Long-term follow-up of auditory performance and speech perception and effects of age on cochlear implantation in children with pre-lingual deafness / 中华医学杂志(英文版)

Background@#The development of auditory and speech perception ability of children with hearing loss is affected by many factors after they undergo cochlear implantation (CI). Age at CI (CI age) appears to play an important role among these factors. This study aimed to evaluate the development of auditory and speech perception ability and explore the impact of CI age on children with prelingual deafness present before 3 years of age.@*Methods@#Two hundred and seventy-eight children with pre-lingual deafness (176 boys and 102 girls) were included in this study, and the CI age ranged from 6 to 36 months (mean age, 19 months). Categorical auditory performance (CAP) was assessed to evaluate auditory ability, and the speech intelligibility rating was used to evaluate speech intelligibility. The evaluations were performed before CI and 1, 3, 6, 12, 18, 24, 36, 48, and 60 months after CI.@*Results@#The auditory ability of the pre-lingually hearing-impaired children showed the fastest development within 6 months after CI (k = 0.524, t = 30.992, P < 0.05); then, the progress started to decelerate (k = 0.14, t = 3.704, P < 0.05) and entered a plateau at the 24th month (k = 0.03, t = 1.908, P < 0.05). Speech intelligibility showed the fastest improvement between the 12th and 24th months after CI (k = 0.138, t = 5.365, P < 0.05); then, the progress started to decelerate (k = 0.026, t = 1.465, P < 0.05) and entered a plateau at the 48th month (k = 0.012, t = 1.542, P < 0.05). The CI age had no statistical significant effect on the auditory and speech abilities starting at 2 years after CI (P < 0.05). The optimal cutoff age for CI was 15 months.@*Conclusions@#Within 5 years after CI, the auditory and speech ability of young hearing-impaired children continuously improved, although speech development lagged behind that of hearing. An earlier CI age is recommended; the optimal cutoff age for CI is at 15 months.

Long-term follow-up of auditory performance and speech perception and effects of age on cochlear implantation in children with pre-lingual deafness / 中华医学杂志(英文版)

BACKGROUND@#The development of auditory and speech perception ability of children with hearing loss is affected by many factors after they undergo cochlear implantation (CI). Age at CI (CI age) appears to play an important role among these factors. This study aimed to evaluate the development of auditory and speech perception ability and explore the impact of CI age on children with pre-lingual deafness present before 3 years of age.@*METHODS@#Two hundred and seventy-eight children with pre-lingual deafness (176 boys and 102 girls) were included in this study, and the CI age ranged from 6 to 36 months (mean age, 19 months). Categorical auditory performance (CAP) was assessed to evaluate auditory ability, and the speech intelligibility rating was used to evaluate speech intelligibility. The evaluations were performed before CI and 1, 3, 6, 12, 18, 24, 36, 48, and 60 months after CI.@*RESULTS@#The auditory ability of the pre-lingually hearing-impaired children showed the fastest development within 6 months after CI (k = 0.524, t = 30.992, P  0.05). The optimal cutoff age for CI was 15 months.@*CONCLUSIONS@#Within 5 years after CI, the auditory and speech ability of young hearing-impaired children continuously improved, although speech development lagged behind that of hearing. An earlier CI age is recommended; the optimal cutoff age for CI is at 15 months.

Chemical constituents from petroleum ether fraction of Swertia chirayita and their activities in vitro / 中国中药杂志

The present work is to study the chemical constituents from petroleum ether fraction of Tibetan medicine Swertia chirayita by column chromatography and recrystallization. The structures were identified by physical and chemical properties and spectral data as swerchirin (1), decussatin (2), 1,8-dihydroxy-3,5,7-trimethoxyxanthone (3), 1-hydroxy-3,5,7,8-tetramethoxyxanthone (4), bellidifolin (5), 1-hydroxy-3, 7-dimethoxyxanthone (6), methylswertianin (7), 1-hydroxy-3,5-dimethoxyxanthone (8), erythrodiol (9), oleanolic acid (10), gnetiolactone (11), scopoletin (12), sinapaldehyde (13), syringaldehyde (14), and β-sitosterol (15). Compounds 3, 4, 9, 11-14 were isolated from S. chirayita for the first time. Compounds 9 and 12 were firstly isolated from the genus Swertia. The cytotoxic activities of compounds 1, 2, 5, 7 and 8 against human pancreatic cancer cell lines SW1990 and BxPC-3,and the protective effects of these compounds against hydrogen peroxide (H2O2)-induced oxidative stress in human endothelium-derived EA.hy926 were investigated in vitro. The results showed no obvious effect at the high concentration of 50 μmol•L⁻¹.

Chemical constituents from Tibetan medicine Swertia chirayita / 中草药

Objective: To investigate the chemical constituents of Swertia chirayita. Methods: Column chromatography, such as silica gel, MCI, Sephadex LH-20 were used to isolate and purify the compounds. Physicochemical properties and spectroscopic methods were used to elucidate their structures. Results: Twelve compounds, including 2 xanthones, 4 triterpenoids, 3 secoiridoids, and 3 other compounds, the chemical constituents were isolated from the ethyl acetate fraction from 85% ethanol extract of S. chirayita, and identified as bellidifolin (1), norbellidifolin (2), oleanolic acid (3), 4-epi-hederagenin (4), 2-epi-corosolic acid (5), ursolic acid (6), amarogentin (7), swerimilegenin I (8), erythrocentaurin (9), pyrocatechol (10), syringic acid (11), and 4-hydroxy-3-methoxybenzoic acid (12). Conclusion: Compounds 4, 5, and 11 are isolated from genus Swertia for the first time, compounds 8 and 9 are found from S. chirayita for the first time.

Correlation study of biological characteristics of non-small cell lung cancer A549 cells after transfecting plasmid by microbubble ultrasound contrast agent

OBJECTIVE@#To explore the role of the abnormal expression of miRNAs in the development process of non-small cell lung cancer and the feasibility of ultrasound microbubble-mediated gene therapy after transfecting antisense miRNA-224 and miRNA-122a plasmids into non-small cell lung cancer A549 cells.@*METHODS@#Antisense miRNA-224 and miRNA-122a plasmids were transfected into non-small cell lung cancer A549 cells on the optimal ultrasound microbubble-mediated condition. We set up a control group. The cell proliferation activity, apoptosis, invasion ability were detected by MTT assay, Annexin V-PE, Transwell invasion experiment and colony formation assay, respectively.@*RESULTS@#The expression of miRNA-224 decreased and the expression of miRNA-122a rose after the plasmids of target genes were transfected into non-small cell lung cancer A549 cells, and there were significant differences when compared with those of the control group (P < 0.05). After the plasmids of target genes were transfected into A549 cells, the growth of antisense miRNA-224 and miRNA-122a were inhibited, and the differences were significant as compared with the control group (P < 0.05). Besides, the inhibition of miRNA-122a group was the most significant and there was statistically significant difference as compared with miRNA-224 group (t = -4.694, P = 0.009). After the plasmids of target genes were transfected into A549 cells, the proportion of apoptotic cells increased, the invasive cells were decreased and the clone ability reduced, and also there was a significant difference as compared with those of the control group (P < 0.05). What's more, the apoptotic peak appeared in miRNA-122a group. Its invasion ability decreased most obviously (40.25 ± 3.97/visual field), the number of clone ability was 104.93 ± 4.87 and the inhibitory effect was the most obviously. There was statistically significant difference as compared with other groups (P < 0.05).@*CONCLUSIONS@#A549 cells transfected by ultrasound microbubble-mediated antisense miRNA-224 and miRNA-122a plasmids possessed good transfection efficiency. The cell growth, invasion and colony-forming abilities of transfected A549 cells were suppressed, which laid a solid foundation for the gene therapy of non-small cell lung cancer.

Correlation study of biological characteristics of non-small cell lung cancer A549 cells after transfecting plasmid by microbubble ultrasound contrast agent

Objective To explore the role of the abnormal expression of miRNAs in the development process of non-small cell lung cancer and the feasibility of ultrasound microbubble-mediated gene therapy after transfecting antisense miRNA-224 and miRNA-122a plasmids into non-small cell lung cancer A549 cells. Methods Antisense miRNA-224 and miRNA-122a plasmids were transfected into non-small cell lung cancer A549 cells on the optimal ultrasound microbubble-mediated condition. We set up a control group. The cell proliferation activity, apoptosis, invasion ability were detected by MTT assay, Annexin V-PE, Transwell invasion experiment and colony formation assay, respectively. Results The expression of miRNA-224 decreased and the expression of miRNA-122a rose after the plasmids of target genes were transfected into non-small cell lung cancer A549 cells, and there were significant differences when compared with those of the control group (P < 0.05). After the plasmids of target genes were transfected into A549 cells, the growth of antisense miRNA-224 and miRNA-122a were inhibited, and the differences were significant as compared with the control group (P < 0.05). Besides, the inhibition of miRNA-122a group was the most significant and there was statistically significant difference as compared with miRNA-224 group (t = −4.694, P = 0.009). After the plasmids of target genes were transfected into A549 cells, the proportion of apoptotic cells increased, the invasive cells were decreased and the clone ability reduced, and also there was a significant difference as compared with those of the control group (P < 0.05). What's more, the apoptotic peak appeared in miRNA-122a group. Its invasion ability decreased most obviously (40.25 ± 3.97/visual field), the number of clone ability was 104.93 ± 4.87 and the inhibitory effect was the most obviously. There was statistically significant difference as compared with other groups (P < 0.05). Conclusions A549 cells transfected by ultrasound microbubble-mediated antisense miRNA-224 and miRNA-122a plasmids possessed good transfection efficiency. The cell growth, invasion and colony-forming abilities of transfected A549 cells were suppressed, which laid a solid foundation for the gene therapy of non-small cell lung cancer.

Effectiveness of cochlear implantation in children with auditory neuropathy and cochlear nerve aplasia / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>The aim of this study is to assess of cochlear implantation in children with auditory neuropathy and cochlear nerve aplasia by using Categories of Auditory Performance (CAP) and Speech Intelligibility Rating (SIR).</p><p><b>METHODS</b>Twenty one children with cochlear implants participated in this study. They all received cochlear implant surgery at our hospital from January 2004 to October 2010. All children had hearing aid trial and hearing and speech rehabilitation before surgery at least three months.Nine children (7 male, 2 female) were diagnosed with auditory neuropathy, twelve (7 male, 5 female) with cochlear nerve aplasia. Twenty children (10 male, 10 female) with sensorineural hearing loss served as a control group. All the children received cochlear implant for more than six months. Forty two children with normal hearing served as another control group which were divided into three subgroups according to their age.Group A included 18 children aged under two yrs, group B consisted of 16 children aged from two to four yrs and group C comprised eight children aged above four yrs. CAP and SIR were used to evaluate among all the children and the scores were compared.</p><p><b>RESULTS</b>The CAP scores of children with auditory neuropathy, cochlear nerve aplasia, sensorial neural hearing loss and the three subgroups children with normal hearing were 4.44 ± 1.50, 4.83 ± 1.69, 4.55 ± 1.66, 5.22 ± 1.11, 6.75 ± 0.45 and 7.00 ± 0.00 respectively, and SIR scores were 2.66 ± 1.11, 2.33 ± 1.15, 2.40 ± 0.75, 2.56 ± 1.04, 4.12 ± 0.81 and 5.00 ± 0.00 respectively. There were significant differences among the six groups for CAP scores(χ(2) = 35.481, P < 0.001) and SIR scores(χ(2) = 40.549, P < 0.001).No significant differences for CAP and SIR scores were observed between children with auditory neuropathy/cochlear nerve aplasia and sensorial neural hearing loss as well as group A (P > 0.05 for each), and there were significant differences were shown between children with auditory neuropathy/cochlear nerve aplasia and group B as well as group C (P < 0.01 for each aplasia).</p><p><b>CONCLUSIONS</b>The auditory and speech capabilities of children with auditory neuropathy and cochlear nerve deficiency can can get benefits from cochlear implants as children with sensorineural hearing loss, however not achieve the level of those with normal hearing after cochlear implantation. The long term effects still need follow-up and evaluation.</p>

A normative study of auditory development in Mandarin-speaking infants / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>The aim of this study is to investigate the development of auditory skills in Mandarin-speaking infants with normal hearing using IT-MAIS, set up normal comparison data for evaluating the auditory performance of children with hearing loss and provide a basis for establishing an appropriate hearing and speech rehabilitation program for them.</p><p><b>METHODS</b>A total of 183 infants with Mandarin-speaking patents participated in this investigation which was conducted in Beijing, China. 160 infants aged from 1 to 36 months were finally included, whose hearing were considered normal according to the history collection, high-risk registers for hearing loss and hearing screening using DPOAE. All infants were divided into 8 groups with 20 infants in each group by their ages. They were 1 month, 2-3 months, 4-6 months, 7-9 months, 10-12 months, 13-18 months, 19-24 months and 25-36 months group. The IT-MAIS/MAIS were administered to evaluate their development of auditory skills. All statistical analyses were executed using the MATLAB R2010a.</p><p><b>RESULTS</b>The detection scores improved with age and reached ceiling at 19 months in infants with normal hearing, the regression function for prediction of scores from age was score = 0.26×ln(age) + 0.23 and prediction of age from score was age = e([score-0.23])/0.26, r(2) = 0.93. The recognition scores also increased with age and reached ceiling at 24 months in infants with normal hearing, the regression function for prediction of scores from age was score = 0.26×ln(age)-0.07 and prediction of age from score was age = e([score+0.07])/0.34, r(2) = 0.93. The overall scores which combine the above two aspects augmented with age and reached ceiling at 22 months. The regression function for prediction of scores from age was score = 0.3×ln(age)+0.09 and prediction of age from score was age = e([score-0.09])/0.3, r(2) = 0.95.</p><p><b>CONCLUSIONS</b>Auditory skills showed a growth trend with age in infants with normal hearing. Scores of different auditory skills can be predicted according to their age. Age can also be predicted according to their scores of different auditory skills.</p>

Evaluation of rhythmic and timbral perception by normal-hearing and postlingually deafened adult cochlear implant users / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To assess the rhythmic and timbral perception ability in musical activities for postlingually deafened adult cochlear implant users.</p><p><b>METHODS</b>Twelve normal-hearing and 12 adult cochlear implant users were recruited in this study. Rhythm discrimination, instrument identification, and instrument number detection measurements in Musical Sounds in Cochlear Implants (MuSIC) test battery were used to assess the rhythmic and timbral perception ability for both normal-hearing and cochlear implant users.</p><p><b>RESULTS</b>Cochlear implant subjects achieved 84.4% correct in rhythm discrimination test on average, which was not significantly different from the performance of normal-hearing subjects (85.1%) (t = 0.116, P > 0.05). The average score of instrument identification and instrument number detection test for cochlear implant subjects were 72.3% and 39.2%, respectively. The performance of cochlear implant subjects in both of two tests were significantly poorer than those of normal-hearing subjects with 88.3% and 73.5% correct, respectively (t = 2.498 and 4.673, P < 0.05).</p><p><b>CONCLUSION</b>Postlingually deafened cochlear implant subjects, on average, performed significantly poorer in timbral perception tasks relative to normal hearing subjects, while close to the ability of normal hearing subjects in rhythmic perception.</p>

Vibrant soundbridge implantation (two cases report) / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of Vibrant Soundbridge (VSB) implantation.</p><p><b>METHODS</b>In accordance with the indications for VSB implantation, surgeries were done for two patients who suffered from either a sensorineural or conductive hearing loss (microtia). Their preoperative auditory thresholds (0.5, 1, 2 and 4 kHz) were 56 dB HL and 61 dB HL. The VSB was turned on and adjusted seven weeks after surgery.</p><p><b>RESULTS</b>Postoperative auditory thresholds of the two patients were improved. Their pure tone thresholds were 32 dB HL and 40 dB HL, and the respective improvement was 24 dB HL and 21 dB HL. There was no facial paralysis, vertigo and tinnitus.</p><p><b>CONCLUSION</b>Patients with a sensorineural or conductive hearing loss may benefit from VSB implantation.</p>

Pituitary adenylate cyclase-activating polypeptide modulates immune function of LPS-stimulatd dendritic cells / 第二军医大学学报

Objective: To investigate whether pituitary adenylate cyclase-activating polypeptide (PACAP) can modulate the immune function of mouse LPS-stimulated dendritic cells. Methods: DCs were derived from bone marrow of C57BL/6 using rmGM-CSF and rmIL-4, then the DCs were exposed to different stimuli for indicated time periods. The stimulated DCs and their culture supernatant were subjected to ELISA and FACS analysis. The total RNA was extracted from the stimulated DC for RT-PCR and RNase Protection Assay. Results: PACAP significantly inhibited the production of IL-2, IL-12,TNF-α and MIP-2 in LPS-stimulated DCs (P<0. 05,P<0. 01,P<0. 01 ,P<0. 01.respectirely), but the inhibition of LPS-induced IL-6, MIP-1α and MIP-1β production was not apparent. Conclusion: PACAP can negatively modulate the immunity of LPS-stimulated DCs.

Analysis of reliability and validity of the Chinese version of Nijmegen Cochlear Implant Questionnaire / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the reliability and validity of the Chinese version of Nijmegen Cochlear Implant Questionnaire (NCIQ).</p><p><b>METHODS</b>There were six subdomains: basic sound perception, advanced sound perception, speech production, self-esteem, activities and social Interactions. The cross-cultural adaptation measures were used to translate the NCIQ into its Chinese version. Ninety-four cochlear implant users no younger than 18 years old were included. Test-retest analysis was administered randomly to 30 users without significant changes in health and social status during a two weeks' interval between test and retest.</p><p><b>RESULTS</b>(1) Reliability: test-retest reliability of the NCIQ was proved to be satisfactory. All domains had coefficients that exceeded 0.70 (P < 0.01). Except for the subdomain, speech production, whose Cronbach's α score was 0.560, other Cronbach's α scores were greater than 0.700. (2) VALIDITY: The correlation coefficients between overall NCIQ scores and the six subdomains were 0.620 - 0.810 (P < 0.01). There were weak or no correlations among the six subdomains. The evaluation of content validity by expert review showed the questionnaire had good content validity. NCIQ total scores in postlingually deafened users were significantly higher than those in prelingually deafened users (Z = 4.350, P = 0.000). This was also true for scores of the following subdomains:advanced sound perception (Z = 4.774, P = 0.000), speech production (Z = 4.416, P = 0.000), self-esteem (Z = 3.718, P = 0.000), activities (Z = 3.228, P = 0.001) and social interactions (Z = 3.001, P = 0.003). There was no significant difference between scores obtained from the two groups in the subdomain of basic sound perception (Z = 1.943, P = 0.052).</p><p><b>CONCLUSIONS</b>The Chinese version of the NCIQ meets many psychometric criteria of a robust instrument. It possesses appropriate validity and good reliability, and can be used to measure the outcome of cochlear implant adults in China.</p>

Effects of epigallocatechin-3-gallate on the proliferation of colonic cancer cell line SW620 and PAK1 gene expression / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of epigallocatechin-3-gallate (EGCG) on the proliferation of SW620 cells and the expression of PAK1 gene.</p><p><b>METHODS</b>Human colonic cancer cell line SW620 was treated with EGCG at 40, 60 and 80 micromol/L and cultured in RPMI 1640 medium for 0, 24, 48 and 72 h. The proliferation of SW620 cells was observed by MTT assay before and after EGCG treatment, and the expression of PAK1 protein was observed by Western blotting.</p><p><b>RESULTS</b>SW620 cells treated with EGCG displayed a slowed growth in comparison with the control cells, and the growth rate decreased with the increase of EGCG concentration. PAK1 protein expression was lowered in SW620 cells after EGCG treatment for 48 h.</p><p><b>CONCLUSION</b>EGCG can inhibit the proliferation and partially reduce the expression of PAK1 protein in SW620 cells.</p>

Application of ultrathin transnasal gastroscopy in percutaneous endoscopic gastrostomy / 南方医科大学学报

<p><b>OBJECTIVE</b>To assess the application of ultrathin transnasal gastroscopy in transnasal percutaneous endoscopic gastrostomy (nPEG) in patients with trimus and/or pharyngeal narrowing.</p><p><b>METHODS</b>Nine consecutive patients underwent PEG with the Introducer method using conventional gastroscopy (5 cases) or ultrathin transnasal gastroscopy (4 cases). Among the 4 patients undergoing nPEG, 3 received radiotherapy for nasopharyngeal cancer, including two with trimus and one with trimus and pharyngeal narrowing. The procedure time, safety, discomfort and complications in these cases were compared and evaluated.</p><p><b>RESULTS</b>The average procedure time of PEG was 17+/-3.5 min in conventional gastroscopy group and 17+/-3.1 min in nPEG group. No complications were observed in these patients, but the patients in nPEG group reported less discomfort associated with the procedure.</p><p><b>CONCLUSIONS</b>Ultrathin transnasal gastroscopy reduces the discomfort of the procedure and is safer than conventional gastroscopy for PEG, especially in patients with trimus and/or pharyngeal narrowing.</p>

Outcome of cochlear implantation in prelingual pediatric auditory neuropathy / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To explore the electrophysiological results and rehabilitation outcome of two prelingually deafened pediatric cochlear implant patients with auditory neuropathy.</p><p><b>METHODS</b>Preoperative audiological evaluation, intra-postoperative electrically evoked auditory brainstem response (EABR) and neural response telemetry (NRT) record for the two cases were conducted in Beijing Tongren Hospital. A one year follow-up was performed. Data collected before and at 6,12-month intervals after implantation were compared with that from control pediatric cochlear implant patients matched for the same duration of implant use as this two cases.</p><p><b>RESULTS</b>The two children implanted had not had any postoperative medical or cochlear implant device complications. Intraoperative EABR and NRT were elicited in case 1 with unrepeatable waveforms. After 12 months of training, Case 1 had shown significant improvements in sound detection, speech perception abilities and communication skills, which was better than the control group, and the electrophysiological results became normal. Case 2 had also benefited from cochlear implantation, even though no recognizable NRT was found until he returned 12 month after the operation. CONTUSIONS: The desynchronization of auditory path had been changed after the electrical stimulation ongoing 12 months for children with auditory neuropathy. The two children had not had any complications postoperatively, and each child had shown improved listening and communication skills. Cochlear implantation could help patients with auditory neuropathy to improve their communication skill and go back to the main stream.</p>

Cochlear implantation in the ossified cochlear / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To explore the problems and describe clinical experiences associated with multichannel cochlear implantation in patients with cochlear ossification.</p><p><b>METHODS</b>Seven cochlear implant cases with bilateral cochlear ossification from 1996 to 2006 in Beijing Tongren Hospital were retrospectively reviewed, 4 of which were the consequence of meningitis. Three patients' cochlear were completely ossified, 1 patient's tympanic scala was completely ossified, 1 patient's cochlear was partially ossified, and 2 patients' cochlear were partially fibrotic. This article addressed the components of the preoperative evaluation, surgical decision-making, and specific techniques for cochlear implant array insertion in all kinds of ossified cochlea.</p><p><b>RESULTS</b>Gusher was found in 1 case but less serious than that with inner ear malformations. The electrodes were inserted in the cochleostomy in full length in 4 cases, 1 case gave up, and the cochlear implant array were partially inserted in the remains. No serious complications occurred after implantation. All patients had auditory sensations. The impedance of the electrodes, the T level, C level and the hearing threshold were slightly higher than that of the normal cochlear implantation.</p><p><b>CONCLUSIONS</b>The key influencing factor of ossified cochlear patients were the degree of the disease and whether the electrode implanted completely or not. So, the profound sensorineural hearing loss patients after meningitis should be paid more attention. Patients with ossified cochlear could be benefit from cochlear implantation.</p>

Construction and identification of recombinant Lactococcus lactis highly expressing human granulocyte-macrophage colony stimulating factor / 南方医科大学学报

<p><b>OBJECTIVE</b>To transfer human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene into Actococcus lactis and obtain recombinant Lactococcus lactis highly expressing hGM-CSF (LL-CSF).</p><p><b>METHODS</b>The optimized hGM-CSF gene sequence capable of expression in Lactococcus lactis was cloned into the vector pNBC1000, which contained P59 promoter, RBS, MCS, USP45 signal peptide and USP45 stop codon, to generate the recombinant plasmid pNCSF. pNCSF was subcloned into a shuttle vector pTR1001c to acquire the plasmid pTRCSF, which was transferred into Lactococcus lactis to obtain LL-CSF by means of electroporation. SDS-PAGE was used to verify the expression of hGM-CSF protein by the constructed LL-CSF.</p><p><b>RESULTS</b>DNA sequencing and restriction enzyme digestion indicated the successful construction of the recombinant plasmid pNCSF, pTRCSF and the recombinant bacterium LL-CSF that was capable of steady and efficient expression of hGM-CSF as shown by SDS-PAGE.</p><p><b>CONCLUSION</b>The recombinant Lactococcus lactis LL-CSF has been successfully constructed, which can be valuable for studying the biological activity of recombinant hGM-CSF and for evaluating the potential clinical application of the protein.</p>

PCR-based assembly of the DNA sequence coding for tetanus toxin C fragment / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop a PCR-based method for gene assembly of tetanus toxin C fragment (TETC) DNA sequence from a large number of oligodeoxyribonucleotides (oligos).</p><p><b>METHODS</b>To allow for its cloning and expression in Lactococcus lactis, the TETC gene sequence was designed according to the known TETC gene sequence (GenBank accession number M12739, 367-1719) and the amino acid coding in Lactococcus lactis. The sequence contained 1383 nucleotides (nt) with Sal I site added to its 5' end and Xho I and Hind III sites to its 3' end. There were 209 synonymous codon substitutions in the designed gene sequence as compared with the sequence reported in GenBank for amino acid coding in Lactococcus lactis and elimination of the restriction site of EcoR I and Kpn I. The 1380 nt of the sequence was divided into 68 oligos designated as TETC 1 to TETC 68, each containing 40 nt. A 16 nt oligos designated as TETC 69 was designed as the downstream primer. The TETC 1-24 fragment was acquired using the oligos TETC 1 to TETC 24 by PCR-based gene assembly method, and the TETC 23-46 and TETC45-68 fragments were assembled similarly. The full-length TETC gene was assembled using TETC 1 and TETC 69 as the primers when the 3 fragments were mixed. The target gene was gel-purified and digested with Sal I and Hind III, followed by ligation to the pBluescript II SK(+) and digestion with the same enzymes. The positive clones were confirmed by restriction enzyme excision and sequencing.</p><p><b>RESULTS</b>Three 500-bp fragments were acquired by PCR-based gene assembly, and the full-length TETC gene was obtained from the 3 fragment mixed at a equal concentration by a second PCR-based gene assembly using TETC 1 and TETC 69 as the primers. The target gene was cloned to pBluescript II SK(+) vector, and sequence analysis of the positive clones indicated that the assembled sequence was identical to the designed coding sequence of TETC gene.</p><p><b>CONCLUSION</b>PCR-based assembly of the synthesized constitutive gene fragments into the complete sequence can be an effective strategy for synthesis of long DNA sequences in vitro.</p>

A comparison study of two different segmentation methods to measure intelligibility in Mandarin / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To confirm alternative methods for Mandarin hearing in noise test (MHINT) to get the performance-intensity (PI) function, and to set up right test rules for scoring sentence intelligibility for subjects.</p><p><b>METHODS</b>Sentence speech reception thresholds were obtained for 30 normally hearing subjects with the MHINT test using several adaptive rules based on either character or word segmentation of the sentences. Each adaptive rule was intended to measure the threshold at a different point on the PI function. By also measuring sentence intelligibility at each threshold, the accuracy with which the PI function was estimated could be evaluated for each type of segmentation. The results of different segmentation were compared.</p><p><b>RESULTS</b>There was no significant difference in the reception threshold of sentences (RTS) between the group which used character segmentation and the group which used word segmentation (P > 0.05); and also no significant difference in their speech recognition score (SRS, P > 0.05). When the same subject used both character segmentation and word segmentation to do the test, there was no significant difference in their RTS between character segmentation and word segmentation (P > 0.05); and also no significant difference in their SRS (P > 0.05).</p><p><b>CONCLUSIONS</b>This method could be used not only with normally hearing individuals and people with hearing loss to evaluate their sentence intelligibility, but also with people who had aided equipment.</p>

Construction of Lactococcus lactis expression vector of recombinant human trefoil factor family 2 / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a Lactococcus lactis expression vector of c-myc-tagged human trefoil factor family 2 (hTFF2) fusion gene to prepare for genetic modification of Lactococcus lactis that can secrete bioactive c-myc-hTFF2 protein.</p><p><b>METHODS</b>Based on the amino sequence of hTFF2 and optimal Lactococcus lactis codon usage, the cDNA of hTFF2 was designed and extended at their 5' ends with a sequence encoding c-myc as the molecular tag. According to the restriction sites of pBluescript II sk (+), the SalI and BamHI sites were arranged at the 5' and 3' ends of the fusion gene respectively. The sequence of the fusion gene c-myc-hTFF2 was designed as 14 oligonucleotides that overlapped with each other, and by means of PCR, all the oligonucleotides were spliced to complete the construction of c-myc-hTFF2 fusion gene. The target gene of c-myc-hTFF2 was inserted into pBluescript II sk (+) to construct the cloning vector pBS-hTFF2 of c-myc-hTFF2 followed by verification by enzyme digestion and DNA sequencing. By digestion of pBS-TFF2 with BamHI/SalI and of pNBC1000 with BamHI/XhoI, we connected c-myc-hTFF2 with pNBC1000 to construct the expression vector c-myc-hTFF2 in E. coli named as pNTFF2. After digestion of pNTFF2 and pTRKH2 with XbaI, the target gene was subcloned into pTRKH2 and the construction of the expression vector pTRTFF2 in Lactococcus lactis was completed. The constructed vector was identified by restriction enzyme digestion.</p><p><b>RESULTS AND CONCLUSION</b>The expression vector pTRTFF2 of c-myc-hTFF2 fusion gene has been successfully constructed. Assembly of oligonucleotides in vitro is an effective means to synthesize the target fusion gene and this prepares the ground for constructing engineered bacterium of Lactococcus lactis.</p>