Discovery of YK-029A, a novel mutant EGFR inhibitor targeting both T790 M and exon 20 insertion mutations, as a treatment for NSCLC.

Although traditional EGFR-TKIs have advanced the treatment landscape of NSCLC with sensitive driver mutations (del19 or L858R), some NSCLC patients with EGFR exon 20 insertion mutations have been left with few effective therapies. The development of novel TKIs is still in progress. Herein, we describe the structure-guided design of a novel selective and orally bioavailable inhibitor, YK-029A, which could overcome both the T790 M mutations and exon 20 insertion of EGFR. YK-029A inhibited EGFR signaling, suppressed sensitive mutations and ex20ins of EGFR-driven cell proliferation, and was largely effective with oral administration in vivo. Furthermore, YK-029A exhibited significant antitumor activity in EGFRex20ins-driven patients-derived xenograft (PDX) models, preventing tumor progression or causing tumor regression at well-tolerated dosages. Based on the outcomes of preclinical efficacy and safety studies, YK-029A will enter phase Ⅲ clinical trials for the treatment of EGFRex20ins NSCLC.

[Relationship between sudden sensorineural hearing loss and facial auditory nerve and anterior inferior cerebellar artery vascular loop in the internal auditory tract MRI].

Objective:To investigate the correlation between the facial auditory nerve and anterior inferior cerebellar artery vascular loop in MRI of the internal auditory meatus and idiopathic sudden sensorineural hearing loss. Methods:This retrospective study enrolled 144 patients with idiopathic sudden sensorineural hearing loss（SSNHL）（experimental group） and 36 healthy subjects with 72 ears（control group）, who attended the Department of Otolaryngology Head and Neck Surgery, the 940th Hospital of the Joint Logistics Support Unit of the Chinese PLA from January, 2019 to January, 2021. The magnetic resonance imaging（MRI） data of the internal auditory meatus and clinical data were collected and compared between the two groups. Results:The distance between the auditory nerve and the peripheral vessels in the unilateral SSNHL-affected ear was significantly different from that in the contralateral ears and that in the healthy ears of the control group （P<0.05）. The distance between the auditory nerve and the peripheral vessels in both ear affected by bilateral SSNHL was significantly different from that in the healthy ears of the control group （P<0.05）. There was no significant difference in radiological grading of vascular loops between the ears affected by unilateral SSNHL and the contralateral ears and the healthy ear of the control group （P>0.05）. No statistically significant differences in radiological grading of vascular loops were found between both ears with bilateral SSNHL and the healthy ears in the control group（P>0.05）. The severity of hearing loss, audiometric configuration, radiological grading of vascular loops and the distance between the facial auditory nerve and peripheral vessels were not significantly different between the affected ears in unilateral SSNHL and both ears in bilateral SSNHL （P>0.05）. Conclusion:SSNHL is associated with the distance between the auditory nerve and the nearest peripheral vessel. SSNHL may occur when the vessel compresses the auditory nerve.

Molecular Dynamics Ensemble Refinement of Intrinsically Disordered Peptides According to Deconvoluted Spectra from Circular Dichroism.

We have developed a computational method of atomistically refining the structural ensemble of intrinsically disordered peptides (IDPs) facilitated by experimental measurements using circular dichroism spectroscopy (CD). A major challenge surrounding this approach stems from the deconvolution of experimental CD spectra into secondary structure features of the IDP ensemble. Currently available algorithms for CD deconvolution were designed to analyze the spectra of proteins with stable secondary structures. Herein, our work aims to minimize any bias from the peptide deconvolution analysis by implementing a non-negative linear least-squares fitting algorithm in conjunction with a CD reference data set that contains soluble and denatured proteins (SDP48). The non-negative linear least-squares method yields the best results for deconvolution of proteins with higher disordered content than currently available methods, according to a validation analysis of a set of protein spectra with Protein Data Bank entries. We subsequently used this analysis to deconvolute our experimental CD data to refine our computational model of the peptide secondary structure ensemble produced by all-atom molecular dynamics simulations with implicit solvent. We applied this approach to determine the ensemble structures of a set of short IDPs, that mimic the calmodulin binding domain of calcium/calmodulin-dependent protein kinase II and its 1-amino-acid and 3-amino-acid mutants. Our study offers a, to our knowledge, novel way to solve the ensemble secondary structures of IDPs in solution, which is important to advance the understanding of their roles in regulating signaling pathways through the formation of complexes with multiple partners.

Histone deacetylase inhibitor sodium butyrate attenuates gentamicin-induced hearing loss in vivo.

OBJECTIVE: Although histone deacetylase (HDAC) inhibition has been shown to protect against gentamicin (GM)-induced hearing loss in vitro, its protective effect has not been proven in vivo. In the present study, the aim was to investigate the protective effect of sodium butyrate (NaB), a specific HDAC inhibitor, on GM-induced ototoxicity in vivo. METHODS: Forty 8-week-old albino guinea pigs were divided into two experimental groups. Group 1 (n=10) underwent bilateral ear surgery to place sponges (0.3mm(3)) permeated with NaB (10µl, 100mg/ml) and physiological saline (10µl; control) in the right and left round window niches, respectively. The sponges were left in place for 15days to evaluate the effects of NaB at the applied concentration. Group 2 (n=30) underwent the same bilateral ear surgery described for Group 1, except three days after surgery, the animals received intramuscular GM injections (200mg/kg/day) for 5 consecutive days. Seven days after the final GM injection, the protective effects of NaB were examined. RESULTS: After 15days of NaB treatment (10µl, 100mg/ml), an increase in histone acetylation was detected in Corti organ samples. Auditory brainstem response (ABR) threshold shifts and hair cell loss were also reduced in NaB-treated ears after GM administration. Furthermore, GM treatment increased HDAC1 expression in outer hair cells (OHCs) in vivo, and NaB blocked this action. CONCLUSION: GM increases HDAC1 expression in OHCs, and NaB is able to block this action. Thus, it appears that the HDAC inhibitor, NaB, attenuates GM-induced hearing loss in guinea pigs.

Conformational frustration in calmodulin-target recognition.

Calmodulin (CaM) is a primary calcium (Ca(2+) )-signaling protein that specifically recognizes and activates highly diverse target proteins. We explored the molecular basis of target recognition of CaM with peptides representing the CaM-binding domains from two Ca(2+) -CaM-dependent kinases, CaMKI and CaMKII, by employing experimentally constrained molecular simulations. Detailed binding route analysis revealed that the two CaM target peptides, although similar in length and net charge, follow distinct routes that lead to a higher binding frustration in the CaM-CaMKII complex than in the CaM-CaMKI complex. We discovered that the molecular origin of the binding frustration is caused by intermolecular contacts formed with the C-domain of CaM that need to be broken before the formation of intermolecular contacts with the N-domain of CaM. We argue that the binding frustration is important for determining the kinetics of the recognition process of proteins involving large structural fluctuations.

MicroRNA-195 chemosensitizes colon cancer cells to the chemotherapeutic drug doxorubicin by targeting the first binding site of BCL2L2 mRNA.

The mechanisms underlying doxorubicin (Dox) resistance in colon cancer cells are not fully understood. MicroRNA (miRNA) play important roles in tumorigenesis and drug resistance. However, the relationship between miRNA and Dox resistance in colon cancer cells has not been previously explored. In this study, we utilized microRNA array and real-time PCR to verify that miR-127, miR-195, miR-22, miR-137 were significantly down-regulated, while miR-21, miR-592 were up-regulated in both HT29/DOX and LOVO/DOX cell lines. In vitro cell viability assay showed that knockdown of miR-195 in HT29 and LOVO cells caused a marked inhibition of Dox-induced cytotoxicity. Moreover, we explored that miR-195 is involved in repression of BCL2L2 expression through targeting its 3'-untranslated region, especially the first binding site within its mRNA. Furthermore, down-regulation of miR-195 conferred DOX resistance in parental cells and reduced cell apoptosis activity, while over-expression of miR-195 sensitized resistant cells to DOX and enhanced cell apoptosis activity, all of which can be partly rescued by BCL2L2 siRNA and cDNA expression. These results may have implications for therapeutic strategies aiming to overcome colon cancer cell resistance to Dox.

Acoustical stimulus changes the expression of stromal cell-derived factor-1 in the spiral ganglion neurons of the rat cochlea.

Neural stem cell (NSC) transplantation into the cochlea has been tested as a treatment for spiral ganglion neuron (SGN) degenerative disease and injury in various animal models. A recent study has shown evidence of functional recovery after transplantation of the stem cells into a degenerated-SGN model. Chemokine stromal cell-derived factor-1 (SDF-1, or known as CXC chemokine ligand-12, CXCL-12) signaling through CXCR4 has previously been identified as a key step in the homing of the stem cells within the injury areas; meanwhile, studies have revealed that the SDF-1/CXCR4 axis is also involved in axon guidance and pathfinding. A study found that transplanted neural precursor cells can migrate to the root of the auditory nerve when animals are subjected to an augmented acoustic environment (AAE). In accordance with these studies, we hypothesize that AAE will up-regulate the expression of SDF-1 in acoustic nerves. We tested our hypothesis by examining the expression of SDF-1 in different acoustic environments, and the results were confirmed by the auditory brainstem response (ABR), immunohistochemical and RT-PCR analyses. The results showed that SDF-1 was expressed at a relatively low level in the SGNs under normal animal unit acoustic conditions (40-50 dB). Moreover, it was significantly up-regulated in the SGNs under the 75 dB (augmented physiological process without hearing loss) and 90 dB AAE (pathological process with light hearing loss) conditions; however, under the 115 dB AAE (pathological process with severe hearing loss) condition, the expression of SDF-1 was not up-regulated. The results confirmed that appropriately augmented acoustical stimuli lead to the up-regulation of SDF-1, which may assist in the migration of the transplanted cells and the subsequent establishment of essential synaptic contacts between the exogenous cells and the host auditory pathway.

Wnt1 from cochlear schwann cells enhances neuronal differentiation of transplanted neural stem cells in a rat spiral ganglion neuron degeneration model.

Although neural stem cell (NSC) transplantation is widely expected to become a therapy for nervous system degenerative diseases and injuries, the low neuronal differentiation rate of NSCs transplanted into the inner ear is a major obstacle for the successful treatment of spiral ganglion neuron (SGN) degeneration. In this study, we validated whether the local microenvironment influences the neuronal differentiation of transplanted NSCs in the inner ear. Using a rat SGN degeneration model, we demonstrated that transplanted NSCs were more likely to differentiate into microtubule-associated protein 2 (MAP2)-positive neurons in SGN-degenerated cochleae than in control cochleae. Using real-time quantitative PCR and an immunofluorescence assay, we also proved that the expression of Wnt1 (a ligand of Wnt signaling) increases significantly in Schwann cells in the SGN-degenerated cochlea. We further verified that NSC cultures express receptors and signaling components for Wnts. Based on these expression patterns, we hypothesized that Schwann cell-derived Wnt1 and Wnt signaling might be involved in the regulation of the neuronal differentiation of transplanted NSCs. We verified our hypothesis in vitro using a coculture system. We transduced a lentiviral vector expressing Wnt1 into cochlear Schwann cell cultures and cocultured them with NSC cultures. The coculture with Wnt1-expressing Schwann cells resulted in a significant increase in the percentage of NSCs that differentiated into MAP2-positive neurons, whereas this differentiation-enhancing effect was prevented by Dkk1 (an inhibitor of the Wnt signaling pathway). These results suggested that Wnt1 derived from cochlear Schwann cells enhanced the neuronal differentiation of transplanted NSCs through Wnt signaling pathway activation. Alterations of the microenvironment deserve detailed investigation because they may help us to conceive effective strategies to overcome the barrier of the low differentiation rate of transplanted NSCs.

Protein recognition and selection through conformational and mutually induced fit.

Protein-protein interactions drive most every biological process, but in many instances the domains mediating recognition are disordered. How specificity in binding is attained in the absence of defined structure contrasts with well-established experimental and theoretical work describing ligand binding to protein. The signaling protein calmodulin presents a unique opportunity to investigate mechanisms for target recognition given that it interacts with several hundred different targets. By advancing coarse-grained computer simulations and experimental techniques, mechanistic insights were gained in defining the pathways leading to recognition and in how target selectivity can be achieved at the molecular level. A model requiring mutually induced conformational changes in both calmodulin and target proteins was necessary and broadly informs how proteins can achieve both high affinity and high specificity.

Stem cell transplantation via the cochlear lateral wall for replacement of degenerated spiral ganglion neurons.

Spiral ganglion neurons (SGNs) are poorly regenerated in the mammalian inner ear. Because of this, stem cell transplantation has been used to replace injured SGNs, and several studies have addressed this approach. However, the difficulty of delivering stem cells into the cochlea and encouraging their migration to Rosenthal's canal (RC), where the SGNs are located, severely restricts this therapeutic strategy. In this study, we attempted to establish a new stem cell transplantation route into the cochlea via the cochlear lateral wall (CLW). First, we tested the precision of this route by injecting Fluorogold into the CLW and next assessed its safety by mock surgeries. Then, using a degenerated SGN animal model, we transplanted neural stem cells (NSCs), derived from the olfactory bulb of C57BL/6-green fluorescent protein (GFP) mice, via the CLW route and examined the cells' distribution in the cochlea. We found the CLW transplantation route is precise and safe. In addition, NSCs migrated into RC with a high efficiency and differentiated into neurons in a degenerated SGN rat model after the CLW transplantation. This result revealed that the basilar membrane (BM) may have crevices permitting the migration of NSCs. The result of this study demonstrates a novel route for cell transplantation to the inner ear, which is important for the replacement of degenerated SGNs and may contribute to the treatment of sensorineural hearing loss.

Up-regulation of stromal cell-derived factor-1 enhances migration of transplanted neural stem cells to injury region following degeneration of spiral ganglion neurons in the adult rat inner ear.

Neural stem cell (NSC) transplantation into the cochlea is widely used for the treatment of spiral ganglion neuron (SGN) degenerative disease and injury in the animal models, but the migration of the transplanted NSCs to the injury region is difficult and the mechanism is still unclear. In this study, we aimed to validate whether the SGN-degenerated cochlear microenvironment plays a role in the NSC migration and investigated whether stromal cell-derived factor-1 (SDF-1) was involved in the NSCs migration. Using a rat SGN degeneration model, we demonstrated that the transplanted NSCs are more likely to migrate to the injury region during the early post-injury (EPI) than the late post-injury (LPI) stage and the control cochlea. We found that the expressions of SDF-1 increased transiently after SGN degeneration. Additionally, we showed that the NSCs express CXCR4, a receptor for SDF-1. We observed that the region to which the transplanted NSC localized coincides with the region where the SDF-1 is highly expressed following the degeneration of SGNs. Finally, we observed that the increased SDF-1 is derived from the Schwann cells in the SGN-degenerated model. These results suggest that SDF-1, which is derived from cochlear Schwann cells and up-regulated in the early injury microenvironment, plays a beneficial role in the NSC migration to the injury region. Optimizing SDF-1 expression in the host microenvironment or increasing the CXCR4 expression of the donor stem cells may improve the migration efficiency of transplanted cells toward the injury region in the cochlea.

Protective role of hydrogen sulfide against noise-induced cochlear damage: a chronic intracochlear infusion model.

BACKGROUND: A reduction in cochlear blood flow plays an essential role in noise-induced hearing loss (NIHL). The timely regulation of cochlear perfusion determines the progression and prognosis of NIHL. Hydrogen sulfide (H(2)S) has attracted increasing interest as a vasodilator in cardiovascular systems. This study identified the role of H(2)S in cochlear blood flow regulation and noise protection. METHODOLOGY/PRINCIPAL FINDINGS: The gene and protein expression of the H(2)S synthetase cystathionine-γ-lyase (CSE) in the rat cochlea was examined using immunofluorescence and real-time PCR. Cochlear CSE mRNA levels varied according to the duration of noise exposure. A chronic intracochlear infusion model was built and artificial perilymph (AP), NaHS or DL-propargylglycine (PPG) were locally administered. Local sodium hydrosulfide (NaHS) significantly increased cochlear perfusion post-noise exposure. Cochlear morphological damage and hearing loss were alleviated in the NaHS group as measured by conventional auditory brainstem response (ABR), cochlear scanning electron microscope (SEM) and outer hair cell (OHC) count. The highest percentage of OHC loss occurred in the PPG group. CONCLUSIONS/SIGNIFICANCE: Our results suggest that H(2)S plays an important role in the regulation of cochlear blood flow and the protection against noise. Further studies may identify a new preventive and therapeutic perspective on NIHL and other blood supply-related inner ear diseases.

[Hearing the impact of MP3 on a survey of middle school students].

OBJECTIVE: To understand the usage of MP3 and effects on hearing of middle school students in Xi'an, and discuss controlling strategies. METHOD: Stratified random cluster sampling method was used in the 1567 middle school students in Xi'an through questionnaire survey, ear examination and hearing examination, data were analysed by the SPSS13.0 statistical software. RESULT: 1) The rate of holding MP3 in the middle school students was 85.2%. Average daily use time was (1.41 +/- 1.11) h. 2) The noise group of pure tone hearing threshold was significantly higher compared with the control group (P<0.01), and increased the detection rate of hearing loss with the increasing use of MP3. 3) The detection rate of symptoms increased with the increasing use of MP3. CONCLUSION: The usage of MP3 can harm hearing in middle school students, which can result in neurasthenic syndrome.

[An epidemiologic study of vertigo among middle school students in the city of Xiran].

OBJECTIVE: To investigate the prevalence of vertigo and related risk factors in middle school students in the city of Xi'an. METHOD: A cross-section study was used to investigate on vertigo among middle school students. Questionnaire ,ear examination and auditory tests were carried out by the staff who received special training. Multiple Logistic regression models were used to analyze the relative influential factors about vertigo. RESULT: There were 1567 middle school students who underwent a complete investigation. The participants comprised 793 males (50.6%) and 774 females (49.4%). The overall prevalence of vertigo was 5.6%. No significant difference of the prevalence was found between males (4.7%) and females (6.5%) (P > 0.05). The use of MP3 or MP4, insomnia and history of middle ear infections or ototoxic drugs ingestion were the main risk factors for vertigo (odds ratio: 2.837, 5.582, 2.808 and 1.695, respectively). CONCLUSION: Vertigo has an influence on the study and living of the students. More researches are urgently needed on prevention and treatment of vertigo.