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Developing vehicles that efficiently deliver genes throughout the human central nervous system (CNS) will broaden the range of treatable genetic diseases. We engineered an adeno-associated virus (AAV) capsid, BI-hTFR1, that binds human transferrin receptor (TfR1), a protein expressed on the blood-brain barrier. BI-hTFR1 was actively transported across human brain endothelial cells and, relative to AAV9, provided 40 to 50 times greater reporter expression in the CNS of human TFRC knockin mice. The enhanced tropism was CNS-specific and absent in wild-type mice. When used to deliver GBA1, mutations of which cause Gaucher disease and are linked to Parkinson's disease, BI-hTFR1 substantially increased brain and cerebrospinal fluid glucocerebrosidase activity compared with AAV9. These findings establish BI-hTFR1 as a potential vector for human CNS gene therapy.
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Antígenos CD , Encéfalo , Capsídeo , Técnicas de Transferência de Genes , Vetores Genéticos , Glucosilceramidase , Receptores da Transferrina , Animais , Humanos , Camundongos , Antígenos CD/metabolismo , Antígenos CD/genética , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Dependovirus , Células Endoteliais/metabolismo , Técnicas de Introdução de Genes , Terapia Genética , Receptores da Transferrina/metabolismo , Receptores da Transferrina/genética , Glucosilceramidase/genética , Doença de Gaucher/genética , Doença de Gaucher/terapia , Doença de Parkinson/genética , Doença de Parkinson/terapiaRESUMO
Overactive bladder (OAB) is a symptomatic complex condition characterised by frequent urinary urgency, nocturia, and urinary incontinence with or without urgency. Gabapentin is an effective treatment for OAB, but its narrow absorption window is a concern, as it is preferentially absorbed from the upper small intestine, resulting in poor bioavailability. We aimed to develop an extended release, intragastric floating system to overcome this drawback. For this purpose, plasticiser-free filaments of PEO (polyethylene oxide) and the drug (gabapentin) were developed using hot melt extrusion. The filaments were extruded successfully with 98% drug loading, possessed good mechanical properties, and successfully produced printed tablets using fused deposition modelling (FDM). Tablets were printed with varying shell numbers and infill density to investigate their floating capacity. Among the seven matrix tablet formulations, F2 (2 shells, 0% infill) showed the highest floating time, i.e., more than 10 h. The drug release rates fell as the infill density and shell number increased. However, F2 was the best performing formulation in terms of floating and release and was chosen for in vivo (pharmacokinetic) studies. The pharmacokinetic findings exhibit improved gabapentin absorption compared to the control (oral solution). Overall, it can be concluded that 3D printing technology is an easy-to-use approach which demonstrated its benefits in developing medicines based on a mucoadhesive gastroretentive strategy, improving the absorption of gabapentin with potential for the improved management of OAB.
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Developing vehicles that efficiently deliver genes throughout the human central nervous system (CNS) will broaden the range of treatable genetic diseases. We engineered an AAV capsid, BI-hTFR1, that binds human Transferrin Receptor (TfR1), a protein expressed on the blood-brain barrier (BBB). BI-hTFR1 was actively transported across a human brain endothelial cell layer and, relative to AAV9, provided 40-50 times greater reporter expression in the CNS of human TFRC knock-in mice. The enhanced tropism was CNS-specific and absent in wild type mice. When used to deliver GBA1, mutations of which cause Gaucher disease and are linked to Parkinson's disease, BI-hTFR1 substantially increased brain and cerebrospinal fluid glucocerebrosidase activity compared to AAV9. These findings establish BI-hTFR1 as a promising vector for human CNS gene therapy.
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BACKGROUND: Changes of toxic metals and essential elements during childhood may be the risk factor of autism spectrum disorder (ASD). This research established an accurate personalized predictive model of ASD behaviors among children by using the blood element detection index of children in Xinjiang, China. METHODS: A total of 1537 children (240 ASD behavior children and 1297 non-ASD behavior children) aged 0-7 were collected from September 2018 to September 2019 in Urumqi Children's Hospital and the health management institute of Xinjiang Medical University. For measuring the copper (Cu), zinc (Zn), magnesium (Mg), iron (Fe), calcium (Ca), lead (Pb), and cadmium (Cd), 80 µL of blood was taken from each participant's ring finger. Univariate logistic regression analysis was used to select predictors, then the multivariate logistic regression was used to establish the predictive model. The discriminability, calibration and clinical validity of the model were evaluated by the receiver operating characteristic (ROC) curve, Hosmer-Lemeshow test and decision curve analysis (DCA). RESULTS: Gender, concentrations of Pb, Ca and Zn in children's blood specimens were found to be the independent risk factors of ASD behaviors and were used to develop the nomogram model. The area under the ROC curve (AUC) in the development group (AUC = 0.778) and the validation group (AUC = 0.775) showed the model had discrimination ability. The calibration curve indicated the model was accurate, and the DCA proved its clinical application value. CONCLUSION: The nomogram model can be used as a reliable tool to predict the risk of ASD behaviors among children.
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Transtorno do Espectro Autista , Nomogramas , Transtorno do Espectro Autista/diagnóstico , Transtorno do Espectro Autista/epidemiologia , Cádmio , Cálcio , Criança , Cobre , Estudos Transversais , Humanos , Ferro , Chumbo , Magnésio , Estudos Retrospectivos , ZincoRESUMO
Precise customer requirements acquisition is the primary stage of product conceptual design, which plays a decisive role in product quality and innovation. However, existing customer requirements mining approaches pay attention to the offline or online customer comment feedback and there has been little quantitative analysis of customer requirements in the analogical reasoning environment. Latent and innovative customer requirements can be expressed by analogical inspiration distinctly. In response, this paper proposes a semantic analysis-driven customer requirements mining method for product conceptual design based on deep transfer learning and improved latent Dirichlet allocation (ILDA). Initially, an analogy-inspired verbal protocol analysis experiment is implemented to obtain detailed customer requirements descriptions of elevator. Then, full connection layers and a softmax layer are added to the output-end of Chinese bidirectional encoder representations from Transformers (BERT) pre-training language model. The above deep transfer model is utilized to realize the customer requirements classification among functional domain, behavioral domain and structural domain in the customer requirement descriptions of elevator by fine-tuning training. Moreover, the ILDA is adopted to mine the functional customer requirements that can represent customer intention maximally. Finally, an effective accuracy of customer requirements classification is acquired by using the BERT deep transfer model. Meanwhile, five kinds of customer requirements of elevator and corresponding keywords as well as their weight coefficients in the topic-word distribution are extracted. This work can provide a novel research perspective on customer requirements mining for product conceptual design through natural language processing.
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Algoritmos , Semântica , Idioma , Processamento de Linguagem Natural , Resolução de ProblemasRESUMO
Tumor innervation has recently been documented and characterized in various settings and tumor types. However, the role that nerves innervating tumors play in the pathogenesis of cancer has not been clarified. In this study, we searched for neural signaling from bulk RNA sequencing from The Cancer Genome Atlas (TCGA) dataset and looked for patterns of interactions between different cell types within the tumor environment. Using a presynapse signature (PSS) as a probe, we showed that multiple stromal cell types crosstalk and/or contribute to neural signals. Based on the correlation and linear regression, we hypothesized that neural signals contribute to an immune-suppressive tumor microenvironment (TME). To test this hypothesis, we performed in vitro dorsal root ganglion (DRG)/macrophage coculture experiments. Compared to the M2 macrophage monoculture, the DRG/M2 macrophage coculture prevented anti-inflammatory M2 to pro-inflammatory M1 polarization by LPS stimulation. Finally, a survey of different TCGA tumor types indicated that higher RNA neural signature is predictive of poor patient outcomes in multiple tumor types.
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Electrically tunable lenses (ETLs) are those with the ability to alter their optical power in response to an electric signal. This feature allows such systems to not only image the areas of interest but also obtain spatial depth perception (depth of field, DOF). The aim of the present study was to develop an ETL-based imaging system for quantitative surface analysis. Firstly, the system was calibrated to achieve high depth resolution, warranting the accurate measurement of the depth and to account for and correct any influences from external factors on the ETL. This was completed using the Tenengrad operator which effectively identified the plane of best focus as demonstrated by the linear relationship between the control current applied to the ETL and the height at which the optical system focuses. The system was then employed to measure amplitude, spatial, hybrid, and volume surface texture parameters of a model material (pharmaceutical dosage form) which were validated against the parameters obtained using a previously validated surface texture analysis technique, optical profilometry. There were no statistically significant differences between the surface texture parameters measured by the techniques, highlighting the potential application of ETL-based imaging systems as an easily adaptable and low-cost alternative surface texture analysis technique to conventional microscopy techniques.
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Hydroxypropyl methyl cellulose, HPMC, a hydrophilic polymer, is widely used for the development of extended release hydrophilic matrices and it is also considered as a good contender for the fabrication of 3D printing of matrix tablets. It is often combined with plasticisers to enable extrusion. The aim of the current project was to develop plasticizer-free 3D printed hydrophilic matrices using drug loaded filaments prepared via HME to achieve an in vitro (swelling, erosion and drug release) and in vivo (drug absorption) performance which is analogous to hydrophilic matrix tablets developed through conventional approaches. Additionally, the morphology of the printed tablets was studied using quantitative 3D surface texture studies and the porosity calculated. Filaments were produced successfully and used to produce matrix tablets with acceptable drug loading (95-105%), mechanical and surface texture properties regardless of the employed HPMC grade. The viscosity of HPMC had a discernible impact on the swelling, erosion, HPMC dissolution, drug release and pharmacokinetic findings. The highest viscosity grade (K100M) results in higher degree of swelling, decreased HPMC dissolution, low matrix erosion, decreased drug release and extended drug absorption profile. Overall, this study demonstrated that the drug loaded (glipizide) filaments and matrix tablets of medium to high viscosity grades of HPMC, without the aid of plasticisers, can be successfully prepared. Furthermore, the in vitro and in vivo studies have revealed the successful fabrication of extended release matrices.
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A technology that simultaneously records membrane potential from multiple neurons in behaving animals will have a transformative effect on neuroscience research1,2. Genetically encoded voltage indicators are a promising tool for these purposes; however, these have so far been limited to single-cell recordings with a marginal signal-to-noise ratio in vivo3-5. Here we developed improved near-infrared voltage indicators, high-speed microscopes and targeted gene expression schemes that enabled simultaneous in vivo recordings of supra- and subthreshold voltage dynamics in multiple neurons in the hippocampus of behaving mice. The reporters revealed subcellular details of back-propagating action potentials and correlations in subthreshold voltage between multiple cells. In combination with stimulation using optogenetics, the reporters revealed changes in neuronal excitability that were dependent on the behavioural state, reflecting the interplay of excitatory and inhibitory synaptic inputs. These tools open the possibility for detailed explorations of network dynamics in the context of behaviour. Fig. 1 PHOTOACTIVATED QUASAR3 (PAQUASAR3) REPORTS NEURONAL ACTIVITY IN VIVO.: a, Schematic of the paQuasAr3 construct. b, Photoactivation by blue light enhanced voltage signals excited by red light in cultured neurons that expressed paQuasAr3 (representative example of n = 4 cells). c, Model of the photocycle of paQuasAr3. d, Confocal images of sparsely expressed paQuasAr3 in brain slices. Scale bars, 50 µm. Representative images, experiments were repeated in n = 3 mice. e, Simultaneous fluorescence and patch-clamp recordings from a neuron expressing paQuasAr3 in acute brain slice. Top, magnification of boxed regions. Schematic shows brain slice, patch pipette and microscope objective. f, Simultaneous fluorescence and patch-clamp recordings of inhibitory post synaptic potentials in an L2-3 neuron induced by electrical stimulation of L5-6 in acute slice. g, Normalized change in fluorescence (ΔF/F) and SNR of optically recorded post-synaptic potentials (PSPs) as a function of the amplitude of the post-synaptic potentials. The voltage sensitivity was ΔF/F = 40 ± 1.7% per 100 mV. The SNR was 0.93 ± 0.07 per 1 mV in a 1-kHz bandwidth (n = 42 post-synaptic potentials from 5 cells, data are mean ± s.d.). Schematic shows brain slice, patch pipette, field stimulation electrodes and microscope objective. h, Optical measurements of paQuasAr3 fluorescence in the CA1 region of the hippocampus (top) and glomerular layer of the olfactory bulb (bottom) of anaesthetized mice (representative traces from n = 7 CA1 cells and n = 13 olfactory bulb cells, n = 3 mice). Schematics show microscope objective and the imaged brain region. i, STA fluorescence from 88 spikes in a CA1 oriens neuron. j, Frames from the STA video showing the delay in the back-propagating action potential in the dendrites relative to the soma. k, Sub-Nyquist fitting of the action potential delay and width shows electrical compartmentalization in the dendrites. Experiments in k-m were repeated in n = 2 cells from n = 2 mice.
Assuntos
Potenciais de Ação , Hipocampo/citologia , Hipocampo/fisiologia , Optogenética/métodos , Algoritmos , Animais , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Bacteriorodopsinas/genética , Bacteriorodopsinas/metabolismo , Células Cultivadas , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/metabolismo , CaminhadaRESUMO
Optical tools for simultaneous perturbation and measurement of neural activity open the possibility of mapping neural function over wide areas of brain tissue. However, spectral overlap of actuators and reporters presents a challenge for their simultaneous use, and optical scattering and out-of-focus fluorescence in tissue degrade resolution. To minimize optical crosstalk, we combined an optimized variant (eTsChR) of the most blue-shifted channelrhodopsin reported to-date with a nuclear-localized red-shifted Ca2+ indicator, H2B-jRGECO1a. To perform wide-area optically sectioned imaging in tissue, we designed a structured illumination technique that uses Hadamard matrices to encode spatial information. By combining these molecular and optical approaches we made wide-area functional maps in acute brain slices from mice of both sexes. The maps spanned cortex and striatum and probed the effects of antiepileptic drugs on neural excitability and the effects of AMPA and NMDA receptor blockers on functional connectivity. Together, these tools provide a powerful capability for wide-area mapping of neuronal excitability and functional connectivity in acute brain slices.SIGNIFICANCE STATEMENT A new technique for simultaneous optogenetic stimulation and calcium imaging across wide areas of brain slice enables high-throughput mapping of neuronal excitability and synaptic transmission.
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Anticonvulsivantes/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Imagem Óptica/métodos , Transmissão Sináptica/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Células HEK293 , Humanos , Camundongos , Rede Nervosa/efeitos dos fármacos , Optogenética , Estimulação Luminosa , RatosRESUMO
The phase slope method which estimates height through fringe pattern frequency and the algorithm which estimates height through the fringe phase are the fringe analysis algorithms widely used in interferometry. Generally they both extract the phase information by filtering the signal in frequency domain after Fourier transform. Among the numerous papers in the literature about these algorithms, it is found that the design of the filter, which plays an important role, has never been discussed in detail. This paper focuses on the filter design in these algorithms for wavelength scanning interferometry (WSI), trying to optimize the parameters to acquire the optimal results. The spectral characteristics of the interference signal are analyzed first. The effective signal is found to be narrow-band (near single frequency), and the central frequency is calculated theoretically. Therefore, the position of the filter pass-band is determined. The width of the filter window is optimized with the simulation to balance the elimination of the noise and the ringing of the filter. Experimental validation of the approach is provided, and the results agree very well with the simulation. The experiment shows that accuracy can be improved by optimizing the filter design, especially when the signal quality, i.e., the signal noise ratio (SNR), is low. The proposed method also shows the potential of improving the immunity to the environmental noise by adapting the signal to acquire the optimal results through designing an adaptive filter once the signal SNR can be estimated accurately.
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Recent advances in optogenetics have enabled simultaneous optical perturbation and optical readout of membrane potential in diverse cell types. Here, we develop and characterize a Cre-dependent transgenic Optopatch2 mouse line that we call Floxopatch. The animals expressed a blue-shifted channelrhodopsin, CheRiff, and a near infrared Archaerhodopsin-derived voltage indicator, QuasAr2, via targeted knock-in at the rosa26 locus. In Optopatch-expressing animals, we tested for overall health, genetically targeted expression, and function of the optogenetic components. In offspring of Floxopatch mice crossed with a variety of Cre driver lines, we observed spontaneous and optically evoked activity in vitro in acute brain slices and in vivo in somatosensory ganglia. Cell-type-specific expression allowed classification and characterization of neuronal subtypes based on their firing patterns. The Floxopatch mouse line is a useful tool for fast and sensitive characterization of neural activity in genetically specified cell types in intact tissue. SIGNIFICANCE STATEMENT: Optical recordings of neural activity offer the promise of rapid and spatially resolved mapping of neural function. Calcium imaging has been widely applied in this mode, but is insensitive to the details of action potential waveforms and subthreshold events. Simultaneous optical perturbation and optical readout of single-cell electrical activity ("Optopatch") has been demonstrated in cultured neurons and in organotypic brain slices, but not in acute brain slices or in vivo Here, we describe a transgenic mouse in which expression of Optopatch constructs is controlled by the Cre-recombinase enzyme. This animal enables fast and robust optical measurements of single-cell electrical excitability in acute brain slices and in somatosensory ganglia in vivo, opening the door to rapid optical mapping of neuronal excitability.
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Potenciais de Ação/fisiologia , Integrases/genética , Neurônios/fisiologia , Optogenética/métodos , Imagens com Corantes Sensíveis à Voltagem/métodos , Animais , Células Cultivadas , Marcação de Genes , Proteínas Luminescentes/genética , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Proteínas Recombinantes/genéticaRESUMO
Mammalian skin is innervated by diverse, unmyelinated C fibers that are associated with senses of pain, itch, temperature, or touch. A key developmental question is how this neuronal cell diversity is generated during development. We reported previously that the runt domain transcription factor Runx1 is required to coordinate the development of these unmyelinated cutaneous sensory neurons, including VGLUT3(+) low-threshold c-mechanoreceptors (CLTMs), MrgprD(+) polymodal nociceptors, MrgprA3(+) pruriceptors, MrgprB4(+) c-mechanoreceptors, and others. However, how these Runx1-dependent cutaneous sensory neurons are further segregated is poorly illustrated. Here, we find that the Runx1-dependent transcription factor gene Zfp521 is expressed in, and required for establishing molecular features that define, VGLUT3(+) CLTMs. Furthermore, Runx1 and Zfp521 form a classic incoherent feedforward loop (I-FFL) in controlling molecular identities that normally belong to MrgprD(+) neurons, with Runx1 and Zfp51 playing activator and repressor roles, respectively (in genetic terms). A knock-out of Zfp521 allows prospective VGLUT3 lineage neurons to acquire MrgprD(+) neuron identities. Furthermore, Runx1 might form other I-FFLs to regulate the expression of MrgprA3 and MrgprB4, a mechanism preventing these genes from being expressed in Runx1-persistent VGLUT3(+) and MrgprD(+) neurons. The evolvement of these I-FFLs provides an explanation for how modality-selective sensory subtypes are formed during development and may also have intriguing implications for sensory neuron evolution and sensory coding.
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Diferenciação Celular/fisiologia , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Mecanorreceptores/fisiologia , Nociceptores/fisiologia , Fatores de Transcrição/fisiologia , Sistemas de Transporte de Aminoácidos Acídicos/fisiologia , Animais , Contagem de Células , Diferenciação Celular/genética , Gânglios Espinais/crescimento & desenvolvimento , Gânglios Espinais/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Neurônios/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
The somatosensory nervous system is critical for the organism's ability to respond to mechanical, thermal, and nociceptive stimuli. Somatosensory neurons are functionally and anatomically diverse but their molecular profiles are not well-defined. Here, we used transcriptional profiling to analyze the detailed molecular signatures of dorsal root ganglion (DRG) sensory neurons. We used two mouse reporter lines and surface IB4 labeling to purify three major non-overlapping classes of neurons: 1) IB4(+)SNS-Cre/TdTomato(+), 2) IB4(-)SNS-Cre/TdTomato(+), and 3) Parv-Cre/TdTomato(+) cells, encompassing the majority of nociceptive, pruriceptive, and proprioceptive neurons. These neurons displayed distinct expression patterns of ion channels, transcription factors, and GPCRs. Highly parallel qRT-PCR analysis of 334 single neurons selected by membership of the three populations demonstrated further diversity, with unbiased clustering analysis identifying six distinct subgroups. These data significantly increase our knowledge of the molecular identities of known DRG populations and uncover potentially novel subsets, revealing the complexity and diversity of those neurons underlying somatosensation.
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Perfilação da Expressão Gênica , Células Receptoras Sensoriais/metabolismo , Transcrição Gênica , Animais , Separação Celular , Análise por Conglomerados , Citometria de Fluxo , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Camundongos , Técnicas de Patch-Clamp , Análise de Componente PrincipalRESUMO
VGLUT3-expressing unmyelinated low-threshold mechanoreceptors (C-LTMRs) are proposed to mediate pleasant touch and/or pain, but the molecular programs controlling C-LTMR development are unknown. Here, we performed genetic fate mapping, showing that VGLUT3 lineage sensory neurons are divided into two groups, based on transient or persistent VGLUT3 expression. VGLUT3-transient neurons are large- or medium-diameter myelinated mechanoreceptors that form the Merkel cell-neurite complex. VGLUT3-persistent neurons are small-diameter unmyelinated neurons that are further divided into two subtypes: (1) tyrosine hydroxylase (TH)-positive C-LTMRs that form the longitudinal lanceolate endings around hairs, and (2) TH-negative neurons that form epidermal-free nerve endings. We then found that VGLUT3-persistent neurons express the runt domain transcription factor Runx1. Analyses of mice with a conditional knock-out of Runx1 in VGLUT3 lineage neurons demonstrate that Runx1 is pivotal to the development of VGLUT3-persistent neurons, such as the expression of VGLUT3 and TH and the formation of the longitudinal lanceolate endings. Furthermore, Runx1 is required to establish mechanosensitivity in C-LTMRs, by controlling the expression of the mechanically gated ion channel Piezo2. Surprisingly, both acute and chronic mechanical pain was largely unaffected in these Runx1 mutants. These findings appear to argue against the recently proposed role of VGLUT3 in C-LTMRs in mediating mechanical hypersensitivity induced by nerve injury or inflammation. Thus, our studies provide new insight into the genetic program controlling C-LTMR development and call for a revisit for the physiological functions of C-LTMRs.
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Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Hiperalgesia/metabolismo , Mecanorreceptores/fisiologia , Terminações Nervosas/fisiologia , Neuralgia/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Animais , Comportamento Animal/efeitos dos fármacos , Capsaicina/farmacologia , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Hiperalgesia/induzido quimicamente , Hiperalgesia/genética , Canais Iônicos/genética , Canais Iônicos/metabolismo , Camundongos , Camundongos Knockout , Neuralgia/induzido quimicamente , Neuralgia/genética , Estimulação Física , Pele/inervação , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Effect of hydraulic load distribution on sewage treatment efficiency of earthworm bio-filter was studied by analyzing influent and effluent of earthworm bio-filter and earthworm behaviors. The results show that when hydraulic load varying from 2.0 m3/(m2 x d) to 6.0 m3/(m2 x d), the concentration of each pollutant in earthworm bio-filter effluent increases slowly and is little effected by hydraulic load. When hydraulic load reaches 6.7 m3/(m2 x d), the concentration of COD, BOD5, SS, NH4(+) -N and TP in earthworm bio-filter effluent increases obviously, but the TN concentration in effluent presents descending tendency. The earthworms become rather inadaptable to the living conditions at this operating mode. As hydraulic load increasing, the earthworms' relative ingestive ability is improved at first, and then decreases. The earthworms' relative ingestive ability comes to the maximum at hydraulic load of 4.8 m3/(m2 x d), with good organic removal efficiency. The relation ships between hydraulic load and average weight, average density, unit-area biomass of the earthworms are significant negative correlation. The hydraulic load of 4.8 m3/(m2 x d) is recommended, but not over 6.7 m3/(m2 x d).