[Comparative study of surgical effects on patients with mixed deafness and otosclerosis with different air bone conduction differences].

Objective: To analyze the surgical efficacy of patients with mixed hearing loss and otosclerosis with different air bone gap (ABG) before surgery, and to provide reference for the prognosis evaluation of otosclerosis surgery. Methods: The clinical data of 108 cases(116 ears) of otosclerosis who had undergone stapes fenestration technique artificial stapes implantation in Xiangya Hospital of Central South University from November 2013 to May 2020 and had mixed hearing loss before surgery were collected, including 71 women(76 ears)and 37 men (40 ears), with an average age of 38.5 years. According to preoperative pure tone audiometry ABG, they were divided into three groups: group S, 15 dB≤ABG<31 dB, a total of 39 ears; group M, 31 dB≤ABG<46 dB, a total of 58 ears; and group L, ABG≥46 dB, 19 ears in total. The hearing outcomes of three groups of patients at 6-12 months after surgery were compared and analyzed using SPSS 24.0 statistical software. Results: A total of 3 patients (group S: 2 cases; group L: 1 case) experienced severe sensorineural hearing loss after surgery and were not included in the statistical analysis. After surgery, the pure tone hearing threshold of patients with otosclerosis in each group was significantly improved compared to before surgery, with an average air conduction threshold improvement of(21.6±13.4) dB. The difference between before and after surgery was statistically significant(t=17.13, P<0.01). The average bone conduction threshold improved by(3.7±7.6) dB, and the difference was statistically significant before and after surgery(t=5.20, P<0.01). The postoperative ABG was(18.3±9.3) dB, which was significantly reduced compared to preoperative(36.2±8.6)dB. Among the three groups of patients, the L group had the highest improvement in air conduction threshold[(29.9±10.8)dB], while the S group had the lowest improvement[(15.7±11.4)dB]. There was no statistically significant difference in post operative pure tone hearing thresholds between the three groups(P>0.05). The postoperative ABG in group S was the smallest[(16.5±9.0)dB], while in group L, the postoperative ABG was the largest[(20.5±10.0)dB]. Compared with group S, group M and group L still had a large residual ABG at 2 000 Hz after surgery. The bone conduction threshold of both S and M groups improved to some extent after surgery compared to before (P<0.01). Conclusions: Surgery can benefit patients with mixed hearing loss and otosclerosis with different preoperative ABG. Patients with small preoperative ABG have better surgical results and ideal ABG closure at all frequencies after surgery. Patients with large preoperative ABG can significantly increase the gas conduction threshold during surgery, but certain frequencies of ABG may still be left behind after surgery. The improvement effect of surgery on bone conduction threshold is not significant. Patients should be informed of treatment methods such as hearing aids based on their actual situation for selection.

[Validation and analysis of Goldengate high-throughput deafness gene chip in detecting the patients with enlarged vestibular aqueduct syndrome].

Objective: To verify the accuracy and effectiveness of Goldengate high-throughput deafness gene chip in detecting the patients with enlarged vestibular aqueduct syndrome(EVAS), and to provide a reference for genetic detection strategy of EVAS. Methods: From August 2016 to February 2018, 15 patients with EVAS and 60 normal controls were detected by Goldengate high-throughput deafness detection chip developed by our team, and the results were verified by Sanger sequencing. SLC26A4 gene sequencing was carried out in all the patients with EVAS. Results: 12/15 of patients with EVAS were detected mutations of SLC26A4 gene. Nine mutations were detected by chip detection and SLC26A4 gene direct sequencing, seven of which were detected by both methods. The chip could detect 93.33%(28/30) of the allele information provided by SLC26A4 gene direct sequencing. In addition to SLC26A4 gene, mutations of GJB2, PCDH15, TMC1, MYO6 and mitochondrial genes were detected in 15 patients with EVAS. These results were verified by Sanger sequencing. Conclusion: Goldengate high-throughput deafness gene chip possesses the traits of wide coverage and high accuracy, which can be used as a preliminary detection method for patients with EVAS.

[Application value of high-throughput gene copy number variation detection in the diagnosis of enlarged vestibular aqueduct].

Objective: To explore the application value of high-throughput gene detection method of copy number variations (CNV) in the diagnosis of enlarged vestibular aqueduct (EVA). Methods: A total of 46 nonsyndromic hearing loss patients with EVA were recruited between May 2014 and December 2016 from Department of Otolaryngology of Xiangya Hospital, Central South University. A high-throughput multiplex analysis method based on double ligation and multiple fluorescent PCR was designed and performed to detect CNV in the three EVA-related genes (SLC26A4, FOXI1 and KCNJ10). The data were analyzed by GeneMapper v4.1. Healthy volunteers (n=100) were selected as normal controls. Results: A total of 46 EVA patients were detected (32 males, 14 females, aged 1 to 26 years). In 4 EVA patients, deletions of exons 1-3 of SLC26A4 gene (4/46, 8.7%) were detected, which were not reported in the database of genomic variants (DGV), and were absent in 100 normal controls. There was no CNV detected in FOXI1 and KCNJ10 in the study. Conclusions: In the current study, three known EVA-related genes were designed as the target area for CNV detection by high-throughput ligation-dependent probe amplification (HLPA) analysis. This method can be used as a supplementary analysis of point mutation detection of hearing loss, which helps achieve the accurate genetic diagnosis of EVA.

[Application of the real-time fluorescence PCR melting curve method in gene screening of non-syndromic hearing loss].

Objective: To detect 20 common deafness gene mutations in non-syndromic hearing loss patients in China using the melting curve method, and analyze and summarize the mutation data to explore the clinical value of this method. Methods: The real-time fluorescence PCR melting curve method was used to detect 20 common mutations of four deafness genes(GJB2,GJB3,SLC26A4 and mtDNA) in 492 patients with non-syndromic hearing loss recruited between March 2014 and September 2016 from the Otolaryngology Department of Xiangya Hospital, Central South University(283 males and 209 females, the age ranged from 1 to 48 years old). The Sanger sequencing method was used to compare the sensitivity, specificity, positive predictive value, negative predictive value, and total coincidence rate of the deafness mutation detected by the real-time fluorescence PCR melting curve method. Results: A total of 492 samples were detected. 193 wild-type samples, 93 homozygous mutant samples, 145 heterozygous mutant samples, 59 composite heterozygous mutant samples and 2 samples with unknown mutations were detected using the real-time fluorescence PCR melting curve method within the range of 20 gene mutations, whichwere identical to the Sanger sequencing results.The two samples were detected as unknown mutations by the real-time fluorescent PCR melting curve method were confirmed by Sanger sequencing, including a composite heterozygous mutant sample and a homogenous mutation sample. GJB2 c.235delC and SLC26A4 c.919-2 A>G were the most common hotspot mutations in this study, followed by mtDNA m.1555 A>G. Compared with the Sanger sequencing method, the sensitivity, specificity, positive predictive value, negative predictive value, and total coincidence rate of the real-time fluorescence PCR melting curve method were 100%, the Youden's index was 1.0, and the Kappa value was 1. Conclusions: The real-time fluorescence PCR melting curve method is suitable for the detection of deafness gene mutations. It has the advantages in terms of simple, rapid, high sensitivity and strong specificity and can accurately detect the 20 gene mutations of 4 common deafness genes in Chinese population, which is expected to be used for the clinical detection of deafness genes in the future.

[Clinical and genetic investigation of families with Waardenburg syndrome type 2].

Objective:To investigate the clinical chacteration and molecular pathology of Waardenburg syndrome type 2 in seven families, and provide genetic diagnosis and hereditary counseling for family members. Method:Clinical data of seven families with WS2（14 patients）were collected. Peripheral blood samples of the probands and related family members were collected and genomic DNA was extracted. The coding sequences of microphthalmia associated transcription factor (MITF), sex-determining region Y-box 10(SOX10), snail family zinc finger 2 (SNAI2) and endothelin receptor type B（EDNRB）were analyzed by polymerase chain reaction and DNA sequencing. Then the raw data was analyzed. Result:The most common manifestations of WS2 are sensorineural hearing loss(10/14,71.4%), freckle(7/14, 50.0%)，heterochromia iridis(6/14, 42.9%) and premature greying(5/14,35.7%). All the deafness phenotype is congenital, bilateral profound sensorineural hearing loss. Freckles phenotype is different from cutaneous pigment abnormalities of WS in Westerners. The heterozygous mutation, c.328C>T in exon 3 of the MITF gene was detected in the proband and all patients of pedigree 2. However, no pathological mutation of the relevant genes (SOX10,SNAI2 and EDNRB) was detected in other pedigrees. Conclusion:There are obvious variations in clinical features of WS, while freckles may be a special subtype of cutaneous pigment disturbances. The MITF gene mutation, R110X,is therefore considered the disease causing mutation in pedigree WS02.However, there are novel disease causing genes or copy number variations in Waardenburg syndrome type 2, which require further research.

[The diversity analysis of spontaneous cerebrospinal fluid otorrhea between young children and adults].

Objective:To assess the diversity of spontaneous cerebrospinal fluid（CSF）otorrhear in clinical manifestation，CT，leakage sites and surgical operation between young children and adults．Method:We conducted a retrospective study of 6 consecutive patients who were all underwent the surgery through the transmastoid approach，including 4 adults patients and 2 children．In the 4 adults patients，two patients' bony defects lay on the tegmen mastoideum，one lay on the tegmen tympani，and another one lay on the sinus meningioma angle．None of the 4 adults patients had abnormal inner ear structures．Materials used in repair included free muscle graft，temporalis fascia，and fibrin glue of the 4 adults patients．The 2 children patients were diagnosed with congenital abnormalities of the lateral inner ear，who had bony defects of the foot plate or fenestra vestibule．Materials used in repair included free muscle graft，temporalis fascia,and musclein sequence. Result:No CSF leaks recurred after the operation except one adults patient，who's left ear recurred two times and experienced three operations. Conclusion:The clinical manifestations of spontaneous CSF otorrhea between young children and adults are different，the HRCT scan on temporal bone before the operation is very important．Especially foradults patients，making sure of theleakagesites and numbers isvaluable and significance for thesurgical procedure selection．.

TNIP1, SLC15A4, ETS1, RasGRP3 and IKZF1 are associated with clinical features of systemic lupus erythematosus in a Chinese Han population.

Systemic lupus erythematosus (SLE) is an autoimmune disease with heterogeneous clinical manifestations influenced by genetic and environmental factors. Five novel susceptibility genes (TNIP1, SLC15A4, ETS1, RasGRP3 and IKZF1) for SLE have been identified in a recent genome-wide association study of a Chinese Han population. This study investigated their relationships with disease subphenotypes, including renal nephritis, photosensitivity, antinuclear antibody (ANA), age at diagnosis, malar rash, discoid rash, immunological disorder, oral ulcer, hematological disorder, neurological disorder, serositis, arthritis and vasculitis. Significant associations were found for the single nucleotide polymorphism rs10036748 of TNIP1 with photosensitivity (odds ratio (OR) = 0.87, p = 0.01) and vasculitis (OR = 1.18, p = 0.04); rs10847697 of SLC15A4 with discoid rash (OR = 1.18, p = 0.02); rs6590330 of ETS1 with SLE of age at diagnosis <20 years (OR = 1.24, p = 8.91 x 10(-5)); rs13385731 of RasGRP3 with malar rash (OR = 1.20, p = 0.01), discoid rash (OR = 0.78, p = 0.02) and ANA (OR = 0.72, p = 0.004); rs4917014 of IKZF1 with renal nephritis (OR = 1.13, p = 0.02) and malar rash (OR = 0.83, p = 0.00038), respectively. The study suggested that these susceptibility genes might not only play important roles in the development of SLE, but also contribute to the complex phenotypes of SLE.

[Detection of the wheat salt-tolerant-mutant using PCR-SSCP combining with direct sequenceing].

gf-2.8 is a gene located on the chromosomal homologous group 4, which was reported to have relationship with salt tolerance of wheat. A pair of primers were designed to amplify the coding region of gf-2.8 in the two salt-tolerant mutants and their parents. Agarose gel electrophoresis showed that the 685 bp band was amplified among all the materials. SSCP analysis suggested that 974915 (one of the mutants) was different from the other materials. The sequencing results showed that Jimai 24 and its salt-tolerant mutant 8901-17 had the same sequence as published, which indicated that the mutation site of 8901-17 was not on the gf-2.8 gene. However, there were at least two single base mutations in the gf-2.8 of 974915, one of which caused the alternation of amino acid, this mutation occurred in the conservative region of gf-2.8.

[Comparative studies of mitochondrial DNA of T-type cytoplasmic male sterile lines and their maintainers in wheat].

mtDNAs of T type wheat cytoplasmic male sterile lines 75-3369A and their maintainers 75-3369B were isolated and digested completely with restriction endonuclease BamH I, EcoR I, Hind III. The mitochondrial genomic difference between CMS line and maintainers were revealed by Southern hybridization with probes of atp6 from Oenothera and nad3/rps12, cox I from wheat mitochondria. Southern hybridization showed the differences between these two lines. Discussion was made on the change of the DNA composition and the male sterile cytoplasm.

Postural vasoregulation and mediators of reperfusion injury in venous ulceration.

PURPOSE: To test the hypothesis that ischemia and reperfusion injury may contribute to the cause or nonhealing of venous ulcers, the effects of postural change on the microcirculation of ulcers and on levels of known mediators of reperfusion injury in their venous effluent were studied. METHODS: A standard protocol of stabilization (20 minutes), limb dependency (1 hour), and reelevation (2 hours) was used in 10 patients with venous leg ulcers as proven by clinical history, examination, ankle-brachial pressure index, and light reflective rheography. Superficial blood flow in and around ulcers was repeatedly examined with a new laser-Doppler scanning technique. Blood samples from the saphenous vein or a tributary adjacent to the ulcer before dependency and at 0, 10, 30, 60, and 120 minutes after reelevation were analyzed for tumor necrosis factor-alpha, interleukin (IL)-1RA, IL-1 beta, IL-6, platelet-activating factor, thromboxane B2, leukotriene B4, and P-selectin. RESULTS: Scans showed a consistent pattern of high ulcer blood flow, which decreased on dependency (p < 0.05) and then returned to baseline levels on reelevation and (in 7 of 10) eventually exceeded initial values. Mediator assays showed that levels of platelet-activating factor, IL-1RA, and IL-6 were significantly higher in resting ulcer venous effluent than in systemic venous samples; the reverse was true for P-selectin. There was no statistically significant change in effluent concentration of any mediator as a function of posture, ulcer size, or healing. CONCLUSIONS: Postural vasoregulation causes relative ischemia and reperfusion in venous leg ulcers. However, this is not associated with changes in release of mediators known to be related to reperfusion injury in internal organs.

Perfusion imaging of skin island flap blood flow by a scanning laser-Doppler technique.

Conventional laser-Doppler perfusion measurements can only obtain information from a single site. Since superficial blood flow is heterogeneous, this is a serious limitation, particularly in studies of methods to improve skin flap survival. A scanning laser-Doppler instrument has been developed which provides both an image and quantitative information about perfusion of the superficial tissue. We have evaluated this instrument in a circumflex iliac artery island flap model in the pig. The validity of the model was demonstrated by fluorescein dye injection and histology. Elevation of flaps was found to increase proximal flap blood flow but to decrease it in the distal portion. In flaps subjected to arterial ischaemia (9 h) and reperfusion we found an early increase in proximal flow, which gradually extended distally over the first 40 to 60 min. 16 h later, flow had declined, compatible with reperfusion damage to the vasculature. We suggest that perfusion imaging may be a valuable technique for investigating the mechanisms and extent of reperfusion injury.

[On the correction of systematic errors of analytical methods used in the content uniformity test].

In this paper, the constant and proportional systematic errors of analytical methods for the content uniformity test were studied with reference to the accurate analytical method for the assay. Seven monographs of tablets admitted to USP(XXII), Ch P (1985, 1990) and JP(XI) were taken as cases in point and the systematic errors of two of them, i.e. digoxin and nitroglycerin tablets, were ascertained by the test of significance. To improve the accuracy of analytical methods for the content uniformity test, a correction formula more reasonable and convincing than that of USP (XXI, XXII) was proposed, and its theoretical basis described briefly.

[Quality assessment of the traditional Chinese medicine gentian by chemical pattern recognition].

Methanol extracts of 38 samples of the traditional Chinese medicine gentian of different origin were prepared. Data reflecting their chemical constituents as a whole were obtained by HPLC. The Chinese Pharmacopoeia (1990) specified four species of the getiana as authentic. Some samples from the authentic and non-authentic gentian were used as the training set of pattern recognition. Chemical features of the gentian samples were selected from among the matching data of HPLC by the SIMCA program. Finally, the selected chemical features were displayed as stars in a semicircular polar coordinate system, resulting in a constellation graph. Quality assessment of the traditional Chinese medicine gentian was accomplished by recognizing the position and path of the stars in the constellation graph. The clusters of the gentian samples in the constellation graph obtained from the HPLC features were consistent with and complement to their plant taxonomy. The vivid graph demonstrated that pattern recognition via chemical constellation promises well a reliable method for assessing traditional Chinese medicines.

[Quality assessment of the Chinese traditional medicine rhubarb by chemical pattern recognition].

Traditionally, the identification of Chinese medicines is performed by the morphological method. As chemical constituents play the role of therapeutic action, it is more rational to identify Chinese traditional medicines by analyzing their chemical constituents. Furthermore, the therapeutic action of a Chinese traditional medicine is usually the result of coordination of its chemical constituents. In this paper, the PRIMA method was adopted to classify 29 samples of the Chinese traditional medicine rhubarb, with 16 samples as the training set and 13 samples as the test set. The recognition ability was 100% and the prediction ability was 92% on HPLC data, while both of them were 100% on UV data. As far as this research is concerned, the PRIMA method is superior to the Bayes classification rule, the SIMCA method and nonlinear mapping in its simplicity, rapidity and correctness. Pharmacologic experiments were carried out to confirm the results.

[A rapid FIA-spectrophotometric method with online extraction for the content uniformity test of atropine sulfate tablets].

A rapid spectrophotometric method based on FIA for the content uniformity test of atropine sulfate tablets has been developed. By means of the self-designed FIA system with an on-line extraction device, many difficulties resulting from extraction, such as slow analytical rate, environmental pollution, poor reproducibility, etc, were overcome, and the routine procedure of the official content uniformity test was tremendously simplified. Results obtained with the proposed method agree well with those obtained with the official test. Furthermore, the proposed method attained a sampling rate of 60 samples per hour, a recovery of 100.0%, a relative standard deviation of 0.7%, and a detection limit of 0.24 micrograms/ml. Uniform design proves effective to optimize complex FIA conditions, and FIA promises a new approach to test the content uniformity of pharmaceutical tablets.

Orthotopic homotransplantation of vascularized composite mandibular tissue in rabbits immunosuppressed with cyclosporine A.

To develop the surgical model, composite mandibular tissue was transplanted at the same site. Revascularization was accomplished by end-to-end anastomosis of the facial vessels using standard microvascular techniques. A total of 33 vascularized composite mandibular tissue allografts were similarly performed between two incompatible strains of rabbit. In a control group of 9 animals, no immunosuppression was administered. All of these allografts were rejected acutely within 10 days after surgery. 7 allograft recipients were immunosuppressed with azathioprine and prednisone at 5 mg/kg.d and 2 mg/kg.d, respectively. Their allografts were also rejected acutely within 10 days. 17 allograft recipients were immunosuppressed with cyclosporine A (CsA). All allografts showed primary wound healing and hair growth and took on normal appearance. 8 of these recipients were given CsA at 5 mg/kg.d i.v. and their allografts were rejected at a mean time of 17.9 days. The remaining 9 recipients given CsA at 10 mg/kg.d i.v. rejected their allografts at a mean time of 36.1 days. In 3 of them, the rejection was reversed with CsA (20 mg/kg.d) injection for five days successfully, and one allograft survived more than 100 days. This pilot study suggests that the surgical model is reliable and that CsA will be useful as an immunosuppressive agent in the study of vascularized composite mandibular tissue allografts.

[The clinical significance of serum creatine phosphokinase measurement in patients with chronic cor pulmonale].

The values obtained from the measurement of serum creatine phosphokinase (SCPK) in 70 cases of chronic cor pulmonale in exacerbation and 72 cases in remission were compared with the normal value as measured in 160 healthy adults at the same period. The results showed that the normal value of SCPK was 46.50 +/- 12.71 u/L in male, while 32.51 +/- 10.8 u/L in female. The differences of the values of SCPK in male and female are highly significant statistically (P less than 0.001). Although the values of SCPK were within normal limits (M +/- 2SD) in most patients with chronic cor pulmonale (72.38-84.10%) in this paper, the values of SCPK of some of them (13.60-22.36%) were higher than normal. But in a few of the patients in exacerbation (2.27-5.26%) the SCPK values were lower than normal. The increased activity of SCPK in patients with chronic cor pulmonale may be related to the vigorous movement of the respiratory muscles. While the level of SCPK decreased to extremely low degree may indicate that the respiratory muscles may be excessively fatigue. So we suggest that the levels of SCPK which are markedly higher or lower than normal could be served as an reference index for the excessive activity or fatigue of the respiratory muscles respectively in chronic cor pulmonale.