Overexpression of mitochondrial histidyl-tRNA synthetase restores mitochondrial dysfunction caused by a deafness-associated tRNAHis mutation.

The deafness-associated m.12201T>C mutation affects the A5-U68 base-pairing within the acceptor stem of mitochondrial tRNAHis The primary defect in this mutation is an alteration in tRNAHis aminoacylation. Here, we further investigate the molecular mechanism of the deafness-associated tRNAHis 12201T>C mutation and test whether the overexpression of the human mitochondrial histidyl-tRNA synthetase gene (HARS2) in cytoplasmic hybrid (cybrid) cells carrying the m.12201T>C mutation reverses mitochondrial dysfunctions. Using molecular dynamics simulations, we demonstrate that the m.12201T>C mutation perturbs the tRNAHis structure and function, supported by decreased melting temperature, conformational changes, and instability of mutated tRNA. We show that the m.12201T>C mutation-induced alteration of aminoacylation tRNAHis causes mitochondrial translational defects and respiratory deficiency. We found that the transfer of HARS2 into the cybrids carrying the m.12201T>C mutation raises the levels of aminoacylated tRNAHis from 56.3 to 75.0% but does not change the aminoacylation of other tRNAs. Strikingly, HARS2 overexpression increased the steady-state levels of tRNAHis and of noncognate tRNAs, including tRNAAla, tRNAGln, tRNAGlu, tRNALeu(UUR), tRNALys, and tRNAMet, in cells bearing the m.12201T>C mutation. This improved tRNA metabolism elevated the efficiency of mitochondrial translation, activities of oxidative phosphorylation complexes, and respiration capacity. Furthermore, HARS2 overexpression markedly increased mitochondrial ATP levels and membrane potential and reduced production of reactive oxygen species in cells carrying the m.12201T>C mutation. These results indicate that HARS2 overexpression corrects the mitochondrial dysfunction caused by the tRNAHis mutation. These findings provide critical insights into the pathophysiology of mitochondrial disease and represent a step toward improved therapeutic interventions for mitochondrial disorders.

Tunable microwave frequency performance of nanocomposite Co2MnSi/PZN-PT magnetoelectric coupling structure.

Nanocrystalline Co2MnSi Heusler alloy films were deposited on the PZN-PT substrates by a composition gradient sputtering method. It is revealed that this multiferroic heterostructure shows very strong magnetoelectric coupling, leading to continuously tunable microwave frequency characteristics by electric field. With the increase of electric field intensity from 0 to 6 kV/cm, the magnetic anisotropy field H(K) increases from 90 Oe to 182 Oe with an increment of 102%, corresponding to a ME coefficient of 15.3 Oe cm/kV; the ferromagnetic resonance frequency f(FMR) shifts from 3.38 to 4.82 GHz with an increment of deltaf(FMR) = 1440 MHz or deltaf(FMR)/f(FMR) = 43%; moreover, the damping constant alpha dramatically decreases from 0.035 to 0.018. These merits demonstrate that this nanocomposite multiferroic structure is promising in fabrication of tunable microwave components.

The dual detection of formaldehydes and sulfenic acids with a reactivity fluorescent probe in cells and in plants.

In order to reveal the inter-relationship between protein sulfenic acid (RSOH) and formaldehyde (FA) in different physiological processes, development of tools that are capable of respective and continuous detection for both species is highly valuable. Herein, we reported an "off-on" sensor NA-SF for dual detection of RSOH and FA in cells and plant tissues. Importantly, the highly desirable attribute of the probe NA-SF combined with TCEP, makes it possible to monitor endogenous both RSOH and FA in living cells and plants tissues. NA-SF has been applied successfully in detecting RSOH and FA at physiological concentrations in HeLa, HepG2, A549 cells. Furthermore, the application of NA-SF in evaluating the RSOH and FA level in Arabidopsis thaliana roots of different growth stages are performed. The results show that the level of RSOH and FA in Arabidopsis thaliana roots correlates well with their growth stages, which suggests that both RSOH and FA might play important roles in promoting plant growth and roots elongation. And it also implied a potential application for the biological and pathological research of RSOH and FA, especially in plant physiology. Therefore, we expect NA-SF could provide a convenient and robust tool for better understanding the physiological and pathological roles of RSOH and FA.

LncRNA ZFAS1 promotes proliferation and migration and inhibits apoptosis in nasopharyngeal carcinoma via the PI3K/AKT pathway in vitro.

BACKGROUND: Increasing evidence shows that long non-coding RNAs (lncRNAs) play a key role in the development of various cancers. Zinc finger antisense 1 (ZFAS1) is a novel lncRNA with previously demonstrated associations with several types of cancer. Here we examined the expression and potential function of the ZFAS1 in nasopharyngeal carcinoma (NPC). METHODS: We detected ZFAS1 expression in GSE12452, a human microarray dataset, and NPC cell lines. Small interfering RNA against ZFAS1 was used to elucidate the cellular functions of ZFAS1 using MTT, colony formation, cell cycle, cell apoptosis, transwell invasion and migration and western blot assays. An activator of the PI3K/AKT signaling pathway (740Y-P) was used to determine the contribution of PI3K/AKT. RESULTS: ZFAS1 was significantly upregulated in NPC tissues and cell lines. Silencing ZFAS1 significantly inhibited cell proliferation and invasion, arrested cell cycle progression and promoted cell apoptosis, as well as reduced epithelial-mesenchymal transition. Moreover, 740Y-P could rescue the effects of ZFAS1 knockdown on proliferation, apoptosis and invasion in 5-8F cells. CONCLUSIONS: ZFAS1 might play an oncogenic role in NPC and facilitate cell proliferation and invasion via the PI3K/AKT signaling pathway in NPC cells.

Full-length spleen tyrosine kinase inhibits the invasion and metastasis of human laryngeal squamous cell carcinoma.

OBJECTIVE: This study aimed to investigate correlation between full-length spleen tyrosine kinase [SYK (L)] expression and clinical characteristics of laryngeal squamous cell carcinoma (LSCC), and explore effects of SYK (L) on invasion and metastasis of LSCC. METHODS: The human laryngeal cancer Hep-2 cells with low SYK (L) expression were transfected with pIRES2-EGFP-SYK (L) vector and empty vector pIRES2-EGFP to generate Hep-2-SYK (L) cells and Hep-2-neo cells. The cell invasion and migration abilities were determined. RESULTS: The SYK (L) positive expression rate in LSCC tissues was significantly lower than in vocal cord dysplasia tissues and normal laryngeal tissues (P < 0.05). There was a significant correlation between SYK (L) expression and LSCC T stage, histopathological grade and lymph node metastasis (P < 0.05). mRNA expression of SYK (L) in Hep-2-SYK (L) cells was significantly higher than in Hep-2-neo cells and Hep-2 cells (P < 0.01). The protein expression of SYK (L) in Hep-2-SYK (L) cells was markedly higher than in Hep-2-neo cells and Hep-2 cells (P < 0.01). The number of invasive cells was significantly lower in Hep-2-SYK (L) group than in Hep-2-neo group and Hep-2 group (P < 0.01). The average number of migrating cells in Hep-2-SYK (L) group also markedly reduced as compared to Hep-2-neo group and Hep-2 group (P < 0.01). CONCLUSION: The SYK (L) expression was down-regulated in LSCC, which was closely correlated with cancer growth and lymph node metastasis. SYK (L) up-regulation was able to inhibit the invasion and metastasis of LSCC, therefore suppressing tumor development. Thus, SYK (L) may be a potential target for the LSCC treatment.

[The microsurgical anatomic research of the internal auditory canal area on the retrosigmold approach].

OBJECTIVE: To evaluate the safety of the circular round window and discus anatomic landmarks of posterior wall of internal auditory canal by investigating the microscopic anatomy of internal auditory canal area of the retrosigmold approach, which can provide the anatomical basis for acoustic neutrinomas surgery. METHOD: Fifteen adult cadaver heads (30 sides) fixed with formalin were used in the study. The retrosigmold approach operations were imitated to dissect the blood vessels and nerves in internal auditory canal area by opening round bony window and removing posterior wall of internal auditory canal. RESULT: Fifteen specimens of 30 sides circular bone window were opened without injury with transverse sinus and sigmoid sinus. The vertical distance between the highest point of bone window margo superior and the lowest point of transverse sinus margo inferior was (4.02 ± 0.32) mm. The vertical distance from the most anterior point of bone window leading edge to the most posterior point of sigmoid sinus trailing edge was (6.31 ± 0.43) mm. The internal auditory canal tubercle located in the anterior superior position of internal auditory canal. The vertical distance from the highest point of internal auditory canal tubercle to the upper margin of internal auditory canal was (2.31 ± 0.32) mm. To expose the whole internal auditory canal, the length and width of the internal auditory canal posterior wall removal was (7.29 ± 0.32) mm, (4.12 ± 0.29) mm. Within this removal range, no case of cochlea, semicircular canal or venous was injured in 30 specimens. CONCLUSION: The method of opening round window through retrosigmold approach is simple, practial and convenient. With little variation and easiness of location, the sinternal auditory canal tubercle can be used in the identification of the internal auditory canal. When exposing the whole internal auditory canal, the removal scope of the posterior wall should be paid more attention to, in order to avoid the damage of cochlea, semicircular canal and jugular bulb.

[Inhibitory effect of full-length spleen tyrosine kinase on invasion and metastasis of human laryngeal squamous cells].

OBJECTIVE: To study the effect of gene transfection of full length spleen tyrosine kinase (Syk (L)) on the biological behavior of malignant cancer cells. METHODS: Eukaryotic expression vector pIRES2-EGFP-Syk (L) was constrauted and sequenc. Laryngeal carcinoma cell line Hep-2 were transfected with pIRES2-EGFP-Syk (L) vectors or blank vectors. The expressions of mRNA and protein were examined by real time fluorescence quantitative polymerase chain reaction (Q-RT-PCR) and Western blot analysis. CCK-8 method was used for evaluating cell proliferation, Transwell for cell invasion capacity in vitro, and tumor formation in nude mice for in vivo tumorigenicity. RESULTS: pIRES2-EGFP-Syk (L) vectors were successfully construct and transfected to Hep-2 cells. Q-PCR showed that mRNA expression level in Hep-2 cells transfected with Sky (L) (28.395 ± 0.067) was higher than those in Hep-2-neo cells transfected with blank vectors (3.891 ± 0.021) and Hep-2 cells with no transfection (1.005 ± 0.012), with statistically significant difference (F = 104.02, P < 0.01). Western blot showed that protein expression level of transfected-Sky (L) cells (0.821 ± 0.047) was significantly higher than those of Hep-2-neo cells (0.558 ± 0.031) and Hep-2 cells (0.468 ± 0.031), and the difference was statistically significant (F = 112.32, P < 0.01) ; CCK-8 assay showed OD value (1.390 ± 0.067) of transfected-Sky (L) cells was lower than those of Hep-2-neo cells (1.830 ± 0.067) and Hep-2 cells (1.920 ± 0.040), and the difference was statistically significant (F = 107.64, P < 0.01). Transwell assay showed average cell number per field of transfected-Sky (L) cells (176.04 ± 22.32) was higher than those of Hep-2-neo cells (301.02 ± 21.45) and Hep-2 cells (336.04 ± 26.01) with statistically significant difference (F = 123.46, P < 0.01). The volume (250.77 ± 34.83) mm(3) tumor formed from transfected-Sky (L) cells in nude mice, was less than those from Hep-2-neo cells (750.77 ± 40.83) mm(3) and Hep-2 cells (770.77 ± 30.83) mm(3), with statistically significant difference (F = 165.78, P < 0.01). CONCLUSION: Down-regulation of Syk in Hep-2 cells is associated with the malignant biological behaviors of the cells. Syk (L) may be a potential target in gene therapy for laryngeal squamous cell carcinoma.

[The effect and mechanism of SU11248 on proliferation of nasopharyngeal carcinoma cell line CNE-2].

OBJECTIVE: To investigate the effect of sunitinib malate (SU11284) on proliferation of nasopharyngeal carcinoma cell line CNE-2 and explore its mechanism. METHODS: Nasopharyngeal carcinoma cell line CNE-2 was treated with SU11248 in vitro, and the change in the proliferation of CNE-2 cell line was observed by MTT assay; Real-time fluorescence quantitative PCR (FQ-PCR) and Western blotting were used to detect the levels of P27(KIP1);, cyclin G1 mRNA and protein in CNE-2 cells after treated with 2.5 µg/mL SU11284 for different durations. RESULTS: SU11248 inhibited the proliferation of CNE-2 cells in a concentration- and time-dependent manner. The 50% inhibiting concentration (IC50) was 2.5 µg/mL. Moreover, SU11248 treatment concentration-dependently decreased cyclin G1 mRNA and protein expressions (P<0.05), in contrast, it time-dependently increased P27(KIP1); mRNA and protein levels (P<0.05). CONCLUSION: SU11248 may significantly inhibit the proliferation of CNE-2 cells through up-regulating P27(KIP1); and down-regulating cyclin G1.

5-aza-CdR induces the demethylation of Syk promoter in nasopharyngeal carcinoma cell.

The present study employed 5-aza-2'-deoxycytidine (5-aza-CdR) to treat nasopharyngeal carcinoma cell line CNE-1, CNE-2 and non-cancerous human nasopharyngeal epithelial cell line NP-69 to understand the effects on spleen tyrosine kinase (Syk) gene promoter methylation. The results showed that the methylation level of Syk gene is negatively associated with the differentiation level of the cell lines, and the 5-aza-CdR treatment decreased the methylation level in nasopharyngeal carcinoma cell lines. Additionally, the drug sensitivity of low-differentiated cell line was significantly higher than the high-differentiated cell line. In conclusion, the Syk gene promoter methylation reflects the cell differentiation status, and 5-aza-CdR treatment could induce the demethylation of Syk gene promoter.

The promoter methylation of the Syk gene in nasopharyngeal carcinoma cell lines.

The aim of this study was to investigate the mRNA and protein expression levels of the Syk gene as well as its promoter methylation in nasopharyngeal carcinoma (NPC) cell lines. The CNE-1 (highly differentiated), CNE-2 (poorly differentiated) and NP69 (non-cancerous human immortalized nasopharyngeal epithelial cells) cell lines were used in the present study. The MS-PCR, Q-RT-PCR and western blotting methods were used to examine the Syk gene promoter methylation levels and mRNA and protein expression in the three cell lines. The promoter methylation levels in CNE-1, CNE-2 and NP69 cells were 36%, 62% and 0, respectively. The mRNA levels in CNE-1 and CNE-2 cells were 42±3.5 and 28±2% of that in NP69, respectively; the protein levels in CNE-1 and CNE-2 cells were 36±4.5 and 16±2.5 of that in NP69, respectively; the statistical differences between groups were significant. The lower differentiation levels of the NPC cell lines correlate with lower levels of mRNA and protein expression of the Syk gene, as well as higher promoter methylation levels.

[Study of demonstrating main operative mark of transmastoid-epitympanum approach of the facial nerve using double oblique multi-planar reconstruction in multi-slice CT].

OBJECTIVE: To explore a method of demonstrating the facial nerve anatomical landmarks under transmastoid and epitympanum approach with multi-slice CT using double oblique multi-planar reconstruction (MPR). METHOD: Two temporal bone of a corpse were dissected, under transmastoid and epitympanum approach, to observe the anatomical landmarks of facial nerve. Based on that, the anatomical landmarks of facial nerve under transmastoid and epitympanum approach in 30 (60 ears) normal temporal bones of adult corpses were reconstructed using double oblique MPR in multi-slice CT. The achievement ratio was calculated and the differences among transverse plane, coronal plane, sagittal plane and double oblique were compared. RESULT: The different part of facial nerve, such as mastoid segment, tympanum segment, pyramid segment, geniculate ganglion and the outer labyrinthine segment could be exposed clearly with the main anatomical landmarks, such as horizontal semicircular canal, epitympanic recess and cochleariform process through transmastoid and epitympanum approach. The image of anatomical landmarks could be showed in the same sections by double oblique multi-planar reconstruction. The double oblique multi-planar reconstruction to show the landmarks of facial nerve displaying on the same imaging is better than transverse plane, coronal plane and sagittal plane. The achievement ratio of every section is 100%. CONCLUSION: Double oblique MPR is a new method to demonstrate anatomical landmarks through transmastoid and epitympanum approach in one slice. Combined with the operative approach and purpose, the reconstructive images with double oblique MPR can provide valuable information for operation.

[The value of induced hypotension in nasal endoscopic surgery].

OBJECTIVE: To observe the value of induced hypotension in nasal endoscopic surgery with local anesthesia. METHOD: Sixty Patients were randomly divided into two groups. Group I : induced hypotension group; Group II: control group. MAP, HR, SaO2 amount of blood loss, clear visual field of operation and the operation time were observed and compared between two groups. RESULT: The difference of MAP, HR, SaO2 data between two groups showed no statistic difference with anatomical measurement (P >0.05). The difference of amount of blood loss, the operation time and clear visual field of operation between two groups was significant (P <0.05). CONCLUSION: The induced hypotension with local anesthesia can produce time of operation and amount of blood loss decreased. It can safely applied to the in clinical practice.