Enhancing nasopharyngeal carcinoma cell radiosensitivity by suppressing AKT/mTOR via CENP-N knockdown.

OBJECTIVE: Investigating the impact of centromere protein N (CENP-N) on radiosensitivity of nasopharyngeal carcinoma (NPC) cells. METHODS: Using immunohistochemistry and immunofluorescence to detect CENP-N expression in tissues from 35 patients with radiosensitive or radioresistant NPC. Assessing the effect of combined CENP-N knockdown and radiotherapy on various cellular processes by CCK-8, colony formation, flow cytometry, and Western blotting. Establishing a NPC xenograft model. When the tumor volume reached 100 mm3, a irradiation dose of 6 Gy was given, and the effects of the combined treatment were evaluated in vivo using immunofluorescence and Western blotting techniques. RESULTS: The level of CENP-N was significantly reduced in radiosensitive tissues of NPC (p < 0.05). Knockdown of CENP-N enhanced NPC radiosensitivity, resulting in sensitizing enhancement ratios (SER) of 1.44 (5-8 F) and 1.16 (CNE-2Z). The combined treatment showed significantly higher levels of proliferation suppression, apoptosis, and G2/M phase arrest (p < 0.01) compared to either CENP-N knockdown alone or radiotherapy alone. The combined treatment group showed the highest increase in Bax and γH2AX protein levels, whereas the protein Cyclin D1 exhibited the greatest decrease (p < 0.01). However, the above changes were reversed after treatment with AKT activator SC79. In vivo, the mean volume and weight of tumors in the radiotherapy group were 182 ± 54 mm3 and 0.16 ± 0.03 g. The mean tumor volume and weight in the combined treatment group were 84 ± 42 mm3 and 0.04 ± 0.01 g. CONCLUSION: Knockdown of CENP-N can enhance NPC radiosensitivity by inhibiting AKT/mTOR.

Specific knockout of Notch2 in Treg cells significantly inhibits the growth and proliferation of head and neck squamous cell carcinoma in mice.

OBJECTIVE: To investigate the effect of Notch2 gene knockout in Treg cells on head and neck squamous cell carcinoma (HNSCC) in mice. METHODS: A mouse model of HNSCC was constructed. Flow cytometry and immunofluorescence were used to examine the numbers of related immune cells and programmed cell death in tumor cells in the spleen and tumor microenvironment of mice. Western blotting was used to measure the expression of related proteins in tumor tissues. RESULTS: The tumor volume of regulatory T (Treg) cell-specific Notch2-knockout mice (experimental group) was significantly smaller than that of control mice (control group) (P < 0.05). Compared with those in the control group, the number of Treg cells and the expression of Ki67 in Treg cells in the spleen and tumor tissue were significantly decreased in the experimental group, while the numbers of CD45+ hematopoietic cells, CD4+ T cells, CD8+ T cells, T helper 1 (Th1) cells, CD11b+ cells (macrophages), and CD11b+CD11c+ cells (dendritic cells) and the expression of Ki67 in CD4+ T cells and CD8+ T cells were significantly increased (P < 0.05). There was no significant difference in the number of Th2 cells between the two groups (P > 0.05). Immunofluorescence analysis showed that the numbers of CD4+ T cells and CD8+ T cells in the tumor tissue in the experimental group were significantly higher than those in the control group (P < 0.05). Compared with that in the control group, programmed cell death in the experimental group was significantly increased (P < 0.05). Moreover, the expression levels of NLRP3, Caspase-1 and GSDMD in the tumor tissues of the experimental group were higher than those in the control group (P < 0.01), while the expression levels of BCL2, Bax, ATG5, LC3 and p62 were not significantly different (P > 0.05). CONCLUSIONS: Specific knockout of the Notch2 gene in Treg cells significantly decreases the function of Treg cells, inhibits the growth of HNSCC and improves the immune microenvironment in mice, thus effectively treating HNSCC.

Successful replantation of amputated facial tissues by supermicrosurgery.

BACKGROUND: Although replantation of amputated facial segments remains challenging in reconstructive surgery, it offers excellent aesthetic and functional outcomes. METHODS: From May 2004 to October 2019, 12 patients underwent replantation of amputated facial tissues by supermicrosurgery. The case details, such as the rationale for replantation, the operation method, and postoperative therapy, are described. Four cases are discussed to demonstrate the replantation of different facial parts. RESULTS: Facial tissue replantation was successful in all 12 patients without secondary surgery. The cases included the nose (1 patient), ears (8 patients), lips (2 patients), and one of the soft tissue segments surrounding the lower jaw. Venous congestion occurred in three patients who received a solitary arterial repair and were treated with bloodletting. All patients expressed satisfaction with the cosmetic and functional results at the final follow-up. CONCLUSIONS: Supermicrosurgical facial tissue replantation is a promising and effective procedure for providing patients with the best aesthetic and functional outcomes.

Overexpression of Notch2 enhances radiosensitivity via inhibition of the AKT/mTOR signaling pathway in nasopharyngeal carcinoma.

Our previous study found that in nasopharyngeal carcinoma (NPC) cells, overexpression of Notch2 can inhibit epithelial-mesenchymal transition (EMT), which plays a vital role in mediating radiosensitivity. The purpose of this study was to explore the radiosensitizing efficacy of the Notch2 gene in NPC cells and its potential mechanism. We used the recombinant plasmid transfection technique to establish Notch2-overexpressing 5-8 F and CNE-2 NPC cells. Cell proliferation, radiosensitivity, apoptosis and cell cycle distribution were assessed by cell counting kit-8 (CCK-8) experiments, colony formation experiments and flow cytometry. The levels of proteins related to cell cycle, apoptosis, and the AKT/mTOR signaling pathway were evaluated by using Western blotting. The results suggested that Notch2 overexpression increased the radiosensitivity of NPC cells, with sensitizing enhancement ratios (SERs) of 1.24 (5-8 F cells) and 1.34 (CNE-2 cells). Flow cytometry indicated that the level of apoptosis and percentage of cells in G2/M-phase were highest in NPC cells overexpressing Notch2 and treated with radiotherapy compared to cells overexpressing Notch2 alone or administered radiotherapy alone. Western blotting showed that compared to that of cells treated with Notch2 overexpression or radiotherapy alone, the levels of γH2AX, Bax, Bcl-2, Cyclin D1 and AKT/mTOR signaling pathway-related proteins were modified in NPC cells overexpressing Notch2 and treated with radiotherapy. These findings showed that overexpression of Notch2 can increase the radiosensitivity of NPC cells by inhibiting the AKT/mTOR pathway.AbbreviationsNPC: Nasopharyngeal carcinoma; EMT: Epithelial-mesenchymal transition; CCK8: Cell counting kit-8; EBV: Epstein-Barr virus; FBS: Fetal bovine serum; PE: Plating efficiency; SF: Survival fraction; SER: Sensitizing enhancement ratio; DSBs: DNA double-strand breaks[Figure: see text].

Preferred Experience of Modifications to Emergency Surgical Treatment for Total Scalp Avulsion With Halo-Vest Head Ring and Quick Hair Removing.

BACKGROUND: Total scalp avulsion is a fairly rear but severe soft tissue injury. Even with microsurgical replantation, the survival rate is still low. In this study, the authors incorporated 2 main modifications (Halo-Vest head ring and quick hair removing) and assessed the surgical outcomes versus those of traditional replantation. METHODS:: Eighteen patients were included in the study who suffered from total scalp avulsion. After consideration of the outcomes from the first 7 patients, the authors modified our surgical procedures and introduced the use of Halo-Vest head ring and quick hair removing in the treatments for the rest 11 patients. The surgical outcomes with both approaches were observed and compared, including the operation time and incidence of scalp necrosis. RESULTS:: The mean debridement time was 3.5 hours in traditional treatment versus 1.68 hours in modified treatment. The mean operative time was 11.14 hours in traditional treatment versus 8.05 hours in modified treatment. After the replantation, in those 7 patients without modifications, there was 1 total scalp necrosis and 6 partial scalp necrosis. In those 11 patients with modifications, there was 1 total scalp necrosis and 1 suffered a partial scalp necrosis, while the scalp survived well in other 9 patients. Classical cases with modified or traditional methods were reported respectively. CONCLUSION: The application of Halo-Vest head ring and quick hair removing provides a reliable method to treat total scalp avulsion. It is safe, technically easy and worth being widely used in the clinical application.

Pulmonary surfactant synthesis in miRNA-26a-1/miRNA-26a-2 double knockout mice generated using the CRISPR/Cas9 system.

Pulmonary surfactant (PS), which is synthesized by type II alveolar epithelial cells (AECIIs), maintains alveolar integrity by reducing surface tension. Many premature neonates who lack adequate PS are predisposed to developing respiratory distress syndrome (RDS), one of the leading causes of neonatal morbidity and mortality. PS synthesis is influenced and regulated by various factors, including microRNAs. Previous in vitro studies have shown that PS synthesis is regulated by miR-26a in fetal rat AECIIs. This study aimed to investigate the role of miR-26a in PS synthesis in vivo. To obtain a miR-26a-1/miR-26a-2 double knockout mouse model, we used the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) system, an important genome editing technology. Real-time PCR was performed to determine the miR-26a levels in various organs, as well as the mRNA levels of surfactant-associated proteins. Moreover, AECIIs and surfactant-associated proteins in lung tissues were analyzed by hematoxylin-eosin staining and immunohistochemistry. Homozygous offspring of miR-26a-1/miR-26a-2 double knockout mice generated using the CRISPR/Cas9 system were successfully obtained, and PS synthesis and the number of AECIIs were significantly increased in the miR-26a knockout mice. These results indicate that miR-26a plays an important role in PS synthesis in AECIIs.

[Application of free flap pedicled with supracarpal cutaneous branch of ulnar artery in repairing of finger replantation].

OBJECTIVE: To evaluate clinical application and clinical outcomes of free flap pedicled with supracarpal cutaneous branch of ulnar artery in repairing of finger replantation with skin defect. METHODS: From April 2007 to March 2013,25 patients affected by finger amputation with skin defect were replanted and repaired by free flap pedicled with supracarpal cutaneous branch of ulnar artery. Among them, 18 patients were male and 7 were female,with an average age of 31.5 years old (ranged 16 to 58). The time of trauma to admission ranged from 45 to 210 min (averaged 105). Fifteen patients were complete separted, and 10 patients were non-complete separated. The area of flaps ranged from 3.5 cm x 2.0 cm to 4.5 cm x 3.0 cm, and the vessels were anastomosed through end-to-end. The functional evaluation standard of finger replantation was used to evaluate the postoperative function. RESULTS: Twenty-four cases were finally survived. Two flaps occurred vascular crisisin within 48 h after operation, one of which was survived after anti-vasospasm treatment and changing dressing,another was replanted finger for failed to survive. One had infection and healed after changing dressing. Twenty-four cases were followed up from 3 to 38 months with an average of 16.5 months. The appearance and texture of flaps were satisfactory, and the superficial senses of pain and touch were recovered,and two-point discrimination was 5.5 to 11 mm (averaged 7.4 mm). According to functional evaluation standard finger replantationissued by Hand Surgery Association of Chinese Medical Association, 8 cases got excellent results, 14 good and 2 poor. CONCLUSION: The free flap pedicled with supracarpal cutaneous branch of ulnar artery can be used in complex finger replantation with skin and vessels defect, which can extend operation indications, recover function and appearance for maximum.

[Clinical application of reverse radial hypothenar flap for finger soft tissue defect].

OBJECTIVE: To investigate the therapeutic effect of reverse radial hypothenar flap for finger soft tissue defect. METHODS: From Mar. 2006 to Mar. 2010, 13 cases (14 fingers) with finger soft tissue defects were treated with reverse radial hypothenar flaps pedicled with ulnar palmar digital artery of little finger. The defects were 1.9 cm x 1.5 cm -4.0 cm x 2.0 cm in size. The flap size ranged from 1.5 cm x 2.0 cm to 4.0 cm x 2.0 cm. RESULTS: All the flaps survived completely with primary healing both in donor and recipient area. 12 cases (13 fingers) were followed up for 1-3 years. The flaps color was similar to the unaffected fingers. Cicatricial contracture happened in one case due to contracture of palmar fascia. The two-point discrimination distance on flap was 3.2-5.3mm. The active and passive movement of finger joints was evaluated as excellent in 12 fingers, good in one finger. There was no complaint about the feeling at the donor site. Two months after operation, all patients could go back to work. CONCLUSIONS: The reverse radial hypothenar flap is very suitable for finger soft tissue defect with less morbidity to donor site.

[Optimization of preparative technique for banxia-houpu effervescent tablets by orthogonal design].

OBJECTIVE: To optimize preparative technique for banxia-houpu effervescent tablets. METHODS: Based on the pH, disintegration time limited, taste, and rigidity of effervescent tablets, the proper proportion between citric acid and sodium bicarbonate, as well as the proper quantity of polyethylene glycol 6000 and sodium cyclamate in the effervescent tablets were determined by using orthogonal design. The content of magnolol and honokiol in effervescent tablets were measured by HPLC. RESULTS: The optimal preparative technique was: cirtic acid: sodium bicarbonate = 0.65:1. The percentage of polyethylene glycol 6000 was 85%, and the percentage of sodium cyclamate was 1.0%. CONCLUSION: The preparative technique is stable, reliable and suitable for practical use.

Adenoviral expression of a truncated S1 subunit of SARS-CoV spike protein results in specific humoral immune responses against SARS-CoV in rats.

The causative agent of severe acute respiratory syndrome (SARS) has been identified as SARS-associated coronavirus (SARS-CoV), but the prophylactic treatment of SARS-CoV is still under investigation. We constructed a recombinant adenovirus containing a truncated N-terminal fragment of the SARS-CoV Spike (S) gene (from--45 to 1469, designated Ad-S(N)), which encoded a truncated S protein (490 amino-acid residues, a part of 672 amino-acid S1 subunit), and investigated whether this construct could induce effective immunity against SARS-CoV in Wistar rats. Rats were immunized either subcutaneously or intranasally with Ad-S(N) once a week for three consecutive weeks. Our results showed that all of the immunized animals generated humoral immunity against the SARS-CoV spike protein, and the sera of immunized rats showed strong capable of protecting from SARS-CoV infection in vitro. Histopathological examination did not find evident side effects in the immunized animals. These results indicate that an adenoviral-based vaccine carrying an N-terminal fragment of the Spike gene is able to elicit strong SARS-CoV-specific humoral immune responses in rats, and may be useful for the development of a protective vaccine against SARS-CoV infection.

Co-transplantation of schwann cells promotes the survival and differentiation of neural stem cells transplanted into the injured spinal cord.

The present study investigates whether Schwann cells (SCs) could promote the survival and differentiation of neural stem cells in the injured spinal cord. Neural stem cells were dissociated and cloned from the hippocampal tissue of newborn rats. SCs were also dissociated and purified simultaneously from the sciatic nerves of 4-day-old rats. The results showed that the number of surviving neural stem cells and differentiated neuron-like cells was significantly increased in the co-grafted (SCs and neural stem cells) group compared with the control group (neural stem cells only). Neuron-like cells that developed axon-like processes were observed more commonly in the co-grafted group. These results demonstrate that SCs can promote the survival and differentiation of transplanted neural stem cells in the injured spinal cord.

Th2 predominance and CD8+ memory T cell depletion in patients with severe acute respiratory syndrome.

UNLABELLED: The immune spectrum of severe acute respiratory syndrome (SARS) is poorly understood. To define the dynamics of the immune spectrum in SARS, serum levels of cytokines, chemokines, immunoglobulins, complement and specific antibodies against SARS-associated coronavirus (SARS-CoV) were assayed by enzyme-linked immunosorbent assay (ELISA), and phenotypes of peripheral lymphocytes were analyzed by flow cytometry in 95 SARS-infected patients. Results showed that interleukin (IL)-10 and transforming growth factor beta (TGF-beta) were continuously up-regulated during the entirety of SARS. Regulated on activation normally T cell-expressed and secreted (RANTES) levels were decreased, while monocyte chemoattractant protein-1 (MCP-1) was elevated in acute patients. Immunoglobulins and complement were elevated during the first month of SARS. Both serum-positive rates and titers of specific IgM and IgG antibodies responding to SARS-CoV peaked at days 41-60 from the onset of SARS. CD4+ and CD8+ T lymphocytes decreased significantly in acute-phase. CD3+CD8+CD45RO+ T lymphocytes were decreased by 36.78% in the convalescent patients. CONCLUSION: SARS-CoV seemed to elicit effective humoral immunity but inhibited cellular immunity, especially CD8+ memory T lymphocytes over time. Prolonged overproduction of IL-10 and TGF-beta may play an important role in the disease.

[A study of gene amplification and expression of cyclin D1 in hepatocellular carcinoma].

OBJECTIVE: To investigate gene amplification of CCND1 and expression of cyclin D1 in hepatocellular carcinoma (HCC) and to explore the possible relationship between CCND1 gene status and carcinogenesis of HCC. METHODS: Differential PCR, RT-PCR and immunohistochemistry were used to detect gene amplification, mRNA and protein expression of cyclin D1 in 20 HCC cases respectively. The relationship between the gene amplification rate and the expression level of cyclin D1 and the histological grades of HCC was analyzed. RESULTS: CCND1 gene amplification was detected in 30% of the cases HCC. An overexpression of cyclin D1 mRNA and protein could be demonstrated in 45% and 70% cases respectively. The expression of cyclin D1 mRNA correlated with its gene amplification status (P < 0.05) and was responsible for the protein expression level (P < 0.05). There was a close relationship between the expression level of cyclin D1 protein and HCC histological grades (P < 0.05). CONCLUSIONS: CCND1 gene amplification is a common phenomenon in HCC and may be directly responsible for the cyclin D1 mRNA and protein overexpression. Cyclin D1 protein expression level is directly related to HCC histological grades. Therefore, CCND1 amplification and cyclin D1 overexpression may play an important role in development and differentiation of HCC.