Minimum Distance Between Two Epitopes in Sandwich Immunoassays for Small Molecules.

The pursuit of the limit between dimensionalities is a scientific goal with high applicability. Sandwich immunoassay, usually based on two antibodies binding two epitopes, is one of the most popular mainstay tools in both academic and industrial fields. Herein, we determined and evaluated the minimum distance of two epitopes in sandwich immunoassays for small molecules. Briefly, nine model analytes comprising two hapten epitopes, that is, melamine (MEL) and p-nitroaniline (NIA), were designed by increasing the linear chain linkers brick by brick. Two groups of monoclonal antibodies (mAbs) were produced with different recognition properties toward MEL and NIA using 12 new haptens with different spacer arms. The results indicated that two epitopes of the analyte with a distance of only 2.4 Å could be simultaneously bound by two mAbs, which is the known limit of epitope distance in sandwich immunoassays thus far. We further found that an epitope distance of below 8.8 Å for the analyte generally induces noticeable steric hindrance of antibodies, preventing a sandwich immunoassay with high probability. These observations were investigated and evaluated by molecular docking, molecular dynamics, and surface plasmon resonance and using model and real analytes. Altogether, we determined the minimum distance of two epitopes and explored the molecular mechanism of the antibody-analyte-antibody ternary complex in sandwich immunoassays, providing a theoretical basis for hapten design, antibody discovery and development, and sandwich immunoassay establishment for small molecules.

Optimization of High Temperature-Resistant Modified Starch Polyamine Anti-Collapse Water-Based Drilling Fluid System for Deep Shale Reservoir.

During drilling in deep shale gas reservoirs, drilling fluid losses, hole wall collapses, and additional problems occur frequently due to the development of natural fractures in the shale formation, resulting in a high number of engineering accidents such as drilling fluid leaks, sticking, mud packings, and buried drilling tools. Moreover, the horizontal section of horizontal well is long (about 1500 m), and the problems of friction, rock carrying, and reservoir pollution are extremely prominent. The performance of drilling fluids directly affects drilling efficiency, the rate of engineering accidents, and the reservoir protection effect. In order to overcome the problems of high filtration in deep shale formations, collapse of borehole walls, sticking of pipes, mud inclusions, etc., optimization studies of water-based drilling fluid systems have been conducted with the primary purpose of controlling the rheology and water loss of drilling fluid. The experimental evaluation of the adsorption characteristics of "KCl + polyamine" anti-collapse inhibitor on the surface of clay particles and its influence on the morphology of bentonite was carried out, and the mechanism of inhibiting clay mineral hydration expansion was discussed. The idea of controlling the rheology and water loss of drilling fluid with high temperature resistant modified starch and strengthening the inhibition performance of drilling fluid with "KCl + polyamine" was put forward, and a high temperature-resistant modified starch polyamine anti-sloughing drilling fluid system with stable performance and strong plugging and strong inhibition was optimized. The temperature resistance of the optimized water-based drilling fluid system can reach 180 °C. Applied to on-site drilling of deep shale gas horizontal wells, it effectively reduces the rate of complex accidents such as sticking, mud bagging, and reaming that occur when resistance is encountered during shale formation drilling. The time for a single well to trip when encountering resistance decreases from 2-3 d in the early stages to 3-10 h. The re-use rate of the second spudded slurry is 100 percent, significantly reducing the rate of complex drilling accidents and saving drilling costs. It firmly supports the optimal and rapid construction of deep shale gas horizontal wells.

Production of highly sensitive monoclonal antibody and development of lateral flow assays for phallotoxin detection in urine.

Phallotoxins, toxic cyclopeptides found in wild poisonous mushrooms, are predominant causes of fatal food poisoning. For the early and rapid diagnosis mushroom toxin poisoning, a highly sensitive and robust monoclonal antibody (mAb) against phallotoxins was produced for the first time. The half-maximum inhibition concentration (IC50) values of the mAb-based indirect competitive ELISAs for phallacidin (PCD) and phalloidin (PHD) detection were 0.31 ng mL-1 and 0.35 ng mL-1, respectively. In response to the demand for rapid screening of the type of poisoning and accurate determination of the severity of poisoning, colloidal gold nanoparticle (GNP) and time-resolved fluorescent nanosphere (TRFN) based lateral flow assays (LFA) were developed. The GNP-LFA has a visual cut-off value of 3.0 ng mL-1 for phallotoxins in human urine sample. The TRFN-LFA provides a quantitative readout signal with detection limit of 0.1 ng mL-1 in human urine sample. In this study, urine samples without pretreatment were used directly for the LFA strip tests, and both two LFAs were able to accomplish analysis within 10 min. The results demonstrated that LFAs based on the newly produced, highly sensitive, and robust mAb were able to be used for both rapid qualitative screening of the type of poisoning and accurate quantitative determination of the severity of poisoning after accidental ingestion by patients of toxic mushrooms.

The potentially therapeutic targets of pediatric anaplastic ependymoma by transcriptome profiling.

Ependymoma (EPN) is a type of tumor that occurs in the central nervous system of children and adults. EPN produces resistance to chemotherapy, and there are no targeted drugs available as a proper cure. Therefore, the use of high-throughput sequencing technologies to elucidate pathogenic mechanisms is of prime importance to identify potential tumor target genes helpful for developing effective therapeutic approaches against EPN. With this objective, we used RNA-seq analysis to identify differentially expressed genes (DEGs) and pathways in 4 pairs of EPN tissues and adjacent tissues. In total, we found 5,445 differentially expressed genes. The synaptic vesicle cycle and extracellular matrix (ECM) receptor interaction pathways were highly enriched in the ependymoma group. Nine differentially expressed genes (SNAP25, GRM4, CELSR1, LAMA1, WNT5A, ROR2, CCND1, EPHB2, FOXJ1) were randomly verified by RT-qPCR, supporting the authenticity of our sequencing results. This study provides global gene information and some new potential biomarkers for the diagnosis and therapeutic targets of ependymoma.

Protein Kinase C Lambda Mediates Acid-Sensing Ion Channel 1a-Dependent Cortical Synaptic Plasticity and Pain Hypersensitivity.

Chronic pain is a serious debilitating disease for which effective treatment is still lacking. Acid-sensing ion channel 1a (ASIC1a) has been implicated in nociceptive processing at both peripheral and spinal neurons. However, whether ASIC1a also contributes to pain perception at the supraspinal level remains elusive. Here, we report that ASIC1a in ACC is required for thermal and mechanical hypersensitivity associated with chronic pain. ACC-specific genetic deletion or pharmacological blockade of ASIC1a reduced the probability of cortical LTP induction and attenuated inflammatory thermal hyperalgesia and mechanical allodynia in male mice. Using cell type-specific manipulations, we demonstrate that ASIC1a in excitatory neurons of ACC is a major player in cortical LTP and pain behavior. Mechanistically, we show that ASIC1a tuned pain-related cortical plasticity through protein kinase C λ-mediated increase of membrane trafficking of AMPAR subunit GluA1 in ACC. Importantly, postapplication of ASIC1a inhibitors in ACC reversed previously established nociceptive hypersensitivity in both chronic inflammatory pain and neuropathic pain models. These results suggest that ASIC1a critically contributes to a higher level of pain processing through synaptic potentiation in ACC, which may serve as a promising analgesic target for treatment of chronic pain.SIGNIFICANCE STATEMENT Chronic pain is a debilitating disease that still lacks effective therapy. Ion channels are good candidates for developing new analgesics. Here, we provide several lines of evidence to support an important role of cortically located ASIC1a channel in pain hypersensitivity through promoting long-term synaptic potentiation in the ACC. Our results indicate a promising translational potential of targeting ASIC1a to treat chronic pain.

Loss of Synaptic Tagging in the Anterior Cingulate Cortex after Tail Amputation in Adult Mice.

Anterior cingulate cortex (ACC) is known to play important roles in key brain functions such as pain perception, cognition, and emotion. Different forms of homosynaptic plasticity such as long-term potentiation (LTP) and long-term depression have been studied in ACC synapses. However, heterosynaptic plasticity such as synaptic tagging has not been reported. Here, we demonstrate synaptic tagging in the ACC of adult male mice by using a 64-channel multielectrode array recording system. Weak theta burst stimulation (TBS), normally inducing early-phase LTP or No-LTP in most of the activated channels, produced late phase-LTP (L-LTP) in a majority of channels when a strong TBS was applied earlier to a separate input within a certain time window. Similar to hippocampus, synaptic tagging in the ACC depends on the synthesis of new proteins. Tail amputation-induced peripheral injury caused a loss of this heterosynaptic L-LTP and occluded strong TBS-evoked L-LTP as well. Together, we provide the first report of the synaptic tagging-like phenomenon in the ACC of adult mice, and the loss of synaptic tagging to amputation may contribute to injury-related cognitive changes and phantom limb sensation and pain.SIGNIFICANCE STATEMENT ACC is an important cortical region involved in many brain functions. Previous studies have dissected the molecular mechanism of multiple types of homosynaptic plasticity of ACC synapses. Here, we report a novel form of heterosynaptic plasticity occurring in the ACC. This newly identified, protein synthesis-dependent neocortical synaptic tagging is sensitive to peripheral tail amputation injury and may provide basic mechanisms for synaptic pathophysiology of phantom pain and related cognitive changes.

Impaired presynaptic long-term potentiation in the anterior cingulate cortex of Fmr1 knock-out mice.

Fragile X syndrome is a common inherited form of mental impairment. Fragile X mental retardation protein (FMRP) plays important roles in the regulation of synaptic protein synthesis, and loss of FMRP leads to deficits in learning-related synaptic plasticity and behavioral disability. Previous studies mostly focus on postsynaptic long-term potentiation (LTP) in Fmr1 knock-out (KO) mice. Here, we investigate the role of FMRP in presynaptic LTP (pre-LTP) in the adult mouse anterior cingulate cortex (ACC). Low-frequency stimulation induced LTP in layer II/III pyramidal neurons under the voltage-clamp mode. Paired-pulse ratio, which is a parameter for presynaptic changes, was decreased after the low-frequency stimulation in Fmr1 wild-type (WT) mice. Cingulate pre-LTP was abolished in Fmr1 KO mice. We also used a 64-electrode array system for field EPSP recording and found that the combination of low-frequency stimulation paired with a GluK1-containing kainate receptor agonist induced NMDA receptor-independent and metabotropic glutamate receptor-dependent pre-LTP in the WT mice. This potentiation was blocked in Fmr1 KO mice. Biochemical experiments showed that Fmr1 KO mice displayed altered translocation of protein kinase A subunits in the ACC. Our results demonstrate that FMRP plays an important role in pre-LTP in the adult mouse ACC, and loss of this pre-LTP may explain some of the behavioral deficits in Fmr1 KO mice.

Minocycline does not affect long-term potentiation in the anterior cingulate cortex of normal adult mice.

It has been reported that activated microglia plays important roles in chronic pain-related sensory signaling at the spinal cord dorsal horn. Less is known about the possible contribution of microglia to cortical plasticity that has been found to be important for chronic pain. In the present study, we used a 64-channel multi-electrode array recording system to investigate the role of microglia in cortical plasticity of the anterior cingulate cortex (ACC) in normal adult mice. We found that bath application of minocycline, an inhibitor of microglial activation, had no effect on postsynaptic LTP (post-LTP) induced by theta burst stimulation in the ACC. Furthermore, presynaptic LTP (pre-LTP) induced by the combination of low-frequency stimulation with a GluK1-containing kainate receptor agonist was also not affected. The spatial distribution of post-LTP or pre-LTP among the cingulate network is also unaltered by minocycline. Our results suggest that minocycline does not affect cingulate plasticity and neurons are the major player in pain-related cortical plasticity.

Loss of long-term depression in the insular cortex after tail amputation in adult mice.

The insular cortex (IC) is an important forebrain structure involved in pain perception and taste memory formation. Using a 64-channel multi-electrode array system, we recently identified and characterized two major forms of synaptic plasticity in the adult mouse IC: long-term potentiation (LTP) and long-term depression (LTD). In this study, we investigate injury-related metaplastic changes in insular synaptic plasticity after distal tail amputation. We found that tail amputation in adult mice produced a selective loss of low frequency stimulation-induced LTD in the IC, without affecting (RS)-3,5-dihydroxyphenylglycine (DHPG)-evoked LTD. The impaired insular LTD could be pharmacologically rescued by priming the IC slices with a lower dose of DHPG application, a form of metaplasticity which involves activation of protein kinase C but not protein kinase A or calcium/calmodulin-dependent protein kinase II. These findings provide important insights into the synaptic mechanisms of cortical changes after peripheral amputation and suggest that restoration of insular LTD may represent a novel therapeutic strategy against the synaptic dysfunctions underlying the pathophysiology of phantom pain.

Assessment of wellbore integrity failure risk and hazardous zones in depleted reservoirs underground gas storage during the operation processes.

Depleted reservoirs are widely used for underground gas storage due to their advantages of low construction cost and easy development. Under the influence of complex geological conditions and frequent operations, the integrity of the wells in depleted reservoirs is prone to failure, which would potentially lead to gas leakage. In this study, by using a finite element-based computational fluid dynamics model, we have developed evaluation criteria for assessing the severity of the occurred wellbore integrity failure and the risk of the un-occurred wellbore integrity failures respectively to identify hazardous zones potentially prone to wellbore integrity failure. The study results indicate that the gas storage wellbore integrity failure is prone to occur inside the wellbore structure in the direction of the minimum ground stress near the lower boundary of the formation interlayer. The wellbore integrity failure hazardous zones are mainly concentrated at the formation interlayer boundaries. The practical guidelines and solutions derived from current research results can provide an accurate direction for monitoring and protecting work of wellbore integrity and avoid environment pollution problems caused by natural gas leakage.

Plasticity of metabotropic glutamate receptor-dependent long-term depression in the anterior cingulate cortex after amputation.

Long-term depression (LTD) is a key form of synaptic plasticity important in learning and information storage in the brain. It has been studied in various cortical regions, including the anterior cingulate cortex (ACC). ACC is a crucial cortical region involved in such emotion-related physiological and pathological conditions as fear memory and chronic pain. In the present study, we used a multielectrode array system to map cingulate LTD in a spatiotemporal manner within the ACC. We found that low-frequency stimulation (1 Hz, 15 min) applied onto deep layer V induced LTD in layers II/III and layers V/VI. Cingulate LTD requires activation of metabotropic glutamate receptors (mGluRs), while L-type voltage-gated calcium channels and NMDA receptors also contribute to its induction. Peripheral amputation of the distal tail impaired ACC LTD, an effect that persisted for at least 2 weeks. The loss of LTD was rescued by priming ACC slices with activation of mGluR1 receptors by coapplying (RS)-3,5-dihydroxyphenylglycine and MPEP, a form of metaplasticity that involved the activation of protein kinase C. Our results provide in vitro evidence of the spatiotemporal properties of ACC LTD in adult mice. We demonstrate that tail amputation causes LTD impairment within the ACC circuit and that this can be rescued by activation of mGluR1.

Long-term potentiation of synaptic transmission in the adult mouse insular cortex: multielectrode array recordings.

The insular cortex (IC) is widely believed to be an important forebrain structure involved in cognitive and sensory processes such as memory and pain. However, little work has been performed at the cellular level to investigate the synaptic basis of IC-related brain functions. To bridge the gap, the present study was designed to characterize the basic synaptic mechanisms for insular long-term potentiation (LTP). Using a 64-channel recording system, we found that an enduring form of late-phase LTP (L-LTP) could be reliably recorded for at least 3 h in different layers of IC slices after theta burst stimulation. The induction of insular LTP is protein synthesis dependent and requires activation of both GluN2A and GluN2B subunits of the NMDA receptor, L-type voltage-gated calcium channels, and metabotropic glutamate receptor 1. The paired-pulse facilitation ratio was unaffected by insular L-LTP induction, and expression of insular L-LTP required the recruitment of postsynaptic calcium-permeable AMPA receptors. Our results provide the first in vitro report of long-term multichannel recordings of L-LTP in the IC in adult mice and suggest its potential important roles in insula-related memory and chronic pain.

N-type voltage gated calcium channels mediate excitatory synaptic transmission in the anterior cingulate cortex of adult mice.

Voltage gated calcium channels (VGCCs) are well known for its importance in synaptic transmission in the peripheral and central nervous system. However, the role of different VGCCs in the anterior cingulate cortex (ACC) has not been studied. Here, we use a multi-electrode array recording system (MED64) to study the contribution of different types of calcium channels in glutamatergic excitatory synaptic transmission in the ACC. We found that only the N-type calcium channel blocker ω-conotoxin-GVIA (ω-Ctx-GVIA) produced a great inhibition of basal synaptic transmission, especially in the superficial layer. Other calcium channel blockers that act on L-, P/Q-, R-, and T-type had no effect. We also tested the effects of several neuromodulators with or without ω-Ctx-GVIA. We found that N-type VGCC contributed partially to (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid- and (R)-Baclofen-induced synaptic inhibition. By contrast, the inhibitory effects of 2-Chloroadenosine and carbamoylcholine chloride did not differ with or without ω-Ctx-GVIA, indicating that they may act through other mechanisms. Our results provide strong evidence that N-type VGCCs mediate fast synaptic transmission in the ACC.

Long-term depression of synaptic transmission in the adult mouse insular cortex in vitro.

The insular cortex (IC) is known to play important roles in higher brain functions such as memory and pain. Activity-dependent long-term depression (LTD) is a major form of synaptic plasticity related to memory and chronic pain. Previous studies of LTD have mainly focused on the hippocampus, and no study in the IC has been reported. In this study, using a 64-channel recording system, we show for the first time that repetitive low-frequency stimulation (LFS) can elicit frequency-dependent LTD of glutamate receptor-mediated excitatory synaptic transmission in both superficial and deep layers of the IC of adult mice. The induction of LTD in the IC required activation of the N-methyl-d-aspartate (NMDA) receptor, metabotropic glutamate receptor (mGluR)5, and L-type voltage-gated calcium channel. Protein phosphatase 1/2A and endocannabinoid signaling are also critical for the induction of LTD. In contrast, inhibiting protein kinase C, protein kinase A, protein kinase Mζ or calcium/calmodulin-dependent protein kinase II did not affect LFS-evoked LTD in the IC. Bath application of the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine produced another form of LTD in the IC, which was NMDA receptor-independent and could not be occluded by LFS-induced LTD. Our studies have characterised the basic mechanisms of LTD in the IC at the network level, and suggest that two different forms of LTD may co-exist in the same population of IC synapses.

Broad-specificity indirect competitive enzyme-linked immunosorbent assay for aristolochic acids: Computer-aided hapten design and molecular mechanism of antibody recognition.

Long-term dietary exposure of aristolochic acids (AAs)-contaminated food proved to be one of the main culprits of Endemic Nephropathy, renal failure; and urothelial cancer. The antibodies utilized in immunoassays for AAs suffer from low affinity and failure of recognition to the family of AAs. This study, we prepared a broad-specificity monoclonal antibody (mAb) 5H5 with highly and uniform affinity for AAs by help of computational chemistry fully exposing the AAs common structures of methoxy and hydroxyl groups. The mAb 5H5 exhibited half inhibitory concentrations of AAA, AAB, AAC, AAD were 0.03, 0.06, 0.05, 0.03 ng/mL. To explain the broad-specificity profile of mAb 5H5, molecular docking was performed. Results shown that multiple conformations of AAs can be flexibly oriented in the spacious cavity of single-chain variable fragment antibody (scFv) 5H5 and the specific hydron bonds were formed by ASN62 and GLY64 of scFV 5H5 to the nitro group of AAs which gave an explanation of the high cross-reactivity of mAb 5H5. The ELISA based on the broad-specificity mAb 5H5with detection limits of 0.04-0.11 µg/kg and 0.02-0.06 µg/kg for four AAs in flour and soil samples, respectively. The study provided a promising method for the family of AAs in environmental and food samples.

Development of epitopephore-based rational hapten design strategy: A combination of theoretical evidence and experimental validation.

Antibody is the key biomolecule that governing the sensitivity and specificity of an immunoassay for chemical compound, also named hapten molecule. Obviously, predication of hapten effectiveness before chemical synthesis is beneficial to boost success, save cost and improve controllability. Here, we proposed and evaluated an epitopephore based rational hapten design (ERHD) to assist antibody production to chemical compound, combining theoretical evidence and then experimental validation by using dinitrocarbanilide (DNC) as a model analyte. Briefly, epitopephores of DNC were firstly generated by HipHop algorithm after features mapping. A homemade drug database also containing reported fragment haptens (HFR) and new designed full hapten (HFU) were constructed, and then was virtually screened by using generated epitopephore followed by structural analysis and visual inspection. The DNC haptens based on the selected hits were further identified by Density Functional Theory before total synthesis. To prove and clarify the usability of the ERHD, two retrieved HFU haptens, one non-retrieved HFU hapten and three non-retrieved HFR haptens were all selected to produce monoclonal antibodies (mAbs) for comparison purpose. A maximal 6000-fold increased affinity of mAb from retrieved HFU than HFR was observed, while, non-retrieved HFU failed to produce antibody to DNC. More importantly, mAbs from HFU haptens provided highly specificity to DNC, while, mAbs from HFR haptens could recognize 15 others analogues. We then constructed antibody structure and investigated molecular recognition of the mAbs to DNC, well supporting the rationality of the ERHD. Lastly, an icELISA was developed for DNC with an IC50 value as low as 0.19 ng mL-1 with high specificity, which has never achieved before.

Representation and control of pain and itch by distinct prefrontal neural ensembles.

Pain and itch are two closely related but essentially distinct sensations that elicit different behavioral responses. However, it remains mysterious how pain and itch information is encoded in the brain to produce differential perceptions. Here, we report that nociceptive and pruriceptive signals are separately represented and processed by distinct neural ensembles in the prelimbic (PL) subdivision of the medial prefrontal cortex (mPFC) in mice. Pain- and itch-responsive cortical neural ensembles were found to significantly differ in electrophysiological properties, input-output connectivity profiles, and activity patterns to nociceptive or pruriceptive stimuli. Moreover, these two groups of cortical neural ensembles oppositely modulate pain- or itch-related sensory and emotional behaviors through their preferential projections to specific downstream regions such as the mediodorsal thalamus (MD) and basolateral amygdala (BLA). These findings uncover separate representations of pain and itch by distinct prefrontal neural ensembles and provide a new framework for understanding somatosensory information processing in the brain.

An Anterior Cingulate Cortex-to-Midbrain Projection Controls Chronic Itch in Mice.

Itch is an unpleasant sensation that provokes the desire to scratch. While acute itch serves as a protective system to warn the body of external irritating agents, chronic itch is a debilitating but poorly-treated clinical disease leading to repetitive scratching and skin lesions. However, the neural mechanisms underlying the pathophysiology of chronic itch remain mysterious. Here, we identified a cell type-dependent role of the anterior cingulate cortex (ACC) in controlling chronic itch-related excessive scratching behaviors in mice. Moreover, we delineated a neural circuit originating from excitatory neurons of the ACC to the ventral tegmental area (VTA) that was critically involved in chronic itch. Furthermore, we demonstrate that the ACC→VTA circuit also selectively modulated histaminergic acute itch. Finally, the ACC neurons were shown to predominantly innervate the non-dopaminergic neurons of the VTA. Taken together, our findings uncover a cortex-midbrain circuit for chronic itch-evoked scratching behaviors and shed novel insights on therapeutic intervention.

Metabotropic glutamate receptor 5 contributes to inflammatory tongue pain via extracellular signal-regulated kinase signaling in the trigeminal spinal subnucleus caudalis and upper cervical spinal cord.

BACKGROUND: In the orofacial region, limited information is available concerning pathological tongue pain, such as inflammatory pain or neuropathic pain occurring in the tongue. Here, we tried for the first time to establish a novel animal model of inflammatory tongue pain in rats and to investigate the roles of metabotropic glutamate receptor 5 (mGluR5)-extracellular signal-regulated kinase (ERK) signaling in this process. METHODS: Complete Freund's adjuvant (CFA) was submucosally injected into the tongue to induce the inflammatory pain phenotype that was confirmed by behavioral testing. Expression of phosphorylated ERK (pERK) and mGluR5 in the trigeminal subnucleus caudalis (Vc) and upper cervical spinal cord (C1-C2) were detected with immunohistochemical staining and Western blotting. pERK inhibitor, a selective mGluR5 antagonist or agonist was continuously administered for 7 days via an intrathecal (i.t.) route. Local inflammatory responses were verified by tongue histology. RESULTS: Submucosal injection of CFA into the tongue produced a long-lasting mechanical allodynia and heat hyperalgesia at the inflamed site, concomitant with an increase in the pERK immunoreactivity in the Vc and C1-C2. The distribution of pERK-IR cells was laminar specific, ipsilaterally dominant, somatotopically relevant, and rostrocaudally restricted. Western blot analysis also showed an enhanced activation of ERK in the Vc and C1-C2 following CFA injection. Continuous i.t. administration of the pERK inhibitor and a selective mGluR5 antagonist significantly depressed the mechanical allodynia and heat hyperalgesia in the CFA-injected tongue. In addition, the number of pERK-IR cells in ipsilateral Vc and C1-C2 was also decreased by both drugs. Moreover, continuous i.t. administration of a selective mGluR5 agonist induced mechanical allodynia in naive rats. CONCLUSIONS: The present study constructed a new animal model of inflammatory tongue pain in rodents, and demonstrated pivotal roles of the mGluR5-pERK signaling in the development of mechanical and heat hypersensitivity that evolved in the inflamed tongue. This tongue-inflamed model might be useful for future studies to further elucidate molecular and cellular mechanisms of pathological tongue pain such as burning mouth syndrome.

Study on anaerobic co-digestion of municipal sewage sludge and fruit and vegetable wastes: Methane production, microbial community and three-dimension fluorescence excitation-emission matrix analysis.

Constantly increased sewage sludge (SS) and fruit and vegetable wastes (FVW) are becoming the major organic solid wastes in human society. Thus, anaerobic digestion is employed as a low carbon energy strategy to reduce their environmental pollution risk. Anaerobic co-digestion system was developed based on the carbon to nitrogen ratio strategy. Results showed that the daily biogas production was higher in co-digester, and the volumetric biogas production rate (VBPR) significantly enhanced for 1.3 âˆ¼ 3 folds, and the highest VBPR was 2.04 L/L • day with optimal OLR of 2.083 Kg L-1 d-1. Analytic results indicated that co-digestion could improve the biodegradable of feedstocks, which transforming to more VFAs and biogas. Compared with mono SS digester, mixed substrates relieved ammonia nitrogen inhibition and enhanced the hydrolytic acidification and methanogenesis. Meanwhile, the excessive humification of organics was suppressed. This study supported the concepts of improving carbon recovery from SS and FVW.