[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.

The effect of Ca2+ ionophores upon the synthesis of proteins in cultured skeletal muscle.

The influence of the Ca2+ ionophores, ionomycin and A23187 upon the incorporation of [35S]methionine into proteins of cultured chicken pectoralis muscle was studied during differentiation of myoblasts into multinucleated myotubes. Fusion was reversibly arrested by growing cells in low-calcium media from the time of plating. Exposure of normal and fusion blocked cultures to 10-6-10-5 M ionomycin or A23187 for 2-6 h on the second to fourth day of growth, resulted in a selective increase in the incorporation of [35S]methionine into two proteins of about 100 000 and 80 000 dalton. When 10-5 M ionomycin or A23187 were added to older cultures, all large myotubes contracted and detached from the plate. Only the adhering myoblasts and small myotubes incorporated [35s[methionine into the muscle proteins and showed increased incorporation of label into 100 000 and 80 000 proteins. After ionophore pulse, the adhering cells retained the ability to differentiate and accumulate myosin. The effect of Ca2+ ionophores upon the rate of protein synthesis is presumably related to increased influx of extracellular Ca2+ with a rise in the Ca2+ concentration of the cytoplasm. We conclude that Ca2+ sensitive mechanisms may regulate the synthesis of a select group of muscle proteins.

Inhibition of synthesis of ribosomal proteins and of ribosome assembly after infection of L cells with vesicular stomatitis virus.

The effect of infection of mouse L cells by vesicular stomatitis virus on the synthesis of ribosomal proteins was investigated using two-dimensional polyacrylamide gel electrophoresis to analyze the ribosomal proteins. It was found that the synthesis of nearly all of the cytoplasmic ribosomal proteins examined was inhibited by infection and mostly to the same extent. Analysis of the ribosomal proteins extracted from intact ribosomes indicated that infection also reduces the incorporation of all the ribosomal proteins tested into assembled ribosomes. The inhibition of ribosome assembly was greater than the inhibition of synthesis of ribosomal proteins, suggesting that some other factor was also limiting the assembly of ribosomes. As shown in this report, infection also inhibits ribosomal RNA production. Thus, the decreased assembly of ribosomes in infected cells probably results from the inhibition of synthesis of both ribosomal proteins and ribosomal RNA.

Insulin depolarization of skeletal muscle in absence of external Na+.

Three mechanisms have been proposed by which insulin might increase the electrical potential difference across the cell membrane of some of its main target cells: stimulation of an electrogenic pump; increased permeability to K+ (PK); and decreased ratio of permeability to Na+ (PNa) compared to PK, with an absolute decrease in permeability to both ions. Our laboratory has reported that insulin-induced hyperpolarization (IIH) of rat skeletal muscle is not due to stimulation of a ouabain-inhibitable pump and that insulin decreases 42K efflux, apparently eliminating the first two candidate mechanisms. If the remaining hypothesis is correct, when Na+ is removed from the bathing solution, insulin should depolarize, not hyperpolarize. It did. With Tris or N-methyl-D-glucamine substituted for Na+, insulin depolarized by approximately 3 mV. Ouabain had no effect. PNa decreased by greater than 90%; PK was reduced by less than 40%. The main component of the immediate mechanism of IIH is the near elimination of PNa. Furthermore, when a poorly permeable cation was substituted for Na+, muscles hyperpolarized in the absence of insulin. This gave us an opportunity to test the hypothesis that hyperpolarization is a link in the insulin-transduction chain. Consistent with this hypothesis, rat muscles hyperpolarized in this manner in the absence of insulin took up more glucose than paired controls in normal Na+ solution.

Calcium currents in rat myoballs and their inhibition by insulin.

Two Ca2+ currents were found in myoballs prepared from primary culture of hindlimb muscles from rat embryos. One whole cell current, not described previously, was early, fast, and transient. It depended on the presence of Na+ in the bathing solution and was blocked by tetrodotoxin. Despite this behavior suggesting that it might be via a Na+ channel, its reversal potential exceeded 80 mV compared to 46 mV for the Na+ equilibrium potential. It was increased by increased Ca2+ concentration in the bathing solution and eliminated by Co2+ and by diltiazem. The other Ca2+ current resembled the slow inward Ca2+ current ICa(si), described in other cell types. Insulin decreased both Ca2+ currents; even at 64 pM insulin reduced ICa(si). When outward K+ currents were prevented, the myoball action potential was altered greatly, owing to the unopposed effect of ICa(si). Its duration was on the order of seconds. Insulin, in a concentration-dependent manner, beginning at 60 pM, reduced the duration of this action potential more effectively than nimodipine. This is the most sensitive response to insulin observed in skeletal muscle.

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.

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.

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.

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.

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.

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.

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.

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.

Venom constituents of Notechis scutatus scutatus (Australian tiger snake) from differing geographic regions.

Column chromatography and polyacrylamide gel electrophoresis of Notechis scutatus scutatus venom showed that the venoms from different geographical locations had variations in their constituents. The venom collected from South Australia region contained both notexin and notechis II-5. The relative quantity of notechis II-5 was about three times that of notexin. On the other hand, the venom from Victoria region contained large amounts of notexin, but lacked notechis II-5. Instead, an unknown nontoxic protein, designated as notechis II-5b, exhibiting weak phospholipase A2 activity appeared in the position of notechis II-5 elution. This protein had an N-terminal sequence of N-L-I-Q-L-S-N-M-I-K-C-A-I-P-G-S-Q-P-L-F, sharing 45% homology with notexin and notechis II-5 and 60% homology with notechis II-1. The antibodies raised against Trp-modified notexin inhibited the enzymatic activities of notexin and notechis II-5 by 88 and 68%, respectively. However, the affinity of notexin for the antibodies was nine-fold greater than that of notechis II-5. This result is contrary to the previous finding (Mollier et al., FEBS Lett. 250, 479-482, 1989) in which notexin and notechis II-5 had similar binding affinities for antibodies raised against native notexin. This observation suggests that the antibodies prepared in this study could differentiate between isoforms of notexin.

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.