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Immunogenic cell death (ICD) is a type of regulated cell death (RCD) which was discovered to activate adaptive immunity. To date, the effect of ICD on lung adenocarcinoma (LUAD) remains unclear. In this research, we will study the role of ICD-related genes (ICDG) in LUAD.RNA sequencing and clinical data were gathered from TCGA-LUAD cohorts and GEO database. Using unsupervised cluster analysis, three clusters were identified with distinctive immune characteristics and significant overall survival based on 18 ICDG. Using LASSO Cox regression, three genes were identified and used to construct the prognosis signature. The association between the 3-ICDG risk signature and immune microenvironment analysis, somatic mutation, and enriched molecular pathways was investigated.Consensus clustering separated the LUAD samples into three clusters (ICDcluster A, B and C), and ICDcluster B had the best prognosis. Different TME cell infiltration characteristics and biological behavior were found in three ICD clusters. Prognostic risk model was contrasted based on the 3 best prognostic ICD-related genes. Subsequently, vitro experiments verified the above analysis results. The high-risk group showed a poor prognosis and enrichment of cancer promoting signal pathway. Multivariate analysis indicated that this 3-ICDG prognostic model might be an accurate prediction parameter for LUAD. Moreover, conducting immune related analysis, we found that the 3-ICDG risk signature was characterized by an immune-active subtype on account of the high infiltration of immune-active cells.This study expands our cognition of ICD in LUAD microenvironment, excavated prognostic biomarkers, and provided potential value for guiding immunotherapy and chemotherapy.

Zhaosheng Jiang,1,* Jiahao Wang,2,* Wei Meng,1,* Youlang Zhou,3 Limin Ma,1 Yangbo Guan1 1Department of Urology, Affiliated Hospital of Nantong University, Medical School of Nantong University, Nantong, People’s Republic of China; 2Department of Urology, Wuxi Hospital Affiliated to the Nanjing University of Chinese Medicine, Wuxi, People’s Republic of China; 3Research Central of Clinical Medicine, Affiliated Hospital of Nantong University, Nantong, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yangbo Guan; Limin Ma, Tel +86 18912888908 ; Tel +86 13404292020, Email guanyangbo123@ntu.edu.cn; ntmalimin@163.comIntroduction: Ureteral stricture caused by iatrogenic ureteral injury induced ureteral injury is more common and challenging to recover quickly. The effective prevention of ureteral stricture due to iatrogenic ureteral injury-induced ureteral damage is a current challenge for urologists. The purpose of this study was to evaluate the effectiveness of nanoparticle/pirfenidone complex-coated ureteral stents with slow-release pirfenidone for the prevention of ureteral stricture in rabbits. In this study, we developed a nanoparticle/pirfenidone complex-coated ureteral stent to deliver pirfenidone into the injured ureter to inhibit ureteral stricture.Methods: Twelve New Zealand rabbits were divided into four groups: Sham, US, US+ Unmodified ureteral stent, and US+NP/PFD ureteral stent; we constructed an irreversible electroporation model of ureteral injury in rabbits and placed unmodified ureteral stents and nanoparticle/pirfenidone complex-coated ureteral stents into the ureter. Two weeks later, we euthanized the rabbits and removed their bilateral kidneys and ureters. We evaluated the effect of ureteral stent prophylaxis by gross specimen observation, section staining, and Western Blot.Results: We found that the nanoparticle/pirfenidone complexes could adhere uniformly to the surface of the ureteral stent. After placement into the ureter, the nanoparticle/pirfenidone complexes were able to remain on the surface of the ureteral stent. We found nanoparticle/pirfenidone complexes could diffuse in the ureteral epithelial tissue two weeks after the order. The study showed that nanoparticle/pirfenidone complex-coated ureteral stents placed into the ureter showed significantly less stenosis due to fibrosis than in US control rabbits and rabbits treated with unmodified ureteral stents.Conclusion: We used a novel platform based on nanoparticle/pirfenidone complex-coated ureteral stents for local and sustained delivery of pirfenidone, which can effectively deliver pirfenidone to the tissue and can slowly control the release of pirfenidone. Therefore, combining ureteral stents with nanoparticle/pirfenidone complexes was an effective measure to prevent ureteral stricture.Keywords: nanoparticles, ureteral stricture, fibrosis, ureteral stents, pirfenidone

Background. Recently, a large number of studies have sought personalized treatment for lung squamous cell carcinoma (LUSC) by dividing patients into different molecular subtypes. Autophagy plays an important role in maintaining the tumor microenvironment and immune-related biological processes. However, the molecular subtypes mediated by autophagy in LUSC are not clear. Methods. Based on 490 LUSC samples, we systematically analyzed the molecular subtype modification patterns mediated by autophagy-related genes. The ssGSEA and CIBERSORT algorithm were utilized to quantify the relative abundance of TME cell infiltration. Principal component analysis was used to construct autophagy prognostic score (APS) model. Results. We identified three autophagy subtypes in LUSC, and their clinical outcomes and TME cell infiltration had significant heterogeneity. Cluster A was rich in immune cell infiltration. The enrichment of EMT stromal pathways and immune checkpoint molecules were significantly enhanced, which may lead to its immunosuppression. Cluster B was characterized by relative immunosuppression and relative stromal activation. Cluster C was activated in biological processes related to repair. Patients with high APS were significantly positively correlated with TME stromal activity and poor survival. Meanwhile, high APS showed an advantage in response to anti-PD1 and anti-CTLA4 immunotherapy. Conclusion. This study explored the autophagy molecular subtypes in LUSC. We also discovered the heterogeneity of TME cell infiltration driven by autophagy-related genes. The established APS model is of great significance for evaluating the prognosis of LUSC patients, the infiltration of TME cells, and the effect of immunotherapy.

Keloid is a pathological result caused by cutaneous injury and irritation, which is on account of excessive deposition of collagen matrix and the growth of fibrous tissue. Long term and uncontrolled chronic inflammation is also one of the main causes. The mechanism of its formation is not clear. Therefore, in this study, we aimed to find key genes for diagnosis. We selected two datasets from the GEO database to find DGEs. Then, we finally obtained one gene, HOXC9, via WGCNA analysis and two algorithms (LASSO and SVM-RFE). Next, we analyzed its expression in normal tissues versus keloid and studied its change over time by Mfuzz time clustering analysis. Finally, through the analysis of this gene and previous studies we realized that, in addition to cell proliferation, immune infiltration also plays a role during the process of keloid formation, and we then explored the infiltration of immune cells in the normal versus keloid groups and the relationship between this gene and them.

To compare the absorbable and nonabsorbable suture knots on the tendon on bone-to-tendon healing during the early phase in a rat rotator cuff tear (RCT) model.Fifty-two male Sprague-Dawley rats (10 weeks old; mean weight, 380 g) were used in this study, and 51 of them were randomly assigned into three groups: absorbable suture group (ASG, n = 22), nonabsorbable suture group (NSG, n = 22), and sham surgery group (SSG, n = 7), and the remaining rat was used to take surgical pictures. Bilateral supraspinatus tendon tears were created and repaired immediately in ASG and NSG. Three rats from ASG and NSG were killed for Western blot and histological evaluation at 3 days, 1 week, and 4 weeks after surgery. At 4 weeks, four rats from each group were killed for biomechanical test, and three rats from SSG were used for histological evaluation.Absorbable suture knots on the tendon completely degraded at 4 weeks. However, nonabsorbable suture knots remained intact between the tendon and articular side. ASG showed a stronger inflammatory reaction at 3 days and 1 week, but a weaker reaction at 4 weeks as confirmed by gross observation and Western blot. Besides, ASG showed superior biomechanical properties in terms of maximum load to failure and stiffness at 4 weeks. Modified Bonar score revealed superior maturity for tissue healing in ASG to that in NSG at 4 weeks. Furthermore, inferior bone-to-tendon interface and weakest link formation were observed in NSG on histologic images.Absorbable suture knots on the tendon contributed to better mechanical properties compared with the nonabsorbable one after rotator cuff repair.

Developing nerve conduits with biological cues is a promising approach for repairing peripheral nerve injuries. Although most biological cues incorporated into conduits generally exert their biological functions at the surface, they could not be released into the on-demand regeneration sites under physiological conditions. Herein, we firstly report a bio-orthogonally functionalized chitosan scaffold with esterase-activatable release for peripheral nerve regeneration. In this study, biological cues are not only selectively conjugated into nerve conduits by bio-orthogonal reaction, but also precisely released in on-demand regeneration sites via esterase-activatable cleavage for peripheral nerve repair. Moreover, this nerve scaffold with esterase-activatable release could promote Schwann cells proliferation. In a rat sciatic nerve defect model, the bio-orthogonally functionalized scaffold with esterase-activatable release significantly increased sciatic nerve function recovery and improved target muscles weight. This strategy of incorporating esterase-activatable bioactive cues into peripheral nerve conduits offers great potential in preclinical studies.

The incidence rate of lung adenocarcinoma (LUAD) is rapidly increasing. Recent studies have reported that histone acetylation modification plays an important role in the occurrence and development of tumors. However, the potential role of modification of histone acetylation modification in the development of tumor immune microenvironment is still unclear.In this study, we comprehensively evaluated the acetylation modification patterns of LUAD samples obtained from various different databases based on 36 histone modification regulators, and constructed a prognostic model based on The Cancer Genome Atlas (TCGA) LUAD cohort using the Cox regression method. The close relationship between histone acetylation and tumor immune characteristics was further studied, including immune infiltration, immune escape and immunotherapy. Finally, we combined three cohort (GSE30219, GSE72094 and GSE50081) from Gene Expression Omnibus (GEO) database to verify the above results.We analyzed the expression, mutation and interaction of 36 histone acetylation regulated genes. After Univariate Cox regression analysis and least absolute shrinkage and selection operator regression (LASSO), 5 genes (Our study provided a robust signature for predicting changing prognosis of patients with LUAD. Thus, it appears to be a potentially useful prognostic tool. Moreover, the important relationship between histone acetylation and tumor immune microenvironment was revealed.

Jinwen Xia,1,2 Quhui Wang,1 Fei Ju,3 Xiang Luo,3 Feng Wang,4 Youlang Zhou,3 Hua Huang,5 Hua Wang,1,2 Xingli Bao6 1Department of Breast and Thyroid Surgery, Affiliated Hospital of Nantong University, Nantong, People’s Republic of China; 2Clinical Medicine, Medical College, Nantong University, Nantong, People’s Republic of China; 3Research Center of Clinical Medicine, Affiliated Hospital of Nantong University, Nantong, People’s Republic of China; 4Department of Laboratory Medicine, Affiliated Hospital of Nantong University, Nantong, People’s Republic of China; 5Department of Pathology, Affiliated Hospital of Nantong University, Nantong, People’s Republic of China; 6Department of Medical Equipment, Affiliated Hospital of Nantong University, Nantong, People’s Republic of ChinaCorrespondence: Hua Wang, Department of Breast and Thyroid Surgery, Affiliated Hospital of Nantong University, No. 20, Xisi Road, Chongchuan District, Nantong City, Jiangsu Province, 226001, People’s Republic of China, Tel +86 137 062 92250, Email wanghua12083@163.comPurpose: Multiple reports have demonstrated that highly expressed chloride intracellular channel 1 (CLIC1) exists in a range of malignant tumors and is involved in proliferation, invasion, and migration of cancer cells. There are few studies on CLIC1 and breast cancer (BC). The purpose of this research was to evaluate the expression level of CLIC1 in BC and its impact on prognosis of BC patients.Patients and Methods: Differences in CLIC1 expression levels in 25 pairs of BC and corresponding paracancerous specimens were tested by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB). Immunohistochemistry (IHC) was performed to discuss the relevance between CLIC1 expression in BC tissue chips and clinicopathological parameters of BC patients. The effect of CLIC1 expression on patient prognosis was evaluated by Kaplan–Meier survival curve and Cox regression analysis. Receiver operating characteristic (ROC) curve assessed the diagnostic performance of CLIC1 for BC.Results: The experimental results of qRT-PCR and WB demonstrated that CLIC1 was highly expressed in BC tissues. IHC results showed that overexpression of CLIC1 was strictly correlated with tumor size, TNM classification, pathological grade, lymph node metastasis and Ki67. Patients with lower CLIC1 expression had longer overall survival (OS) and progression-free survival (PFS). Cox regression analysis and ROC curve confirmed that CLIC1 could independently influence the prognosis of BC patients and might have diagnostic efficiency.Conclusion: Overexpressed CLIC1 is closely related to the progression of BC and the poor prognosis of the patients, suggesting that it may act as a potential biological diagnostic index for BC.Keywords: CLIC1, clinical pathological parameter, prognosis, diagnosis

Background Lung adenocarcinoma (LUAD) is a major subtype of lung cancer and closely associated with poor prognosis. N6-methyladenosine (m6A), one of the most predominant modifications in mRNAs, is found to participate in tumorigenesis. However, the potential function of m6A RNA methylation in the tumor immune microenvironment is still murky. Methods The gene expression profile cohort and its corresponding clinical data of LUAD patients were downloaded from TCGA database and GEO database. Based on the expression of 21 m6A regulators, we identified two distinct subgroups by consensus clustering. The single-sample gene-set enrichment analysis (ssGSEA) algorithm was conducted to quantify the relative abundance of the fraction of 28 immune cell types. The prognostic model was constructed by Lasso Cox regression. Survival analysis and receiver operating characteristic (ROC) curves were used to evaluate the prognostic model. Result Consensus classification separated the patients into two clusters (clusters 1 and 2). Those patients in cluster 1 showed a better prognosis and were related to higher immune scores and more immune cell infiltration. Subsequently, 457 differentially expressed genes (DEGs) between the two clusters were identified, and then a seven-gene prognostic model was constricted. The survival analysis showed poor prognosis in patients with high-risk score. The ROC curve confirmed the predictive accuracy of this prognostic risk signature. Besides, further analysis indicated that there were significant differences between the high-risk and low-risk groups in stages, status, clustering subtypes, and immunoscore. Low-risk group was related to higher immune score, more immune cell infiltration, and lower clinical stages. Moreover, multivariate analysis revealed that this prognostic model might be a powerful prognostic predictor for LUAD. Ultimately, the efficacy of this prognostic model was successfully validated in several external cohorts (GSE30219, GSE50081 and GSE72094). Conclusion Our study provides a robust signature for predicting patients’ prognosis, which might be helpful for therapeutic strategies discovery of LUAD.

Extrachromosomal circular DNA (eccDNA) is considered a circular DNA molecule that exists widely in nature and is independent of conventional chromosomes. eccDNA can be divided into small polydispersed circular DNA (spcDNA), telomeric circles (t-circles), microDNA, and extrachromosomal DNA (ecDNA) according to its size and sequence. Multiple studies have shown that eccDNA is the product of genomic instability, has rich and important biological functions, and is involved in the occurrence of many diseases, including cancer. In this review, we focus on the discovery history, formation process, characteristics, and physiological functions of eccDNAs; the potential functions of various eccDNAs in human cancer; and the research methods employed to study eccDNA.

A major challenge in tendon injury is the weak intrinsic healing capacity of tendon that may cause rupture of the repair after surgery. Growth factors are believed to be critical during tendon healing. This study aimed to investigate the effects of vascular endothelial growth factor (VEGF) genes delivered by adeno-associated virus (AAV) vectors on tendon healing and molecular events involved in a chicken model. A total of 128 deep flexor tendons in the long toes of chickens were completely transected and injected with 2 × 109 particles of AAV2-VEGF or saline before surgically repaired. At postoperative 4, 6 and 8 weeks, the gliding excursions of tendon were recorded and adhesions around the repair site scored. At 2, 4, 6 and 8 weeks, the ultimate strengths of the healing tendons were tested. Terminal deoxynucleotide transferase dUTP nick end labeling assay were performed to detect cellular apoptosis and immunofluorescence staining to detect type III collagen and matrix metalloprotease-2 (MMP2) expression in tendon tissues. The gliding excursion and adhesion score were similar between AAV2-VEGF-treated tendons and the control tendons. Delivery of AAV2-VEGF significantly increased ultimate strength of the healing tendons at postoperative 4, 6 and 8 weeks (P

Docosahexaenoic acid (DHA) is the most abundant n-3 polyunsaturated fatty acid in the human brain and works as an anticancer agent to induce cell cycle arrest and apoptosis in glioblastoma multiforme (GBM) cell lines. However, little is known about the connection between DHA and autophagy in GBM cells. We found that high-dose DHA caused cellular autophagy in cultured U251 and U118 GBM cell lines, but there was no effect with a low dose. Moreover, after treatment with a high dose of DHA at 12, 24, and 48 h, the protein expression of SQSTM1/p62 decreased in DHA-treated U251 cells at 12 and 24 h, but increased at 48 h, while in DHA-treated U118 cells, the protein expression increased at all time points. Interestingly, the level of SQSTM1/p62 mRNA was elevated in both DHA-treated U251 and U118 cells at all time points, indicating that DHA activated SQSTM1/p62 transcription in both cell lines. Furthermore, downregulation of SQSTM1/p62 by siRNA attenuated DHA-induced cellular autophagy in both cell lines. This report confirms that high-dose DHA induces cellular autophagy in GBM cells, and demonstrates that SQSTM1/p62 acts as a regulator and participates in DHA-induced autophagy.

Recently, chemotherapy-based polymeric nanoparticles have been extensively investigated for solid tumor treatment. Tumor targeted nanoparticles demonstrated great potential for improved accumulation in the tumor tissue, superior anticancer activity and reduced side effects. Thus, inulin-ibuprofen polymer was synthesized by esterification between inulin and ibuprofen, and RGD targeted epirubicin (EPB) loaded nanoparticles were prepared by the self-assembly of inulin-ibuprofen polymer and in situ encapsulation of EPB. RGD conjugated EPB loaded nanoparticles were characterized by dynamic light scattering (DLS) and transmission electron microscope (TEM). The EPB release from the nanoparticles showed pH-dependent profile and accelerated by the decreased pH value, which would favor the effective drug delivery in vivo. Intracellular uptake analysis suggested that RGD conjugated nanoparticles could be easily internalized by the cancer cells. In vitro cytotoxicity revealed that RGD conjugated EPB loaded nanoparticles exhibited the better antitumor efficacy compared with non-conjugated nanoparticles. More importantly, RGD conjugated EPB loaded nanoparticles showed superior anticancer effects and reduced toxicity than free EPB and non-conjugated nanoparticles by in vivo antitumor activity, EPB biodistribution and histology analysis.

Background aims Sustained gene delivery could be particularly necessary in modulating gene expression in healing intrasynovial tendons and may be a promising approach for preventing adhesions. Methods Polylactic-co-glycolic acid nanospheres were prepared and were then incorporated with plasmids expressing enhanced green fluorescence protein and miRNA for inhibiting the transforming growth factor-β1 gene expression. Results We demonstrated that cultured tenocytes could be effectively transfected by means of nanosphere/plasmid. The expression of transforming growth factor-β1 is significantly downregulated in healing chicken flexor tendon treated with nanosphere/plasmid. Histology analysis did not demonstrate any significant inflammation or necrosis in tendons injected with nanosphere/plasmid after surgery. Conclusions Nanosphere/plasmid may be a promising non-viral vector for gene therapy of the injured flexor tendon.

Heparanase (HPSE) is a type of endoglycosidase that decomposes the heparan sulfate (HS) lateral chains of heparan sulfate proteoglycans (HSPGs), releases related growth factors and participates in angiogenesis and bone formation. HPSE is expressed in osteoblasts and is involved in fracture healing. However, the role of HPSE in osteogenic differentiation requires in-depth investigation. To investigate the expression of HPSE in the osteogenic differentiation of rat marrow stromal cells (MSCs), the protein and mRNA expression levels of HPSE on days 0, 1, 3, 7, 10, 14 and 21 of osteogenic differentiation of MSCs in 2- and 10-month-old rats were detected using western blotting and reverse transcription-polymerase chain reaction (RT-PCR), respectively. From the third day of osteogenic differentiation onwards, all HPSE protein and mRNA expression levels in 2-month-old rats were significantly increased compared with basal levels (days 0 and 1; P0.05). The protein and mRNA levels of HPSE in the 2-month-old rats were significantly higher compared with the respective levels in the 10-month-old rats (P

miR-155 (microRNA-155) is an important non-coding RNA in regulating host crucial biological regulators. However, its regulatory function in mycobacterium infection remains unclear. Our study demonstrates that miR-155 expression is significantly increased in macrophages after Mycobacterium marinum (M.m) infection. Transfection with anti-miR-155 enhances nitric oxide (NO) synthesis and decreases the mycobacterium burden, and vice versa, in interferon γ (IFN-γ) activated macrophages. More importantly, miR-155 can directly bind to the 3'UTR of CCAAT/enhancer binding protein β (C/EBPβ), a positive transcriptional regulator of nitric oxide synthase (NOS2), and regulate C/EBPβ expression negatively. Knockdown of C/EBPβ inhibit the production of nitric oxide synthase and promoted mycobacterium survival. Collectively, these data suggest that M.m-induced upregulation of miR-155 downregulated the expression of C/EBPβ, thus decreasing the production of NO and promoting mycobacterium survival, which may provide an insight into the function of miRNA in subverting the host innate immune response by using mycobacterium for its own profit. Understanding how miRNAs partly regulate microbicidal mechanisms may represent an attractive way to control tuberculosis infectious.

The cancer stem cell theory hypothesizes that cancer stem cells (CSCs), which possess self-renewal and other stem cell properties, are regarded as the cause of tumor formation, recurrence and metastasis. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. In this study, we enriched gastric cancer stem cells through spheroid body formation by cultivating the human gastric cancer cell line MKN-45 in defined serum-free medium. The stemness characteristics of spheroid body-forming cells, including self-renewal, proliferation, chemoresistance, tumorigenicity of the MKN-45 spheroid body-forming cells were evaluated, and the expression levels of stemness genes and related proteins in the MKN-45 spheroid body-forming cells were assessed. Furthermore, immunofluorescence staining for the stem cell markers on spheroid body-forming cells was examined to evaluate the association between stemness factors (Oct4, Sox2, Nanog) and the proposed CSC marker CD44. Our data demonstrated that non-adherent spheroid body-forming cells from the gastric cancer cell line MKN-45 cultured in stem cell-conditioned medium possessed gastric CSC properties, such as persistent self-renewal, extensive proliferation, drug resistance, high tumorigenic capacity and overexpression of CSC-related genes and proteins (Oct4, Sox2, Nanog and CD44), compared with the parental cells. More importantly, CD44-positive cells co-expressing the pluripotency genes Oct4, Sox2 and Nanog may represent gastric CSCs. Further experiments using more refined selection criteria such as a combination of two or multiple markers would be useful to specifically identify and purify CSCs.

The growth-associated protein 43 (GAP-43) gene of Gekko japonicus was obtained from a brain and spinal cord cDNA library. The results of northern blot analysis showed the gecko GAP-43 gene transcript is 1.7 kb in length, and it was abundantly expressed in tissues of brain, spinal cord and ovary. Gecko GAP-43 promoted the outgrowth of Gsn3 cells and PC12 cell in vitro, and phosphorylation at serine 42 modulated the effect of GAP-43 on cell spreading and morphology. The change in GAP-43 expression in the spinal cord after tail amputation was examined by reverse transcription polymerase chain reaction (RT-PCR). The level of GAP-43 in the spinal cord was increased during the time course we examined, indicating a possible correlation between GAP-43 expression and the spinal cord injury and regeneration.

Although both porcine flexor and extensor tendons have been used in tendon repair research, no studies have specifically studied the anatomical differences and repair strengths in both types of tendons. We used 12 pig trotters to observe the anatomy of these tendons and compared the 2 mm gap and ultimate strengths of flexor and extensor tendons. There were four annular (A1, A2, A3, and A4) pulleys and one oblique pulley, which form a fibro-osseous tunnel for the flexor tendons, but the anatomy of the porcine extensor tendons was markedly different from the human flexor or extensor tendons. The diameter of flexor tendons was significantly greater than that of the extensors. The 2 mm gap and ultimate strengths of the flexor tendon with either two-strand or four-strand repairs were significantly greater than those of the extensor tendon. We conclude that the porcine flexor tendon systems are similar to those in the human, but the extensor tendons are not similar to either the flexor or extensor tendons in humans. Flexor and extensor tendons have different repair strengths which should be taken into account when interpreting findings from investigations using these tendons.

Multi-strand repairs are often used in flexor tendon surgery. We evaluated the strength of four four-strand tendon repairs with different locks and suture geometry. Forty-eight pig flexor tendons were repaired with the following methods: a cross-lock four-strand repair; a U-shaped repair with circle-locks; a modified Kessler repair with Pennington locks (with longitudinal sutures located more centrally; and another modified Kessler (with longitudinal sutures located more laterally). The tendons were loaded to complete failure of the repairs. The two Kessler repairs showed a 35% lower 2 mm gap force, and 15% lower ultimate strength compared with the other two repairs; the differences were of statistical significance. The failure pattern was breakage of sutures in almost all tendons. These four-strand repairs differed in gapping and ultimate strengths. The Kessler-type repairs with different geometry in the longitudinal sutures produced identical strengths. The Kessler-type repairs with Pennington locks were weaker than the two repairs with either cross-locks or circle-locks.

The platelet-derived growth factor-C (PDGF-C) gene of Gekko japonicus was obtained from a brain and spinal cord cDNA library. The results of Northern blot showed that transcript of PDGF-C gene of gecko is 2.8 kb in length, and it was abundantly expressed in tissues of heart, lung, kidney, and ovary. In situ hybridization (ISH) revealed that positive hybridization signals were present in both gray matter and white matter of the spinal cord. The change of PDGF-C expression in the spinal cord after tail amputation was examined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. The expression of PDGF-C in the spinal cord showed highest level at 1 day after tail amputation, and gradually decreased until 2 weeks, which indicated that the expression level of PDGF-C might be associated with the process of spinal cord injury and regeneration.

One major obstacle in the application of drug delivery systems for cancer chemotherapy is their poor penetration in tumor tissues. Conjugating active ligand moieties to the surface of nanoparticles may be a promising approach for enhancing the tumor accumulation and penetration of nanoparticles. Herein, the cell-penetrating peptide twin-arginine translocation (Tat)-conjugated epirubicin-loaded poly(lactic-glycolic acid) nanoparticles were prepared to achieve deep tumor penetration. The morphology and size of nanoparticles were characterized by scanning electron microscopy and dynamic light scattering, and the biological behaviors of nanoparticles were evaluated. It is demonstrated that Tat-conjugated nanoparticles have a significant improvement in antitumor activity and biodistribution compared with nonconjugated nanoparticles. Importantly, Tat conjugated on the surface of nanoparticles could facilitate the encapsulated drug penetration into deeper tumor tissue. Additionally, Tat-conjugated nanoparticles have good biocompatibility, as demonstrated by hemolytic tests, in vitro cytotoxicity, and histology study. These results suggested that the Tat-conjugated nanoparticles, as a powerful delivery system for chemotherapeutic drug, would have a promising application in human cancer therapy.

Adhesion formation after digital flexor tendon injury greatly affects gliding function of the tendon, which is a major clinical complication after hand surgery. Transforming growth factor beta 1 (TGF-β1) has a critical role in adhesion formation during tendon healing. Persistent regulation of TGF-β1 through application of microRNA (miRNA) specifically inhibiting the function of TGF-β1 (TGF-β1-miRNA) holds promise for treatment of such a complication. Adeno-associated virus (AAV) was used to transfer TGF-β1-miRNA to the chicken digital flexor tendons, which had been injured and surgically repaired. Four doses of AAV2-TGF-β1-miRNA (2 × 10¹¹, 2 × 10¹⁰, 2 × 10⁹ and 2 × 10⁸ vector genomes (vg)) were used to determine the transfection efficiency. At postoperative 3 weeks, we found a positive correlation between the administered AAV2-TGF-β1-miRNA doses and transfection efficiency. The transfection rate ranged from 10% to 77% as the doses increased. Production of TGF-β1 protein in the tendons decreased on increasing vector dosage. When 2 × 10¹¹ and 2 × 10¹⁰) vg were injected into the tendon, gliding excursion of the repaired tendon and work of flexion of chicken toes were significantly increased and adhesion score decreased 6 and 8 weeks later, indicating the improvement of tendon gliding and decreases in adhesion formations. However, the ultimate strength of the tendons transfected at the dose of 2 × 10¹⁰ vg was 12-24% lower than that of the control tendons. The results of this study demonstrate that application of TGF-β1-miRNA had a mixed impact on tendon healing: adhesion around the tendon is reduced but strength of the tendon healing is adversely affected. Future studies should aim at maintaining the beneficial effects of reducing tendon adhesions, while eliminating the adverse effects of decreasing the healing strength.

The cancer stem cell (CSC) theory hypothesizes that CSCs are regarded as the cause of tumor formation, recurrence and metastasis. This study aimed to investigate whether spheroid body-forming cells in human gastric cancer cell were enriched for CSC properties, and to assess the expression of candidate CSC markers, cluster of differentiation 44 (CD44) and adenosine triphosphate binding cassette transporter G 2 (ABCG2) in the MKN45 spheroid body cells.Human gastric cancer cell line MKN45 were plated in stem cell conditioned culture system allowed for spheroid body forming. The expression levels of CD44 and ABCG2 in the spheroid body cells were assessed by quantitative real-time PCR, western blot analysis and immunofluorescence staining, and the tumorigenicity of the spheroid body-forming cells were assessed by in vivo xenograft studies in nude mice.The MKN45 cells could form spheroid bodies cultured in stem cell conditioned medium. The spheroid body-forming cells showed a significantly greater (p0.05) expression of CD44 and ABCG2 than the parental cells.Spheroid body cells from gastric cancer cell line MKN45 cultured in stem cell conditioned medium possessed gastric CSC properties. The cells co-expressed of CD44 and ABCG2 might represent a subpopulation of gastric CSCs.

Treatment of the disrupted digital flexor tendon is troublesome because of the lack of sufficient healing capacity and the formation of adhesions. Sustained gene delivery may be a promising approach of modulating gene expression in enhancing tendon healing and decreasing adhesions. In this study, a microRNA-based RNAi plasmid was used to specifically silence the expression of TGF-β1 gene associated with scar and adhesion formation in the flexor tendons. The miRNA plasmids were complexed with polylactic-co-glycolic acid (PLGA) nanoparticles to form nanoparticle/TGF-β1 miRNA plasmid (nanoparticle/plasmid) complexes. In vitro and in vivo transfection efficiencies experiments against tenocytes revealed that nanoparticle/plasmid complexes have significantly superior transfection efficiency over the lipofectamine/plasmid complexes. The gene and protein expression associated with adhesion of tendon treated with nanoparticle/plasmid complexes were evaluated by real-time PCR and immunoblotting. The grading of adhesions for tendons treated with nanoparticle/plasmid complexes was less severe than that treated with the nanoparticle/mock plasmid complexes. However, the ultimate strength of repaired tendons treated with nanoparticle/plasmid complexes was significantly lower than that of tendons treated with the nanoparticle/mock plasmid complexes.

The enolase2 gene is usually expressed in mature neurons and also named neuron specific enolase (NSE). In the present study, we first obtained the NSE gene cDNA sequence by using the RACE method based on the expressed sequence tag (EST) fragment from the cDNA library of Gekko japonicus and identified one transcript of about 2.2 kb in central nervous system of Gekko japonicus by Northern blotting. The open reading frame of NSE is 1305 bp, which encodes a 435 amino-acid protein. We further investigated the multi-tissue expression pattern of NSE by RT-PCR and found that the expression of NSE mRNA was very high in brain, spinal cord and low in heart, while it was not detectable in other tissues. The real-time quantitative PCR was used to investigate the time-dependent change in the expression of the NSE mRNA level after gecko spinal cord transection and found it significantly increased at one day, reaching its highest level three days post-injury and then decreasing at the seventh day of the experiment. The recombinant plasmid of pET-32a-NSE was constructed and induced to express His fused NSE protein. The purified NSE protein was used to immunize rabbits to generate polyclonal antisera. The titer of the antiserum was more than 1:65536 determined by ELISA. Western blotting showed that the prepared antibody could specifically recognize the recombinant and endogenous NSE protein. The result of immunohistochemistry revealed that positive signals were present in neurons of the brain and the spinal cord. This study provided the tools of cDNA and polyclonal antibody for studying NSE function in Gekko japonicus.

Gekko japonicus undergoes dramatic changes in the caudal spinal cord after tail amputation. The amputation induces cell proliferation in the caudal ependymal tube. We performed hematoxylin and eosin staining at different time points in the regeneration process to investigate the morphological characterization of the regenerated appendages. The central canal extended to the blastema post-amputation and the cartilage and muscle tissue appeared 3 weeks after injury. We performed the bromodeoxyuridine (BrdU) incorporation assay to detect proliferating cells during the regeneration process. BrdU positive cells were detected in the peri-central canal. Furthermore, nestin and neuron-specific enolase (NSE) immunocytochemistry were applied to detect neural stem/progenitor cells and neurons. Two weeks after injury, nestin-positive cells undergoing proliferation were located outside of the ependymal tube, and NSE positive cells appeared after 3 weeks of amputation. These data suggest that neurogenesis is an early event during caudal spinal cord regeneration in gecko.

Jun Qin, Jing Ke, Junfei Xu, Feiran Wang, Youlang Zhou, Yasu Jiang, Zhiwei Wang Department of General Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, People’s Republic of China Abstract: microRNAs (miRNAs) are small, noncoding RNAs that are involved in many biological processes, and aberrant regulation of miRNAs is always associated with cancer progression and development. Abnormal expression of miRNA-132 (miR-132) has been found in some types of cancer, but the effects and potential mechanisms of miR-132 in colorectal cancer (CRC) have not been explored to date. In this study, quantitative real-time polymerase chain reaction was used to investigate the level of miR-132 in CRC tissues and their paired adjacent normal tissues. Bioinformatics analysis indicated that the mechanism underlying the tumor suppressor role of miR-132 in CRC cells may play a role in tumor suppression by targeting paxillin. Furthermore, methylation-specific polymerase chain reaction was performed to evaluate the methylation status of the miR-132 regulatory region. A DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, was used to activate the expression of miR-132 in CRC cells in vitro. Downregulation of miR-132 may occur as a result of hypermethylation and implies a poor prognosis in CRC; therefore, triggering miR-132 reexpression by using DNA methyltransferase inhibitors may be a potential molecular therapeutic target for CRC. Keywords: microRNA-132, colorectal cancer, DNA methylation, invasion