CircLPAR3 Acts as an Oncogene in Oral Squamous Cell Carcinoma Through Regulating the miR-643/HMGB2 Network.

The malignant progression of oral squamous cell carcinoma (OSCC) has been confirmed to be mediated by a variety of factors, including circular RNA (circRNA). However, the role of circLPAR3 in OSCC development is still unclear. 70 paired OSCC tissues and normal control tissues were obtained from 70 OSCC patients. Quantitative real-time PCR was used to detect the expression of circLPAR3, microRNA (miR)-643, and high-mobility group box 2 (HMGB2). Cell proliferation, apoptosis, metastasis and stemness were assessed using cell counting kit 8 assay, colony-formation assay, flow cytometry, transwell assay and sphere formation assay. Marker protein expression and HMGB2 protein expression were determined by western blot analysis. The interaction between miR-643 and circLPAR3 or HMGB2 was confirmed by RNA pull-down assay, dual-luciferase reporter and RIP assay. The role of circLPAR3 in OSCC tumorigenesis was explored by constructing the xenograft models. Our data showed that circLPAR3 was highly expressed in OSCC tissues and cells. CircLPAR3 silencing suppressed OSCC cell proliferation, metastasis and stemness, while promoted apoptosis. On the mechanism, we discovered that circLPAR3 could sponge miR-643 to positive regulate HMGB2. MiR-643 overexpression had an inhibition effect on OSCC progression, and its inhibitor could reverse the negative regulation of circLPAR3 knockdown on OSCC progression. In addition, overexpressed HMGB2 also reversed the suppressive effect of circLPAR3 silencing on OSCC progression. Animal experiments results showed that downregulated circLPAR3 repressed OSCC tumorigenesis in vivo. Taken together, our data showed that circLPAR3 contributed to OSCC malignant progression through regulating the miR-643/HMGB2 axis.

Stromal B Lymphocytes Affecting Prognosis in Triple-Negative Breast Cancer by Opal/TSA Multiplexed Immunofluorescence.

Objective: Immune cells play a key role in tumor microenvironment. The purpose of this study was to investigate the infiltration and clinical indication of immune cells including their combined prognostic value in microenvironment of triple negative breast cancer. Methods: We investigated 100 patients with triple negative breast cancer by Opal/Tyramide Signal Amplification multispectral immunofluorescence between 2003 and 2017 at Zhejiang Provincial people's Hospital. Intratumoral and stromal immune cells of triple negative breast cancer were classified and quantitatively analyzed. Survival outcomes were compared using the Kaplan-Meier method and further analyzed with multivariate analysis. Results: Infiltration level of stromal B lymphocytes, stromal and intratumoral CD8+ T cells, stromal CD4+ T cells, stromal PD-L1 and intratumoral tumor associated macrophages 2 cells were shown as independent factors affecting disease-free survival and overall survival in univariate analysis. Stromal B lymphocytes, T stage, N stage and pathological type were independent predictive factors for both DFS and OS in multivariate analysis. We firstly found that patients with B lymphocytes-enriched subtypes have a better prognosis than those with T lymphocytes-enriched subtypes and tumor-associated macrophage-enriched subtypes. Conclusion: The present study identified a bunch of immune targets and subtypes, which could be exploited in future combined immunotherapy/chemotherapy strategies for triple negative breast cancer patients.

A Study of Feature Construction Based on Least Squares and RBF Neural Networks in Sports Training Behaviour Prediction.

This paper examines the problem of athletes' training in sports, exploring the methods and means by which athletes can perform difficult movements in which they normally make minor training errors in order to achieve better competition results and placements. To this end, we test the explanatory and predictive effects of a theoretical model starting with planned behaviour and then use exercise planning, self-efficacy, and support as variables to develop a partial least squares regression model of sports to improve the explanation and prediction of sporting athletes' intentions and behaviour. An improved RBF network-based method for player behaviour prediction is proposed. On the basis of the RBF analysis, the number of layers and the number of neurons in the hidden layer of the network are adjusted and optimised, respectively, to improve its generalisation and learning abilities, and the athlete behaviour prediction model is given. The results demonstrate the advantages of the improved algorithm, which in turn provides a more scientific approach to the current basketball training.

Dosimetric parameters and absolute monocyte count can predict the prognosis of acute hematologic toxicity in cervical cancer patients undergoing concurrent chemotherapy and volumetric-modulated arc therapy.

PURPOSE: To explore clinical and dosimetric predictors of acute hematologic toxicity (HT) in cervical cancer patients treated with concurrent chemotherapy and volumetric-modulated arc therapy (VMAT). METHODS AND MATERIALS: We retrospectively reviewed the clinical data of 184 cervical cancer patients who had concurrent chemotherapy and VMAT. Hematological parameters were collected during the treatment period. The total pelvic bone (TPB) was delineated retrospectively for dose-volume calculations. To compare the differences between two groups, the normality test findings were used to run a paired-samples t-test or Wilcoxon signed-rank test. Pearson's correlation analysis or Spearman's correlation was used to testing the correlation between the two variables. Binary logistic regression analysis was used to analyze associations between HT and possible risk factors. The receiver operating characteristic curve(ROC) was used to evaluate the best cut-off point for dosimetric planning constraints. RESULTS: The nadir of absolute monocyte count (AMC) was found to be positively correlated with the nadir of absolute white blood cells (WBC) count (r = 0.5378, 95% CI 0.4227-0.6357, P < 0.0001) and the nadir of absolute neutrophil count(ANC) (r = 0.5000, 95% CI 0.3794-0.6039, P < 0.0001). The AMC decreased and increased before the ANC and WBC. In multivariate logistic regression analysis, the chemotherapy regimens and the TPB_V20 were independent risk factors for developing grade ≥ 3 HT. The optimal TPB_V20 cut-off value identified by ROC curves and the Youden test was 71% (AUC = 0.788; 95% CI 0.722-0.845; P value < 0.001). CONCLUSIONS: The changing trend of AMC can be used as an effective predictor for the timing and severity of the ANC/WBC nadirs and prophylactic G-CSF administration. Maintain TPB_V20 < 71% and selecting single-agent cisplatin or carboplatin could significantly reduce grade ≥ 3 HT in cervical cancer patients undergoing concurrent chemoradiotherapy.

Baseline Amide Proton Transfer Imaging at 3T Fails to Predict Early Response to Induction Chemotherapy in Nasopharyngeal Carcinoma.

BACKGROUND: Early identification of nasopharyngeal carcinoma (NPC) patients with high risk of failure to induction chemotherapy (IC) would facilitate prompt individualized treatment decisions and thus reduce toxicity and improve overall survival rate. This study aims to investigate the value of amide proton transfer (APT) imaging in predicting short-term response of NPC to IC and its potential correlation with well-established prognosis-related clinical characteristics. METHODS AND MATERIALS: A total of 80 pathologically confirmed NPC patients receiving pre-treatment APT imaging at 3T were retrospectively enrolled. Using asymmetry analysis, APT maps were calculated with mean (APTmean), 90th percentile (APT90) of APT signals in manually segmented NPC measured. APT values were compared among groups with different histopathological subtypes, clinical stages (namely, T, M, N, and overall stages), EBV-related indices (EBV-DNA), or responses to induction chemotherapy, using Mann-Whitney U test or Kruskal-Wallis H test. RESULTS: NPC showed significantly higher APTmean than normal nasopharyngeal tissues (1.81 ± 0.62% vs.1.32 ± 0.56%, P <0.001). APT signals showed no significant difference between undifferentiated and differentiated NPC subtypes groups, different EBV-DNA groups, or among T, N, M stages and overall clinical stages of II, III, IVA and IVB (all P >0.05). Similarly, baseline APT-related parameters did not differ significantly among different treatment response groups after IC, no matter if evaluated with RECIST criteria or sum volumetric regression ratio (SVRR) (all P >0.05). CONCLUSION: NPC showed significantly stronger APT effect than normal nasopharyngeal tissue, facilitating NPC lesion detection and early identification. However, stationary baseline APT values exhibited no significant correlation with histologic subtypes, clinical stages and EBV-related indices, and showed limited value to predict short-term treatment response to IC.

A nomogram concerning immune infiltration and radiosensitivity to predict biochemical recurrence after radical radiation therapy in prostate cancer.

PURPOSE: To construct a nomogram concerning immune infiltration and radiosensitivity to predict biochemical recurrence (BCR) after radical radiation therapy in prostate cancer (PCa). METHODS: The Affymetrix microarray GSE116918 was acquired from the Gene Expression Omnibus (GEO) database. This cohort was grouped into biochemical recurrence ("BCR" group), among whom some patients developed metastatic recurrence ("MET" group), while the other patients were free from biochemical recurrence ("NO" group). Gene set enrichment analysis (GSEA) was performed. Immune infiltration was quantified by CIBERSORT, and infiltration score (IFS) and radiosensitivity score (RSS) were constructed. Cox multivariate regression coefficients were used to generate a nomogram. RESULTS: Compared to patients in the NO group, patients in the BCR group tended to be in a higher T stage (56.85% IN T1-2 VS. 43.15% IN T3-T4; <0.05). IFS was calculated based on the infiltration level of neutrophils, macrophages, plasmacytoid dendritic cells (pDC), activated dendritic cells (aDC), and CD56 bright NK cells. Patients in the IFS-low group had a significantly longer BCR-free survival than those in the IFS-high group (p<0.0001). RSS was calculated based on the expression levels of BRCA2, IGF1, BCL2L1, MAPK1, MAPK6, and MAPK13. Patients in the RSS-low group had a significantly longer BCR-free survival than those in the RSS-high group (p<0.0001). A nomogram predicting BCR after radical radiation therapy in PCa showed a 95% CI of [0.6584, 0.7928] for C-index, an AUC of 0.741 at 5 years, and fine calibration. CONCLUSIONS: In this study, we constructed a visual nomogram to predict BCR after radical radiation therapy in PCa with fine discriminatory and calibration capacity, which took elements, such as immune infiltration and radiosensitivity, into consideration for the first time.

Image-guided, intensity-modulated radiotherapy for the treatment of diffuse-type tenosynovial giant cell tumor of the knee: Case report and review of the literature.

RATIONALE: Tenosynovial giant cell tumor (TGCT) is a neoplastic, inflammatory disease with a benign but aggressive course that often presents as localized (TGCT-L) and diffuse (TGCT-D) forms based on the growth pattern and clinical behavior. For TGCT-L, simple excision of the diseased synovial tissue is the preferred treatment option, while for TGCT-D, adequate synovectomy is usually tricky but is essential. However, approximately 44% of TGCT-D cases will relapse after surgery alone. Thus, the optimal treatment strategy in patients with TGCT-D is evolving, and standalone surgical resection can no longer be regarded as the only treatment. The previous studies have shown that postoperative adjuvant radiotherapy can reduce recurrence in TGCT, especially in patients with incomplete synovectomy. PATIENT CONCERNS: In the first case, a 54-year-old male presented with recurrent pain and swelling of the right knee with a protracted disease course (≥10 years). The other patient is a 64-year-old male who developed swelling, pain, abnormal bending, and limited movement of the left knee without obvious inducement. DIAGNOSES: Clinical and imaging examinations can provide a definitive diagnosis, and pathology is the gold standard. TGCT-D was confirmed by postoperative pathology. After the operation, the patients underwent an MRI re-examination and showed that the lesions of the knee were not completely resected. INTERVENTIONS: Arthroscopic synovectomy was performed on the patients, and postoperative pathology was confirmed as TGCT-D. Because of incomplete synovectomy, the 2 cases received image-guided, intensity-modulated radiotherapy (IG-IMRT) after the operation. OUTCOMES: The follow-up time was 1 year, no evidence of disease progression was found in MRI. No obvious adverse effects associated with radiotherapy were detected during the follow-up period. LESSONS: These cases and reviews illustrate the necessity of radiotherapy for TGCT-D and that IG-IMRT is a safe and effective method for treating TGCT-D of the knee.

ATF2-Induced Overexpression of lncRNA LINC00882, as a Novel Therapeutic Target, Accelerates Hepatocellular Carcinoma Progression via Sponging miR-214-3p to Upregulate CENPM.

BACKGROUND: Long intergenic non-protein coding RNA 882 (LINC00882) are abnormally expressed in several tumors. Our research aimed to uncover the functions and the potential mechanisms of LINC00882 in hepatocellular carcinoma (HCC) progression. METHODS: RT-qPCR was applied to identify LINC00882 and miR-214-3p levels in HCC specimens and cells. Luciferase reporter was applied for the exploration of whether activating transcription factor 2 (ATF2) could bind to the promoter region of LINC00882. Cell proliferation, invasion, and migration were evaluated. In vivo tumor xenograft models were constructed to assess tumorigenicity. RT-PCR, Western blot and Luciferase reporter assays were conducted to examine the regulatory relationships among LINC00882, miR-214-3p and ATF2. RESULTS: LINC00882 was markedly upregulated in HCC cells and clinical specimens. Additionally, ATF2 could bind directly to the LINC00882 promoter region and activate its transcription. Loss-of-function studies further demonstrated that LINC00882 knockdown inhibited proliferation, invasion, and migration of HCC cells. Mechanistically, LINC00882 adsorbed miR-214-3p, thus promoting the expressions of CENPM. Rescue assays demonstrated that functions of LINC00882 deficiency in HCC cells were reversed through suppressing miR-214-3p. CONCLUSION: Our group identified a novel regulatory axis of ATF2/LINC00882/miR-214-3p/CENPM, which may provide potential therapeutic targets for HCC.

Impaired Metabolic Pathways Related to Colorectal Cancer Progression and Therapeutic Implications.

BACKGROUND: Risk of colorectal cancer (CRC) is defined by genetic predisposition and environmental factors that often co-occur and interact, resulting in diversiform biological reactions. The present study attempted to investigate transcriptome alteration and adaptation associated with CRC progression. METHODS: The study consisted of patients who presented at Memorial Sloan-Kettering Cancer Center, Guangzhou, China with a colonic neoplasm in 1992-2004. Microarray GSE41258 of the study was acquired from Gene Expression Omnibus and 253 included microarrays were categorized by groups of normal colon, early primary tumor, lymph node metastases primary tumor, advanced primary tumor and distant metastases. Short Time-series Expression Miner (STEM) was applied to discover tumor grade-dependent gene expression patterns. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were carried out to explore functional enrichment of differential expression genes (DEGs). RESULTS: Overall, 2870 significant DEGs were screened out on all groups. Six significant grade-dependent gene expression patterns were statistically significant. DEGs in all significant patterns were mainly assembled in GO terms of metastases and deterioration of tumor, epithelial proteins and cytokines, and protein binding and bridging. DEGs in profile 0 down-regulated with higher tumor grade, prominently enriched in KEGG pathways of metabolism. CONCLUSION: Besides many well-known colorectal cancer-related pathways, DEGs of profiles especially those down-regulated with CRC progression, clustered in various metabolic pathways including starch and sucrose metabolism, fatty acid metabolism, nitrogen metabolism, as well as xenobiotics biotransformation that link to tumorigenesis, demonstrating the impairment of physiological metabolic pathways in the context of tumor progression. These results gave a high potential for therapeutic strategies.

Factors associated with de novo metastatic disease in invasive breast cancer: comparison of artificial neural network and logistic regression models.

BACKGROUND: De novo metastasis of breast cancer is a complex clinical issue to be identified. This study was the first to construct artificial neural networks (ANN) and logistic regression (LR) models with comparison to find out important factors associated with occurrence of de novo metastasis in invasive breast cancer. METHODS: A total of 40,899 patients diagnosed with de novo metastatic breast cancer in 2010 from Surveillance, Epidemiology and End Results (SEER) Cancer database were enrolled. ANN models and LR models were constructed based on thirteen relevant factors by 10-fold cross-validation approach respectively. Evaluation indexes as well as processing time were compared. RESULTS: Overall area under ROC curve (AUC) value of ANN models was significantly higher than that of LR models (0.917±0.01 vs. 0.844±0.011, P<0.001). In ANN models, number of positive ipsilateral axillary lymph nodes, tumor size, lymph node ratio (LNR) and regional lymph nodes status were important associated factors. While under the same experiment environment, ANN models obviously took much more processing time than LR models did (14,400 vs. 15 minutes for 10-fold cross-validation). CONCLUSIONS: ANN models outperformed traditional LR models in identifying de novo metastasis of breast cancer. On the other hand, the much longer processing time of ANN models should also be considered.

Double kissing mini-culotte versus mini-culotte stenting: insights from micro-computed tomographic imaging of bench testing.

AIMS: This study aimed to evaluate the morphologic characteristics of double kissing (DK) mini-culotte and mini-culotte stenting through imaging of bench testing. METHODS AND RESULTS: DK mini-culotte and mini-culotte stenting were performed in two silicone bifurcated phantoms with branch vessel diameter differences of 0.5 mm (Model 1) and 1.25 mm (Model 2), and their morphologic characteristics were evaluated by micro-computed tomography. In Model 1, metal carina length (0.25±0.13 mm vs 0.55±0.15 mm), area stenosis of the side branch ostium (SBO) (4.65±3.24% vs 12.5±3.93%), and maximum distance of malapposed struts for the wall facing the SBO (0.27±0.08 mm vs 0.49±0.15 mm) were lower in the DK mini-culotte group than in the mini-culotte group. In Model 2, metal carina length (0.21±0.47 mm vs 0.47±0.12 mm), SBO area stenosis (5.13±3.37% vs 15.00±6.18%), and maximum distance of malapposed struts (0.32±0.13 mm vs 0.68±0.10 mm) were also lower in the DK mini-culotte group. The results of factorial analysis showed that maximum distance of malapposed struts tended to be shorter in Model 1 (F=4.226, p=0.062). CONCLUSIONS: Compared with mini-culotte stenting, DK mini-culotte stenting was associated with shorter metal carina length, shorter maximum distance of malapposed struts, and smaller SBO area stenosis. Thus, DK mini-culotte stenting may obtain better morphologic characteristics.

Discovery of substituted maleimides as liver X receptor agonists and determination of a ligand-bound crystal structure.

Substituted 3-(phenylamino)-1H-pyrrole-2,5-diones were identified from a high throughput screen as inducers of human ATP binding cassette transporter A1 expression. Mechanism of action studies led to the identification of GSK3987 as an LXR ligand. GSK3987 recruits the steroid receptor coactivator-1 to human LXRalpha and LXRbeta with EC(50)s of 40 nM, profiles as an LXR agonist in functional assays, and activates LXR though a mechanism that is similar to first generation LXR agonists.

[Genetic analysis in a Chinese deaf-mute family with X linked recessive inheritance].

In studying genetic factors in hearing loss among Chinese hearing-impaired population, a Chinese family with deaf-mute that had been reversion inherited through five generations was found (named pedigree L021). X linked recessive inheritance was hypothesized to be the transmission in this family. A total of 64 members in this family were investigated. Of these, audiometric evaluation was performed on 31 members, including 8 males with deaf-mute. Most affected individuals showed deafness or profound sensorineural hearing loss. Blood samples were obtained from 31 consented individuals in this family. Pedigree analysis indicates a X-linked recessive inheritance pattern in pedigree L021. The pedigree described herein provides an excellent model for further study on the molecular mechanism of congenital deaf-mute.

A G-quadruplex based label-free fluorescent biosensor for lead ion.

Many Pb(2+) biosensors based on Pb(2+)-specific RNA-cleaving DNAzyme have been developed in the past years. However, many of them have limited practical use because of high cost (e.g., enzymes), complicated processing and the use of unstable molecules (e.g., RNA). In this study, a novel label-free fluorescent biosensor for Pb(2+) was proposed based on Pb(2+)-induced allosteric G-quadruplex (PS2.M). In the presence of K(+), N-methyl mesoporphyrin IX (NMM) could bind to K(+)-stabilized G-quadruplexes, giving rise to high fluorescence. On addition of Pb(2+), Pb(2+) competitively binded to K(+)-stabilized G-quadruplexes to form more compact DNA folds. The Pb(2+)-stabilized G-quadruplexes did not bind to NMM, which resulted in fluorescence decrease. This allowed us to utilize PS2.M for quantitative analysis of Pb(2+) using the NMM-G-quadruplex system by convenient "mix-and-detect" protocol. The fluorescence emission ratio (F(0)/F) showed a good linear response toward Pb(2+) over the range from 5.0 nM to 1.0 µM with a limit of detection of 1.0 nM. This proposed biosensor was simple and cost efficiency in design and in operation with high sensitivity and selectivity. We validated the practicality of this biosensor for the determination of Pb(2+) in lake water samples.

[Development and evaluation of standardized Mandarin monosyllable audiometric materials].

OBJECTIVE: To develop a set of Mandarin monosyllabic list for the goal to use as a standardized speech recognition assessment tool in China with sufficient validity, reliability and sensitivity. METHODS: Thirty lists were designed based on the following criteria: efficiency, phonemic-balance, familiarity and coverage, while each list was designed corresponding to 25 monosyllables. These lists were read by a male broadcaster, recorded digitally and composed into compact disc. Our work consisted of three phases. Phase I: Sixty adults with normal hearing were recruited from Beijing to repeat as many syllables which they heard as possible. According to the randomized block design, 30 lists were presented with 6 intensities including -1 dB, 5 dB, 11 dB, 15 dB, 21 dB and 27 dB HL(speech). The lists and intensities were counterbalanced across all participants. Recognition scores in individual intensities for each list were calculated, and then logistic regression was utilized to fit Performance-Intensity (P-I) function. Two-way (list No. and Intensity) repeated measurement analysis of variance and Post-Hoc Tukey HSD test indicated that 22 lists were equivalent. Phase II: Twenty-two oral/aural normal adults were recruited to assess monosyllable recognition scores with the 22 equivalent lists at 10 dB HL(Speech), according to the Latin-Square design. Tests were administered twice for all participants with the same procedure and situation during 6 to 35 day intervals. The differences in scores (after a "rationalized" arcsine transformation) among 22 lists across over the two sessions is 9.3%, the data were collected from 22 participants, the measurement error was calculated by SD (standard deviation), the critical difference (CD) for test score improvement was 18.3% (determined as SD x 1.96, in 95% confidence level). Phase III: Eighteen participants with sensorineural hearing loss were recruited to assess recognition perception using 18 equivalent monosyllable lists at 30 dB suprathreshold based on Latin-Square design. Tests were administered twice by using the same procedure and situation within 1 to 16 day intervals. The same approach in Phase II was utilized to calculate SD (8.3%). The CD was calculated as 16.3% (in 95% confidence level). RESULTS: A set of standardized Mandarin recognition assessment material had been developed and it consisted of 22 equivalent phonemic-balanced lists with 25 monosyllables each. Approximately, every single list took 2 minutes, and thus it might be appropriate for clinical assessment. The P-I functions reveal that the recognition threshold was (8.30 +/- 0.84) dB HL(speech) and the slope of PI functions was (4.0 +/- 0.3)%/dB for adults with normal hearing. When a set of Mandarin monosyllable lists was utilized as an assessment tool, the critical difference of 18.3% (for normal-hearing adults) and 16.3% (for hearing-impaired adults) would be a key for clinicians to assess the improvement of speech recognition ability appropriately with statistically significance. CONCLUSION: In this study, a new Mandarin monosyllabic lists has been successfully developed with a sufficient validity, reliability and sensitivity for clinical evaluations, thus it might be convenience and helpful to be used as a standardized speech recognition assessment tool in China.

[Audiological and vestibular evaluation of new coagulation factor C homology mutation carriers in a Chinese family].

OBJECTIVE: To analyze the clinical features of audiological and vestibular function in a Chinese family with late onset autosomal dominant nonsyndromic sensorineural hearing loss. METHODS: Comprehensive audiological and vestibular evaluation including pure tone audiometry, auditory brainstem response (ABR), electrocochleogram (EcochG), oculomotor testing, caloric tests, rotational testing, computerized dynamic posturography and vestibular evoked myogenic potentials (VEMP) were conducted to identify the hearing and vestibular impairment. RESULTS: All affected family members shared sensorineural hearing loss with full penetrance starting between the second and fifth decade of life as a high frequency loss which progresses to a severe to profound loss at the sixth to seventh decade. The extensive vestibular evaluation indicated that all affected members performed normally in computerized dynamic posturography and caloric testing. Impairment of the saccular otolith in all of six affected members was suggested by results of the VEMP test. The velocity step test generated abnormal time constants and sinusoidal oscillation test generated abnormal gains and phase in affected members indicated that horizontal canal vestibular hyporeflexia in history. All affected subjects examined in this family showed completely normal ocular motor responses in oculomotor testing, including smooth pursuit, optokinetic nystagmus, gaze and saccade. CONCLUSIONS: The predominant feature of the Chinese DFNA9 family was that all the affected subjects harboring COCH mutation in the vWFA2 domain didn't suffer the vestibular symptoms during their life time and comprehensive vestibular assessment revealed only subtle vestibular hypofunction in affected members of this family. There is a genotype-phenotype correlation in DFNA9.

The kinetic model of the shikimate pathway as a tool to optimize enzyme assays for high-throughput screening.

Four-enzyme section of the shikimate pathway (Aro B, D, E, and K) of Streptococcus pneumoniae has been studied. Kinetic properties of the individual enzymes and three- and four-enzyme linked reactions have been characterized in vitro. On the basis of the data measured in spectrophotometric and LC-MS experiments, kinetic mechanisms of the enzymes have been suggested and all kinetic parameters have been identified. Kinetic models for these three- and four-enzyme sections of the shikimate pathway have been constructed and validated. The model of the four-enzyme section of shikimate pathway has been employed to design an inhibition-sensitive reconstituted pathway for a high-throughput screening effort on the shikimate pathway. It was demonstrated that using the model it was possible to optimize this reconstituted pathway in such a way to provide equal sensitivity of the enzymes to inhibition.