Loss of Cochlear Ribbon Synapse Is a Critical Contributor to Chronic Salicylate Sodium Treatment-Induced Tinnitus without Change Hearing Threshold.

Tinnitus is a common auditory disease worldwide; it is estimated that more than 10% of all individuals experience this hearing disorder during their lifetime. Tinnitus is sometimes accompanied by hearing loss. However, hearing loss is not acquired in some other tinnitus generations. In this study, we injected adult rats with salicylate sodium (SS) (200 mg/kg/day for 10 days) and found no significant hearing threshold changes at 2, 4, 8, 12, 14, 16, 20, or 24 kHz (all p > 0.05). Tinnitus was confirmed in the treated rats via Behaviour Testing of Acoustic Startle Response (ASR) and Gap Prepulse Inhibition Test of Acoustic Startle Reflex (GPIAS). A immunostaining study showed that there is significant loss of anti-CtBP2 puncta (a marker of cochlear inner hair cell (HC) ribbon synapses) in treated animals in apical, middle, and basal turns (all p < 0.05). The ABR wave I amplitudes were significantly reduced at 4, 8, 12, 14, 16, and 20 kHz (all p < 0.05). No significant losses of outer HCs, inner HCs, or HC cilia were observed (all p > 0.05). Thus, our study suggests that loss of cochlear inner HC ribbon synapse after SS exposure is a contributor to the development of tinnitus without changing hearing threshold.

The red distribution width and the platelet distribution width as prognostic predictors in gastric cancer.

BACKGROUND: Increasing attention is focused on the relationship of inflammation biomarkers with malignant tumors. The purpose of the present study was to detect whether the preoperative the red distribution width (RDW) and the platelet distribution width (PDW) can be used to distinguish patients with gastric cancer (GC) or early stage GC from the healthy controls and predict the progression and prognosis of the GC. METHODS: The RDW and PDW values of 227 patients with GC and 164 patients with early GC were retrospectively analyzed comparing with 101 healthy controls. In addition, the clinicopathological features, survival curves and prognosis of the patients with GC were compared between the high and low groups according to the RDW and PDW values. RESULTS: Significant higher RDW and lower PDW were detected in patients with GC and early GC compared to the healthy controls. A higher RDW was significantly associated with older age, a larger tumor diameter, deeper tumor infiltration, and lymph node metastasis while a lower PDW was significantly associated with male, older age, a larger tumor diameter, deeper tumor infiltration, elevated CEA and CA125. Increased RDW was significantly associated with worse overall survival (OS) and disease-free survival (DFS) for GC (P = 0.042 and P = 0.033, respectively) and early GC (P = 0.037 and P = 0.009, respectively) while decreased PDW indicated a significantly association with poor DFS for early GC (P = 0.006). Univariate and multivariate survival analysis showed that RDW and PDW can act as independent prognostic factors for DFS (P = 0.028 and P = 0.020) in patients with early GC. CONCLUSION: The preoperative RDW and PDW were simple and convenient predictive factors for the progression and prognosis of patients with GC.

Cancer-associated fibroblast-secreted miR-421 promotes pancreatic cancer by regulating the SIRT3/H3K9Ac/HIF-1α axis.

This study was designed to explore the effects of exosomal miR-421 secreted by cancer-associated fibroblasts (CAFs) on pancreatic cancer (PC) progression and the mechanisms involved. CAFs and exosomes (exos) were isolated and identified. PC cells were treated with CAF-derived exos (CAF-exos). Western blotting and quantitative polymerase chain reaction (qPCR) were used to measure miR-421, sirtuin-3 (SIRT3), and hypoxia duciblefactors-1 alpha (HIF-1α) levels. Cell counting kit-8 (CCK-8), wound-healing, and transwell migration assays were used to measure proliferation, migration, and invasion abilities of the cells. Dual-luciferase assay and RNA immunoprecipitation (RIP) experiment analyzed the relationship between miR-421 and SIRT3. Chromatin immunoprecipitation (f)-verified H3K9Ac enrichment in the HIF-1α promoter region. In vivo tumorigenesis experiments were performed to further explore the effects of exosomal miR-421 from CAFs on PC. CAFs and exos were successfully isolated. CAF-exo-treated PC cells highly expressed miR-421 and had increased cell proliferation, migration, and invasion abilities. Knocking down miR-421 increased the expression of SIRT3. SIRT3 is a target of miR-421, and inhibiting the expression of SIRT3 reversed the negative effects of miR-421 knockdown on PC cell. Knocking down miR-421 in CAF-exo inhibited the expression of HIF-1α in PC cells. Moreover, SIRT3-mediated HIF-1α expression by regulating H3K9Ac. HIF-1α overexpression reversed the inhibiting effects of SIRT3 overexpression on PC progression and counteracted the inhibiting effects of miR-421 knockdown on glycolysis. Moreover, in vivo tumorigenesis experiments showed that knocking down miR-421 attenuated CAF-exo induced tumor growth. Exosomal miR-421 from CAFs promoted PC progression by regulating the SIRT3/H3K9Ac/HIF-1α axis. This study provided insights into the molecular mechanism of PC.

LINC00960 regulates cell proliferation and glycolysis in pancreatic cancer through the miR-326-3p/TUFT1/AKT-mTOR axis.

Pancreatic cancer (PC) is a common malignant cancer characterized by high mortality and poor prognosis. LINC00690 was involved in the occurrence and progression of PC, but the underlying mechanisms require further investigation. The goal of this study was to figure out how LINC00960 mediates glycolysis in PC. LINC00960, miR-326-3p, and Tuftelin 1 (TUFT1) expression levels were detected in PC cell lines. LINC00960 and TUFT1 expression levels were increased in PC cells when compared with normal pancreatic cells, whereas miR-326-3p expression levels were decreased. The expression levels of LINC00690 affected glycolysis in PC, and inhibition of LINC00960 inhibited tumor growth in vivo. LINC00690 targeted and suppressed the expression of miR-326-3p. MiR-326-3p bound to TUFT1, and miR-326-3p inhibited AKT-mTOR pathway activation via TUFT1. In conclusion, the depletion of LINC00960 repressed cell proliferation and glycolysis in PC by mediating the miR-326-3p/TUFT1/AKT-mTOR axis. Thus, we present a novel mechanism underlying the progression of PC that suggests LINC00960 is a potential therapeutic target for this cancer.

Erratum: Expression of Tim-3 in breast cancer tissue promotes tumor progression.

[This corrects the article on p. 1157 in vol. 11, PMID: 31938210.].

Cellular origin and response of flat epithelium in the vestibular end organs of mice to Atoh1 overexpression.

A flat epithelium (FE) may be found in the vestibular end organs of humans and mice with vestibular dysfunction. However, the pathogenesis of FE is unclear and inducing hair cell (HC) regeneration is challenging, as both HCs and supporting cells (SCs) in vestibular FE are damaged. To determine the cellular origin of vestibular FE and examine its response to Atoh1 overexpression, we fate-mapped vestibular epithelial cells in three transgenic mouse lines (vGlut3-iCreERT2:Rosa26tdTomato, GLAST-CreERT2:Rosa26tdTomato, and Plp-CreERT2:Rosa26tdTomato) after inducing a lesion by administering a high dose of streptomycin. Atoh1 overexpression in vestibular FE was mediated by an adeno-associated virus serotype 8 (AAV8) vector. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, was administered with AAV8 to enhance Atoh1 overexpression. The transduction efficiency and population of myosin VIIa-positive cells were analyzed. A small number of HCs were present in vestibular FE. FE did not show broad GLAST-Cre or Plp-Cre expression, unlike the original SCs. SAHA dramatically enhanced AAV8-mediated exogenous gene overexpression, and Atoh1 overexpression plus SAHA promoted myosin VIIa expression in FE cells. Our data provide insight into FE formation and will facilitate studies of gene therapy for vestibular FE.

Age-related insult of cochlear ribbon synapses: An early-onset contributor to D-galactose-induced aging in mice.

Presbycusis results from age-related degeneration of the auditory system. D-galactose (D-gal)-induced aging is an ideal and commonly used animal model in aging research. Previous studies demonstrate that administration of D-gal can activate mitochondria-dependent apoptosis in the cochlear stria vascularis. However, D-gal-induced changes to cochlear inner (IHCs) and outer (OHCs) hair cells, spiral ganglion cells (SGCs), and ribbon synapses connecting IHCs and SGCs have not been systematically reported. The current study investigated changes in the numbers of hair cells, SGCs, and ribbon synapses in the mouse model of aging. We found that in comparison to control mice, the numbers of ribbon synapses and their nerve fibers were significantly decreased in D-gal-treated mice, whereas the numbers of OHCs, IHCs, and SGCs were almost unchanged. Moreover, hair cell stereocilia were also not obviously influenced by D-gal administration. Although D-gal-induced aging did not significantly shift the auditory brainstem response (ABR) thresholds in the 8, 16, and 32 kHz frequency bands, the amplitude and latency of the ABR wave I, reflecting ribbon synapse functions, were abnormal in D-gal-treated mice compared to control mice. We also found that 8-hydroxy-2-deoxyguanosine, a marker of oxidative DNA damage, was significantly increased in mitochondria of cochleae from mice exposed to D-gal-induced aging in comparison to control mice. Moreover, D-gal administration increased the levels of H2O2 and mitochondrial 3860-bp common deletion, and decreased superoxide dismutase activity and ATP production in the cochlea. Furthermore, compared with control mice, the protein levels of NADPH oxidase 2 and uncoupling protein 2 were significantly increased in the cochlea of D-gal-treated mice. Taken together, these findings support that the cochlear ribbon synapse is the primary insult site in the early stage of presbycusis, and mitochondrial oxidative damage and subsequent dysfunctions might be responsible for this insult.

NADPH oxidase inhibitor apocynin decreases mitochondrial dysfunction and apoptosis in the ventral cochlear nucleus of D-galactose-induced aging model in rats.

Presbycusis has become a common sensory deficit in humans. Oxidative damage to mitochondrial DNA and mitochondrial dysfunction is strongly associated with the aging of the auditory system. A previous study established a mimetic rat model of aging using D-galactose (D-gal) and first reported that NADPH oxidase-dependent mitochondrial oxidative damage and apoptosis in the ventral cochlear nucleus (VCN) might contribute to D-gal-induced central presbycusis. In this study, we investigated the effects of apocynin, an NADPH oxidase inhibitor, on mitochondrial dysfunction and mitochondria-dependent apoptosis in the VCN of D-gal-induced aging model in rats. Our data showed that apocynin decreased NADPH oxidase activity, H2O2 levels, mitochondrial DNA common deletion, and 8-hydroxy-2-deoxyguanosine (8-OHdG) expression and increased total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) activity in the VCN of D-gal-induced aging model in rats. Moreover, apocynin also decreased the protein levels of phospho-p47phox (p-p47phox), tumor necrosis factor alpha (TNFα), and uncoupling protein 2 (UCP2) in the VCN of D-gal-induced aging model in rats. Meanwhile, apocynin alleviated mitochondrial ultrastructure damage and enhanced ATP production and mitochondrial membrane potential (MMP) levels in the VCN of D-gal-induced aging model in rats. Furthermore, apocynin inhibited cytochrome c (Cyt c) translocation from mitochondria to the cytoplasm and suppressed caspase 3-dependent apoptosis in the VCN of D-gal-induced aging model in rats. Consequently, our findings suggest that neuronal survival promoted by an NADPH oxidase inhibitor is a potentially effective method to enhance the resistance of neurons to central presbycusis.

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea.

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear "synaptopathy", which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

Expression of Tim-3 in breast cancer tissue promotes tumor progression.

T-cell immunoglobulin mucin-3 (Tim-3) plays a pivotal role in immune regulation and tolerance induction as a negative regulatory molecule on innate versus adaptive immune cells, especially in antitumor immunity. However, the mechanism of Tim-3 expression on tumor cells and the mechanism that inhibits anti-tumor immunity are obscure. In this present study, we aimed to investigate the functions of Tim-3 in breast cancer and to explore its correlation to tumor prognosis. In a total of 42 clinical samples of invasive breast cancer, Tim-3 was semiquantitatively scored based on both distribution and intensity of immunohistochemistry staining and was found to correlate with clinicopathological parameters. Western blotting was used to detect the expression of Tim-3 in breast cancer cells. Furthermore, the effect of Tim-3 in breast cancer cells was evaluated after overexpression by ADV-Tim-3 and downregulation by Tim-3-siRNA. High immunoreactivity of Tim-3 was found to be significantly correlated with clinical stage, metastasis, KI67, and a lower 5-year survival rate. We supported this finding by confirming the presence of Tim-3 protein in the breast cell lines. Downregulation of Tim-3 significantly inhibited the proliferation, migration, and invasion of breast cancer cells while it promoted apoptosis. Overexpression of Tim-3 promoted the proliferation, migration, and invasion of breast cancer cells and inhibited apoptosis. Taken together, as a valuable marker of breast cancer prognosis, Tim-3 in breast cancer cells play an important role in the progression of breast cancer and may be an effective novel target in tumor prevention and treatment.

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice.

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

T Cell Immunoglobulin- and Mucin-Domain-Containing Molecule 3 Gene Polymorphisms and Susceptibility to Invasive Breast Cancer.

The T-cell immunoglobulin and mucin domain containing molecule 3 (TIM-3) gene is an important immune regulatory molecule. In fact, studies have shown that polymorphisms in the (TIM-3) gene may be associated with various cancers. The goal of this study was to investigate whether the -1516G/T, -574G/T, or +4259T/G single-nucleotide polymorphisms (SNPs) in the TIM-3 gene contribute to a genetic susceptibility to invasive breast cancer in the Han ethnicity of northern China. Genotyping of the TIM-3 -1516G/T, -574G/T and +4259T/G were performed in 301 patients with invasive breast cancer and in 151 healthy individuals via a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The results showed that the prevalence of the +4259T/G genotype and the +4259G allele were significantly increased in the breast cancer patients compared to the controls [odds ratio (OR)=7.641, 95% confidence interval (CI), 1.795-32.522, P=0.001; OR=7.317, 95%CI, 1.731-30.925, P=0.001, respectively]. The GGG haplotype had a significantly different distribution between patients and controls (OR=5.421, 95%CI, 1.263-23.275, P=0.011). In addition, the prevalence of the +4259T/G polymorphism was higher in patients with metastasis than those without metastasis (13.6% vs. 4.8%, respectively, OR=3.158, 95%CI, 1.300-7.672, P=0.011). Furthermore, results showed that the prevalence of the +4259T/G genotype was correlated with the intensity of Ki-67 by immunohistochemical staining (P=0.022). Overall, these results suggested that the +4259T/G SNP in the TIM-3 gene may play an important role as a genetic risk factor for the progression and prognosis of invasive breast cancer in these patients. in Han ethnicity of northern China.

Cochleovestibular gene transfer in neonatal mice by canalostomy.

Impairments of the inner ear result in sensorineural hearing loss and vestibular dysfunction in humans. A large proportion of these disorders are congenital, and involve both auditory and vestibular systems. Therefore, genetic interventions to correct deficits must be administered during early developmental stages. In this study, we evaluated inner ear gene transfer in neonatal mice by canalostomy using an adeno-associated virus serotype 8 (AAV8) vector. AAV8 with the green fluorescence protein (GFP) gene was inoculated into the inner ear of the neonatal mice through the posterior semicircular canal (canalostomy). At 30 days following surgery, animals were subjected to swim tests and auditory brainstem response measurements. Then, the animals were euthanized and temporal bones were harvested for whole-mount preparation. GFP expression and morphological changes in the inner ear were assessed by immunohistochemistry. After surgery, no signs of vestibular dysfunction were found, and there were no significant differences in the auditory brainstem response threshold between AAV8-inoculated ears and nonsurgery ears. In the surgery ears, extensive GFP expression and no morphological lesions were detected in the cochlear and vestibular end organs. Robust GFP expression was found in inner hair cells, marginal cells, vestibular hair cells, and vestibular supporting cells. In conclusion, AAV8 inoculation through canalostomy into the inner ears of neonatal mice led to extensive overexpression of exogenous genes in the inner ear without affecting hearing or vestibular function. It serves as a promising approach for gene therapy of congenital cochleovestibular diseases.

Investigation of T-cell immunoglobulin- and mucin-domain-containing molecule-3 (TIM-3) polymorphisms in essential thrombocythaemia (ET).

OBJECTIVES: T-cell immunoglobulin- and mucin-domain-containing molecule-3 (TIM-3) is preferentially expressed on terminally differentiated Th1 cells and inhibits their IFN-γ production. It has been reported that chronic inflammation may be an important driving force for myeloproliferative neoplasms (MPNs). Therefore, we hypothesized that as an important inflammation regulator, TIM-3 may be involved in essential thrombocythaemia (ET). The goal of this study was to investigate whether the -1516G > T, -574G > T and +4259T > G single-nucleotide polymorphisms (SNPs) within the TIM-3 gene contribute to the genetic susceptibility of individuals to ET. METHODS: Genotyping of the TIM-3 -1516G > T, -574G > T and + 4259T > G SNPs was performed in 175 patients with ET and in 151 controls via a polymerase chain reaction-restriction fragment length polymorphism assay. We also investigated the relationships between the genotypes of each SNP and the risk factors of ET such as routine blood indexes, age and JAK2 V617F mutation. RESULTS: The genotype and allele frequencies of the -1516G > T SNP (p = 0.016 and 0.019, respectively), the -574G > T SNP (p = 0.035 and 0.038, respectively) and the +4259T > G SNP (p = 0.036 and 0.038, respectively) of the ET patients and the controls were significantly different. A haplotype analysis found that the GGT and TGT haplotypes had significantly different distributions between ET and controls (p = 0.041 and 0.041, respectively). However, no significant differences were detected between the genotypes of all SNPs and routine blood indexes, age and JAK2V617F mutation. CONCLUSION: The -1516G > T, -574G > T and +4259T > G SNPs within TIM-3 gene might play an important role as a genetic risk factor in the pathogenesis of ET.