Degradative autophagy regulates the homeostasis of miRnas to control cancer development.

Macroautophagy/autophagy acts as an anti-tumor mechanism in early cancer stages but promotes growth in established tumors. Similarly, miRNAs function as tumor suppressors or oncogenes, depending on their target genes. This reciprocal relationship between autophagy and miRNAs is a well-studied area, primarily focused on how miRNAs regulate autophagy-related genes. Our research provides innovative insights into how autophagy selectively controls miRNAs. For instance, MIR224 is preferentially degraded within autophagosomes, leading to the upregulation of SMAD4 and suppressing hepatocellular carcinoma (HCC) tumorigenesis. Conversely, autophagy positively regulates MIR449A by degrading EP300/p300 to activate FOXO1 and facilitate MIR449A transcription in colorectal cancer (CRC). In conclusion, our findings reveal the role of autophagy in maintaining the cellular balance of two miRNAs to mitigate tumorigenic stresses and highlight that autophagy-regulated miRNA profiles may serve as diagnostic and therapeutic markers for cancer development.

Treatment of kyphotic deformity in Kümmell's disease through vertebral body screw fixation and intertransverse process grafting: A case report.

RATIONALE: Kümmell's disease, also well acknowledged as delayed posttraumatic vertebral body collapse, it is a rare condition which mainly occurs in elderly people more than 50 years old, with the thoracolumbar junction being mostly affected. PATIENT CONCERNS: In this research, we employed posterior short-segment screw fixation within the injured vertebral region, coupled with intertransverse process bone grafting, to address Kümmell's disease. A 57-year-old female was admitted to our institution with incapacitating back pain and obvious kyphotic deformity. DIAGNOSES: The diagnosis of Kummell disease was mainly depended on clinical symptoms and imaging examinations. INTERVENTIONS: In this research, we employed posterior short-segment screw fixation within the injured vertebral region, coupled with intertransverse process bone grafting, to address Kümmell's disease. OUTCOMES: The patient could walk independently with the help of a thoracolumbosacral orthosis brace on postoperative Day 2. No pains, kyphotic deformity and neurological deficits were observed during the 36 months of postoperative follow-up. These improvements can be visualized through postoperative magnetic resonance imaging and CT scans. Short-segment screw fixation provides short-term stability to the fracture site and accelerates fracture healing. Subsequently, the healed intervertebral and transverse process grafts offer long-term stability, a fact corroborated by postoperative CT scans. LESSONS: In summary, for Kümmell's disease patients exhibiting kyphotic deformity without neurological deficits or compression, posterior short-segment vertebral screw fixation with intertransverse process bone grafting stands as a viable alternative treatment approach.

The Critical Role of The Piezo1/ß-catenin/ATF4 Axis on The Stemness of Gli1+ BMSCs During Simulated Microgravity-Induced Bone Loss.

Disuse osteoporosis is characterized by decreased bone mass caused by abnormal mechanical stimulation of bone. Piezo1 is a major mechanosensitive ion channel in bone homeostasis. However, whether intervening in the action of Piezo1 can rescue disuse osteoporosis remains unresolved. In this study, a commonly-used hindlimb-unloading model is employed to simulate microgravity. By single-cell RNA sequencing, bone marrow-derived mesenchymal stem cells (BMSCs) are the most downregulated cell cluster, and coincidentally, Piezo1 expression is mostly enriched in those cells, and is substantially downregulated by unloading. Importantly, activation of Piezo1 by systemically-introducing yoda1 mimics the effects of mechanical stimulation and thus ameliorates bone loss under simulated microgravity. Mechanistically, Piezo1 activation promotes the proliferation and osteogenic differentiation of Gli1+ BMSCs by activating the ß-catenin and its target gene activating transcription factor 4 (ATF4). Inhibiting ß-catenin expression substantially attenuates the effect of yoda1 on bone loss, possibly due to inhibited proliferation and osteogenic differentiation capability of Gli1+ BMSCs mediated by ATF4. Lastly, Piezo1 activation also slightly alleviates the osteoporosis of OVX and aged mice. In conclusion, impaired function of Piezo1 in BMSCs leads to insufficient bone formation especially caused by abnormal mechanical stimuli, and is thus a potential therapeutic target for osteoporosis.

Synthetic lethality in human bladder cancer cells by curcumin via concurrent Aurora A inhibition and autophagy induction.

Combination therapies to induce mixed-type cell death and synthetic lethality have the potential to overcome drug resistance in cancer. In this study, we demonstrated that the curcumin-enhanced cytotoxicity of cisplatin/carboplatin in combination with gemcitabine was associated with Aurora A suppression-mediated G2/M arrest, and thus apoptosis, as well as MEK/ERK-mediated autophagy in human bladder cancer cells. Animal study data confirmed that curcumin combined with cisplatin/gemcitabine reduced tumorigenesis of xenograft in mice and this phenomenon was associated with elevated expressions of p-ERK and reduced p-Aurora A in tumors. Gene analyses using data repositories further revealed that reduced Aurora A expression alone did not significantly elevate the sensitivity of human bladder carcinoma cells to these anticancer drugs. Unlike other major cancer types, human bladder urothelial carcinoma tissue coexpressed higher AURKA and lower MAP1LC3B than normal tissue, and reduced Aurora A and induction of autophagy have been clinically associated with a better prognosis in patients with early but not advanced stage bladder cancer. Therefore, our results suggest that treatment strategies can utilize the synthetic lethal pair to concurrently suppress oncogenic Aurora A and induce autophagy by coadministrating curcumin with anticancer drugs for early-stage bladder cancer with high expression of Aurora A.

Secretory autophagy-promoted cargo exocytosis requires active RAB37.

RAB37 GTPase regulates cargo exocytosis by cycling between an inactive GDP-bound form and an active GTP-bound form. We reveal that RAB37 simultaneously regulates autophagy activation and tissue inhibitor of metalloproteinase 1 (TIMP1) secretion in lung cancer cells under starvation conditions. TIMP1, an inflammatory cytokine, is a known inhibitory molecule of matrix metalloproteinases matrix metalloproteinase 9 and suppresses the mobility of lung cancer cells both in vitro and in vivo through conventional exocytosis under serum-free conditions. Notably, we disclosed that secretory autophagy participates in TIMP1 secretion in a RAB37- and Sec22b-dependent manner. Sec22b, a SNARE family protein, participates in vesicle and membrane fusion of secretory autophagy. Knockdown of Sec22b decreased TIMP1 secretion and cell motility but did not affect cell proliferation under starvation conditions. We confirmed that starvation-activated RAB37 accompanied by Sec22b is essential for secretory autophagy to further enhance TIMP1 exocytosis. We further use an off-label drug amiodarone to demonstrate that autophagy induction facilitates TIMP1 secretion and suppresses the motility and metastasis of lung cancer cells in a RAB37-dependent manner in the lung-to-lung mouse model. In conclusion, we demonstrated that the RAB37 activation plays a pivotal regulatory role in secretory autophagy for TIMP1 secretion in lung cancer.Abbreviations: ATG: autophagy-related gene; GDP: guanosine diphosphate; GTP: guanosine triphosphate; LC3: microtubule-associated protein 1A/1B-light chain 3; SNARE: soluble N-ethylmaleimide-sensitive-factor attachment protein receptor; TIMP1: tissue inhibitor matrix metalloproteinase 1.

A Recombinant Parathyroid Hormone-Related Peptide Locally Applied in Osteoporotic Bone Defect.

The local application of drug-loaded bioactive scaffold materials is one of the important directions to solve the clinical problem of osteoporotic (OP) bone defects. This study retains the advantages of drug loading and mechanical properties of natural 3D bioactive scaffolds. The scaffolds are functionally modified through chemical and self-assembly approaches with application of polydopamine (PDA) nanoparticles and parathyroid hormone-related peptide-1 (PTHrP-1) for efficient local drug loading. This study investigates the effects of the novel bioactive scaffolds on ossification, osteoclastogenesis, and macrophage polarization. This work elucidates the effects of the scaffolds in regulating osteoclastic activity and new bone formation in vitro. Further studies on the establishment and repair of OP bone defects in small animals are conducted, and the potential of natural bioactive porous scaffold materials to promote the repair of OP bone defects is initially verified. The preparation of safe and economical anti-OP bone repair material provides a theoretical basis for clinical translational applications.

Formosanin C suppresses cancer cell proliferation and migration by impeding autophagy machinery.

Formosanin C (FC) is a natural compound extracted from Paris formosana Hayata with anticancer activity. FC induces both autophagy and apoptosis in human lung cancer cells. FC-induced depolarization of mitochondrial membrane potential (MMP) may trigger mitophagy. In this study, we clarified the effect of FC on autophagy, mitophagy, and the role of autophagy in FC-related cell death and motility. We found FC caused the continuous increase of LC3 II (representing autophagosomes) from 24 to 72 h without degradation after treatment of lung and colon cancer cells, indicating that FC blocks autophagic progression. In addition, we confirmed that FC also induces early stage autophagic activity. Altogether, FC is not only an inducer but also a blocker of autophagy progression. Moreover, FC increased MMP accompanied by overexpression of COX IV (mitochondria marker) and phosphorylated Parkin (p-Parkin, mitophagy marker) in lung cancer cells, but no colocalization of LC3 with COX IV or p-Parkin was detected under confocal microscopy. Moreover, FC could not block CCCP (mitophagy inducer)-induced mitophagy. These results imply that FC disrupts mitochondria dynamics in the treated cells, and the underlying mechanism deserves further exploration. Functional analysis reveals that FC suppresses cell proliferation and motility through apoptosis and EMT-related pathway, respectively. In conclusion, FC acts as an inducer as well as a blocker of autophagy that results in cancer cell apoptosis and decreased motility. Our findings shed the light on the development of combined therapy with FC and clinical anticancer drugs for cancer treatment.

Secretory autophagy promotes RAB37-mediated insulin secretion under glucose stimulation both in vitro and in vivo.

High blood glucose is one of the risk factors for metabolic disease and INS (insulin) is the key regulatory hormone for glucose homeostasis. Hypoinsulinemia accompanied with hyperglycemia was diagnosed in mice with pancreatic ß-cells exhibiting autophagy deficiency; however, the underlying mechanism remains elusive. The role of secretory autophagy in the regulation of metabolic syndrome is gaining more attention. Our data demonstrated that increased macroautophagic/autophagic activity leads to induction of insulin secretion in ß-cells both in vivo and in vitro under high-glucose conditions. Moreover, proteomic analysis of purified autophagosomes from ß-cells identified a group of vesicular transport proteins participating in insulin secretion, implying that secretory autophagy regulates insulin exocytosis. RAB37, a small GTPase, regulates vesicle biogenesis, trafficking, and cargo release. We demonstrated that the active form of RAB37 increased MAP1LC3/LC3 lipidation (LC3-II) and is essential for the promotion of insulin secretion by autophagy, but these phenomena were not observed in rab37 knockout (rab37-/-) cells and mice. Unbalanced insulin and glucose concentration in the blood was improved by manipulating autophagic activity using a novel autophagy inducer niclosamide (an antihelminthic drug) in a high-fat diet (HFD)-obesity mouse model. In summary, we reveal that secretory autophagy promotes RAB37-mediated insulin secretion to maintain the homeostasis of insulin and glucose both in vitro and in vivo.

Secretory autophagy promotes Rab37-mediated exocytosis of tissue inhibitor of metalloproteinase 1.

BACKGROUND: Rab37-mediated exocytosis of tissue inhibitor of metalloproteinase 1 (TIMP1), an inflammatory cytokine, under serum-depleted conditions which leads to suppression of lung cancer cell metastasis has been reported. Starvation is also a stimulus of autophagic activity. Herein, we reveal that starvation activates Rab37 and induces autophagy. METHODS: We used an overexpression/knockdown system to determine the relationship between autophagy and Rab37 in vitro and in vivo. The autophagy activity was detected by immunoblotting, transmission electron microscope, autophagosome purification, and immunofluorescence under the confocal microscope. Lung-to-lung metastasis mouse model was used to clarify the role of autophagy and Rab37 in lung cancer. Clinical lung cancer patient specimens and an online big database were analyzed. RESULTS: Initially, we demonstrated that active-form Rab37 increased LC3-II protein level (the marker of autophagosome) and TIMP1 secretion. Accordingly, silencing of Rab37 gene expression alleviated Rab37 and LC3-II levels as well as TIMP1 secretion, and induction of autophagy could not increase TIMP1 exocytosis under such conditions. Moreover, silencing the Atg5 or Atg7 gene of lung cancer cells harboring active-mutant Rab37 (Q89L) led to decreased autophagy activity and TIMP1 secretion. In the lung-to-lung metastasis mouse model, increased TIMP1 expression accompanied by amiodarone-induced autophagy led to decreased tumor nodules and cancer cell metastasis. These phenomena were reversed by silencing the Atg5 or Atg7 gene. Notably, increasing autophagy activity alone showed no effect on TIMP1 secretion under either Rab37 or Sec22b silencing conditions. We further detected colocalization of LC3 with either Rab37 or TIMP1, identified Rab37 and Sec22b proteins in the purified autophagosomes of the lung cancer cells harboring the active-form Rab37 gene, and confirmed that these proteins are involved in the secretion of TIMP1. We reveal that autophagic activity was significantly lower in the tumors compared to the non-tumor parts and was associated with the overall lung cancer patient survival rate. CONCLUSIONS: We are the first to report that autophagy plays a promoting role in TIMP1 secretion and metastasis in a Rab37-dependent manner in lung cancer cells and the lung-to-lung mouse model.

m6A-Related lncRNAs Predict Overall Survival of Patients and Regulate the Tumor Immune Microenvironment in Osteosarcoma.

Background: m6A-related lncRNAs have demonstrated great potential tumor diagnostic and therapeutic targets. The goal of this work was to find m6A-regulated lncRNAs in osteosarcoma patients. Method: The Cancer Genome Atlas (TCGA) database was used to retrieve RNA sequencing and medical information from osteosarcoma sufferers. The Pearson's correlation test was used to identify the m6A-related lncRNAs. A risk model was built using univariate and multivariable Cox regression analysis. Kaplan-Meier survival analysis and receiver functional requirements were used to assess the risk model's performance (ROC). By using the CIBERSORT method, the associations between the relative risks and different immune cell infiltration were investigated. Lastly, the bioactivities of high-risk and low-risk subgroups were investigated using Gene Set Enrichment Analysis (GSEA). Result: A total of 531 m6A-related lncRNAs were obtained from TCGA. Seven lncRNAs have demonstrated prognostic values. A total of 88 OS patients were separated into cluster 1, cluster 2, and cluster 3. The overall survival rate of OS patients in cluster 3 was more favorable than that of those in cluster 1 and cluster 2. The average Stromal score was much higher in cluster 1 than in cluster 2 and cluster 3 (P < 0.05). The expression levels of lncRNAs used in the construction of the risk prediction model in the high-risk group were generally lower than those in the low-risk group. Analysis of patient survival indicated that the survival of the low-risk group was higher than that of the high-risk group (P < 0.0001) and the area under the curve (AUC) of the ROC curve was 0.719. Using the CIBERSORT algorithm, the results revealed that Macrophages M0, Macrophages M2, and T cells CD4 memory resting accounted for a large proportion of immune cell infiltration. By GSEA analysis, our results implied that the high-risk group was mainly involved in unfolded protein response, DNA repair signaling, and epithelial-mesenchymal transition signaling pathway and glycolysis pathway; meanwhile, the low-risk group was mainly involved in estrogen response early and KRAS signaling pathway. Conclusion: Our investigation showed that m6A-related lncRNAs remained tightly connected to the immunological microenvironment of osteosarcoma tumors, potentially influencing carcinogenesis and development. The immune microenvironment and immune-related biochemical pathways can be changed by regulating the transcription of M6A modulators or lncRNAs. In addition, we looked for risk-related signaling of m6A-related lncRNAs in osteosarcomas and built and validated the risk prediction system. The findings of our current analysis will facilitate the assessment of outcomes and the development of immunotherapies for sufferers of osteosarcomas.

Mechanosensitive Piezo1 is crucial for periosteal stem cell-mediated fracture healing.

The biomechanical environment plays a dominant role in fracture healing, and Piezo1 is regarded as a major mechanosensor in bone homeostasis. However, the role of Piezo1 in fracture healing is not yet well characterized. In this study, we first delineated that Piezo1 is highly expressed in periosteal stem cells (PSCs) and their derived osteoblastic lineage cells and chondrocytes. Furthermore, downregulation of Piezo1 in callus leads to impaired fracture healing, while activation by its specific agonist promotes fracture healing through stimulation of PSC-modulated chondrogenesis and osteogenesis, along with accelerated cartilage-to-bone transformation. Interestingly, vascular endothelial growth factor A is upregulated after Yoda1 treatment of PSCs, indicating an indirect role of Piezo1 in angiogenesis. Mechanistically, activation of Piezo1 promotes expression of Yes-associated protein (YAP) and its nuclear localization in PSCs, which in turn increases the expression and nuclear localization of ß-catenin. In detail, YAP directly interacts with ß-catenin in the nucleus and forms a transcriptional YAP/ß-catenin complex, which upregulates osteogenic, chondrogenic and angiogenic factors. Lastly, Yoda1 treatment significantly improves fracture healing in a delayed union mouse model generated by tail suspension. These findings indicate that Piezo1 is a potential therapeutic target for fracture delayed union or nonunion.

Autophagy Upregulates miR-449a Expression to Suppress Progression of Colorectal Cancer.

Many studies reported that microRNAs (miRNAs) target autophagy-related genes to affect carcinogenesis, however, autophagy-deficiency-related miRNA dysfunction in cancer development remains poorly explored. During autophagic progression, we identified miR-449a as the most up-regulated miRNA. MiR-449a expression was low in the tumor parts of CRC patient specimens and inversely correlated with tumor stage and metastasis with the AUC (area under the curve) of 0.899 and 0.736 as well as poor overall survival rate, indicating that miR-449a has the potential to be a prognostic biomarker. In the same group of CRC specimens, low autophagic activity (low Beclin 1 expression and high p62 accumulation) was detected, which was significantly associated with miR-449a expression. Mechanistic studies disclosed that autophagy upregulates miR-449a expression through degradation of the coactivator p300 protein which acetylates the transcription factor Forkhead Box O1 (FoxO1). Unacetylated FoxO1 translocated to the nucleus and bound to the miR-449a promoter to drive gene expression. Either activation of autophagy by the inducer or overexpression of exogenous miR-449a decreases the expression of target gene LEF-1 and cyclin D1, which lead to decreased proliferation, colony formation, migration, and invasion of CRC cells. Autophagy-miR-449a-tartet genes mediated suppression of tumor formation was further confirmed in the xenograft mouse model. In conclusion, this study reveals a novel mechanism wherein autophagy utilizes miR-449a-LEF1-cyclin D1 axis to suppress CRC tumorigenesis. Our findings open a new avenue toward prognosis and treatment of CRC patients by manipulating autophagy-miR-449a axis.

The Autophagosomes Containing Dengue Virus Proteins and Full-Length Genomic RNA Are Infectious.

Autophagic machinery is involved in selective and non-selective recruitment as well as degradation or exocytosis of cargoes, including pathogens. Dengue virus (DENV) infectioninduces autophagy that enhances virus replication and vesicle release to evade immune systemsurveillance. This study reveals that DENV2 induces autophagy in lung and liver cancer cells andshowed that DENV2 capsid, envelope, NS1, NS3, NS4B and host cell proinflammatory high mobilitygroup box 1 (HMGB1) proteins associated with autophagosomes which were purified by gradientcentrifugation. Capsid, NS1 and NS3 proteins showing high colocalization with LC3 protein in thecytoplasm of the infected cells were detected in the purified double-membrane autophagosome byimmunogold labeling under transmission electron microscopy. In DENV infected cells, the levels ofcapsid, envelope, NS1 and HMGB1 proteins are not significantly changed compared to the dramaticaccumulation of LC3-II and p62/SQSTM1 proteins when autophagic degradation was blocked bychloroquine, indicating that these proteins are not regulated by autophagic degradation machinery.We further demonstrated that purified autophagosomes were infectious when co-cultured withuninfected cells. Notably, these infectious autophagosomes contain DENV2 proteins, negativestrandand full-length genomic RNAs, but no viral particles. It is possible that the infectivity ofthe autophagosome originates from the full-length DENV RNA. Moreover, we reveal that DENV2promotes HMGB1 exocytosis partially through secretory autophagy. In conclusion, we are the firstto report that DENV2-induced double-membrane autophagosomes containing viral proteins andfull-length RNAs are infectious and not undergoing autophagic degradation. Our novel findingwarrants further validation of whether these intracellular vesicles undergo exocytosis to becomeinfectious autophagic vesicles.

MicroRNA-146a suppresses tumor malignancy via targeting vimentin in esophageal squamous cell carcinoma cells with lower fibronectin membrane assembly.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is widely prevalent in Taiwan, and high metastatic spread of ESCC leads to poor survival rate. Fibronectin (FN) assembly on the cell membrane may induce ESCC mobility. MicroRNAs (MiRNAs) are abundant in and participate in tumorigenesis in many cancers. However, the role of MiRNA in FN assembly-related ESCC mobility remains unexplored. METHODS: We divided ESCC CE81T cells into high-FN assembly (CE81FN+) and low-FN assembly (CE81FN-) groups by flow cytometry. MiRNA microarray analysis identified miR-146a expression as the most down-regulated miRNA in comparison of CE81FN+ and CE81FN- cells. RESULTS: Cell proliferation and migration were decreased when CE81FN+ cells overexpressed transgenic miR-146a compared to the parental cells, indicating an inverse correlation between low miR-146a expression and high proliferation as well as motility of FN assembly ESCC cells. Furthermore, vimentin is the target gene of miR-146a involved in ESCC tumorigenesis. MiR-146a suppressed cell proliferation, migration and invasion of CE81FN+ cells through the inhibition of vimentin expression, as confirmed by real-time PCR, Western blotting and Transwell™ assay. Analysis of one hundred and thirty-six paired ESCC patient specimens revealed that low miR-146a and high vimentin levels were frequently detected in tumor, and that the former was associated with late tumor stages (III and IV). Notably, either low miR-146a expression or high vimentin level was significantly associated with poor overall survival rate among ESCC patients. CONCLUSIONS: This is the first report to link FN assembly in the cell membrane with miR-146a, vimentin and ESCC tumorigenesis both in vitro and in ESCC patients.

Pterostilbene Sensitizes Cisplatin-Resistant Human Bladder Cancer Cells with Oncogenic HRAS.

Analysis of various public databases revealed that HRAS gene mutation frequency and mRNA expression are higher in bladder urothelial carcinoma. Further analysis revealed the roles of oncogenic HRAS, autophagy, and cell senescence signaling in bladder cancer cells sensitized to the anticancer drug cisplatin using the phytochemical pterostilbene. A T24 cell line with the oncogenic HRAS was chosen for further experiments. Indeed, coadministration of pterostilbene increased stronger cytotoxicity on T24 cells compared to HRAS wild-type E7 cells, which was paralleled by neither elevated apoptosis nor induced cell cycle arrest, but rather a marked elevation of autophagy and cell senescence in T24 cells. Pterostilbene-induced autophagy in T24 cells was paralleled by inhibition of class I PI3K/mTOR/p70S6K as well as activation of MEK/ERK (a RAS target) and class III PI3K pathways. Pterostilbene-induced cell senescence on T24 cells was paralleled by increased pan-RAS and decreased phospho-RB expression. Coadministration of PI3K class III inhibitor 3-methyladenine or MEK inhibitor U0126 suppressed pterostilbene-induced autophagy and reversed pterostilbene-enhanced cytotoxicity, but did not affect pterostilbene-elevated cell senescence in T24 cells. Animal study data confirmed that pterostilbene enhanced cytotoxicity of cisplatin plus gemcitabine. These results suggest a therapeutic application of pterostilbene in cisplatin-resistant bladder cancer with oncogenic HRAS.

All-Inside Arthroscopic Anterior Talofibular Ligament Anatomic Reconstruction With a Gracilis Tendon Autograft for Chronic Ankle Instability in High-Demand Patients.

The goal of this study was to evaluate the surgical technique and clinical outcome of all-inside arthroscopic anterior talofibular ligament anatomic reconstruction with a gracilis tendon autograft for chronic ankle instability in high-demand patients. Fifteen consecutive patients (14 [93.3%] males and 1 [6.7%] female, mean age 31.9 ± 7.8 [range 21 to 48] years) with chronic ankle instability were enrolled in this study. Under direct arthroscopic visualization, bone tunnels were created in the fibula and talus by a 4.5-mm cannulated drill system. The gracilis tendon autograft was passed through the tunnels and secured by 5.0-mm interference screws. At the final follow-up, functional evaluation was carried out according to the Ankle-Hindfoot Score by the American Orthopaedic Foot and Ankle Society, Sefton grading system, and visual analog scale score. Complications were also recorded. Mean follow-up was 19.5 ± 1.8 (range 18 to 24) months. No complications of wound infection and nerve injury were noted. No patients experienced recurrent ankle instability. Radiologically, the mean varus tilting angle was 15.2° ± 1.5° before surgery and 4.3° ± 1.2° at the last follow-up (p ≤ .001). The anterior drawer distance was 13.2 ± 1.5 mm before surgery and 4.8 ± 1.1 mm at last follow-up (p ≤ .001). The mean American Orthopaedic Foot and Ankle Society and visual analog scale scores were 56.8 ± 10.5 and 5.7 ± 1.3 before surgery, which became 90.2 ± 6.2 and 0.5 ± 0.8 after surgery. Fourteen (93.3%) patients reported excellent/good functional results according to the Sefton grading system (6 [40.0%] excellent, 8 [53.3%] good, and 1 [6.7%] fair). From our clinical experience, all-inside arthroscopic anterior talofibular ligament anatomic reconstruction with a gracilis tendon is an effective treatment for chronic ankle instability in high-demand patients.

MiR-338-5p promotes metastasis of colorectal cancer by inhibition of phosphatidylinositol 3-kinase, catalytic subunit type 3-mediated autophagy pathway.

BACKGROUND: In our preliminary screening, expression of miR-338-5p was found to be higher in primary colorectal cancer (CRC) with metastasis. The autophagy related gene- phosphatidylinositol 3-kinase, catalytic subunit type 3 (PIK3C3) appeared to be targeted by miR-338-5p. Here, we provide solid evidence in support of PIK3C3 involved in miR-338-5p related metastasis of CRC in vitro and in vivo. METHODS: The potential clinical relevance of miR-338-5p and its target gene was analysed on benign colorectal polyps and primary CRCs by QPCR. Mouse spleen xenograft experiment was performed to examine the importance of miR-338-5p for metastasis. FINDINGS: PIK3C3 was one of target genes of miR-338-5p. In primary CRCs, expression of miR-338-5p is positively related to tumour staging, distant metastasis and poor patient survival. Patients with higher ratios of miR-338-5p/PIK3C3 also had significantly poor overall survival, supporting their significance in the progression of CRC. Over-expression of miR-338-5p promotes CRC metastasis to the liver and lung in vivo, in which PIK3C3 was down-regulated in the metastatic tumours. In contrast, overexpression of PIK3C3 in miR-338-5p stable cells inhibited the growth of metastatic tumours. Both migration and invasion of CRC in vitro induced by miR-338-5p are mediated by suppression of PIK3C3. Using forward and reverse approaches, autophagy was proved to involve in CRC migration and invasion induced by miR-338-5p. INTERPRETATION: MiR-338-5p induces migration, invasion and metastasis of CRC in part through PIK3C3-related autophagy pathway. The miR-338-5p/PIK3C3 ratio may become a prognostic biomarker for CRC patients. FUND: NCKU Hospital, Taiwan, Ministry of Science and Technology, Taiwan.

Degradative autophagy selectively regulates CCND1 (cyclin D1) and MIR224, two oncogenic factors involved in hepatocellular carcinoma tumorigenesis.

Overexpressed CCND1 (cyclin D1) is associated with hepatocellular carcinoma (HCC) and we used 147 tumor tissue samples from HCC patients and 3 murine models to reveal an inverse correlation between low autophagic activity and high CCND1 expression. These 2 phenomena in combination correlated with poor overall survival in HCC patients. Mechanistic analysis showed that activated autophagy triggered CCND1 ubiquitination followed by SQSTM1 (sequestosome 1)-mediated selective phagophore recruitment, autophagosome formation, fusion with a lysosome, and degradation. Functional studies revealed that autophagy-selective degradation of CCND1 suppresses DNA synthesis, cell proliferation, and colony, and liver tumor formation by arresting the cell cycle at the G1 phase. Most importantly, diverse pharmacological inducers (rapamycin and amiodarone) effectively suppress tumor growth in orthotopic liver tumor and subcutaneous tumor xenograft models. In conclusion, we have demonstrated a link between degradative autophagy and the cell cycle regulator CCND1, and have discovered the underlying mechanism by which the autophagic degradation machinery regulates the turnover of the cell-cycle regulator CCND1, which in turn affects HCC tumorigenesis. Abbreviations: CCDN1: cyclin D1; HBV: hepatitis B virus; HCC: hepatocellular carcinoma; HCV: hepatitis C virus; SQSTM1: sequestosome 1.

Vancomycin-impregnated electrospun polycaprolactone (PCL) membrane for the treatment of infected bone defects: An animal study.

There is no consensus for the management of critical infected bone defects. The purpose of this study was to produce a vancomycin-impregnated electrospun polycaprolactone (PCL) membrane for the treatment of infected critical bone defects, and test it in a rabbit model. Electrospinning produced a resorbable PCL fiber membrane containing vancomycin approximately 1 mm in thickness, with a pore diameter of <10 µm. Femur defects were made in the limbs of 18 rabbits and infected with Staphylococcus aureus. The rabbits were divided into three groups according to treatment: (1) Experimental group: rabbit freeze-dried allogeneic bone graft and the vancomycin-PCL membrane. (2) Control group 1: bone graft. (3) Control group 2: vancomycin-PCL membrane only. Culture showed no difference in osteoclast activity between the three groups. Transwell testing showed that almost no fibroblasts passed through the membrane during the first 24 h, but some fibroblasts were able to pass it after 72 h. At 12 weeks after surgery, there was significantly less inflammatory cell infiltration in the experimental compared to the control groups. New bone formation and fracture bone callus were greater in the experimental group than control groups. We thus conclude the resorbable electrospun vancomycin-impregnated PCL membrane was effective at controlling bone infection, and in the regeneration of bone in a critical bone defect animal model.

Hepatocellular carcinoma-related cyclin D1 is selectively regulated by autophagy degradation system.

Dysfunction of degradation machineries causes cancers, including hepatocellular carcinoma (HCC). Overexpression of cyclin D1 in HCC has been reported. We previously reported that autophagy preferentially recruits and degrades the oncogenic microRNA (miR)-224 to prevent HCC. Therefore, in the present study, we attempted to clarify whether cyclin D1 is another oncogenic factor selectively regulated by autophagy in HCC tumorigenesis. Initially, we found an inverse correlation between low autophagic activity and high cyclin D1 expression in tumors of 147 HCC patients and three murine models, and these results taken together revealed a correlation with poor overall survival of HCC patients, indicating the importance of these two events in HCC development. We found that increased autophagic activity leads to cyclin D1 ubiquitination and selective recruitment to the autophagosome (AP) mediated by a specific receptor, sequestosome 1 (SQSTM1), followed by fusion with lysosome and degradation. Autophagy-selective degradation of ubiquitinated cyclin D1 through SQSTM1 was confirmed using cyclin D1/ubiquitin binding site (K33-238 R) and phosphorylation site (T286A) mutants, lentivirus-mediated silencing autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and Sqstm1 knockout cells. Functional studies revealed that autophagy-selective degradation of cyclin D1 plays suppressive roles in cell proliferation, colony, and liver tumor formation. Notably, an increase of autophagic activity by pharmacological inducers (amiodarone and rapamycin) significantly suppressed tumor growth in both the orthotopic liver tumor and subcutaneous tumor xenograft models. Our findings provide evidence of the underlying mechanism involved in the regulation of cyclin D1 by selective autophagy to prevent tumor formation. CONCLUSION: Taken together, our data demonstrate that autophagic degradation machinery and the cell-cycle regulator, cyclin D1, are linked to HCC tumorigenesis. We believe these findings may be of value in the development of alternative therapeutics for HCC patients. (Hepatology 2018;68:141-154).