Engineering of the AAV-Compatible Hair Cell-Specific Small-Size Myo15 Promoter for Gene Therapy in the Inner Ear.

Adeno-associated virus (AAV)-mediated gene therapy is widely applied to treat numerous hereditary diseases in animal models and humans. The specific expression of AAV-delivered transgenes driven by cell type-specific promoters should further increase the safety of gene therapy. However, current methods for screening cell type-specific promoters are labor-intensive and time-consuming. Herein, we designed a "multiple vectors in one AAV" strategy for promoter construction in vivo. Through this strategy, we truncated a native promoter for Myo15 expression in hair cells (HCs) in the inner ear, from 1,611 bp down to 1,157 bp, and further down to 956 bp. Under the control of these 2 promoters, green fluorescent protein packaged in AAV-PHP.eB was exclusively expressed in the HCs. The transcription initiation ability of the 2 promoters was further verified by intein-mediated otoferlin recombination in a dual-AAV therapeutic system. Driven by these 2 promoters, human otoferlin was selectively expressed in HCs, resulting in the restoration of hearing in treated Otof -/- mice for at least 52 weeks. In summary, we developed an efficient screening strategy for cell type-specific promoter engineering and created 2 truncated Myo15 promoters that not only restored hereditary deafness in animal models but also show great potential for treating human patients in future.

A protocol for measuring the sexual receptivity of female Drosophila.

Female receptivity in mating is crucial for successful copulation, but protocols for quantifying female behaviors reflecting receptivity are scarce compared to the analysis of male behaviors. Here, we present a protocol for assessing the sexual receptivity of female Drosophila that considers behaviors from both sexes. We describe steps for preparing and loading flies into a courtship chamber, video recording the behaviors of the pairs, and analyzing their behavioral displays. This protocol includes behavior recognition criteria suitable for typical laboratory settings. For complete details on the use and execution of this protocol, please refer to Yang et al. (2023).1.

Sex peptide regulates female receptivity through serotoninergic neurons in Drosophila.

The courtship ritual is a dynamic interplay between males and females. Courtship successfully leading to copulation is determined by the intention of both parties which is conveyed by complex action sequences. In Drosophila, the neural mechanisms controlling the female's willingness to mate, or sexual receptivity, have only recently become the focus of investigations. Here, we report that pre-mating sexual receptivity in females requires activity within a subset of serotonergic projection neurons (SPNs), which positively regulate courtship success. Of interest, a male-derived sex peptide, SP, which was transferred to females during copulation acted to inhibit the activity of SPN and suppressed receptivity. Downstream of 5-HT, subsets of 5-HT7 receptor neurons played critical roles in SP-induced suppression of sexual receptivity. Together, our study reveals a complex serotonin signaling system in the central brain of Drosophila which manages the female's desire to mate.

Hearing of Otof-deficient mice restored by trans-splicing of N- and C-terminal otoferlin.

Mutations to the OTOF gene are among the most common reasons for auditory neuropathy. Although cochlear implants are often effective in restoring sound transduction, there are currently no biological treatments for individuals with variants of OTOF. Previous studies have reported the rescue of hearing in DFNB9 mice using OTOF gene replacement although the efficacy needs improvement. Here, we developed a novel dual-AAV-mediated gene therapy system based on the principles of protein trans-splicing, and we show that this system can reverse bilateral deafness in Otof -/- mice after a single unilateral injection. The system effectively expressed exogenous mouse or human otoferlin after injection on postnatal day 0-2. Human otoferlin restored hearing to near wild-type levels for at least 6 months and restored the release of synaptic vesicles in inner hair cells. Our study not only provides a preferential clinical strategy for the treatment of OTOF-related auditory neuropathies, but also describes a route of development for other large-gene therapies and protein engineering techniques.

Serotonin Signals Overcome Loser Mentality in Drosophila.

Traumatic experiences generate stressful neurological effects in the exposed persons and animals. Previous studies have demonstrated that in many species, including Drosophila, the defeated animal has a higher probability of losing subsequent fights. However, the neural basis of this "loser effect" is largely unknown. We herein report that elevated serotonin (5-HT) signaling helps a loser to overcome suppressive neurological states. Coerced activation of 5-HT neurons increases aggression in males and promotes losers to both vigorously re-engage in fights and even defeat the previous winners and regain mating motivation. P1 neurons act upstream and 5-HT1B neurons in the ellipsoid body act downstream of 5-HT neurons to arouse losers. Our results demonstrate an ancient neural mechanism of regulating depressive behavioral states after distressing events.

The Effect of Inner Friction on Concrete Fracture Behavior under Biaxial Compression: A 3D Mesostructure Study.

The mechanical behavior of concrete under biaxial loading condition (especially biaxial compression) is one of the most important indexes to evaluate the quality of concrete. To study the mechanical behavior of concrete under biaxial compression at mesoscale, we adopted our recently developed 3D numerical model based on Voronoi tessellation and cohesive elements. A constitutive model considering the friction effect is used in the model to characterize the fracture behavior of all potential fracture surfaces inside the concrete. A series of numerical experiments with different biaxial compression stress ratios were carried out. It was found that with the increase of the biaxial compression ratio, the proportion of energy increment caused by friction stress increases. The effect of inner friction coefficient on the biaxial relative strength was also investigated, and this kind of study is hard to be carried out through laboratory experiments. The results show that the inner friction coefficient has a great influence on the biaxial relative strength of concrete, and there is a positive correlation between these two parameters. Based on the above rules, a conservative biaxial relative compression strength envelope is obtained by setting the inner friction coefficient as zero.

[Study on difference of flavonoids content in stems and leaves of Mentha Haplocalycis Herba in different harvest periods].

The dynamic changes of active components in stems and leaves of Mentha Haplocalycis Herba(mint) at different harvest periods were investigated, and the optimum harvest time of mint was explored. In this study, hesperidin, diosmin, didymin and buddleoside were selected as flavonoids index components of mint, and the QAMS method was established to measure the contents of these flavonoids in mint. The contents of 4 flavonoid glycosides in the mint stems and leaves from three habitats harvested in different time were studied and evaluated comprehensively using statistical analysis and principal component analysis (PCA). The results showed that the contents of 4 components in the leaves are higher than that in the stems despite of habitats and harvest time, and they all exhibited dynamic changes along with the harvest periods within the same habitat. Three harvest periods in mid April, mid September and late October scored higher in comprehensive evaluation in Jiangsu region, the genuine producing area of Mentha Haplocalycis Herba. Combined with the yield and contents of active compounds, the optimum harvest time of mint in Jiangsu region was mid September and late October, which is basically consistent with the traditional harvesting periods.

Regulation effect of zinc fingers and homeoboxes 2 on alpha-fetoprotein in human hepatocellular carcinoma.

Aim. To investigate the relationship between alpha-fetoprotein and zinc fingers and homeoboxes 2 in hepatocellular carcinoma. Materials and Methods. The expressions of zinc fingers and homeoboxes 2, nuclear factor-YA, and alpha-fetoprotein mRNA in 63 hepatocellular carcinoma were detected by reverse transcriptase-polymerase chain reaction and compared with the clinical parameters of the patients. Selectively, silence of zinc fingers and homeoboxes 2 in HepG2 cells was detected by RNA interference technique. Results. Alpha-fetoprotein mRNA expression was detected in 60.3% of hepatocellular carcinoma cases. Zinc fingers and homeoboxes 2 mRNA expression (36.5%) was significantly negatively correlated with serum alpha-fetoprotein concentration and mRNA expression. A strong positive correlation was found between zinc fingers and homeoboxes 2 and nuclear factor-YA mRNA expression (42.9%), while the latter was negatively correlated with serum alpha-fetoprotein concentration and mRNA expression. Treatment with zinc fingers and homeoboxes 2 small interfering RNA led to 85% and 83% silence of zinc fingers and homeoboxes 2 mRNA and protein expression and 60% and 61% reduction of nuclear factor-YA mRNA and protein levels in the HepG2 cells, respectively. Downregulation of zinc fingers and homeoboxes 2 also induced a 2.4-fold increase in both alpha-fetoprotein mRNA and protein levels. Conclusions. Zinc fingers and homeoboxes 2 can regulate alpha-fetoprotein expression via the interaction with nuclear factor-YA in human hepatocellular carcinoma and may be used as an adjuvant diagnostic marker for alpha-fetoprotein-negative hepatocellular carcinoma.

A new convenient technique for making cell blocks.

Cell block sections serve as an important diagnostic annex for cytological smears, liquid-based SurePath cytology and the Liquid-based Thin-prep Cytology Test (TCT). A variety of methods for the preparation of cell blocks are described in the literature and the techniques in cell blocks are in continuous improvement. A new technique for making cell blocks was introduced in the present study. We first used pregelatinized starch as the frame for the cell block, which is a really simple and economic method, because it can be carried out at room temperature without additional special instruments. We have performed hematoxylin and eosin (HE) staining, immunohistochemistry analysis and fluorescence in situ hybridization (FISH) in the cell block sections in 122 cytological specimens. The results demonstrated in this article show that pregelatinized starch is a useful frame for cell blocks. The pregelatinized starch can effectively collect even a few cells with powerful adhesiveness. Therefore, this new technique for making cell blocks is especially useful for cytologic samples with low cellularity, such as cerebrospinal fluid specimens.

[Coupling anaerobic baffled reactor and membrane-aerated biofilm reactor].

Based on the consistent anaerobic status of outer layer of membrane-aerated biofilm reactor (MABR) and internal anaerobic baffled reactor (ABR), MABR and ABR were started up separately. The aerating membrane module was installed into a compartment of anaerobic baffled bioreactor to form the Hybrid MAB-ABR (HMABR). After the installation of membrane module, total COD and VFA concentrations in the HMABR effluent were deceased by 59.5% and 68.1% respectively, with increased nitrogenous pollutant remove efficiency by 83.5%, at influent COD concentration of 1600 mg/L and NH4+ -N concentration of 80 mg/L. When organic loading rate was increased by 50%, the effluent COD concentration was still below the level of 60 mg/L, indicating its good capability of counteracting influent organic loading fluctuation. Due to the decreased COD concentration and increased nitrate concentration in the third compartment after installing the membrane module, the biogas volume and methane contents in the third compartment were decreased, resulting in the steady and excellent effluent quality. In this hybrid process, the improved simultaneous removal of carbon and nitrogen for high-strength nitrogenous organic pollutants was realized in a single reactor.

[Influence of controlling factors on partial nitrification performance in membrane bioreactor].

A membrane bioreactor (MBR) was developed successfully to carry out partial nitrification process. Temperature, and dissolved oxygen (DO) were investigated as the factors which may affect the results. It has been proved that the optimal operational parameters were at 35 degrees C, ammonia loading 0.45 kg x (m3 x d)(-1) and < 0.5 mg/L, respectively, with the effluent NO3(-) -N concentration below 20 mg x L(-1) and rho(NO2(-) -N)/rho(NH4(+) -N) ratio being close to 1.0. It is not observed severe membrane fouling during all the experiment. Fluorescence in situ hybridization analysis indicated that aerobic ammonium oxidizers were the dominant population, and nitrite-oxidizing bacteria were inhibited. The microbiological community analysis further provided the necessary biological information for the realization of partial nitrification.

[Simultaneous nitrification and denitrification of membrane-aerated biofilm reactor].

A carbon membrane-aerated biofilm reactor was developed to treat municipal wastewater, in which the carbon and nitrogen were removed simultaneously. The results showed that COD removal, NH4+-N removal and TN removal efficiency could reach 82.5%, 95.1% and 84.2%, respectively, under the conditions of intra-membrane pressure of 13.6 kPa, HRT of 14 h, influent COD and NH4+-N concentrations of 338 mg/L and 75 mg/L. However, in the last period during the operation of the reactor, the TN removal efficiency dramatically decreased because of the excessive growth of biomass on the nonwoven fiber, which also had serious negative effect on nitrification course. The microbiological community and spatial profiles were observed by fluorescence in situ hybridization and scanning electron microscopy. The anaerobic and anoxic bacteria were mainly located in the outer anaerobic region of the biofilm, while the aerobic ammonium oxidizing bacteria were mainly located in the inner aerobic region of the biofilm. The co-existing and coupling effect of aerobic nitrifying bacteria and anaerobic denitrifying bacteria provided the large biological potential for the simultaneous nitrification and denitrification in the carbon membrane-aerated biofilm reactor.

[Promoting high-temperature hydrolysis under alkaline condition].

The effect of alkaline thermal hydrolysis method on excess sludge and its characteristic parameters(such as soluble chemical oxygen demand, volatile fatty acids, ammonia-nitrogen, pH value, sludge concentration, etc.) were investigated. The results show that the addition of alkali can reduce the resistance of the sludge cells to high temperature and enhance the release and the hydrolysis of organic materials from the sludge cells with the increase of temperature, pH value and reaction time. The concentration of SCOD reached 17,950 mg/L and SCOD/TCOD was 0.65 on the conditions of pH 13, 170 degrees C and 75 min. The maximal dissolving rate of suspended solids (SS) was 67% and 72% for volatile suspended solids (VSS) at pH 13 with 60 min. The concentration of SCOD after alkaline thermal hydrolysis treatment increased with the increase of sludge concentration, in which the nice linear relation was reached and the correlation coefficient (R2) was above 0.97. The increasing rate of the SCOD was improving with the increase of the pH value and the SCOD of unit sludge reached 672 mg/g at pH 13. The effect of three factors on the SCOD was investigated through orthogonal experiment at the temperature of 170 degrees C, the importance was sludge concentration,pH value and reaction time in turn.

[Innovative application of carbon tube aerated membranes to enhance anaerobic baffled reactor for wastewater treatment].

New type hybrid biological reactor was developed by initiating the carbon membrane aeration in ABR which was augmented with carbon aerated membranes to improve wastewater treatment by removing COD, NH4(+) -N and TN. The results showed that on the conditions of intra-membrane pressure of 0.025 MPa, HRT 24 h, influent COD 2000 mg/L, NH4(+) -N 50 mg/L, simultaneous nitrification and denitrification was happened, and COD and NH4(+) -N decreased from 156 mg/L, 36 mg/L to 45 mg/L, 6 mg/L respectively after the membrane aeration with biofilm was initiated in the third compartment of ABR.TN removal reached 87.66%, and VFA and biogas was decreased by 77.12% and 30%, respectively.

[Nitrification performance and microbial community analysis in carbon membrane-aerated biofilm reactor].

A carbon membrane-aerated biofilm reactor was developed to treat nitrogenous inorganic wastewater. Influent NH; -N concentrations and HRT were changed to investigate nitrification performance of reactor,oxygen utilization and NH4+ -N's removal loading. Biofilm's surface characteristics and dominant bacteria of nitrifier were analyzed. The results show that under the conditions of intra-membrane pressure of 0.017 MPa, influent NH4+ -N of 50 mg/L and HRT of 8 h NH4+ -N removal efficiency reaches 96% and effluent average nitrite is 17 mg/L, which benefits short-cut nitrification to a certain extent. The bacteria within biofilm consume all oxygen supplied through carbon membrane. The maximum specific removal rate of NH4+ -N is 9.7 g/(m2 x d), which is limited by the amount of bacteria grown onto carbon membrane's surface. Fluorescent in situ hybridization analysis indicates that within the biofilm Nitrosomonas and Nitrosospira are main ammonia-oxidizing bacteria and occupy about 19% and 21% of the total bacteria number, respectively. The Nitrobacter are not observed and Nitrospira are dominant nitrite-oxidizing bacteria, the fraction of which is 20% of total bacteria.

[Study on performance characteristics of carbon membrane-aerated biofilm reactor treating municipal wastewater].

A carbon membrane-aerated biofilm reactor was developed to treat municipal wastewater. Tests were conducted to investigate oxygen transfer ability of carbon membrane, the bacteria adhesion and reactor's set-up performance. The optimum parameters were determined in terms of intra-membrane pressure, COD and nitrogen ratio and hydraulic retention time (HRT). The results showed that compared with that of other hollow fibres, bacterial suspended exhibited a high degree of adhesion onto carbon membrane and that oxygen transfer coefficient of carbon membrane was 0.36 m/h, so that it was feasible to serve as both biofilm carrier and aerator. NH4(+) -N removal, denitrification and COD removal efficiency could reach 95%, 92% and 88%, respectively, under the conditions of intra-membrane pressure of 0.025 MPa, carbon nitrogen ratio of 5 and HRT of 8 h, and effluent quality could be up to A standard in the national discharge standard of pollutants for municipal wastewater treatment plant (GB 18918-2002) . Furthermore, the system displayed better resistance to shock loads.