The entry of unclosed autophagosomes into vacuoles and its physiological relevance.

It is widely stated in the literature that closed mature autophagosomes (APs) fuse with lysosomes/vacuoles during macroautophagy/autophagy. Previously, we showed that unclosed APs accumulated as clusters outside vacuoles in Vps21/Rab5 and ESCRT mutants after a short period of nitrogen starvation. However, the fate of such unclosed APs remains unclear. In this study, we used a combination of cellular and biochemical approaches to show that unclosed double-membrane APs entered vacuoles and formed unclosed single-membrane autophagic bodies after prolonged nitrogen starvation or rapamycin treatment. Vacuolar hydrolases, vacuolar transport chaperon (VTC) proteins, Ypt7, and Vam3 were all involved in the entry of unclosed double-membrane APs into vacuoles in Vps21-mutant cells. Overexpression of the vacuolar hydrolases, Pep4 or Prb1, or depletion of most VTC proteins promoted the entry of unclosed APs into vacuoles in Vps21-mutant cells, whereas depletion of Pep4 and/or Prb1 delayed the entry into vacuoles. In contrast to the complete infertility of diploid cells of typical autophagy mutants, diploid cells of Vps21 mutant progressed through meiosis to sporulation, benefiting from the entry of unclosed APs into vacuoles after prolonged nitrogen starvation. Overall, these data represent a new observation that unclosed double-membrane APs can enter vacuoles after prolonged autophagy induction, most likely as a survival strategy.

Disulfiram Improves Fat Graft Retention by Modulating Macrophage Polarization With Inhibition of NLRP3 Inflammasome-Mediated Pyroptosis.

BACKGROUND: Macrophage-mediated inflammatory response in the early post-grafting period restricts fat graft retention. Pyroptosis is a novel type of programmed cell death that extensively participates in inflammatory pathologies. OBJECTIVES: This study sought to determine whether macrophage pyroptosis was activated during the inflammatory phase after fat grafting and to investigate the efficacy of a pyroptosis inhibitor, disulfiram (DSF), in fat graft retention. METHODS: We established a C57BL/6 mice fat grafting model and then analyzed macrophage pyroptosis. DSF (50 mg/kg, every other day) was intraperitoneally injected starting 1 hour before fat grafting and continued for 14 days. An in vitro co-culture system was established in which mouse RAW264.7 macrophages were co-cultured with apoptotic adipocytes to further validate the findings of the in vivo studies and to explore the underlying mechanisms. RESULTS: Here we reported that macrophage pyroptosis was activated in both fat grafts and in vitro co-culture models. DSF was found to be a potent pyroptosis inhibitor, promoting M2 macrophage polarization. In addition, DSF was demonstrated to enhance vascularization and graft retention. CONCLUSIONS: Our results suggested that pyroptosis plays a crucial role in the inflammatory cascade within fat grafts. DSF, being a clinically available drug, could be translated into a clinically effective drug for improving fat graft survival by inhibiting macrophage pyroptosis, therefore inducing M2 macrophage polarization and promoting neovascularization.

The ESCRT-III complex contributes to macromitophagy in yeast.

Mitochondria play important roles in energy generation and homeostasis maintenance in eukaryotic cells. The damaged or superfluous mitochondria can be nonselectively or selectively removed through the autophagy/lysosome pathway, which was referred as mitophagy. According to the molecular machinery for degrading mitochondria, the selectively removed mitochondria can occur through macromitophagy or micromitophagy. In this study, we show that the endosomal sorting complex required for transport III (ESCRT-III) in budding yeast regulates macromitophagy induced by nitrogen starvation, but not by the post-logarithmic phase growth in lactate medium by monitoring a mitochondrial marker, Om45. Firstly, loss of ESCRT-III subunit Snf7 or Vps4-Vta1 complex subunit Vps4, two representative subunits of the ESCRT complex, suppresses the delivery and degradation of Om45-GFP to vacuoles. Secondly, we show that the mitochondrial marker Om45 and mitophagy receptor Atg32 accumulate on autophagosomes marked with Atg8 (mitophagosomes, MPs) in ESCRT mutants. Moreover, the protease-protection assay indicates that Snf7 and Vps4 are involved in MP closure. Finally, Snf7 interacts with Atg11, which was detected by two ways, glutathione-S-transferase (GST) pulldown and bimolecular fluorescence complementation (BiFC) assay, and this BiFC interaction happens on mitochondrial reticulum. Therefore, we proposed that the ESCRT-III machinery mediates nitrogen starvation-induced macromitophagy by the interaction between Snf7 and Atg11 so that Snf7 is recruited to Atg32-marked MPs by the known Atg11-Atg32 interaction to seal them. These results reveal that the ESCRT-III complex plays a new role in yeast on macromitophagy.

U-type soft tissue lifting: A new composited technique for reversing lower eyelid and midfacial aging.

BACKGROUND: Facial rejuvenation is becoming more and more popular, particularly among middle-aged persons. There are currently many techniques for improving the aforementioned situations, but each has its drawbacks. Our study aimed to discuss the treatment effect of a composited technique for reversing both lower eyelid and midface aging. METHODS: The patient's face was designed and measured before surgery. During surgery, a traditional lower blepharoplasty incision was made. The layer between the orbital septum and the orbicularis oculi muscle was separated to approximately 4-5 mm below the infraorbital, then the orbital septum and orbicularis retaining ligament were found to be released. A self-made suspension curving needle subconsciously passed through the brim of the superficial cheek fat pad via the "U-type" path and raised them to the proper location. Then sutured them to the infraorbital rim periosteum, as well as the suborbicularis oculi fat (SOOF) and the orbital septum fat. Secured the outside canthus to keep the lower lid position stable. RESULTS: From February 2020 to November 2022, 106 patients underwent the new surgical procedure and were successfully followed up for 20 ± 6.5 months postoperatively. The mean GAIS score was 2.42 ± 0.78, patient satisfaction rate was 95%. All of the Barton grades were decreased. The nasal base level suspension points were elevated to a level of 5 ± 2 mm. 3D measurement data revealed significant improvements. CONCLUSIONS: The composited technique is a safe and effective way to reverse the aging of the lower eyelid and midface.

Vps21 Directs the PI3K-PI(3)P-Atg21-Atg16 Module to Phagophores via Vps8 for Autophagy.

Phosphatidylinositol 3-phosphate (PI(3)P) serves important functions in endocytosis, phagocytosis, and autophagy. PI(3)P is generated by Vps34 of the class III phosphatidylinositol 3-kinase (PI3K) complex. The Vps34-PI3K complex can be divided into Vps34-PI3K class II (containing Vps38, endosomal) and Vps34-PI3K class I (containing Atg14, autophagosomal). Most PI(3)Ps are associated with endosomal membranes. In yeast, the endosomal localization of Vps34 and PI(3)P is tightly regulated by Vps21-module proteins. At yeast phagophore assembly site (PAS) or mammalian omegasomes, PI(3)P binds to WD-repeat protein interacting with phosphoinositide (WIPI) proteins to further recruit two conjugation systems, Atg5-Atg12·Atg16 and Atg8-PE (LC3-II), to initiate autophagy. However, the spatiotemporal regulation of PI(3)P during autophagy remains obscure. Therefore, in this study, we determined the effect of Vps21 on localization and interactions of Vps8, Vps34, Atg21, Atg8, and Atg16 upon autophagy induction. The results showed that Vps21 was required for successive colocalizations and interactions of Vps8-Vps34 and Vps34-Atg21 on endosomes, and Atg21-Atg8/Atg16 on the PAS. In addition to disrupted localization of the PI3K complex II subunits Vps34 and Vps38 on endosomes, the localization of the PI3K complex I subunits Vps34 and Atg14, as well as Atg21, was partly disrupted from the PAS in vps21∆ cells. The impaired PI3K-PI(3)P-Atg21-Atg16 axis in vps21∆ cells might delay autophagy, which is consistent with the delay of early autophagy when Atg21 was absent. This study provides the first insight into the upstream sequential regulation of the PI3K-PI(3)P-Atg21-Atg16 module by Vps21 in autophagy.

Long-term oncologic safety of immediate reconstructive surgery in patients with invasive breast cancer: a retrospective matched-cohort study.

OBJECTIVE: Immediate reconstruction (IR) is a safe and effective surgical treatment for patients with breast cancer. We aimed to assess the prognosis, aesthetic outcomes, and patient satisfaction of IR compared with breast conservation surgery (BCS) and total mastectomy (TM). METHODS: This retrospective matched-cohort study was conducted between May 2005 and December 2014. We established two cohorts according to the tumor (T) size of breast cancer. In the T≤3cm group, cases (IR) and controls (BCS or TM) were matched for age, pathological tumor size, and pathologic nodal status in a 1:1:1 ratio. In the T>3cm group, cases (IR) and controls (TM) were matched with the same factors and ratio. The primary outcome was the 5-year disease-free survival (DFS). The secondary outcome was patient satisfaction and quality of life. RESULTS: A total of 12,678 breast cancer patients were assessed for eligibility, of which 587 were included (T≤3 cm group: 155 IR vs 155 BCS vs 155 TM; T>3cm group: 61 IR vs 61 TM). In the T≤3 cm cohort, patients who underwent IR had no difference compared with those who underwent BCS or TM regarding the 5-year DFS (P=0.539); however, an improved aesthetic satisfaction, psychosocial, and sexual well-being were achieved in the IR group (P<0.001). In the T>3 cm cohort, the IR group had a worse median 5-year DFS (P=0.044), especially for Her2+ or triple-negative breast carcinoma (TNBC) subtypes compared with the TM group. CONCLUSIONS: IR improves aesthetic satisfaction, psychosocial, and sexual well-being for breast cancer patients with T≤3 cm. For patients with T > 3 cm invasive breast cancer, TM is superior to IR as it predicts a better 5-year DFS.

New Design for Axillary Dual-Plane Endoscopic Breast Augmentation for Asians: the Feasibility of Two Types of Dual-Plane Implant Pockets in 70 Patients as Measured by the BREAST-Q.

BACKGROUND: The design methods for dual-plane implant pockets for axillary endoscopic breast augmentation vary among different countries. We applied a modified approach for an Asian population. METHODS: Seventy patients with micromastia underwent our modified approach between 2011 and 2014. Breasts were divided into two types according to the soft-tissue pinch thickness of the lower pole: type I (thickness >2 cm; Group I) and type II (thickness ≤2 cm; Group II). The levels at which the pectoralis major (PM) was severed were 6-6.5 cm and 3-4 cm below the nipple for type I and II pockets, respectively. Then, dissection of the retromammary space was continued from the severance level downward to the new inframammary fold for type I pockets, whereas no dissection was made for type II pockets. All patients completed the pre- and post-operative BREAST-Q augmentation modules. RESULTS: During a mean follow-up of 10 months (range, 6-12 months), patients reported higher satisfaction with breasts after surgery than before surgery (satisfaction scores of 64.9 ± 5.6 vs. 14.7 ± 11.0). The mean satisfaction score for the overall outcome was 91.3 ± 17.3. However, there was no significant difference in physical well-being (87.1 ± 10.4 vs. 85.2 ± 11.7). No complications such as severe capsular contracture or displacement occurred. CONCLUSION: Distinguishing the need for a type I or II dual-plane pocket can lead to good outcomes and optimal soft-tissue coverage. The higher satisfaction and quality of life reported by our patients indicate that our new design is feasible and safe for most Asians with a medium build. LEVEL OF EVIDENCE II: 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 the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Effects of Medical Chitosan on Capsular Formation Following Silicone Implant Insertion in a Rabbit Model.

BACKGROUND: Capsular contracture is a serious complication that occurs after breast implant surgery. This study was performed to confirm that medical chitosan (MC) affects capsule formation and elucidates a possible mechanism. MATERIALS AND METHODS: In this study, we used 18 female adult New Zealand White rabbits. In each rabbit, two silicone implants were placed under the pectoralis muscle layer on both sides (one side was included in the experimental group and the other side was included in the control group). MC was applied around the silicone implant of the experiment group, while the control group received no treatment. The capsular thickness was calculated by Masson's trichrome stain. The expression of MMPs and TIMPs were determined by real-time PCR, Western blotting, and immunohistochemistry. RESULTS: Compared to the control group, the capsular thickness of the MC group was significantly reduced at 4, 8, and 12 weeks after the operation (4 week: 229.3 ± 72.2 vs 76.1 ± 12.6 µm, p < 0.05; 8 week: 326.0 ± 53.8 vs 155.4 ± 61.7 µm, p < 0.0.5; 12 week: 151.2 ± 52.5 vs 60.0 ± 22.0 µm, p < 0.05). Compared to the control group, the MC group had significantly lower expressions of TIMP-1 and TIMP-2 (p < 0.05). However, compared to the control group, there was no statistically significant difference in the expressions of MMP-2 and MMP-9 in the experiment group (p > 0.05). CONCLUSION: MC reduced the risk of developing capsular contracture around silicone implants, possibly by blocking the signaling pathway of TIMPs. NO LEVEL ASSIGNED: 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.

Scarless Epicanthoplasty and Concomitant Double Eyelidplasty in Chinese Eyelids.

BACKGROUND: The presence of mild or moderate medial epicanthus is typical in Asian patients. Numerous epicanthoplasty techniques have been described previously. However, these methods usually leave obvious scars in the medial canthal area. The aim of this report is to introduce a novel epicanthoplasty technique and a concomitant double eyelidplasty which avoid leaving scars in the medial canthal region. METHODS: From July 2013 to July 2015, 252 patients received epicanthoplasty and concomitant double eyelidplasty with this new technique. The medial epicanthus was corrected through the medial end of the eyelid crease incision. One hundred eighteen of these patients were followed up for 3-24 months (8 months in average). The preoperative and postoperative interepicanthal distances were measured at pre, 3 and 6 months post-operation. The aesthetic results were evaluated with patient visual analog scale (VAS) scores. RESULTS: The average intercanthal distance significantly decreased 3 months after the operation (32.7 ± 2.3 mm vs 36.5 ± 2.6 mm, p < 0.05, paired t test). Little retraction was noticed at 6 months after the operation (33.0 ± 2.4 vs 32.7 ± 2.3 mm, p < 0.05, paired t test). The mean patient VAS score associated with satisfaction of overall outcome was 4.2 at 6 months after operation (range 2.5-5.0). CONCLUSION: This new method provides an effective way to correct the medial epicanthus without leaving any scar in the medial canthal region. Patients with mild to moderate medial epicanthus are good candidates for this procedure. LEVEL OF EVIDENCE V: 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 the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Adipose tissue-derived stem cells treated with estradiol enhance survival of autologous fat transplants.

Autologous fat transplantation (AFT) is a common and important operation in plastic surgery for soft tissue defects and adipose tissue-derived stem cells (ADSCs) are considered as a promising supplement to decrease absorption and subsequent side effects due to the ability of multiple differentiation and production of vascular endothelial growth factor (VEGF). The capacities of ADSCs can be further enhanced by treatment with 17-ß estradiol (E2). Therefore, we hypothesized that E2 may promote the potential of ADSCs for AFT. In this study, ADSCs were extracted from three female patients by liposuction. In vitro studies showed that E2 supplementation at an optimal concentration of 10(-8) M resulted in enhanced proliferation, VEGF production, and adipogenic differentiation of human ADSCs, and reduced apoptosis rate in a serum-free environment. In addition, a nude mice model of fat transplantation was utilized to demonstrate the efficacy of ADSC for survival ratio in vivo. These results using the volume of fat tissues after 12 weeks compared original volume, revealed that the addition of E2-treated ADSCs induced a significantly higher tissue survival ratio (76.9 ± 1.9%) when compared with the ADSC-free system (55.5 ± 1.5%). Furthermore, increased capillary formation stained with hematoxylin-eosin (H&E) was observed in ADSCs systems after treatment with E2. Therefore, this study demonstrated E2 could promote the capacities of ADSCs about aspects of adipogenic differentiation, growth factor secretion and apoptosis reduction in vitro, vascularization improvement in vivo, and then enhanced the survival ratio of AFT.

MAPK/ERK-CBP-RFPL-3 Mediates Adipose-Derived Stem Cell-Induced Tumor Growth in Breast Cancer Cells by Activating Telomerase Reverse Transcriptase Expression.

Adipose-derived stem cells (ASCs) improve the self-renewal and survival of fat grafts in breast reconstruction after oncology surgery. However, ASCs have also been found to enhance breast cancer growth, and its role in tumor proliferation remains largely elusive. Here, we explored a novel mechanism that mediates hTERT reactivation during ASC-induced tumor growth in breast cancer cells. In this study, we found the proliferative ability of breast cancer cells markedly increased with ASC coculture. To explore the molecular mechanism, we treated cells with anibody/inhibitor and found that the activation of MEK-ERK pathway was triggered in breast cancer cells by SCF secreted from ASCs, leading to the nuclear recruitment of CBP. As a coactivator of hTERT, CBP subsequently coordinated with RFPL-3 upregulated hTERT transcription and telomerase activity. The inhibition of CBP and RFPL-3 abrogated the activation of hTERT transcription and the promotion of proliferation in breast cancer cells with cocultured ASCs in vitro and in vivo. Collectively, our study findings indicated that CBP coordination with RFPL-3 promotes ASC-induced breast cancer cell proliferation by anchoring to the hTERT promoter and upregulating telomerase activity, which is activated by the MAPK/ERK pathway.

Adipose derived stem cells promote tumor metastasis in breast Cancer cells by stem cell factor inhibition of miR20b.

Breast cancer (BC) metastasis after surgery is associated with the tumor microenvironment and especially with adipose tissue-derived mesenchymal stem cells (ASCs) that have been shown to promote the BC progression. To better understand the role of ASCs in tumor metastasis, our study explored a novel mechanism that mediates the negative regulation of miR20b during ASC-induced tumor metastasis of BC cells. In this study, we found that the migration and invasion abilities of BC cells are markedly increased coculture with ASCs. By studying the regulatory mechanism, we found that miR20b biogenesis in BC cells can be attenuated by ASC-released stem cell factor (SCF) through the downstream c-Kit/MAPK-p38/E2F1 signaling cascade and that miR-20b acts as a tumor suppressor miRNA in the inhibition of BC migration and invasion. HIF-1α and VEGFA are the target genes of miR20b and miR20b downregulation activated HIF-1α-mediated VEGFA transcription and ASC-induced BC migration and invasion. The upregulation of miR20b abrogated the activation of EMT and lung metastasis of breast cancer cells cocultured with ASCs by the inhibition of N-cadherin, vimentin and Twist expression in vitro and in vivo. Collectively, our findings indicate that downregulation of miR20b by ASCs/SCF activates HIF-1α/VEGFA and induces BC cell EMT and metastasis, suggesting that this process is activated by the p-c-Kit/MAPK-p38/E2F1 pathway.

Rab5-dependent autophagosome closure by ESCRT.

In the conserved autophagy pathway, autophagosomes (APs) engulf cellular components and deliver them to the lysosome for degradation. Before fusing with the lysosome, APs have to close via an unknown mechanism. We have previously shown that the endocytic Rab5-GTPase regulates AP closure. Therefore, we asked whether ESCRT, which catalyzes scission of vesicles into late endosomes, mediates the topologically similar process of AP sealing. Here, we show that depletion of representative subunits from all ESCRT complexes causes late autophagy defects and accumulation of APs. Focusing on two subunits, we show that Snf7 and the Vps4 ATPase localize to APs and their depletion results in accumulation of open APs. Moreover, Snf7 and Vps4 proteins complement their corresponding mutant defects in vivo and in vitro. Finally, a Rab5-controlled Atg17-Snf7 interaction is important for Snf7 localization to APs. Thus, we unravel a mechanism in which a Rab5-dependent Atg17-Snf7 interaction leads to recruitment of ESCRT to open APs where ESCRT catalyzes AP closure.

c-Kit-Positive Adipose Tissue-Derived Mesenchymal Stem Cells Promote the Growth and Angiogenesis of Breast Cancer.

BACKGROUND: Adipose tissue-derived mesenchymal stem cells (ASCs) improve the regenerative ability and retention of fat grafts for breast reconstruction in cancer patients following mastectomy. However, ASCs have also been shown to promote breast cancer cell growth and metastasis. For the safety of ASC application, we aimed to identify specific markers for the subpopulation of ASCs that enhance the growth of breast cancer. METHODS: ASCs and bone marrow-derived vascular endothelial progenitor cells (EPCs) were isolated from Balb/c mice. c-Kit-positive (c-Kit+) or c-Kit-negative (c-Kit-) ASCs were cocultured with 4T1 breast cancer cells. Orthotropic murine models of 4T1, EPCs + 4T1, and c-Kit+/-ASCs + 4T1/EPCs were established in Balb/c mice. RESULTS: In coculture, c-Kit+ ASCs enhanced the viability and proliferation of 4T1 cells and stimulated c-Kit expression and interleukin-3 (IL-3) release. In mouse models, c-Kit+ASCs + 4T1/EPCs coinjection increased the tumor volume and vessel formation. Moreover, IL-3, stromal cell-derived factor-1, and vascular endothelial growth factor A in the c-Kit+ASCs + 4T1/EPCs coinjection group were higher than those in the 4T1, EPCs + 4T1, and c-Kit-ASCs + 4T1/EPCs groups. CONCLUSIONS: c-Kit+ ASCs may promote breast cancer growth and angiogenesis by a synergistic effect of c-Kit and IL-3. Our findings suggest that c-Kit+ subpopulations of ASCs should be eliminated in fat grafts for breast reconstruction of cancer patients following mastectomy.

Transcriptome Profiling of Petal Abscission Zone and Functional Analysis of an Aux/IAA Family Gene RhIAA16 Involved in Petal Shedding in Rose.

Roses are one of the most important cut flowers among ornamental plants. Rose flower longevity is largely dependent on the timing of petal shedding occurrence. To understand the molecular mechanism underlying petal abscission in rose, we performed transcriptome profiling of the petal abscission zone during petal shedding using Illumina technology. We identified a total of 2592 differentially transcribed genes (DTGs) during rose petal shedding. Gene ontology term enrichment and pathway analysis revealed that major biochemical pathways the DTGs were involved in included ethylene biosynthesis, starch degradation, superpathway of cytosolic glycolysis, pyruvate dehydrogenase and TCA cycle, photorespiration and the lactose degradation III pathway. This suggests that alterations in carbon metabolism are an important part of rose petal abscission. Among these DTGs, approximately 150 genes putatively encoding transcription factors were identified in rose abscission zone. These included zinc finger, WRKY, ERF, and Aux/IAA gene families, suggesting that petal abscission involves complex transcriptional reprogramming. Approximately 108 DTGs were related to hormone pathways, of which auxin and ethylene related DTGs were the largest groups including 52 and 41 genes, respectively. These also included 12 DTGs related to gibberellin and 6 DTGs in jasmonic acid pathway. Surprisingly, no DTGs involved in the biosynthesis/signaling of abscisic acid, cytokinin, brassinosteroid, and salicylic acid pathways were detected. Moreover, among DTGs related to auxin, we identified an Aux/IAA gene RhIAA16 that was up-regulated in response to petal shedding. Down-regulation of RhIAA16 by virus-induced gene silencing in rose promoted petal abscission, suggesting that RhIAA16 plays an important role in rose petal abscission.