[Knockout notch1 gene can enhanced radiosensitivity of nasopharyngeal carcinoma cells].

Objective:To investigate the effect of Notch1 gene on radiosensitivity of nasopharyngeal carcinoma cells and its molecular mechanism. Method:A Notch1-knockout CNE-2 cell line was constructed using CRISPR/Cas9 system, and the expression of Notch1 gene was detected by RT-PCR and Western blot. After treatment with different doses of radiation, the survival fraction （SF） of each group was calculated, and used the GraphPad Prism 6.0 software and the Linear quadratic model were used to calculate the fitted dose survival curve and the sensitivity enhancement ratio（SER）. Taking 6 Gy as radiation dose, the experiment was divided into four groups: Notch1（+） group, Notch1（-） group, IR+Notch1（+） group and IR+Notch1（-） group. CCK-8 assay was used to detect cell proliferation in each group. Annexin V-FITC/PI double staining assay was used to detect the changes of apoptosis in each group. The expression of H2AX, CyclinD1, Bax, Bcl-2 and GAPDH proteins were detected by Western blot. Result:The CNE-2 cell line with Notch1 gene knockout was successfully constructed. The clonogenic assay showed knockout of Notch1 enhanced the radiosensitivity of NPC cells. The CCK-8 assay showed that cell proliferation and cell viability were significantly reduced in the IR+Notch1（-） group compared with the IR+Notch1（+） group（P<0.05）. Annexin V-FITC/PI double staining assay showed that the IR+Notch1（-） group had the highest apoptosis rate compared with the other groups （P<0.05）. Western blotting demonstrated that the expression of γH2AX was significantly increased after irradiation of Notch1 nasopharyngeal carcinoma cells, the expression of Cyclin-D1 was increased, and the ratio of Bax:Bcl-2 was higher. Conclusion:Knockout of Notch1 signaling molecule can effectively improve the radiosensitivity of NPC cells cultured in vitro, which may be a potential target for radiosensitization of NPC.

Role of the RUNX2 p.R225Q mutation in cleidocranial dysplasia: a rare presentation and an analysis of the RUNX2 protein structure.

Cleidocranial dysplasia (CCD; MIM 119600) is an autosomal dominant hereditary disorder of skeletal features whose characteristic clinical symptoms are caused by mutations in the RUNX2 gene. Varying degrees of clavicular hypoplasia and dental abnormalities are the most prominent features of this disorder. In this study, we presented a Chinese family that included 4 individuals with a p.R225Q mutation in the RUNX2 gene and characteristic CCD phenotypes. Through structural analysis of the p.R225Q mutation in the RUNX2 protein, we determined that the location of this mutation has the potential to affect DNA binding by RUNX2. The proband in this CCD-affected family showed a specific clinical phenotype of CCD that included a median pseudo-cleft palate, which is a presentation of this mutation that has not been reported previously. On the basis of the structural analysis, this study further demonstrated that the p.R225Q mutation abolished DNA binding by RUNX2 and its results also suggested that other genetic and/or environmental factors could affect the CCD phenotypes.

Gene cloning of porcine adiponectin gene from adipose tissue and construction of its eukaryotic expression vector.

To clone adiponectin (ADPN) gene from Shaziling porcine adipocyte and construct its eukaryotic expression vector, total RNA was extracted from subcutaneous fatty tissue. One pair of specific primers was designed by Primer 5.0 software according to the sequence of ADPN gene of porcine available in GenBank. The ADPN gene was amplified by PCR from cDNA and cloned into pMD18-T vector to construct recombinant clonal vector pMD-ADPN, sequenced and analysed. A recombinant expression plasmid pPICZaA-ADPN was constructed by subcloning the cloned ADPN gene into the linearized pPICZaA vector. Then, the plasmid pPICZaA-ADPN was expressed in Pichia pastoris (GS115) by electrotransformation. Western blot and Bradford analysis were used to determine the target protein induced by methanol. Results showed that the genome size of ADPN was 732 bp and encoded 244 amino acid, the nucleotide sequence of ADPN shared 100% identity with that of porcine available in GenBank. Western blot and Bradford analysis showed that the recombinant ADPN was expressed in GS115 correctly and has certain immune activity. The expression level of ADPN was 28.5 µg/ml. In conclusion, the recombinant ADPN could express in eukaryotic expression vector pPICZaA-ADPN constructed in this study effectively.

Cotransplant of neural stem cells and NT-3 gene modified Schwann cells promote the recovery of transected spinal cord injury.

STUDY DESIGN: An animal model of transected spinal cord injury (SCI) was used to test the hypothesis that cografted neural stem cells (NSCs) and NT-3-SCs promote morphologic and functional recoveries of injured spinal cord. OBJECTIVE: To explore whether cotransplant of NSCs and NT-3-SCs could promote the injured spinal cord repair. SETTING: Zhongshan Medical College, Sun Yat-sen University, PR China. METHODS: Female Sprague-Dawley (SD) rats weighing on 200-220 g were used to prepare SCI models. The spinal cord was transected between T(9) and T(10), then NSCs, SCs+NSCs, LacZ-SCs+NSCs, or NT-3-SCs+NSCs were grafted into the transected site. RESULTS: (1) Part of NSCs could differentiate to neuron-like cells in the transected site and the percentage of differentiation was NT-3-SCs+NSCs group>SCs+NSCs group>NSCs group. (2) In the grafted groups, there were 5-HT, CGRP, and SP positive nerve fibres within the transected site. Some fluorogold (FG)-labeled cells were found in the spinal cord rostral to the transected site, the red nuclei and the inner pyramidal layer of sensorimotor cortex. (3) The cells grafted could enhance the injured neurons survival in inner pyramidal layer of sensorimotor cortex, red nuclei of midbrain, and Clark's nuclei of spinal cord's L1 segment, could decrease the latency and increase the amplitude of cortical somatosensory evoked potential (CSEP) and cortical motor evoked potential (CMEP), and could promote partly structural and functional recovery of the SCI rats. CONCLUSION: These results demonstrate that cografted NT-3-SCs and NSCs is a potential therapy for SCI. SPONSORSHIP: This research was supported by Chinese National Key Project for Basic Research (G1999054009), Chinese National Natural Science Foundation (30270700) and Social Developmental Foundation of Guangdong Province (2003C33808) to YS Zeng; Natural Science Foundation of Guangdong Province (04300468) and Medical Science Research Grant of Guangdong Province (A2004081) to JS Guo.

Luminescence and photocatalytic properties of a platinum(II)-quaterpyridine complex incorporated in Nafion membrane.

The non-emissive platinum(II)-quaterpyridine complex shows strong photoluminescence at room temperature upon incorporation into Nafion membrane; this complex is stabilized toward photochemical decomposition in Nafion even in the presence of oxygen, and can be used as a sensitizer to generate singlet oxygen to oxidize alkenes.

Spinal kappa-opioid system plays an important role in suppressing morphine withdrawal syndrome in the rat.

To explore the possible involvement of spinal kappa-opioid receptor in modulating morphine withdrawal syndrome, rats were made dependent on morphine by multiple injections of morphine HCl for 5 days. They were then given intrathecal administration (i.t.) of a kappa-opioid receptor agonist trans-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzenacetamide hydrochloride (U-50,488H, 2.5-10 microg) or its antagonist nor-binaltorphimine (nor-BNI, 1.25-5 microg), followed by intraperitoneal administration (i.p.) of naloxone (0.5 mg/kg), and the withdrawal syndrome was scored for 60 min. U-50,488H produced a dose-dependent suppression, whereas nor-BNI a dose-dependent potentiation in withdrawal syndrome. The latter result implies that an endogenous kappa receptor agonist, most probably dynorphin, exerts a tonic suppressive effect on morphine syndrome at spinal level.

Suppression of morphine withdrawal by electroacupuncture in rats: dynorphin and kappa-opioid receptor implicated.

Our previous work has demonstrated that 100-Hz electroacupuncture (EA) or 100-Hz transcutaneous electrical nerve stimulation (TENS) was very effective in ameliorating the morphine withdrawal syndrome in rats and humans. The mechanism was obscure. (1) Rats were made dependent on morphine by repeated morphine injections (5-140 mg/kg, s.c., twice a day) for eight days. They were then given 100-Hz EA for 30 min 24 h after the last injection of morphine. A marked increase in tail flick latency (TFL) was observed. This effect of 100-Hz EA could be blocked by naloxone (NX) at 20 mg/kg, but not at 1 mg/kg, suggesting that 100-Hz EA-induced analgesia observed in morphine-dependent rats is mediated by kappa-opioid receptors. (2) A significant decrease of the concentration of dynorphin A (1-17) immunoreactivity (-ir) was observed in the spinal perfusate in morphine-dependent rats, that could be brought back to normal level by 100-Hz EA. (3) 100-Hz EA was very effective in suppressing NX-precipitated morphine withdrawal syndrome. This effect of EA could be prevented by intrathecal administration of nor-BNI (2.5 micrograms/20 microliters), a kappa-opioid receptor antagonist, or dynorphin A (1-13) antibodies (25 micrograms/20 microliters) administered 10 min prior to EA. In conclusion, while the steady-state spinal dynorphin release is low in morphine-dependent rats, it can be activated by 100-Hz EA stimulation, which may be responsible for eliciting an analgesic effect and ameliorating morphine withdrawal syndrome, most probably via interacting with kappa-opioid receptor at spinal level.

[Correlation between motor paralysis and neurotoxicity induced by intrathecal dynorphin A (1-17) in rats].

Intrathecal(i.t.) injection of 10 microliters of dynorphin A(1-17) 20 nmol.L-1 per rat resulted in irreversible hind limb paralysis and suppression of the tail-flick reflex lasting for up to 40 h. The dual effects of dynorphin appeared 5-10 min after the i.t. administration. Histologic examination of the spinal cord in the rats demonstrated dead and/or dying and degenerated motor-neurons in the ventral horn located predominately in the lumbar segment(a 87.2% reduction of the number of motor neurons, P < 0.01) and also in a lesser degree in sacral segment(-69.6%, P < 0.05). The thoracic segment was essentially normal(-8.2%, P > 0.05).

Clinical visual electrophysiology in China.

With the development of modern ophthalmology in the past 40 years, great progress has been made in visual electrophysiology in terms of clinical application, basic research and academic exchange in China. A series of visual electrophysiological measurements, such as electroretinography (ERG), evoked early response (ERP), electrooculography (EOG), visual evoked potential (VEP), which are assisted with various stimuli and computer analysis, have been used in testing different layers of the visual system. Some new relevant techniques have been developed and the academic organizations established. These demonstrate that clinical visual physiology has entered a new period of development.

Changing seroepidemiology of hepatitis A virus infection between two regions in Taiwan differing in socioeconomic status.

In Taiwan, hepatitis A virus (HAV) infection is hyperendemic, and prior to the late 1970s more than 85% of the population was infected by 15 years of age. In this study, the prevalence of HAV infection in two regions of Taiwan with differing standards of living is evaluated. Serum antibody to HAV (anti-HAV) was determined by enzyme immunoassay. A total of 4,218 subjects under the age of 16 years were enrolled. The first group of 1,581 subjects were residents of the prosperous western plain region where the socioeconomic status and living environment have improved greatly in the past 20 years; the second group of 2,637 subjects were aborigines inhabiting the eastern and central mountain regions where improvement in the standard of living lags far behind the western region. All serum samples were collected in 1991 except for 1,100 samples collected in 1989 from elementary and junior middle school students in Tainan city. The prevalence of HAV infection was 11.9% for the western region and 81.0% for the eastern and central regions. In the eastern and central regions, 80% of the subjects had been infected with HAV before the age of six years, in contrast to 0% in the western region. By the age of 13 years, only 6% of the subjects were infected in the western region; however 97% had been infected in the eastern and central regions. Among children under the age of 10 years, only 10.6% (10/94) of the IgM-anti-HAV positive cases had clinical symptoms. These findings indicate that HAV infection has a close relationship with socioeconomic status and environmental sanitation.

[A study on sexual differences of postero-anterior cephalometrics among Chinese in Taiwan with different occlusions].

The main purpose of this study is to investigate the characteristics of cranio-dento-facial morphology among Chinese in Taiwan with different occlusions by the postero-anterior cephalograms and to examine the differences between sexes in the cranio-dento-facial width as well as morphology. Our study comprised three hundred and seventy three patients with preorthodontic treatment records. They were divided into 12 groups with respect to the Angle's classification, sex, and age. Summarizing the differences of the various cephalometric analyses, 31 kinds of measurement, including 20 values of width dimension and 11 values of the width ratio, were employed. The results of using the Student t-test to study the sex differences of cranio-dento-facial width and morphology are summarized as follows: (1) The sexual dimorphism of the cranio-dento-facial width occurs as early as the age of 9-12. The deviation is augmented continuously with growth and, hence, it is more pronounced in adults. (2) Regardless of the class of occlusion or the stage of maturity, the cranio-dento-facial size of the male is significantly larger than that of the female. However, their cranio-dento-facial morphology is similar. (3) The sexual dimorphism of the cranio-dento-facial width dimensions is most pronounced in the zygoma, the mandibular angle and the condyles.

[The characteristic electroretinogram anomalies in age-related macular degeneration].

The color stimulated electroretinogram (ERG) was found abnormal in 104 eyes of 64 cases of age-related macular degeneration (AMD). The number of a-wave troughs with dark adapted red stimulation decreased in 69 eyes, of which 27 eyes also showed diminished a- and b-waves under dark and light adaptation. Among the remaining 35 eyes that demonstrated no decrease in the number of a-wave troughs, there were 6 eyes with a- and b-wave diminution under dark and light adaptation. The findings indicated that the ERG anomalies in AMD were detected more readily with dark adapted red stimulation, particularly evident in the wet type of AMD, thus conducive to the judgement of visual impairment and prognosis.

Model parameters of the smooth pursuit eye movement system with electrooculogram.

The visual pursuit test is a method that collects and analyzes the characteristics of pursuit eye movements and examines the function of the eye movement system. This paper analyzes the model parameters of the smooth pursuit eye movement system in order to explore a method for improving the analysis. The input-output relationship of the smooth pursuit system can be expressed by a quasilinear model. We compute the model parameters (gain, phase, spectral purity, cross covariance) by digital signal processing. Eye movement is recorded by electrooculogram. Both eyes are tested individually. The visual target moves at frequencies of 0.2, 0.4, 0.8, 1.2, and 1.6 Hz. Ranges are gain, 1.01 to 0.70; phase, -0.1 degree to -66 degrees; spectral purity, 0.97 to 0.70; and cross covariance, 0.99 to 0.26. We tested 40 normal subjects as well as patients with ataxia (8), vertigo (18), and ophthalmoplegia (9). The oculomotor system of normal subjects functions as a linear system in the performance of this test at 0.2 to 0.8 Hz. The spectral purity dropped to about 0.70 at 1.6 Hz. The variability of all measures increases greatly at 1.6 Hz, which indicates that this target motion exceeds the tracking ability of many normal subjects and that the oculomotor system of normal subjects functions as a nonlinear system in this condition. Statistical tests show no significant differences between sex, age, and the two eyes. The model parameters tentatively proved effective in clinical application.

Objective measurement of contrast sensitivity using the steady-state visual evoked potential.

Contrast sensitivity functions (CSFs) were determined electrophysiologically with the steady-state visual evoked potential (VEP). Psychophysical CSFs obtained by the method of increasing contrasts were also measured concurrently with the VEP trials. The VEP contrast thresholds were obtained using a rapid recording technique in which the contrast of a counterphase sinewave modulated at a temporal frequency of 7.5 Hz was swept from 0.5% to 40% over a period of 22 s in 39 equal logarithmic steps. For this pattern reversal stimulus, the amplitude and phase of the second harmonic response as a function of contrast were measured using a discrete Fourier transform (DFT). Contrast sensitivities at five spatial frequencies ranging from 0.5 to 14.9 cpd were measured. The VEP contrast thresholds were determined by a linear extrapolation to zero amplitude. The contrast threshold obtained by the two methods correlated at 0.816 for 14 subjects. For all five spatial frequencies there were no significant differences between the contrast sensitivities derived from the two methods.

[Study of aging macular degeneration].

Aging macular degeneration (AMD) is an important ocular disease causing blind. The study of epidemiology, pathogenetic factors and visual function of AMD show that it has become a disease worth noticing in aged. Although most AMD cases were of the dry type and the patients had rather good visual acuity, various determination of visual function showed different degrees of impairment. Controlling light exposure and improving trace element metabolism may be helpful for early prevention and treatment of AMD. It will also be an important factor in the prevention of blindness in Asian nations.

[Early abnormalities of visual functions in aging macular degeneration].

Visual functions in aging macular degeneration were observed by contrast sensitivity, color vision (FM 100-hue, Panel D-15), visual field (dynamic perimetry, suprathreshold static perimetry and static perimetry profile), with simultaneous recordings of pattern electroretinogram and pattern evoked potential. The results showed that even in AMD eyes with normal visual acuity, 34.0-79.1% had abnormal visual psychophysical function or electrophysiological measurements, suggesting that many abnormalities in visual function appeared in early AMD. The factors of abnormal early visual function changes in AMD were discussed.

The electroretinogram in Marfan's syndrome.

Marfan's syndrome (MS) is an inherited disease of connective tissue. The most characteristic ocular change is dislocation of lens, but myopia and retinal detachment also can occur. We examined 11 MS patients using electroretinography (ERG). 6 cases showed normal ERG. 5 cases had abnormal ERG. One had retinal detachment. 3 had myopia and 1 case suffered from secondary glaucoma. The defects of visual function were shown by electrophysiological test in those MS patients.

Clinical study of low vision. Part I: Refraction and the aided distance vision.

Eighty three cases of low vision patients (144 eyes) were examined with distant visual aids. The average visual acuity of the 144 eyes before refraction was approximately 0.07, while the counterpart afterwards was about 0.1 (p less than 0.01). With the 4x monocular focusable aid, acuity improved in 142 eyes out of the 144 eyes. The combined use of clip-on distance aid and the spectacle reading aid was observed to be especially suitable for partially sighted students. The mean MEF of 142 eyes was 0.9435 +/- 0.2068. The paired T-test between the difference of actual magnification and theoretical magnification was not significant at the 5% level. The factors contributing to the variance of MEF were discussed.

Visual field versus visual evoked potentials in maculopathies and optic neuropathies.

Both kinetic and static visual fields and visual evoked potential (VEP) were tested in 52 patients with maculopathies and in 29 patients with axial optic neuropathies. The results of central static quantitative perimetry and pattern reversal VEP were compared. The amplitude and latency of P100 were correlated with the total loss of central visual threshold in maculopathies. While the P100 latency was not correlated with the total loss of the central visual threshold in neuropathies, their latency delay was more obvious than that in maculopathies. We arrived at the conclusion that both VEP and visual field are necessary for assessing function loss and reflecting different pathological mechanisms in macular and optic nerve diseases.