"I Sometimes Pretended to Get Groceries": Restrictive Deterrence in Drug Dealing.

Despite steady progress in drug control work, drug-related crimes are ranked third in the total number of criminal cases in China. Based on in-depth interviews with 24 offenders who are now incarcerated for drug offenses, this paper examines the strategies employed by drug offenders to evade detection and mitigate their punishment for drug dealing. Their most frequently-used strategies focus on filtering out risky people, ensuring information asymmetry and knowing when to stop. Exploring how offenders adapt their behavior in response to punishment helps to further our understanding of crime commission and control.

Co-transduction of dual-adeno-associated virus vectors in the neonatal and adult mouse utricles.

Adeno-associated virus (AAV)-mediated gene transfer is an efficient method of gene over-expression in the vestibular end organs. However, AAV has limited usefulness for delivering a large gene, or multiple genes, due to its small packaging capacity (< 5 kb). Co-transduction of dual-AAV vectors can be used to increase the packaging capacity for gene delivery to various organs and tissues. However, its usefulness has not been well validated in the vestibular sensory epithelium. In the present study, we characterized the co-transduction of dual-AAV vectors in mouse utricles following inoculation of two AAV-serotype inner ear (AAV-ie) vectors via canalostomy. Firstly, co-transduction efficiencies were compared between dual-AAV-ie vectors using two different promoters: cytomegalovirus (CMV) and CMV early enhancer/chicken ß-actin (CAG). In the group of dual AAV-ie-CAG vectors, the co-transduction rates for striolar hair cells (HCs), extrastriolar HCs, striolar supporting cells (SCs), and extrastriolar SCs were 23.14 ± 2.25%, 27.05 ± 2.10%, 57.65 ± 7.21%, and 60.33 ± 5.69%, respectively. The co-transduction rates in the group of dual AAV-ie-CMV vectors were comparable to those in the dual AAV-ie-CAG group. Next, we examined the co-transduction of dual-AAV-ie-CAG vectors in the utricles of neonatal mice and damaged adult mice. In the neonatal mice, co-transduction rates were 52.88 ± 3.11% and 44.93 ± 2.06% in the striolar and extrastriolar HCs, respectively, which were significantly higher than those in adult mice. In the Pou4f3+/DTR mice, following diphtheria toxin administration, which eliminated most HCs and spared the SCs, the co-transduction rate of SCs was not significantly different to that of normal utricles. Transgene expression persisted for up to 3 months in the adult mice. Furthermore, sequential administration of two AAV-ie-CAG vectors at an interval of 1 week resulted in a higher co-transduction rate in HCs than concurrent delivery. The auditory brainstem responses and swim tests did not reveal any disruption of auditory or vestibular function after co-transduction with dual-AAV-ie vectors. In conclusion, dual-AAV-ie vectors allow efficient co-transduction in the vestibular sensory epithelium and facilitate the delivery of large or multiple genes for vestibular gene therapy.

AAV8-mediated Atoh1 overexpression induces dose-dependent regeneration of vestibular hair cells in adult mice.

Vestibular hair cells (HCs) are mechanoreceptors for the detection of head movement. Vestibular HCs of adult mammals never completely regenerate after damage, resulting in vestibular dysfunction. Overexpression of Atoh1 is effective for inducing HC regeneration. However, method of clinical feasibility and improvement of regenerative extent are both in need. Here we used an adeno-associated virus (AAV) serotype 8 vector of two different titers to overexpress Atoh1 in the injured utricles of adult mice. One month after virus inoculation, abundant myosin VIIa-positive cells and immature stereocilia were observed. Quantitative analyses revealed that Atoh1 overexpression replenished vestibular HCs in a dose-dependent manner. Vectors of a higher titer increased the number of myosin VIIa-positive cells compared to those of lower titer. Moreover, only Atoh1 overexpression in the higher titer group enhanced stereocilium regeneration, which is an important step in the maturation of regenerated HCs. Although the current treatment failed to initiate functional recovery of the animals, our results prompt further improvements in the recovery of vestibular dysfunction by AAV.

Epithelial-Mesenchymal Transition Participates in the Formation of Vestibular Flat Epithelium.

The vestibular sensory epithelium of humans and mice may degenerate into a layer of flat cells, known as flat epithelium (FE), after a severe lesion. However, the pathogenesis of vestibular FE remains unclear. To determine whether the epithelial-mesenchymal transition (EMT) participates in the formation of vestibular FE, we used a well-established mouse model in which FE was induced in the utricle by an injection of streptomycin into the inner ear. The mesenchymal and epithelial cell markers and cell proliferation were examined using immunofluorescence staining and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The function of the EMT was assessed through transcriptome microarray analysis. The results demonstrated that mesenchymal cell markers (α-SMA, S100A4, vimentin, and Fn1) were upregulated in vestibular FE compared with the normal utricle. Robust cell proliferation, which was absent in the normal status, was observed in the formation of FE. Microarray analysis identified 1,227 upregulated and 962 downregulated genes in vestibular FE. Gene Ontology (GO) analysis revealed that differentially expressed genes (DEGs) were highly associated with several EMT-related GO terms, such as cell adhesion, cell migration, and extracellular matrix. Pathway enrichment analysis revealed that DEGs were enriched in the EMT-related signaling pathways, including extracellular matrix (ECM)-receptor interaction, focal adhesion, PI3K/Akt signaling pathway and cell adhesion molecule. Protein-protein interaction networks screened 20 hub genes, which were Akt, Casp3, Col1a1, Col1a2, Fn1, Hgf, Igf1,Il1b, Irs1, Itga2, Itga5, Jun, Mapk1, Myc, Nras, Pdgfrb, Tgfb1, Thbs1, Trp53, and Col2a1. Most of these genes are reportedly involved in the EMT process in various tissues. The mRNA expression level of hub genes was validated using qRT-PCR. In conclusion, the present study indicates that EMT plays a significant role in the formation of vestibular FE and provides an overview of transcriptome characteristics in vestibular FE.

Suggestion of a Modified Classification for Congenital Middle Ear Cholesteatoma: Based on the Clinical Characteristics and Staging of Fifty-Seven Patients.

Objective: To explore more refined classification methods of congenital middle ear cholesteatoma (CMEC) based on two existing staging systems. Subjects and Methods: This study involved a retrospective data review of 57 patients (61 ears involved) with CMEC requiring the surgical treatment. Patients were classified into different stages according to Nelson, Potsic, and Modified Nelson staging system. Preoperative data and intraoperative findings were recorded. Results: The mean age at operation was 15 ± 15.04 years with a median of 10 years. The main clinical manifestation was hearing loss (72.13%). CMEC mass was mainly located in the posterior portion of the tympanic cavity (65.57%). No patient was classified into Potsic stage II. The erosion of incus happened in all cases. Patients with Nelson type 2 and type 3 had erosions to the structures out of middle ear, such as dura mater, lateral semicircle canal, and facial canal. Postoperative follow-up time was more than 24 months. Recurrence occurred in four patients (6.56%), all of them in Nelson type 2, who had received canal wall down mastoidectomy (three cases) and canal wall up mastoidectomy (one case). Conclusions: Nelson staging system was more suitable for advanced CMEC patients than Potsic staging system. The rare case of Potsic stage II restricted the application of Potsic staging system. Moreover, since both of two staging systems do not distinguish the type of involved ossicles, the authors recommended to subdivide Nelson type 2 into type 2a and type 2b based on the erosion of the ossicular chain, as well as subdivide Nelson type 3 into type 3a and 3b based on the erosion of structures out of middle ear, which was named as Modified Nelson staging system.

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.

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.

The histone deacetylase inhibitor sodium butyrate protects against noise-induced hearing loss in Guinea pigs.

Noise-induced hearing loss (NIHL) severely impacts the quality of life of affected individuals. Oxidative stress resulting from noise exposure is a significant cause of NIHL. Although histone deacetylase (HDAC) inhibitors were shown to protect against NIHL, the underlying mechanism remains unclear, and it is not known how they act on noise-induced oxidative stress. In the current study, we investigated the expression levels of acetyl-histone H3 (Lys9) (H3-AcK9), histone deacetylase 1 (HDAC1), and 3-nitrotyrosine (3-NT), an oxidative stress marker, in a guinea pig model of NIHL using immunohistology and Western blotting. We then assessed the effects of systemic administration of the HDAC inhibitor, sodium butyrate (SB), on noise-induced permanent threshold shifts (PTS), hair cell (HC) loss, and changes in the above mentioned markers. The results showed that SB attenuated noise-induced PTS and outer hair cell loss. SB treatment promoted H3-AcK9 expression and repressed HDAC1 expression in the nuclei of HCs and Hensen's cells after noise exposure. Furthermore, SB attenuated the noise-induced increase of 3-NT expression in HCs and Hensen's cells. These findings suggest that SB protects against NIHL by reversing the noise-induced histone acetylation imbalance and inhibiting oxidative stress in cochlear HCs and Hensen's cells. SB treatment may represent a potential strategy to prevent and treat NIHL.

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.

E2F1-CDK1 pathway activation in kanamycin-induced spiral ganglion cell apoptosis and the protective effect of CR8.

Cochlear hair cell loss results in the secondary loss of spiral ganglion cells (SGCs). The death of these SGCs is due to apoptosis. The E2F1-cyclin dependent kinase 1 (CDK1) pathway is believed to represent an important mechanism of neuronal cell death. However, the role of this pathway in spiral ganglion neuronal apoptosis has not yet been reported. In this study, we deafened guinea pigs with a subcutaneous injection of kanamycin followed by an intravenous infusion of furosemide and then assayed the expression levels of cleaved caspase-3, E2F1, CDK1 and cleaved caspase-9 during the induced SGC apoptosis. Our results revealed that co-administration of kanamycin and furosemide rapidly induced hair cell loss in the guinea pigs and then resulted in a progressive loss of SGCs. Expression levels of E2F1 and CDK1 were obviously up-regulated at 1 and 3 days after deafening. Cleaved caspase-9 also increased robustly 1 or 2 weeks after the deafening procedure. The up-regulation of E2F1, CDK1 and cleaved caspase-9 was significantly attenuated by the systemic injection of CR8 (1mg/kg/day, intraperitoneally) starting at 5min after deafening. These findings indicate that the activation of the E2F1-CDK1 pathway and cell cycle re-entry contributes to the apoptosis of SGCs and that the selective inhibition of this signaling cascade may represent an attractive therapeutic strategy. CR8 has the potential to protect SGCs from apoptosis.

Tumor Necrosis Factor-α-Induced Ototoxicity in Mouse Cochlear Organotypic Culture.

Tumor necrosis factor (TNF)-α is a cytokine involved in acute inflammatory phase reactions, and is the primary upstream mediator in the cochlear inflammatory response. Treatment of the organ of Corti with TNF-α can induce hair cell damage. However, the resulting morphological changes have not been systematically examined. In the present study, cochlear organotypic cultures from neonatal mice were treated with various concentrations and durations of TNF-α to induce inflammatory responses. Confocal microscopy was used to evaluate the condition of hair cells and supporting cells following immunohistochemical staining. In addition, the ultrastructure of the stereocilia bundle, hair cells, and supporting cells were examined by scanning and transmission electron microscopy. TNF-α treatment resulted in a fusion and loss of stereocilia bundles in hair cells, swelling of mitochondria, and vacuolation and degranulation of the endoplasmic reticulum. Disruption of tight junctions between hair cells and supporting cells was also observed at high concentrations. Hair cell loss was preceded by apoptosis of Deiters' and pillar cells. Taken together, these findings detail the morphological changes in the organ of Corti after TNF-α treatment, and provide an in vitro model of inflammatory-induced ototoxicity.

A review of prophylactic antibiotics use in plastic surgery in China and a systematic review.

The purpose of this study was to investigate the use of antibiotic prophylaxis for plastic surgical procedures at our hospital, and to perform a systematic literature review of randomized controlled trials evaluating the use of prophylactic antibiotics in plastic surgery. The records of patients who received plastic surgical procedures with Class I surgical incisions between 2009 and 2010 were retrospectively reviewed. A systematic literature review was conducted for studies examining the use of prophylactic antibiotics for Class I surgical wounds. A total of 13,997 cases with Class I surgical incisions were included. Prophylactic antibiotics were given in 13,865 cases (99.1%). The antibiotics used were primarily cefuroxime, clindamycin, metronidazole, cefoxitin sodium, and gentamicin. The average duration of administration was 4.84 ± 3.07 (range, 1-51) days. Antibiotics were administered postoperatively in >99% of cases while preoperative antibiotic administration was only given in 32 cases (0.23%). Wound infections occurred in 21 cases for an overall infection rate of 0.15%. Fourteen studies met the inclusion criteria of the systematic review. There was marked variation in the timing of antibiotic administration with antibiotics given pre-, peri-, and postoperatively. Of studies that compared the use of prophylactic antibiotics with placebo, a reduction in wound infections was noted in 4 trials and no difference was noted in 6 trials. No significant difference in infection rates was shown between the prophylactic and postoperative arms. In conclusion, prophylactic antibiotics are overused in plastic surgical procedures. Evidence-based guidelines for the use of prophylactic antibiotics in plastic surgical procedures are needed.

Temporospatial expression and cellular localization of VGLUT3 in the rat cochlea.

Vesicular glutamate transporter 3 (VGLUT3) plays an important role in hearing, and VGLUT3 knockout mice are deaf. However, the mechanisms whereby VGLUT3 exerts its effects in the cochlea are not well established. Elucidating the developmental and aging dynamics of VGLUT3 localization and expression in the cochlea would aid a functional understanding of auditory glutamatergic transmission. In this study, we characterized the expression of VGLUT3 in rat auditory epithelium and spiral ganglion cells (SGCs) during postnatal development and with age using immunohistochemistry and quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). We observed VGLUT3 expression in cochlea inner hair cells (IHCs) and SGCs as early as postnatally, and its expression increased gradually with maturity. However, the expression of VGLUT3 in the IHCs decreased in the aging rats compared to postnatal day 60 (P60), and VGLUT3 was still expressed in the remaining SGCs in the aging rats. VGLUT3-immunopositive punctate structures were only found in the cytoplasm of SGCs, not IHCs, at P7, or later. Variation in VGLUT3 mRNA expression evaluated by quantitative real time RT-PCR was consistent with the immunohistochemical data except for no statistical difference between P60 and aging rats in the whole cochlea level. Our data suggest that VGLUT3 likely has developmental and physiological roles in the rat cochlea during postnatal development as well as later in life. VGLUT3 may have additional unknown roles in SGCs besides glutamate transport.