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Introduction: The dissemination of strains producing tetracyclines monooxygenase Tet(X) from breeding farms to the natural environment poses a potential threat to public health. Methods: Antimicrobial susceptibility testing and WGS were performed to identify resistance phenotypes and genotypes. Cloning experiments, sequence alignment, and homology modeling were used to characterize the function and formation mechanisms of the recombinant variant. The mobilization potential of Tet(X) was assessed by collinearity analysis, conjugation experiments, and phylogenetic analysis. Results: Three tet(X)-producing Elizabethkingia meningoseptica strains were isolated from bullfrog breeding ponds, the sewage outlet, and downstream river in Zhejiang Province, China. These strains carry a novel Tet(X) variant, differing from Tet(X6) by seven residues, and possess the ability to degrade tetracyclines. Interestingly, the novel Tet(X) is a recombinant variant formed by homologous recombination of Tet(X6) and the C-terminal of Tet(X2). Further analysis revealed that Tet(X6) formed several Tet(X) variants, including Tet(X5), through homologous recombination. The novel tet(X) gene is located on a circularizable integrative and conjugative element (ICEEmeChn3), with ISwz1 participating in the recombination of its multi-drug resistance region, potentially facilitating the mobilization and recombination of tet(X) in early hosts. These three strains were clonally transmitted and shared a close genetic relationship (SNP < 62) with a clinically-sourced strain isolated from the same province. Discussion: To our knowledge, this is the first report of homologous recombination between Tet(X) variants with differing activities. These clonal strains provide evidence of the transmission of tet(X)-positive strains from aquaculture sewage to the natural environment, highlighting the need to strengthen the monitoring and management of this emerging farming model.
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Transglutaminase 2 (Tgm2) plays an essential role in hepatic repair following prolonged toxic injury. During cholestatic liver injury, the intrahepatic cholangiocytes undergo dynamic tissue expansion and remodelling, referred to as ductular reaction (DR), which is crucial for liver regeneration. However, the molecular mechanisms governing the dynamics of active cells in DR are still largely unclear. Here, we generated Tgm2-knockout mice (Tgm2-/-) and Tgm2-CreERT2-Rosa26-mTmG flox/flox (Tgm2CreERT2-R26T/Gf/f) mice and performed a three-dimensional (3D) collagen gel culture of mouse hepatocytes to demonstrate how Tgm2 signalling is involved in DR to remodel intrahepatic cholangiocytes. Our results showed that the deletion of Tgm2 adversely affected the functionality and maturity of the proliferative cholangiocytes in DR, thus leading to more severe cholestasis during DDC-induced liver injury. Additionally, Tgm2 hepatocytes played a crucial role in the regulation of DR through metaplasia. We unveiled that Tgm2 regulated H3K4me3Q5ser via serotonin to promote BMP signalling activation to participate in DR. Besides, we revealed that the activation or inhibition of BMP signalling could promote or suppress the development and maturation of cholangiocytes in DDC-induced DR. Furthermore, our 3D collagen gel culture assay indicated that Tgm2 was vital for the development of cholangiocytes in vitro. Our results uncovered a considerable role of BMP signalling in controlling metaplasia of Tgm2 hepatocytes in DR and revealed the phenotypic plasticity of mature hepatocytes.
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Autosomal recessive (AR) non-syndromic hearing loss (NSHL) is the most common form of hereditary deafness. Mutations in the gap junction protein beta 2 (GJB2) gene encoding connexin 26 (Cx26) account for about 50% of cases of ARNSHL. In the current study, a combination of exome sequencing and Sanger sequencing in a Chinese Dong family with ARNSHL allowed identification of a novel compound heterozygous mutation c.240G>C(p. Q80H)/C.109G>A(p.V37I) in exon 2 of the GJB2 gene, which co-segregated with the disease phenotype in this family and was not evident in 100 healthy controls. Bioinformatic analysis revealed that the two mutations in the GJB2 gene were probably pathogenic. Results indicated that the compound heterozygous variants, p.Q80H and p.V37I, in the GJB2 gene are associated with ARNSHL. The Q80H variant was initially identified in patients of Dong Chinese origin with NSHL. The current results broaden the spectrum of GJB2 mutations responsible for NSHL and have important implications for molecular diagnosis, treatment, and genetic counseling for this family.
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Conexinas/genética , Sordera/genética , Mutación , Adulto , Niño , China , Conexina 26 , Análisis Mutacional de ADN , Exoma , Salud de la Familia , Femenino , Asesoramiento Genético , Heterocigoto , Humanos , Masculino , Linaje , Análisis de Secuencia de ADNRESUMEN
OBJECTIVE: To investigate the biochemical characteristic of the neurons associated Zusanli (ST 36) in the rat by using Alexa Fluor 594 conjugated cholera toxin subunit B (AF594-CTB) neural tracing and calcitonin gene-related peptide (CGRP) fluorescent immunohistochemical techniques. METHODS: Four male Sprague Dawley rats were injected with AF594-CTB into the corresponding area of the Zusanli in the human body. After 3 surviving days, the rat's spinal cord and dorsal root ganglia (DRGs) at lumbar segments were dissected following perfusion with 4% paraformaldehyde, cut into sections, and then stained with CGRPfluorescent immunohistochemical method. RESULTS: AF594-CTB labeled sensory neurons were detected in the L3-L6 DRGs with high concentration in L4 DRG, and the labeled motor neurons located in the dorsolateral and intermediate regions of lamina IX from L3-L5 segments with high concentration at L4. Meanwhile, CGRPpositive neural labeling distributed symmetrically on both sides of DRGs, anterior and dorsal horns of spinal cord. In the AF594-CTB labeled neurons, 37% sensory neurons and 100% motor neurons expressed CGRPpositive. CONCLUSION: These findings present the morphological evidence to demonstrate that the sensory and motor neurons associated Zusanli in the rat distributed with segmental and regional patterns, and contained CGRP-expression.
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Puntos de Acupuntura , Péptido Relacionado con Gen de Calcitonina/metabolismo , Neuronas/metabolismo , Animales , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: To observe the distribution of neurons and their axonal projection associated with "Yongquan" (KI 1) area by neural tracing technique with cholera toxin subunit B (CTB). METHODS: A total of 5 microL of 1% CTB solution was injected into the front central part of hind foot plantar (corresponding to the KI 1 region in the human body) using a Hamilton microsyringe. After 3 survival days, the rats were deeply anesthetized and transcardically perfused for collecting the dorsal root ganglia (DRGs), spinal cord and brain tissues. Following fixing in 4% paraforldehyde containing phosphate buffer solution (PBS) and incubation in PBS containing 25% glucose solution, the aforementioned tissues were sectioned to be stained with immunofluorescence or immunohistochemistry for revealing the labeled sensory neurons and their tansganglionic projection and motor neurons. RESULTS: All the labeled neurons (sensory and motor neurons) and transganglionic axonal projection appeared ipsilaterally to the injection side. The labeled sensory neurons were located in the DRGs of lumbar 3-5 segments (L 3-L 5) with a higher concentration at L 4, while motor neurons distributed in the dorsolateral portion of spinal ventral horn from L 3 to L 6 with a higher concentration at L 5. In addition, transganglionic axonal projections were found to situate in the medial part of laminae III - IV from L 3 to L 5, as far as in the gracile nucleus. CONCLUSION: Acupoint KI 1 area is innervated by sensory neurons in L 3-L 5 DRGs and motor neurons from the dorsolateral ventral horns of L 3-L 6. The axonal projection of the primary sensory neurons distributes in the medial part of laminae III - IV of L 3-L 5 and gracile nucleus, respectively.
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Puntos de Acupuntura , Axones/química , Rastreo Celular/métodos , Toxina del Cólera/análisis , Neuronas/química , Animales , Humanos , Masculino , Neuronas/citología , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/química , Células Receptoras Sensoriales/citología , Coloración y EtiquetadoRESUMEN
The purpose of this study is to investigate the specific innervations on "Weizhong" (BL40) and "Huantiao" (GB30) by using a dual neural tracing technique. After Alexa Fluor 488 and 594 conjugates of cholera toxin subunit B (AF488/594-CTB) were, respectively, injected into BL40 and GB30 in the same rat, the labeled sensory and motor neurons were examined in the rat's dorsal root ganglia (DRGs) and spinal cord at thoracic (T) and lumbar (L) segments with a laser scanning confocal microscope. In the cases of BL40 injection, AF488-CTB labeled sensory and motor neurons were located in L2-6 DRGs and on the mediolateral part of spinal ventral horn from L3 to L5 segments, respectively. By contrast, in the cases of GB30 injection, AF594-CTB labeled sensory and motor neurons were distributed in T13-L6 DRGs and on the anterolateral part of spinal ventral horn from L1 to L5 segments, respectively. These results indicate that the sensory and motor neurons associated with BL40 and GB30 are located in different spinal segments and regions in the nervous system, providing the neuroanatomical evidence to serve the specificity of acupoints.
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OBJECTIVES: This study was performed to investigate the innervations related to acupuncture point PC8 in rats using a neural tracing technique. METHODS: After 6 µL of 1% cholera toxin subunit B (CTB) was injected into the site between the second and third metacarpal bone in rats, a corresponding site to acupuncture point PC8 in the human body, CTB labelling was examined with immunofluorescence and immunohistochemistry in the dorsal root ganglia (DRG), spinal cord and brainstem. RESULTS: All CTB labelling appeared on the ipsilateral side of the injection. The labelled sensory neurons distributed from cervical (C)6 to thoracic (T)1 DRG, while the labelled motor neurons were located on the dorsolateral part of the spinal ventral horn ranging from the C6 to T1 segments. In addition, the transganglionically-labelled axonal terminals were found to be dense in the medial part of laminae 3-4 from C6 to the T1 spinal dorsal horn, as far as in the cuneate nucleus. CONCLUSIONS: These results indicate that sensory and motor neurons associated with PC8 distribute in a distinct segmental pattern. The sensory information from PC8 could be transganglionically transported to the spinal dorsal horn and cuneate nucleus.
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Puntos de Acupuntura , Toxina del Cólera/metabolismo , Animales , Toxina del Cólera/química , Ganglios Espinales/anatomía & histología , Ganglios Espinales/química , Ganglios Espinales/metabolismo , Masculino , Neuronas Motoras/química , Neuronas Motoras/metabolismo , Neuroanatomía , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/química , Células Receptoras Sensoriales/metabolismo , Médula Espinal/anatomía & histología , Médula Espinal/química , Médula Espinal/metabolismo , Coloración y EtiquetadoRESUMEN
BACKGROUND: The afferent projections from the auricular branch of the vagus nerve (ABVN) to the nucleus tractus solitaries (NTS) have been proposed as the anatomical basis for the increased parasympathetic tone seen in auriculo-vagal reflexes. As the afferent center of the vagus nerve, the NTS has been considered to play roles in the anticonvulsant effect of cervical vagus nerve stimulation (VNS). Here we proposed an "auriculo-vagal afferent pathway" (AVAP), by which transcutaneous auricular vagus nerve stimulation (ta-VNS) suppresses pentylenetetrazol (PTZ)-induced epileptic seizures by activating the NTS neurons in rats. RESULTS: The afferent projections from the ABVN to the NTS were firstly observed in rats. ta-VNS increased the first grand mal latency of the epileptic seizure and decreased the seizure scores in awake rats. Furthermore, when the firing rates of the NTS neurons decreased, epileptiform activity manifested as electroencephalogram (EEG) synchronization increased with 0.37±0.12 s delay in anaesthetized rats. The change of instantaneous frequency, mean frequency of the NTS neurons was negative correlated with the amplitude of the epileptic activity in EEG traces. ta-VNS significantly suppressed epileptiform activity in EEG traces via increasing the firing rates of the neurons of the NTS. In comparison with tan-VNS, the anticonvulsant durations of VNS and ta-VNS were significantly longer (P<0.01). There was no significant difference between the anticonvulsant durations of VNS and ta-VNS (P>0.05). The anticonvulsant effect of ta-VNS was weakened by reversible cold block of the NTS. CONCLUSIONS: There existed an anatomical relationship between the ABVN and the NTS, which strongly supports the concept that ta-VNS has the potential for suppressing epileptiform activity via the AVAP in rats. ta-VNS will provide alternative treatments for neurological disorders, which can avoid the disadvantage of VNS.
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Vías Aferentes/fisiología , Convulsiones/terapia , Estimulación del Nervio Vago/métodos , Nervio Vago/fisiología , Potenciales de Acción/fisiología , Anestésicos/farmacología , Animales , Toxina del Cólera/metabolismo , Convulsivantes/toxicidad , Modelos Animales de Enfermedad , Electroencefalografía , Masculino , Neuronas/fisiología , Pentilenotetrazol/toxicidad , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Convulsiones/inducido químicamente , Núcleo Solitario/citología , Núcleo Solitario/fisiología , Corteza Somatosensorial/fisiología , Médula Espinal/fisiología , Ganglio Cervical Superior/fisiología , VigiliaRESUMEN
Rat models of adjuvant arthritis were established, and anti-corticotropin release hormone serum injection in the lateral ventricles and electroacupuncture at right Jiaji (EX-B2) were performed. The pain threshold was decreased at 45 and 60 minutes after injection of the anti-corticotropin release hormone serum. Electroacupuncture at Jiaji can resist this effect. Immunohistochemical staining results showed that the expression of corticotropin release hormone in the hypothalamic paraventricular nucleus was greater in the electroacupuncture + anti-corticotropin release hormone serum group compared with the anti-corticotropin release hormone serum group. The expression of corticotropin release hormone was correlated with the pain threshold. The effect of endogenous corticotropin release hormone in pain modulation can be obstructed by anti-corticotropin release hormone serum. The analgesia of electroacupuncture can partially resist the depressed pain threshold caused by injection of anti-corticotropin release hormone serum. The analgesic effect of electroacupuncture is associated with the corticotropin release hormone content in the hypothalamus.
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OBJECTIVE: To study the specific correlation between "Jinggu" (BL 64) and "Dazhong" (KI 4) in the nervous system by using a double-labeling of cholera toxin subunit B conjugated with Alexa Fluor 488 and 594 (CTB-Alexa 488, 594) in rats, so as to investigate its neuroanatomical basis for clinical joint-application of Yuan-Source and Luo acupoints. METHODS: Three male SD rats were used in the present study. Under anesthesia (10% urethane), 0.1% CTB-Alexa 488 (5 microL) and CTB-Alexa 594 (5 microL) were respectively injected into the border area between the red and white flesh, distal to the tuberosity of the fifth metatarsal bone, and the depression anterior to the medial attachment of the calcaneal tendon, the corresponding sites of the acupoints Jinggu (BL 64) and Dazhong (KI 4) in the human body. After 3 surviving days, the rat's brain, spinal cord and dorsal root ganglia (DRGs) of L3-L6 were dissected following perfusion with 4% paraformaldehyde, cut into sections and observed under fluorescent microscope equipped with a digital camera. The labeled neurons were recorded and counted. RESULTS: It was found under fluorescent microscope that the single-labeled neurons and the dual-labeled neurons were ipsilaterally located on the injected side. Among the single-labeled neurons, the labeled sensory neurons related to "Jinggu" (BL 64) and "Dazhong" (KI 4) were found to be in the DRGs of L3-L6, with a higher concentration in the DRGs of L.4 (27/162, 102/332) and L5 (130/162, 204/332). The dual-labeled 7 neurons were found to be in DRGs of L4 and L5. In addition, the labeled motoneurons related to "Jinggu" (BL 64) and "Dazhong" (KI 4) distributed in the dorsolateral portion of lamina IX, forming a longitudianal column from L3-L6 with a higher concentration at L4 and L5. CONCLUSION: The labeled sensory and motor neurons innervating Yuan-acupoint "Jinggu" (BL 64) and Luo-acupoint "Dazhong" (KI 4) distribute in DRGs of the same spinal segments and spinal ventral horns from L3-L6.
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Puntos de Acupuntura , Toxina del Cólera , Colorantes Fluorescentes , Ganglios Espinales/anatomía & histología , Neuronas Motoras/citología , Células Receptoras Sensoriales/citología , Animales , Masculino , Neuroanatomía , Compuestos Orgánicos , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: To investigate neuroanatomical characteristics of the primary sensory afferent and the motor neurons coming from and innervating acupoint "Chengshan" (BL 57) area in the rat by using cholera toxin subunit B conjugated with Alexa Fluor 488 (CTB-Alexa 488), a new generation of fluorescent neural tracing reagent. METHODS: Four male SD rats were used in the present study. Under anesthesia, 0.05% CTB-Alexa 488 (5 ML) was injected into the central part of the rear of the hind leg, a corresponding site of "Chengshan" (BL 57) in the human body. After 40-48 surviving hours, the rat's brain, spinal cord and dorsal root ganglia (DRGs) of the lumbar segments (L1-L6) were dissected following perfusion with 4% paraformaldehyde, cut into sections and observed under fluorescent microscope equipped with a digital camera. The neurons labeled by CTB-Alexa 488 were counted. RESULTS: All CTB-Alexa 488 labeled neurons appeared in green under fluorescent filters of 450-490 and were located ipsilaterally on the injection side. The labeled primary sensory neurons were found in the DRGs at L4 (11 neurons) and L5 (35 neurons). Among them, 29 neurons (63.04%) were bigger, with their cell body diameters being 35-50 microm and 17 (36.96%) smaller, with their body diameters being lower than 35 microm. The labeled motor neurons were found to distribute in the mediolateral portion of lamina IX, forming a longitudinal column from L4 to L5. Of the observed 316 motor neurons, 259 (81.96%) belong to alpha type with their body diameters being 25-40 microm and 57 (18.04%) to gamma type with their body diameters being lower than 25 microm. CONCLUSION: The CTB-Alexa 488-labeled primary sensory and motor neurons innervating acupoint "Cheng-shan" (BL 57) distribute in the DRGs of L4-L5. The present fluorescent tracing technique may be quite useful for investigating the neural characteristics of acupoints.