstitution of Stem-Cell-Derived Extracellular Vesicles.

Stem-cell-derived extracellular vesicles (EVs) are emerging as an alternative approach to stem cell therapy. Successful lyophilization of EVs could enable convenient storage and distribution of EV medicinal products at room temperature for long periods, thus considerably increasing the accessibility of EV therapeutics to patients. In this study, we aimed to identify an appropriate lyoprotectant composition for the lyophilization and reconstitution of stem-cell-derived EVs. MSC-derived EVs were lyophilized using different lyoprotectants, such as dimethyl sulfoxide, mannitol, trehalose, and sucrose, at varying concentrations. Our results revealed that a mixture of trehalose and sucrose at high concentrations could support the formation of amorphous ice by enriching the amorphous phase of the solution, which successfully inhibited the acceleration of buffer component crystallization during lyophilization. Lyophilized and reconstituted EVs were thoroughly evaluated for concentration and size, morphology, and protein and RNA content. The therapeutic effects of the reconstituted EVs were examined using a tube formation assay with human umbilical vein endothelial cells. After rehydration of the lyophilized EVs, most of their generic characteristics were well-maintained, and their therapeutic capacity recovered to levels similar to those of freshly collected EVs. The concentrations and morphologies of the lyophilized EVs were similar to the initial features of the fresh EV group until day 30 at room temperature, although their therapeutic capacity appeared to decrease after 7 days. Our study suggests an appropriate composition of lyoprotectants, particularly for EV lyophilization, which could encourage the applications of stem-cell-derived EV therapeutics in the health industry.

Salvage percutaneous transluminal angioplasty of the superficial femoral artery in the diabetic foot with deteriorating limb ischemia.

INTRODUCTION: PAD frequently co-occurs with diabetes, often leading to chronic nonhealing wounds. Foot gangrene and amputation are common outcomes of untreated CLI. CASE REPORT: A 67-year-old male with diabetes and deteriorating limb ischemia following surgical stress underwent successful surgical repair after emergency PTA of the SFA for extensive heel necrosis. After surgical debridement of necrotic heel tissue, the ABI on the affected side suddenly reduced to 0.36, but it improved to 1.06 at 4 weeks following stenting angioplasty, allowing the subsequent flap surgery to repair the heel defect. At the 1-year follow-up visit, the patient exhibited durable heel coverage and the restoration of weightbearing function. No signs or symptoms indicative of restenosis were evident in the blood vessel treated with stent angioplasty. CONCLUSION: This case highlights the importance of proper evaluation of critical ischemic conditions and the need for prompt endovascular interventions in preserving the at-risk diabetic foot.

The Fate of Chronic Burn Wounds Suspected as Marjolin's Ulcers.

Marjolin's ulcer is the malignant transformation of chronic nonhealing ulcers that have developed in burn scars or in any other chronic wound. Development of this malignancy tends to be slow and insidious, but it becomes more aggressive when the Marjolin's ulcer breaks free of the scar. We reviewed 24 cases of patients who complained of chronic burn wounds suspected to be Marjolin's ulcers. Histologically, chronic ulcer and pseudoepitheliomatous hyperplasia were 21%, respectively, and malignancy, including squamous cell carcinoma and leiomyosarcoma, were 58%. The mean latency period was 31.6 ± 13.0 years, and the majority of lesions occurred in the extremities. Pseudoepitheliomatous hyperplasia in chronic burn wounds, which is difficult to distinguish from squamous cell carcinoma and considered as a transitional state to becoming a malignant tumor, should be treated as a malignancy. An aggressive excision and reconstruction with free tissue transfer or regional flap transposition should be adopted for adequate ablation and definitive coverage, rather than skin graft and regular surveillance.

Anatomical reconstruction of the fourth brachymetatarsia with one-stage iliac bone and cartilage cap grafting.

BACKGROUND: We present a one-stage procedure for lengthening the fourth brachymetatarsia with autogenous iliac bone and cartilage cap grafting for the anatomical reconstruction of the metatarsophalangeal (MTP) joint METHODS: During the last 8 years, 56 feet in 41 patients with congenital brachymetatarsia of the fourth toe were corrected with a one-stage operation to reposition the articular cartilage cap to the distal part of interpositional iliac bone graft at the metatarsal epiphysis. RESULTS: The length of the harvested iliac bone graft was 22.9 mm on average. The mean fixation period was 58.5 days, and the mean gain in length and percentage increase was 20.9 mm and 39%, respectively. MRI showed a stable MTP joint over viable cartilage cap in 83.3% of the cases. Mean postoperative American Orthopedic Foot and Ankle Society lesser MTP-interphalangeal score was 82.0. Neither neurovascular impairment nor recurrence of brachymetatarsia occurred in the mean follow-up period of 43.6 months. All patients were satisfied with the postoperative cosmetic results. Thirteen patients (23.2%) complained of limited active dorsiflexion of the fourth toe, and extensor adhesion was released by extensor tenolysis in only one patient. In a single case of nonunion at the bone graft site, additional surgery was not necessary. CONCLUSIONS: Anatomical reconstruction of the fourth brachymetatarsia with one-stage interpositional iliac bone and cartilage cap grafting resulted in excellent cosmetic results and a physiologic MTP joint, providing the benefits of one-stage lengthening with a low complication rate. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Identification of Alternative Splicing and Fusion Transcripts in Non-Small Cell Lung Cancer by RNA Sequencing.

BACKGROUND: Lung cancer is the most common cause of cancer related death. Alterations in gene sequence, structure, and expression have an important role in the pathogenesis of lung cancer. Fusion genes and alternative splicing of cancer-related genes have the potential to be oncogenic. In the current study, we performed RNA-sequencing (RNA-seq) to investigate potential fusion genes and alternative splicing in non-small cell lung cancer. METHODS: RNA was isolated from lung tissues obtained from 86 subjects with lung cancer. The RNA samples from lung cancer and normal tissues were processed with RNA-seq using the HiSeq 2000 system. Fusion genes were evaluated using Defuse and ChimeraScan. Candidate fusion transcripts were validated by Sanger sequencing. Alternative splicing was analyzed using multivariate analysis of transcript sequencing and validated using quantitative real time polymerase chain reaction. RESULTS: RNA-seq data identified oncogenic fusion genes EML4-ALK and SLC34A2-ROS1 in three of 86 normal-cancer paired samples. Nine distinct fusion transcripts were selected using DeFuse and ChimeraScan; of which, four fusion transcripts were validated by Sanger sequencing. In 33 squamous cell carcinoma, 29 tumor specific skipped exon events and six mutually exclusive exon events were identified. ITGB4 and PYCR1 were top genes that showed significant tumor specific splice variants. CONCLUSION: In conclusion, RNA-seq data identified novel potential fusion transcripts and splice variants. Further evaluation of their functional significance in the pathogenesis of lung cancer is required.

Penoscrotal reconstruction using groin and bilateral superomedial thigh flaps: a case of penile vaselinoma causing Fournier's gangrene.

Penile augmentation by the injection of mineral oil provokes many serious, undesirable effects. Although there are reports of complications such as deformity, ulceration, necrosis, and erectile dysfunction, Fournier's gangrene resulting from the injection of petroleum jelly into the penis has not been reported. Here, we present a 42-year-old man with penile vaselinoma causing Fournier's gangrene which was treated successfully with aggressive surgical debridement, followed by penoscrotal reconstruction using groin and bilateral superomedial thigh flaps.