Objective:To investigate the effects of pulsed electric field (PEF) combined with gemcitabine (GEM) on the viability and stemness of HCCC-9810 cholangiocarcinoma stem cells.Methods:HCCC-9810 cholangiocarcinoma stem cells were established in serum-free, cytokine-rich medium and divided into four groups: the control group, GEM group, PEF group, and the pulsed electric field combined with gemcitabine (PEF+ GEM) group. Cell proliferation was detected using the Cell Counting Kit-8 (CCK8) assay. Cell viability and apoptosis rate were measured by flow cytometry. Cell invasion ability was assessed using the Transwell assay. The expression of stemness marker proteins CD133 and Octamer-binding transcription factor 4 (OCT4), as well as the expression of β-catenin, was detected by Western blotting.Results:Regarding cell viability, the GEM, PEF, and PEF+ GEM groups showed significantly lower cell viability and higher apoptosis rate than the control group at 24 h, 48 h, and 72 h (all P<0.05). At 48 h and 72 h, the PEF+ GEM group showed significantly lower cell viability (7.2%±0.3% and 5.9%±0.8%, respectively) than the GEM group (50.7%±0.6% and 31.0%±1.2%, respectively) and the PEF group (12.2%±0.2% and 12.8%±0.2%, respectively) (all P<0.05). Regarding stemness inhibition, the PEF+ GEM groups showed significantly lower expression levels of CD133 and OCT4 at 24 h, 48 h, and 72 h compared with the control group (all P<0.05). Notably, at 48 h, the PEF+ GEM group showed a significantly lower expression level of the OCT4 (0.61±0.02) than the GEM group (0.87±0.08) and the PEF group (1.00±0.10) ( P<0.01). Furthermore, at 24 h and 48 h, the GEM, PEF, and PEF+ GEM groups showed significantly lower expression levels of β-catenin compared with the control group (all P<0.05). Conclusion:Pulsed electric field combined with gemcitabine therapy demonstrated more effective anti-proliferation and cancer stemness inhibition effects on HCCC-9810 cholangiocarcinoma stem cells compared with either monotherapy.

Objective:To investigate the effects of pulsed electric field (PEF) combined with gemcitabine (GEM) on the viability and stemness of HCCC-9810 cholangiocarcinoma stem cells.Methods:HCCC-9810 cholangiocarcinoma stem cells were established in serum-free, cytokine-rich medium and divided into four groups: the control group, GEM group, PEF group, and the pulsed electric field combined with gemcitabine (PEF+ GEM) group. Cell proliferation was detected using the Cell Counting Kit-8 (CCK8) assay. Cell viability and apoptosis rate were measured by flow cytometry. Cell invasion ability was assessed using the Transwell assay. The expression of stemness marker proteins CD133 and Octamer-binding transcription factor 4 (OCT4), as well as the expression of β-catenin, was detected by Western blotting.Results:Regarding cell viability, the GEM, PEF, and PEF+ GEM groups showed significantly lower cell viability and higher apoptosis rate than the control group at 24 h, 48 h, and 72 h (all P<0.05). At 48 h and 72 h, the PEF+ GEM group showed significantly lower cell viability (7.2%±0.3% and 5.9%±0.8%, respectively) than the GEM group (50.7%±0.6% and 31.0%±1.2%, respectively) and the PEF group (12.2%±0.2% and 12.8%±0.2%, respectively) (all P<0.05). Regarding stemness inhibition, the PEF+ GEM groups showed significantly lower expression levels of CD133 and OCT4 at 24 h, 48 h, and 72 h compared with the control group (all P<0.05). Notably, at 48 h, the PEF+ GEM group showed a significantly lower expression level of the OCT4 (0.61±0.02) than the GEM group (0.87±0.08) and the PEF group (1.00±0.10) ( P<0.01). Furthermore, at 24 h and 48 h, the GEM, PEF, and PEF+ GEM groups showed significantly lower expression levels of β-catenin compared with the control group (all P<0.05). Conclusion:Pulsed electric field combined with gemcitabine therapy demonstrated more effective anti-proliferation and cancer stemness inhibition effects on HCCC-9810 cholangiocarcinoma stem cells compared with either monotherapy.

Objective:To systematically evaluate the efficacy of Fuzheng Huayu (FZHY) tablets/capsules on hepatitis B-associated liver fibrosis or cirrhosis based on randomized controlled trials (RCTs) in order to provide more accurate evidence-based medicine for clinical rational drug use.Methods:Randomized controlled clinical trial research reports related to the treatment of hepatitis B-associated liver fibrosis or cirrhosis with FZHY published in SCI and statistical source core journals were retrieved from databases such as PubMed, Cochrane Library, and China National Knowledge Infrastructure (CNKI). RevMan 5.3 and Stata18.0 software were used to conduct a meta-analysis of the improvement rate of liver tissue inflammatory activity (HAI) and Ishak stage of liver fibrosis, the decrease value of liver stiffness measurement (LSM), hyaluronic acid (HA), laminin (LN), type Ⅲ procollagen (PC-Ⅲ), type Ⅳ collagen (IV-C), total bilirubin (TBil), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (Alb). The Q test was used for the heterogeneity test, with a random-effect model selected for large heterogeneity and a fixed-effect model for less heterogeneity.Results:A total of 852 articles were retrieved. Duplicate articles, non-RCT articles, non-SCI/statistical source/core journal articles, and other articles that did not meet the inclusion criteria were sequentially excluded. Finally, a total of 2 746 cases (1 382 cases in the FZHY group and 1 364 cases in the control group) were included from 25 studies. The results of statistical analysis showed that the improvement rates of HAI grade of liver inflammation were 75.56% (68/90) and 42.22% (38/90, P<0.001) in the FZHY group and the control group at 24-48 weeks of treatment, while the improvement rates of Ishak stage of liver fibrosis were 67.90% (110/162) and 40.91% (63/154, P=0.005), respectively. Compared with the control group (95% CI -5.10-1.77, P<0.001) the mean △LSM of the FZHY group decreased by 3.43 kPa ( P<0.001)and 0.30 kPa ( P=0.93) at 48 and 72 weeks of treatment. The standardized mean differences (SMDs) of △HA, △LN, △PC-Ⅲ and △Ⅳ-C were -1.12, -1.00, -0.89 and -1.10 ( P<0.001) after 24 weeks of treatment between the FZHY group and the control group. The SMDs of △HA, △IV-C, △LN and △PC-Ⅲ were -1.13 ( P=0.01), -1.51 ( P<0.001), -0.53 ( P=0.14) and -0.42 ( P=0.19) after 48 weeks of treatment between the two groups. The △TBil, △ALT, △AST, and △ALB was -12.99 μmol/L ( P=0.007), -36.91 U/L ( P<0.001), -22.05 U/L ( P=0.12), and 6.09 g/L ( P=0.05) after 24 weeks of treatment between the two groups. The observation on indicators such as aspartate aminotransferase and platelet ratio index, fibrosis-4 index, and hepatocellular carcinoma incidence in current RCT studies remained deficient. Conclusion:FZHY can significantly improve the degree of histologic liver inflammation and fibrosis, LSM values, reduce serum liver fibrosis indexes, and serum bilirubin and transaminase levels in patients with hepatitis B-associated liver fibrosis or cirrhosis. Therefore, it is necessary to further explore the optimal course of FZHY and its long-term effects on the risk of complications of cirrhosis such as hepatocellular carcinoma, ascites, and esophageal varicose bleeding, through prospective large-sample multicenter real-world cohort studies.

Background and purpose:Silent information regulator proteins(sirtuins,SIRT)are a class Ⅲ histone deacetylases with nicotinamide adenine dinucleotide(NAD+)as coenzyme.YME1 like 1 ATPase(YME1L1)is essential for the maintenance of mitochondrial morphology,function and plasticity.Optic atrophy 1(OPA1)mainly mediates mitochondrial fusion.The aim of this study was to explore the expression of SIRT3 in the endocrine resistance of breast cancer,the relationship between SIRT3 and YME1L1 and OPA1,and the mechanism of SIRT3 in the endocrine resistance of breast cancer.Methods:4-hydroxytamoxifen was used to induce tamoxifen-resistant MCF-7/TAM cells.cell counting kit-8(CCK-8)was used to detect cell proliferation and verify drug resistance.The mitochondrial morphology was observed by transmission electron microscopy(TEM)and immunofluorescence staining.The expressions of SIRT3 and OPA1 were detected by real-time fluorescent quantitative polymerase chain reaction(RTFQ-PCR)and Western blot.JC-1 staining was used to detect mitochondrial membrane potential,and dihydroethidium(DHE)staining was used to detect reactive oxygen species(ROS)to verify mitochondrial function.SIRT3 was knocked down in drug-resistant cells by RNA interference,and SIRT3 and YME1L1 wild type(WT),simulated acetylation state mutant(MUT K237Q),and simulated deacetylation state mutant(MUT K237R)were overexpressed in parental cells by overexpression plasmid.Immunoprecipitation assay(IP)and immunofluorescence(IF)were used to verify the interaction between SIRT3 and YME1L1.Results:RTFQ-PCR and Western blot results showed that SIRT3 gene expression and protein level was significantly higher in drug-resistant cells than in parental cells.Overexpression of SIRT3 in parental cells decreased the sensitivity of breast cancer cells to tamoxifen.Knockdown of SIRT3 in drug-resistant cells enhanced the sensitivity of drug-resistant cells to tamoxifen.DHE staining showed that the ROS level was lower in tamoxifen resistant cells than in parental cells at the same concentration.Transmission electron microscopy and fluorescence staining showed that the mitochondria of the drug-resistant cells were elongated compared with the parental cells.Western blot results showed that the expression level of L-OPA1 protein was higher in drug-resistant cells than in parental cells.Overexpression of SIRT3 in the parental cells resulted in enhanced mitochondrial function and longer mitochondrial morphology compared with the control cells.Western blot showed that the expression of L-OPA1 was upregulated.When SIRT3 was knocked down in drug-resistant cells,the opposite result was obtained.We further verified how SIRT3 regulated OPA1 protein,affected the morphology and function of mitochondria,and promoted drug resistance of breast cancer.Overexpression of YME1L1(wild-type and mutant plasmids)in parental cells showed that overexpression of YME1L1 in the simulated deacetylation state resulted in similar results as overexpression of SIRT3,and overexpression of YME1L1 in the acetylated state resulted in similar results as knockdown of SIRT3.IP assay confirmed the interaction between SIRT3 and YME1L1 in breast cancer cells.The acetylation level of YME1L1 was different at different SIRT3 expression levels.IF assay showed that YME1L1 was co-localized with SIRT3 in MCF-7 cells.Conclusion:SIRT3 is highly expressed in tamoxifen-resistant breast cancer cells.SIRT3 upregulates L-OPA1 expression by deacetylating YME1L1,thereby promoting mitochondrial fusion and enhancing mitochondrial function,and promotes tamoxifen resistance in breast cancer.

Objective:To retrospectively analyze the viral levels and associated factors in patients with hepatitis B virus (HBV)-related primary liver cancer (PHC) in real-world settings and further explore the correlation between low viral load (LVL) and/or low-level viremia (LLV) and PHC.Methods:Five hundred twenty-four cases with HBV-related PHC with complete pathologically confirmed data from 2013 to 2020 were included. Percentages (%) were used to express their viral load, antiviral (oral) status, patient compliance, presence or absence of cirrhosis, family history of liver cancer, and others. LVL definition: After excluding detection errors by PCR method, serum HBV DNA <50-2 000 IU/ml, and those who had received antiviral drug treatment were called LLV. Antiviral treatment (AVT) rate definition: As of the confirmed diagnosis of PHC, those who had been regularly treated using oral antiviral drugs for six months or more (≥6 months).Results:General situation: The ratio of male to female enrolled patients was 15.90:1 (493/31). Patients aged >40 years accounted for 91.98% (482 cases). Hepatitis B surface antigen (HBsAg) positivity condition: The ratio of HBsAg-positive to HBsAg-negative/anti-HBc-positive (HBsAg-/anti-HBc+) PHC patients was 5.89:1 (448/76). Among the 76 HBsAg-/anti-HBc+patients, the ratio of HBsAg-/anti-HBs+/anti-HBc+ to HBsAg-/anti-HBs-/anti-HBc+ patients was 0.95:1 (37/39). Hepatitis B e antigen (HBeA) positivity condition: The ratio of HBeAg-negative to HBeAg-positive cases was 3.23:1 (400/124). HBV DNA level condition: The medical history records of 75.00% of patients (393/524) had traceable HBV DNA test reports. Out of 393 patients, 45.04% (177/393) accounted for undetectable HBV DNA, 13.49% (53/393) accounted for LVL, 41.48% (163/393) accounted for HBV DNA exceeding the upper limit of LVL, and 4.07% (16/393) accounted for LLV. Among HBsAg-positive and HBsAg-/anti-HBc+ patients, the HBV DNA positivity rates were 59.12% (214/362) and 6.45% (2/31), respectively. Antiviral treatment condition: Among the 448 HBsAg-positive PHC patients, the total AVT rate was 18.08% (81/448), of which seven patients did not have their HBV DNA results traced back. Among them, the AVT rate of 148 patients with HBV DNA lower than the lowest detection value was 41.22% (61/148); the AVT rate of 53 patients with LVL was 18.87% (10/53); and the AVT rate of 163 patients with HBV DNA≥LVL upper limit was 1.84% (3/163). Liver cirrhosis and family history condition: 348 patients (66.41%) had liver cirrhosis. 67 patients (12.79%) had a distinct family history of HBV-related liver cirrhosis and liver cancer. Alpha-fetoprotein (AFP) condition: 514 patients underwent AFP testing, with 30.93% of the patients had normal AFP levels, and 69.07% had AFP levels exceeding the upper limit of normal values (355/514). Among them, 10 μg/L400 μg/L accounted for 34.44% (177/514) and 34.63% (178/514), respectively. Tissue typing condition: 99.05% (519/524) were hepatocellular carcinoma, and well-differentiated and moderately differentiated cases accounted for only 8.59%.Conclusions:In the real world, comprehensive, timely screening, standardized, and effective antiviral treatment have not yet been achieved for patients with chronic hepatitis B, resulting in the persistence of HBsAg and/or HBV DNA positivity (especially LVL and/or LLV). Although LVL does not account for a high proportion among all HBV-related PHC populations, the vast majority of LVL populations have not received any antiviral treatment. In addition, some PHC populations with HBV DNA>2 000 IU/ml have not received standard antiviral treatment before the onset of the disease, which should be paid attention to. At the same time, HBsAg-/anti-HBc+ populations may have latent HBV infection and may even progress to PHC. Notably, PHC may still occur after HBeAg seronegative conversion.

Objective:To systematically evaluate the efficacy of Fuzheng Huayu (FZHY) tablets/capsules on hepatitis B-associated liver fibrosis or cirrhosis based on randomized controlled trials (RCTs) in order to provide more accurate evidence-based medicine for clinical rational drug use.Methods:Randomized controlled clinical trial research reports related to the treatment of hepatitis B-associated liver fibrosis or cirrhosis with FZHY published in SCI and statistical source core journals were retrieved from databases such as PubMed, Cochrane Library, and China National Knowledge Infrastructure (CNKI). RevMan 5.3 and Stata18.0 software were used to conduct a meta-analysis of the improvement rate of liver tissue inflammatory activity (HAI) and Ishak stage of liver fibrosis, the decrease value of liver stiffness measurement (LSM), hyaluronic acid (HA), laminin (LN), type Ⅲ procollagen (PC-Ⅲ), type Ⅳ collagen (IV-C), total bilirubin (TBil), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (Alb). The Q test was used for the heterogeneity test, with a random-effect model selected for large heterogeneity and a fixed-effect model for less heterogeneity.Results:A total of 852 articles were retrieved. Duplicate articles, non-RCT articles, non-SCI/statistical source/core journal articles, and other articles that did not meet the inclusion criteria were sequentially excluded. Finally, a total of 2 746 cases (1 382 cases in the FZHY group and 1 364 cases in the control group) were included from 25 studies. The results of statistical analysis showed that the improvement rates of HAI grade of liver inflammation were 75.56% (68/90) and 42.22% (38/90, P<0.001) in the FZHY group and the control group at 24-48 weeks of treatment, while the improvement rates of Ishak stage of liver fibrosis were 67.90% (110/162) and 40.91% (63/154, P=0.005), respectively. Compared with the control group (95% CI -5.10-1.77, P<0.001) the mean △LSM of the FZHY group decreased by 3.43 kPa ( P<0.001)and 0.30 kPa ( P=0.93) at 48 and 72 weeks of treatment. The standardized mean differences (SMDs) of △HA, △LN, △PC-Ⅲ and △Ⅳ-C were -1.12, -1.00, -0.89 and -1.10 ( P<0.001) after 24 weeks of treatment between the FZHY group and the control group. The SMDs of △HA, △IV-C, △LN and △PC-Ⅲ were -1.13 ( P=0.01), -1.51 ( P<0.001), -0.53 ( P=0.14) and -0.42 ( P=0.19) after 48 weeks of treatment between the two groups. The △TBil, △ALT, △AST, and △ALB was -12.99 μmol/L ( P=0.007), -36.91 U/L ( P<0.001), -22.05 U/L ( P=0.12), and 6.09 g/L ( P=0.05) after 24 weeks of treatment between the two groups. The observation on indicators such as aspartate aminotransferase and platelet ratio index, fibrosis-4 index, and hepatocellular carcinoma incidence in current RCT studies remained deficient. Conclusion:FZHY can significantly improve the degree of histologic liver inflammation and fibrosis, LSM values, reduce serum liver fibrosis indexes, and serum bilirubin and transaminase levels in patients with hepatitis B-associated liver fibrosis or cirrhosis. Therefore, it is necessary to further explore the optimal course of FZHY and its long-term effects on the risk of complications of cirrhosis such as hepatocellular carcinoma, ascites, and esophageal varicose bleeding, through prospective large-sample multicenter real-world cohort studies.

New drug clinical trials have been considered as a positive way for treating cancer by cancer patients and doctors, and the extended dosing is a special way for patients' withdrawal from antitumor clinical trials to obtain investigational new drugs. However, neither the regulations of expanded dosing nor the detail documents for expanded dosing have been officially published in China. At present, expanded dosing of investigational drugs is still at the exploratory stage in various medical institutions, and a complete management system has not been established to meet patients' urgent needs for drug use. Based on the practical experience of extended dosing in Hunan Cancer Hospital, this paper preliminarily explored the application procedures and ethical review requirements of extended dosing for subjects in antitumor clinical trials. It is necessary to clarify the responsibilities of all patients in the procedure and establish a patient-medical institution-sponsor joint application system. In the process of ethical review, it is recommended that all parties fully consider the risks and benefits of extended dosing for patients, and then the ethics committee makes a comprehensive assessment to decide whether to approve extended dosing.
Humans ; China ; Physicians ; Antineoplastic Agents/therapeutic use*

OBJECTIVETo explore the effects of electrical stimulation at acupoints in the distribution area of auricular vagus nerve combined with sound masking on auditory brainstem response (ABR) and contents of neurotransmitters of γ-aminobutyric acid (γ-GABA), 5-hydroxytryptamine (5-HT) and acetyl choline (Ach) in inferior colliculus of tinnitus rats.

METHODSTwenty-four male adult SD rats were randomized into a control group, a model group, a 7-d treatment group and a 15-d treatment group. Except the control group, rats in the remaining groups were treated with intraperitoneal injection of 10% salicylate sodium at a dose of 350 mg/kg to establish tinnitus model. Rats in the control group were treated with injection of 0.9% NaCl. Rats in the 7-d treatment group and 15-d treatment group were treated with electrical stimulation at "Shenmen (TF₄)" and "Yidan (CO₁₁)" in the distribution area of auricular vagus nerve combined with sound masking, once a day, for 7 days and 15 days. The SigGenRP software of TDT system was applied to provide voice for single ear and collect the signal, and the voice threshold of ABR was tested. The levels of γ-GABA, 5-HT and Ach in inferior colliculus of rats were detected by enzyme linked immunosorbent assay (ELISA) and compared.

RESULTSCompared with the model group, the threshold values of ABR in 12 kHz and 16 kHz voice stimulation in the 7-d treatment group were significantly lower all P < 0.05); the threshold values of ABR from 4 kHz to 28 kHz voice stimulation in the 15-d treatment group were signally reduced (P < 0.05, P < 0.01), which was more significant than those in the 7-d treatment group. The level of γ-GABA in the model group was significantly lower than that in the control group (P < 0.05), and that in the 15-d treatment group was apparently higher than that in the model group (P < 0.05). The level of 5-HT in the model group was markedly higher than that in the control group (P < 0.05), and that in the 7-d treatment group was lower than that in the model group (P < 0.05), while that in the 15-d treatment group was apparently higher than that in the model group (P < 0.05). The level of Ach in the model group was obviously; lower than that in the control group (P < 0.05), and that in the 7-d treatment group was higher than that in the model group (P < 0.05).

CONCLUSIONElectrical stimulation at auricular vagus nerve combined with sound masking) could regulate the threshold of ABR, especially in the 15-d treatment group. This may be ascribed to modulating the levels of neurotransmitter of γ-GABA, 5-HT and Ach in inferior colliculus.

Acupuncture Points ; Animals ; Brain Stem ; physiopathology ; Electric Stimulation ; Evoked Potentials, Auditory, Brain Stem ; Humans ; Inferior Colliculi ; physiopathology ; Male ; Neurotransmitter Agents ; metabolism ; Rats ; Rats, Sprague-Dawley ; Serotonin ; metabolism ; Tinnitus ; physiopathology ; therapy ; Vagus Nerve ; physiopathology ; gamma-Aminobutyric Acid ; metabolism

Objective To explore the clinical characteristics of patients with olfactory dysfunction after upper respiratory tract infection.Methods Through clinical specialist examination and imaging examination,95 cases of patients with olfactory dysfunction after upper respiratory tract infection were confirmed,58 cases in anosmia group and 37 cases in hyposmia group.All were performed by a subjective olfactometry (Sniffin'Sticks test) and a subjective taste function tests.The results were statistically analyzed by SPSS 17.0 software.Results In 58 cases of anosmia group,21 cases of male,37 cases of female;Twenty-six cases of youth,23 cases of middle age,9 cases of old age;Twenty-seven cases occurs in spring,11 cases in summer,12 in autumn and 8 in winter.Among 37 cases of hyposmia group,12 cases of male,25 cases of female;Eighteen cases of youth,16 cases of middle age,3 cases of old age;Fourteen cases occurs in spring,8 cases in summer,7 in autumn and 8 in winter.There was no statistically significant difference in gender,age and the onset season between the two groups (x2 =0.142,P ＞ 0.05;x2 =1.124,P ＞0.05;x2 =1.335,P ＞ 0.05).In anosmia group,with 4 cases of ageusia,22 cases of hypogeusia,32 cases of normal taste;in hyposrnia group,with 0 cases of ageusia,10 cases of hypogeusia,27 cases of normal taste.There were significant differences between the two groups with different types of taste disorder (Pearson correlation coefficient r =0.210,P ＜ 0.05),it was positive correlation.Conclusions It is suggested that after the upper respiratory tract infection,the olfactory dysfunction is often accompanied by the sense of taste dysfunction,the more severe the damage of olfactory function,the degree of damage to the taste function is also increased.Olfactory impairment degree exhibited no relationship with gender,age or onset seasons.

Objective To explore the rule of traditional Chinese medicine in treating tinnitus based on literature and Logistic multiple regression analysis. Methods Articles about tinnitus treatment using traditional Chinese medicine were searched in several databases, i.e. CNKI (1984-2013), VIP (1989-2013), CBM (1990-2013), and PubMed (1984-2013), and medication frequency was analyzed. Then, models for tinnitus medication were established through metrological method and Logistic multiple regression. Results The common prescriptions with highest frequency usage were Liuwei Dihuang Pill, Longdan Xiegan Decoction, and Erlong Zuoci Pill. Corni Fructus, Psoraleae Fructus, and Lycii Fructus were commonly used for kidney-deficiency type of tinnitus;Astragali Radix, Atractylodis Macrocephalae Rhizoma, and Codonopsis Radix were commonly used for the spleen-qi-deficiency type of tinnitus;Gardeniae Fructus, Gentianae Radix et Rhizoma, and Cyperi Rhizoma were commonly used for the liver-qi-dysfunction type of tinnitus;Pinelliae Rhizoma, Aurantii Fructus Immaturus, and Stephaniae Tetrandrae Radix were commonly used for the phlegm-fire disturbance type of tinnitus;Forsythiae Fructus, Menthae Haplocalycis Herba, and Mori Cortex were commonly used for the wind-fire-invasion type of tinnitus. Conclusion Analysis of medication frequency and Logistic multiple regression analysis can provide evidence and reference for the treatment of tinnitus and syndrome differentiation.