[Mechanism of noise induced hidden hearing loss based on proteomics].

Objective: To explore the mechanism of noise-induced hidden hearing loss by proteomics. Methods: In October 2022, 64 SPF male C57BL/6J mice were divided into control group and noise exposure group with 32 mice in each group according to random sampling method. The noise exposure group was exposed to 100 dB sound pressure level, 2000-16000 Hz broadband noise for 2 h, and the mouse hidden hearing loss model was established. Auditory brainstem response (ABR) was used to test the change of hearing threshold of mice on the 7th day after noise exposure, the damage of basal membrane hair cells was observed by immunofluorescence, and the differentially expressed proteins in the inner ear of mice in each group were identified and analyzed by 4D-Label-free quantitative proteomics, and verified by Western blotting. The results were statistically analyzed by ANOVA and t test. Results: On the 7th day after noise exposure, there was no significant difference in hearing threshold between the control group and the noise exposure group at click and 8000 Hz acoustic stimulation (P>0.05) . The hearing threshold in the noise exposure group was significantly higher than that in the control group under 16000 Hz acoustic stimulation (P<0.05) . Confocal immunofluorescence showed that the basal membrane hair cells of cochlear tissue in noise exposure group were arranged neatly, but the relative expression of C-terminal binding protein 2 antibody of presynaptic membrane in middle gyrus and basal gyrus was significantly lower than that in control group (P<0.05) . GO enrichment analysis showed that the functions of differentially expressed proteins were mainly concentrated in membrane potential regulation, ligand-gated channel activity, and ligand-gated ion channel activity. KEGG pathway enrichment analysis showed that differentially expressed proteins were significantly enriched in phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt) signaling pathway, NOD-like receptor signaling pathway, calcium signaling pathway, etc. Western blotting showed that the expression of inositol 1, 4, 5-trisphosphate receptor 3 (Itpr3) was increased and the expression of solute carrier family 38 member 2 (Slc38a2) was decreased in the noise exposure group (P<0.05) . Conclusion: Through proteomic analysis, screening and verification of the differential expression proteins Itpr3 and Slc38a2 in the constructed mouse noise-induced hidden hearing loss model, the glutaminergic synaptic related pathways represented by Itpr3 and Slc38a2 may be involved in the occurrence of hidden hearing loss.

[Developmental characteristics of various types of hand bones of Poland's syndrome].

OBJECTIVE: To explore development status in different types of the hand bone and its developmental characteristics with Poland syndrome. METHODS: There were 32 cases with Poland's syndrome who accepted bilateral hand X-ray examination in Department of Hand Surgery, Beijing Jishuitan Hospital from February 2013 to August 2014.There were 24 male and 8 female patients aged from 1.0 to 15.0 years with median age of 2.4 years. Right hand deformity was 23 cases and left hand deformity was 9 cases. According to Tanner-Whitehouse skeletal age scoring system, 20 bones (radius and ulna, 7 carpal bones, 11 metacarpal and phalangeal bones) selected from the affected and contralateral limb respectively, were evaluated. Besides, hand deformity of the cases was classified into 5 types based on relevant literature. Each bone was given an individual age using the references of Greulich-Pyle chart. The average of all individual ages was taken as gross bone age, the average of individual ages of radius and ulna was taken as bone age of long bones, the average of individual ages of carpal bone was taken as bone age of carpal bones, and the average of individual age of metacarpal and phalangeal bones was taken as bone age of short bones.The delay of bone age was evaluated by correlation test, while the curve of cubic equation was used for analyzing the variance of skeletal development with age. RESULTS: The delay of long bone age of patients with Poland's syndrome in this study were 0-1.9 years ((0.5±0.5) years), 0-2.2 years ((0.7±0.5)years) for carpal bone, 0.5-2.0 years((0.6±0.4) years)for short bone and 0.1-1.7 years((0.6±0.4)years) for gross bone.Twelve cases in type Ⅱ hand deformity, 15 cases in type Ⅲ and 5 cases in type Ⅳ. The delay of bone ages, including long bone age, carpal bone age, short bone age and gross bone age, was not related with gender and side(all P>0.05), but related with degree of deformity(F=3.663-12.971, P=0.000-0.038). CONCLUSION: Compared with normal upper limb, the bone age in the affected limb in Poland's syndrome is delayed and it is correlated with gender, age and the extent of hand deformity and negative with side.

A randomized phase II study of the MEK1/MEK2 inhibitor trametinib (GSK1120212) compared with docetaxel in KRAS-mutant advanced non-small-cell lung cancer (NSCLC)†.

BACKGROUND: KRAS mutations are detected in 25% of non-small-cell lung cancer (NSCLC) and no targeted therapies are approved for this subset population. Trametinib, a selective allosteric inhibitor of MEK1/MEK2, demonstrated preclinical and clinical activity in KRAS-mutant NSCLC. We report a phase II trial comparing trametinib with docetaxel in patients with advanced KRAS-mutant NSCLC. PATIENTS AND METHODS: Eligible patients with histologically confirmed KRAS-mutant NSCLC previously treated with one prior platinum-based chemotherapy were randomly assigned in a ratio of 2 : 1 to trametinib (2 mg orally once daily) or docetaxel (75 mg/m(2) i.v. every 3 weeks). Crossover to the other arm after disease progression was allowed. Primary end point was progression-free survival (PFS). The study was prematurely terminated after the interim analysis of 92 PFS events, which showed the comparison of trametinib versus docetaxel for PFS crossed the futility boundary. RESULTS: One hundred and twenty-nine patients with KRAS-mutant NSCLC were randomized; of which, 86 patients received trametinib and 43 received docetaxel. Median PFS was 12 weeks in the trametinib arm and 11 weeks in the docetaxel arm (hazard ratio [HR] 1.14; 95% CI 0.75-1.75; P = 0.5197). Median overall survival, while the data are immature, was 8 months in the trametinib arm and was not reached in the docetaxel arm (HR 0.97; 95% CI 0.52-1.83; P = 0.934). There were 10 (12%) partial responses (PRs) in the trametinib arm and 5 (12%) PRs in the docetaxel arm (P = 1.0000). The most frequent adverse events (AEs) in ≥20% of trametinib patients were rash, diarrhea, nausea, vomiting, and fatigue. The most frequent grade 3 treatment-related AEs in the trametinib arm were hypertension, rash, diarrhea, and asthenia. CONCLUSION: Trametinib showed similar PFS and a response rate as docetaxel in patients with previously treated KRAS-mutant-positive NSCLC. CLINICALTRIALSGOV REGISTRATION NUMBER: NCT01362296.

Functional and symptom impact of trametinib versus chemotherapy in BRAF V600E advanced or metastatic melanoma: quality-of-life analyses of the METRIC study.

BACKGROUND: In a randomized phase III study, trametinib prolonged progression-free survival and improved overall survival versus chemotherapy in patients with BRAF V600 mutation-positive melanoma. PATIENTS AND METHODS: Patients' quality of life (QOL) was assessed at baseline and follow-up visits using the European Organisation for Research and Treatment of Cancer Core QOL questionnaire. RESULTS: In the primary efficacy population (BRAF V600E+, no brain metastases) from baseline to weeks 6 and 12, patients' global health status scores worsened by 4-5 points with chemotherapy but improved by 2-3 points with trametinib. Rapid and substantive reductions in QOL functionality (e.g. role functioning, 8-11 points at weeks 6 and 12) and symptom exacerbation (e.g. fatigue, 4-8 points; nausea and vomiting, 5 points, both at weeks 6 and 12) were observed in chemotherapy-treated patients. In contrast, trametinib-treated patients reported small improvements or slight worsening from baseline at week 12, depending on the functional dimension and symptom. The mean symptom-scale scores for chemotherapy-treated patients increased from baseline (symptoms worsened) for seven of eight symptoms at week 6 (except insomnia) and six of eight symptoms at week 12 (except dyspnea and insomnia). In contrast, at weeks 6 and 12, the mean symptom-scale scores for trametinib decreased from baseline (symptoms improved) for pain (11-12 points), insomnia (10-12 points), and appetite loss (1-5 points), whereas those for diarrhea worsened (15-16 points). Mixed-model repeated-measures analyses showed significant (P < 0.05) and/or clinically meaningful improvements (small to moderate) from baseline in favor of trametinib for global health; physical, role, and social functioning; fatigue; pain; insomnia; nausea and vomiting; constipation; dyspnea; and appetite at weeks 6 and/or 12. QOL results for the intent-to-treat population were consistent. CONCLUSIONS: This first QOL assessment for a MEK inhibitor in metastatic melanoma demonstrated that trametinib was associated with less functional impairment, smaller declines in health status, and less exacerbation of symptoms versus chemotherapy.

Let-7c microRNA expression and clinical significance in hepatocellular carcinoma.

MicroRNAs (miRNAs) are small non-coding regulatory RNAs that are often dysregulated during carcinogenesis. Downregulation of let-7 miRNA in many human cancers indicates its role in tumourigenesis. This study evaluated the levels of let-7c miRNA, using real-time reverse transcription-polymerase chain reaction, between 32 hepatocellular carcinoma (HCC) tissues and matched normal adjacent tumour tissues within the context of the patient's clinical pathology. Levels of let-7c miRNA were significantly lower in HCC tissues than in corresponding normal adjacent tumour tissues and there was a correlation between the downregulation of let-7c and poor tissue differentiation in HCC. There was no correlation between let-7c miRNA levels and other clinicopathological factors, such as patient age, sex, hepatitis B virus status, α-fetoprotein levels, tumour size, tumour number, the presence of cirrhosis, liver envelope invasion or portal vein thrombosis. These data suggested that let-7c microRNA may play a role in regulating HCC cell differentiation.

Noncompetitive inhibition of the glycine receptor-mediated current by melatonin in cultured neurons.

The effect of melatonin on the glycine receptor-mediated response was studied in cultured chick spinal cord neurons using the whole-cell voltage-clamp recording technique. Melatonin rapidly and reversibly inhibited the glycine-induced current in a dose-dependent fashion, with an EC(50) of 934 microM and a maximal inhibition of 100%. Furthermore, melatonin noncompetitively inhibited the glycine response by an agonist-independent mechanism that was distinct from that of an open-channel blocker.

Non-competitive inhibition of 5-HT3 receptor-mediated currents by progesterone in rat nodose ganglion neurons.

The effect of progesterone on the serotonin type 3 (5-HT3) receptor-mediated response was studied in acutely dissociated rat nodose ganglion neurons by using the whole-cell voltage-clamp technique. Progesterone rapidly and reversibly inhibited 5-HT-induced currents in a dose-dependent manner, with an EC50 of 31 microM and a maximal inhibition of 75%. Neither the 5-HT response nor inhibition of the 5-HT response by extracellularly applied progesterone was significantly affected by inclusion of a saturating concentration of progesterone in the electrode buffer, arguing that progesterone acted at the extracellular surface of the membrane. Progesterone also inhibited the 5-HT response non-competitively by a voltage- and agonist-independent mechanism that was distinct from that of open-channel blockers.

The tumor suppressor p53 can reduce stable transfection in the presence of irradiation.

The tumor suppressor p53 is believed to play an essential role in maintaining genome stability. Although it is currently unknown how p53 is involved in this important biological safeguard, several previous publications indicate that p53 can help to maintain genome integrity through the recombination-mediated DNA repair process. The integration of linearized plasmid DNA into the host chromosome utilizes the same repair process, and the frequency can be measured by clonogenic assays in which cells that were stably transfected by plasmid integration can be scored by their colony-forming abilities. To gain insight into whether p53 has a direct role in plasmid integration into the host chromosome, we determined the frequency of stable transfection with CHO cells expressing either wild-type or mutant p53 in the presence and absence of irradiation. We found that low-dose irradiation ( approximately 50 to 100 cGy) increased stable transfection frequencies in CHO cells regardless of their p53 status. However, the increase of transfection frequency was significantly lower in CHO cells expressing wild-type p53. Our data thus suggest that wild-type p53 can suppress plasmid DNA integration into the host genome. This p53 function may play a direct and significant role in maintaining genome stability.

Melatonin potentiates the GABA(A) receptor-mediated current in cultured chick spinal cord neurons.

The effect of melatonin on the gamma-aminobutyric acidA (GABA(A)) receptor-mediated response was studied in cultured chick spinal cord neurons using the whole-cell voltage-clamp recording technique. Melatonin rapidly and reversibly potentiated the GABA-induced current in a dose-dependent fashion, with an EC50 of 766 microM and a maximal potentiation of 148%. Potentiation of the GABA response by melatonin was mediated by increasing the potency of GABA rather than the efficacy. Prolonged exposure to a saturating concentration of the disulfide-reducing agent dithiothreitol did not attentuate the effect of melatonin on the GABA response, indicating that melatonin does not act through the redox site. Furthermore, our results demonstrate that melatonin and 5alpha-pregnan-3alpha-ol-20-one (a positive steroid modulator of the GABA(A) receptor) act through different sites.

Pregnenolone sulfate exacerbates NMDA-induced death of hippocampal neurons.

Excessive stimulation of the N-methyl-d-aspartate (NMDA)-type glutamate receptor has been implicated in the neuronal death resulting from focal hypoxia-ischemia. Certain neurosteroids, steroids synthesized de novo in the central nervous system (CNS), have been shown to modulate the action of neurotransmitters at their cellular receptors. Pregnenolone sulfate (PS) is an abundant neurosteroid that enhances the current evoked by NMDA. Using the Ca2+-sensitive fluorescent dye, Fluo-3, AM, and a trypan blue exclusion assay, we evaluated the ability of PS to modulate NMDA-induced changes in intracellular free calcium concentration ([Ca2+]i) and neuronal death in primary cultures of rat hippocampal neurons. The results demonstrate that PS potentiates NMDA-induced increases in [Ca2+]i by 150%. Further, PS exacerbates the MK-801-sensitive neuronal death produced by acute (PS EC50=37 microM) or chronic NMDA exposure, reducing the EC50 of NMDA from 13 to 4 microM under chronic exposure conditions, whereas pregnenolone is ineffective. Our results show that PS, or related sulfated neurosteroids, may play a role in the onset of excitotoxic neuronal death in vivo.

Mechanism underlying potentiation by progesterone of the kainate-induced current in cultured neurons.

The effect of the neurosteroid progesterone on the kainate receptor-mediated response was studied in cultured chick spinal cord neurons using the whole-cell voltage-clamp recording technique. Progesterone rapidly and reversibly potentiates the kainate-induced current in a dose-dependent manner, with an EC50 of 35 microM and a maximal potentiation of 30%. Potentiation of the kainate response by extracellularly applied progesterone is not significantly affected by inclusion of a saturating concentration of progesterone in the electrode buffer, indicating that progesterone acts at the extracellular surface of the membrane. Furthermore, progesterone enhances the kainate maximal response with little effect on the kainate EC50.

Distinct sites for inverse modulation of N-methyl-D-aspartate receptors by sulfated steroids.

Steroid sulfation occurs in nervous tissue and endogenous sulfated steroids can act as positive or negative modulators of N-methyl-D-aspartate (NMDA) receptor function. In the current study, structure-activity relationships for sulfated steroids were examined in voltage-clamped chick spinal cord and rat hippocampal neurons in culture and in Xenopus laevis oocytes expressing NR1(100) and NR2A subunits. The ability of pregnenolone sulfate (a positive modulator) and epipregnanolone sulfate (a negative modulator) to compete with each another, as well as with other known classes of NMDA receptor modulators, was examined. The results show that steroid positive and negative modulators act at specific, extracellularly directed sites that are distinct from one another and from the spermine, redox, glycine, Mg2+, MK-801, and arachidonic acid sites. Sulfated steroids are effective as modulators of ongoing glutamate-mediated synaptic transmission, which is consistent with their possible role as endogenous neuromodulators in the CNS.

Competitive inhibition of the glycine-induced current by pregnenolone sulfate in cultured chick spinal cord neurons.

The effect of the neurosteroid pregnenolone sulfate (PS) on the glycine receptor-mediated response was studied in cultured chick spinal cord neurons using the whole-cell voltage-clamp recording technique. PS rapidly and reversibly inhibits the glycine-induced current in a dose-dependent manner, with an EC50 of 3.7 microM and a maximal inhibition of 100%. The fact that antagonism of the glycine response by PS is neither voltage- nor agonist-dependent indicates that PS does not act as an open-channel blocker. Furthermore, inhibition by PS of the glycine-induced current appears to be of a competitive type since the drug induces a parallel, rightward shift of the glycine dose-response curve.

Mechanism underlying the effect of pregnenolone sulfate on the kainate-induced current in cultured chick spinal cord neurons.

The effect of the neurosteroid pregnenolone sulfate on the kainate receptor-mediated response was studied in cultured chick spinal cord neurons using the whole-cell voltage-clamp recording technique. Pregnenolone sulfate rapidly and reversibly inhibits the kainate-induced current in a dose-dependent manner, with an EC50 of 67 microM and maximal inhibition of 38%. Inhibition by pregnenolone sulfate of the kainate response is not attenuated by increasing concentrations of kainate, suggesting that the blocking action of pregnenolone sulfate is non-competitive. Antagonism of the kainate response by pregnenolone sulfate is neither agonist- nor voltage-dependent, indicating that pregnenolone sulfate does not act as an open-channel blocker. Furthermore, our results demonstrate that pregnenolone sulfate and 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466; a potent non-competitive kainate antagonist) do not act through a common site.

3 alpha-Hydroxy-5 beta-pregnan-20-one sulfate: a negative modulator of the NMDA-induced current in cultured neurons.

We have shown previously that the neurosteroid pregnenolone sulfate acts as a positive allosteric modulator at the N-methyl-D-aspartate (NMDA) receptor while inhibiting the kainate, the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), the glycine, and the gamma-aminobutyric acid (GABA) responses of chick spinal cord neurons. Here, we report that 3 alpha-hydroxy-5 beta-pregnan-20-one sulfate (5 beta 3 alpha S), a sulfated form of naturally occurring 5 beta 3 alpha, inhibits both the NMDA and the non-NMDA receptor-mediated responses as measured by whole cell voltage clamp recordings. 100 microM 5 beta 3 alpha S rapidly and reversibly inhibits the response to 30 microM NMDA by 66%, 50 microM kainate by 37%, and 25 microM AMPA by 29%. Application of 50 microM nonsulfated 5 beta 3 alpha does not produce any significant effect on the NMDA response, demonstrating that the sulfate moiety is important for the effect of 5 beta 3 alpha S on the NMDA response. The effect of 5 beta 3 alpha S on the NMDA response is concentration dependent, with an EC50 of 62 microM. 5 beta 3 alpha S reduces the maximum NMDA response with little effect on the NMDA EC50, indicating that antagonism of the NMDA response by 5 beta 3 alpha S is noncompetitive. The fact that 5 beta 3 alpha S inhibition of the NMDA response is neither agonist nor voltage dependent demonstrates that 5 beta 3 alpha S does not act as an open channel blocker. Furthermore, inhibition of the NMDA response by 5 beta 3 alpha S is not reduced by the addition of a maximal concentration (10 microM) of glycine, indicating that 5 beta 3 alpha S does not act via the glycine recognition site. The inhibitory action of 5 beta 3 alpha S on the NMDA and non-NMDA receptors may provide a basis for inhibiting glutamate receptor-induced seizures and excitotoxic cell death.

Dual activation of GABAA and glycine receptors by beta-alanine: inverse modulation by progesterone and 5 alpha-pregnan-3 alpha-ol-20-one.

The differential sensitivity of the glycine and GABAA receptors to modulation by progesterone and 5 alpha-pregnan-3 alpha-ol-20-one (5 alpha 3 alpha) was used to determine whether beta-alanine acts through its own receptor, or through the glycine and/or GABAA receptor(s). The response to beta-alanine resembles the glycine response as it is inhibited by strychnine (a competitive glycine antagonist) or progesterone (a negative modulator of the glycine response). Significantly, the response to beta-alanine also resembles the GABA response in that it is inhibited by 2-(carboxy-3'-propyl)-3-amino-6-paramethoxy-phenylpyridazinium+ ++ bromide (SR-95531; a competitive GABA antagonist) and potentiated by 5 alpha 3 alpha (a positive modulator of the GABA response). The efficacy of beta-alanine at the GABAA receptor is comparable to that of GABA. Similarly, the efficacy of beta-alanine at the glycine receptor is comparable to that of glycine. The greater potency of beta-alanine at the glycine receptor indicates that, if beta-alanine is a neurotransmitter, its effects are more likely to be mediated by glycine receptors than by GABAA receptors. However, activation of the GABAA receptor by beta-alanine may become important in the presence of steroid modulators such as progesterone or 5 alpha 3 alpha.

An early outward transient K+ current that depends on a preceding Na+ current and is enhanced by insulin.

A whole-cell early transient outward current occurs in rat myoballs if and only if there is an immediately preceding current of large amplitude through the voltage-gated, tetrodotoxin-inhibitable Na+ channel. This early outward transient is a K+ current, designated IK(Na+). Under the conditions in which IK(Na+) appears, simultaneous measurement of voltage and current, under voltage clamp, demonstrates that there is transient voltage escape to depolarized levels, peaking at about the time of peak inward Na+ current and resembling an action potential. IK(Na+) was never seen in the absence of this breach of the voltage clamp, suggesting that IK(Na+) might be an artefact due to transient depolarization from the clamp. However, when the voltage escape was mimicked by voltage commands under conditions in which the Na+ channel was not activated, there was no IK(Na). Insulin increased or produced IK(Na+) even though insulin had no effect on INa or on the delayed rectifier K+ current or on the escape from voltage clamp. It is concluded that there is a population of rat myoballs in which there is an early outward K+ current that requires an immediately preceding current through the voltage-gated, tetrodotoxin-inhibitable Na+ channel and is enhanced by insulin.