Metabolomics reveals arbuscular mycorrhizal fungi-mediated tolerance of walnut to soil drought.

BACKGROUND: Arbuscular mycorrhizal fungi (AMF) have a positive effect on drought tolerance of plants after establishing reciprocal resymbiosis with roots, while the underlying mechanism is not deciphered. Metabolomics can explain the mechanism of plant response to environmental stress by analyzing the changes of all small molecular weight metabolites. The purpose of this study was to use Ultra High Performance Liquid Chromatography Q Exactive Mass Spectrometer to analyze changes in root metabolites of walnut (Juglans regia) after inoculation with an arbuscular mycorrhizal fungus Diversispora spurca under well-watered (WW) and drought stress (DS). RESULTS: Sixty days of soil drought significantly inhibited root mycorrhizal colonization rate, shoot and root biomass production, and leaf water potential in walnut, while AMF inoculation significantly increased biomass production and leaf water potential, accompanied by a higher increase magnitude under DS versus under WW. A total of 3278 metabolites were identified. Under WW, AMF inoculation up-regulated 172 metabolites and down-regulated 61 metabolites, along with no changes in 1104 metabolites. However, under DS, AMF inoculation up-regulated 49 metabolites and down-regulated 116 metabolites, coupled with no changes in 1172 metabolites. Among them, juglone (a quinone found in walnuts) as the first ranked differential metabolite was up-regulated by AMF under WW but not under DS; 2,3,5-trihydroxy-5-7-dimethoxyflavanone as the first ranked differential metabolite was increased by AMF under DS but not under WW. The KEGG annotation showed a large number of metabolic pathways triggered by AMF, accompanied by different metabolic pathways under WW and DS. Among them, oxidative phosphorylation and phenylalanine metabolism and biosynthesis were triggered by AMF in response to WW and DS, where N-acetyl-L-phenylalanine was induced by AMF to increase under DS, while decreasing under WW. CONCLUSION: This study provides new insights into the metabolic mechanisms of mycorrhiza-enhanced drought tolerance in walnuts.

The potent analgesia of intrathecal 2R, 6R-HNK via TRPA1 inhibition in LF-PENS-induced chronic primary pain model.

BACKGROUND: Chronic primary pain (CPP) is an intractable pain of unknown cause with significant emotional distress and/or dysfunction that is a leading factor of disability globally. The lack of a suitable animal model that mimic CPP in humans has frustrated efforts to curb disease progression. 2R, 6R-hydroxynorketamine (2R, 6R-HNK) is the major antidepressant metabolite of ketamine and also exerts antinociceptive action. However, the analgesic mechanism and whether it is effective for CPP are still unknown. METHODS: Based on nociplastic pain is evoked by long-term potentiation (LTP)-inducible high- or low-frequency electrical stimulation (HFS/LFS), we wanted to develop a novel CPP mouse model with mood and cognitive comorbidities by noninvasive low-frequency percutaneous electrical nerve stimulation (LF-PENS). Single/repeated 2R, 6R-HNK or other drug was intraperitoneally (i.p.) or intrathecally (i.t.) injected into naïve or CPP mice to investigate their analgesic effect in CPP model. A variety of behavioral tests were used to detect the changes in pain, mood and memory. Immunofluorescent staining, western blot, reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and calcium imaging of in cultured dorsal root ganglia (DRG) neurons by Fluo-8-AM were used to elucidate the role and mechanisms of 2R, 6R-HNK in vivo or in vitro. RESULTS: Intrathecal 2R, 6R-HNK, rather than intraperitoneal 2R, 6R-HNK or intrathecal S-Ketamine, successfully mitigated HFS-induced pain. Importantly, intrathecal 2R, 6R-HNK displayed effective relief of bilateral pain hypersensitivity and depressive and cognitive comorbidities in a dose-dependent manner in LF-PENS-induced CPP model. Mechanically, 2R, 6R-HNK markedly attenuated neuronal hyperexcitability and the upregulation of calcitonin gene-related peptide (CGRP), transient receptor potential ankyrin 1 (TRPA1) or vanilloid-1 (TRPV1), and vesicular glutamate transporter-2 (VGLUT2) in peripheral nociceptive pathway. In addition, 2R, 6R-HNK suppressed calcium responses and CGRP overexpression in cultured DRG neurons elicited by the agonists of TRPA1 or/and TRPV1. Strikingly, the inhibitory effects of 2R, 6R-HNK on these pain-related molecules and mechanical allodynia were substantially occluded by TRPA1 antagonist menthol. CONCLUSIONS: In the newly designed CPP model, our findings highlighted the potential utility of intrathecal 2R, 6R-HNK for preventing and therapeutic modality of CPP. TRPA1-mediated uprgulation of CGRP and neuronal hyperexcitability in nociceptive pathways may undertake both unique characteristics and solving process of CPP.

Evaluation of macular microvascular density using optical coherence tomography angiography in patients with Posner-Schlossman syndrome.

BACKGROUND: Optical coherence tomography angiography (OCTA) is a novel technology that provides a noninvasive, dye-less method to visualize the blood vessels of the retina. In the present study, we investigate macular microvascular density and the correlation of ocular and demographic factors using OCTA in Posner-Schlossman syndrome (PSS) patients. METHODS: This is a prospective observational study. All PSS patients and age- and sex-matched healthy subjects underwent complete ophthalmologic examination, and RE, BCVA, IOP, CCT, AL, CMT, GCIPI, RNFL, C/D ratio were recorded. The whole-image vessel density (wiVD) and whole-image perfusion density (wiPD), three-circle (1 mm central ring, 3 mm inner ring, 6 mm outer ring), and four-quadrant segmental VD and PD were calculated. RESULTS: Seventeen PSS patients and 17 healthy subjects were enrolled in this study. The mean age was 42.65 ± 11.22 years in PSS patients and 42.71 ± 10.50 years in healthy controls. IOP, CCT, and C/D ratio were higher in PSS-attacked eyes, and BCVA, OPP and RNFL thickness was lower than those in the fellow eyes (p < 0.05). BCVA and OPP were improved in the PSS-attacked eyes in intermittent period (p < 0.05). The wiVD and wiPD were lower in the PSS-affected eyes than in the fellow eyes and in the control eyes in the PSS-attacked period (p < 0.05). All segmental VD and PD was lower in the PSS affected eyes than in the healthy control eyes (p < 0.05). In intermittent period, the wiVD and wiPD were lower in the PSS-affected eyes than in the fellow eyes (p < 0.05). Age, CCT, and SSI were associated with macular wiVD and wiPD in PSS attacked period. Age and CCT were associated with macular wiVD and wiPD in PSS intermittent period. CONCLUSION: Decreased macular superficial VD and PD was found in patients with Posner-Schlossman syndrome in attacked period and in remission. Macular wiVD and wiPD were associated with age, CCT and SSI in PSS patients.

The long noncoding RNA NKILA protects against myocardial ischaemic injury by enhancing myocardin expression via suppressing the NF-κB signalling pathway.

BACKGROUND: The lncRNA NKILA has been reported to interact with NF-κB and has an important role in various human diseases. However, the role of NKILA in myocardial ischaemic injury is still unknown. METHODS: We established cell and animal models of myocardial ischaemic injury. We confirmed our findings by overexpressing NKILA, silencing myocardin and using an NF-κB pathway inhibitor in a hypoxia/reoxygenation (H/R) model of H9c2 cells. An animal model of ischaemia-reperfusion (I/R) injury was established by LAD ligation. Overexpression of NKILA was achieved by adeno-associated virus (AAV) injection through the tail vein. Annexin-V/PI staining and flow cytometric analysis were performed to test cell apoptosis. ELISAs were used to determine the secretion of inflammatory factors. TTC, HE and TUNEL staining were performed to study myocardial pathological injury. qRT-PCR or Western blotting were used to test the expression levels of NKILA, myocardin, the NF-κB pathway and apoptosis-related proteins. RESULTS: H/R and I/R treatment significantly suppressed the expression of NKILA and activated the NF-κB pathway, resulting in the loss of myocardin. Overexpressing NKILA led to the suppression of the NF-κB pathway and successfully prevented the cell apoptosis and inflammatory responses caused by H/R stimulation in H9c2 cells. Silencing myocardin reversed the protective effect of NKILA and led to severe injury in the H9c2 cells that underwent H/R. Furthermore, the NF-κB pathway inhibitor BAY11-7028 reduced the H/R injury in H9c2 cells with little effect on NKILA expression. Similar results were confirmed in an animal model of myocardial I/R injury and showed that overexpression of NKILA inhibited I/R-triggered myocardial injury in vivo. CONCLUSION: NKILA enhanced the expression of myocardin via inhibiting the NF-κB signalling pathway and preventing cell apoptosis and the inflammatory response of cardiomyocytes, thus ameliorating myocardial I/R injury.

Quantitative assessment and determinants of the papillary microvasculature in healthy subjects.

BACKGROUND: It is critical to monitor the optic disc's vessel density using Optical coherence tomography angiography (OCTA) and evaluate its determinants. In the current study, we investigate the superficial vessel density (VD) of the papillary microvasculature and its determinants in healthy subjects of Southern China. METHODS: This was a prospective, cross-sectional study. Superficial VD in healthy individuals' optic disc region was measured by OCTA. The factors associated with ocular and systemic parameters were analyzed using a generalized estimation equation (GEE) model. RESULTS: A total of 510 eyes of 260 healthy subjects were analyzed in the study. The total VD in the optic disc area was 17.21 ± 2.15 mm- 1 (95% CI, 17.02-17.40 mm- 1). The VD in the inner ring and the outer ring of the optic disc were significantly higher compared with the central ring, while the VD of the superior quadrant and inferior quadrant was significantly higher compared with the temporal and nasal quadrant. After adjusting for the ocular factors and systemic factors, AL (ß = - 0.4917, P = 0.0003), disc area (ß = - 0.3748, P = 0.0143), CMT (ß = - 0.0183, P = 0.0003) and SSI (ß = 1.0588, P < 0.001) were significantly associated with total VD of the optic disc. CONCLUSION: The mean total VD in the optic disc area was 17.21 ± 2.15 mm- 1 in healthy subjects, and the superior and inferior VD was significantly higher than the temporal and nasal VD. AL, disc area, CMT, and SSI may affect the total VD in the optic disc area and should be considered in clinical practice.

Tissue concentrations, trophic transfer and human risks of antibiotics in freshwater food web in Lake Taihu, China.

The objective of this study was to evaluate the tissue distributions of antibiotics in the fish, the bioaccumulation and trophic transfer in freshwater food web in Taihu Lake, a large shallow freshwater lake. Twenty four out of 41 antibiotics were detected in the biotas of the food web; and antibiotic concentrations followed the orders: fish plasma ~ fish muscle < fish liver ~ fish bile and fish < invertebrates ~ plankton. Antibiotic concentrations in the liver of piscivores were higher than those in omnivores and planktivores. Most bioaccumulation factors (BAFs) of sulfonamides (SAs), macrolides (MLs), ionophores (IPs) and lincomycin (LIN) were less than 2000 L/kg, indicating low bioaccumulation ability of these compounds in fish. Fluoroquinolones (FQs) were frequently detected in fish liver, invertebrates and plankton with much of BAFs great than 5000 L/kg, indicating that FQs have the potential of bioaccumulation in fish. Relationship analysis between BAFs and physicochemical properties of antibiotics showed that the bioaccumulation of antibiotics in the biota was related with their adsorption ability. Generally, the antibiotics in the food web of Lake Taihu including plankton, invertebrates and fish showed trophic dilution. The normalized estimated daily intake (EDI) values are less than the acceptable daily intake (ADI) values, and then hazard quotients were much less than 1. This result suggests the consumption of fish, crab and shrimp in Lake Taihu would probably not pose direct detrimental effects on humans.

Differential regulation of GSK-3ß in spinal dorsal horn and in hippocampus mediated by interleukin-1beta contributes to pain hypersensitivity and memory deficits following peripheral nerve injury.

Accumulating evidence shows that inhibition of glycogen synthase kinase-3beta (GSK-3ß) ameliorates cognitive impairments caused by a diverse array of diseases. Our previous work showed that spared nerve injury (SNI) that induces neuropathic pain causes short-term memory deficits. Here, we reported that GSK-3ß activity was enhanced in hippocampus and reduced in spinal dorsal horn following SNI, and the changes persisted for at least 45 days. Repetitive applications of selective GSK-3ß inhibitors (SB216763, 5 mg/kg, intraperitoneally, three times or AR-A014418, 400 ng/kg, intrathecally, seven times) prevented short-term memory deficits but did not affect neuropathic pain induced by SNI. Surprisingly, we found that the repetitive SB216763 or AR-A014418 induced a persistent pain hypersensitivity in sham animals. Mechanistically, both ß-catenin and brain-derived neurotrophic factor (BDNF) were upregulated in spinal dorsal horn but downregulated in hippocampus following SNI. Injections of SB216763 prevented the BDNF downregulation in hippocampus but enhanced its upregulation in spinal dorsal horn in SNI rats. In sham rats, SB216763 upregulated both ß-catenin and BDNF in spinal dorsal horn but affect neither of them in hippocampus. Finally, intravenous injection of interleukin-1beta that induces pain hypersensitivity and memory deficits mimicked the SNI-induced the differential regulation of GSK-3ß/ß-catenin/BDNF in spinal dorsal horn and in hippocampus. Accordingly, the prolonged opposite changes of GSK-3ß activity in hippocampus and in spinal dorsal horn induced by SNI may contribute to memory deficits and neuropathic pain by differential regulation of BDNF in the two regions. GSK-3ß inhibitors that treat cognitive disorders may result in a long-lasting pain hypersensitivity.

Trends in the occurrence and risk assessment of antibiotics in shallow lakes in the lower-middle reaches of the Yangtze River basin, China.

Antibiotics have become a global public concern because of their extensively usage and high toxicity on aquatic organisms, especially leading to the widespread of antibiotic resistance genes. The objective of this study was to evaluate the occurrence, spatial distribution and ecological risks of multi-classes commonly used human and veterinary antibiotics in both aqueous and sedimentary phases of 65 shallow lakes in the lower-middle reaches of the Yangtze River, China. In the target area, antibiotic concentrations in most of lakes (<20 ng/L in the water of 22 lakes and <20 ng/g in the sediments of 43 lakes) were generally lower than those documented in previous studies in China and other countries, and these differences were probably due to less pollutant sources, high temperatures and heavy rainfall in summer. The concentrations of antibiotics in water (>100 ng/L) or sediments (>100 ng/g) of nine lakes, such as Dianshan Lake, Ge Lake and Ce Lake, were comparable to those in rivers and lakes that were seriously polluted by urban and livestock wastewater in China. The Taihu lakes showed relatively higher antibiotic concentrations, followed by the Huaihe River lakes, Poyang lakes and Dongting lakes. The composition of antibiotics showed that agricultural source might be the main source of antibiotics in most of the lakes in the lower-middle reaches of the Yangtze River basin, China. The pseudo distribution coefficient (P-Kd) and significant relationship between antibiotics and environmental factors in the present study suggested the spatial of antibiotics in the lakes might be affected by antibiotics' physiochemical properties and environmental factors. The environmental risk assessment results showed that in general, sulfamethoxazole (SMX), erythromycin (ETM) and ofloxacin (OFX) in the surface water could pose medium risks to algae or bacteria in the aquatic ecosystem, while antibiotics ETM, roxithromycin (RTM), enrofloxacin (EFX) and sulfadiazine (SDZ) in the sediment might pose medium risks to algae or bacteria populations. High potential risk might occur in winter in most lakes due to lower water storage and less degradation. Overall, our study reveals the pollution trends and potential sources of antibiotics in shallow lakes in the lower-middle reaches of the Yangtze River basin.

Multi-phase distribution and risk assessment of endocrine disrupting chemicals in the surface water of the Shaying River, -Huai River Basin, China.

Herein we investigated the multi-phase distribution and estrogenic effects of endocrine disrupting chemicals (EDCs) in suspended particulate matter (SPM), colloids, and soluble phases from the Shaying River to assess the composition of estrogenic compounds and associated estrogenic risk. The yeast two hybrid (YES) method, cross-flow ultrafiltration (CFUF), and LC-MS/MS were employed. Risk quotient (RQ) values ranged from 0.72 to 3.88, revealing that the Shaying River posed high estrogenic risk to aquatic organisms. The contribution ratios of the target EDCs to the EEQYES ranged from 62.7% to 92.5%, indicating that these chemicals were major contributors of estrogenic effects in the Shaying River. Further, 54.0-77.8% of the detected EDCs were distributed in the soluble phase, 15.1-31.7% were bound to colloidal substances, and 3.90-19.4% EDCs were associated with SPM. Significant correlation between total EDC abundance and COD contents was detected, and the concentrations of endogenous estrogens (E1, E2, and E3) were positively correlated with total nitrogen (TN) and total phosphorus (TP). In addition, the in-situ SPM-soluble (Kpoc) and colloid-soluble partition (Kcoc) coefficients were calculated. The log Kpoc values of target compounds varied from 4.10 to 5.19, while log Kcoc values ranged from 4.25 to 5.56. Their Kcoc values were larger than the Kpoc values, indicating that organic colloids were the most important carriers of EDCs in the aquatic environment.

[A modified protocol of mouse hippocampal primary microglia culture by using manual dissociation, magnetic activated cell sorting and TIC medium].

In this study, we improved the culture method of mouse hippocampal primary microglia to obtain hippocampal ramified microglia with high activity and purity, which were resemble to the resting status of normal microglia in healthy brain in vivo. Hippocampal tissue was excised from 2-4-week-old SPF C57BL/6J mice and cut into pieces after PBS perfusion, and then manually dissociated into the single-cell suspension by using Miltenyi Biotec's Adult Brain Dissociation Kit. The tissue fragments such as myelin in the supernatant were removed by debris removal solution in the kit. The cell suspension was incubated with CD11b immunomagnetic beads for 15 min at 4 °C. To obtain high-purity microglia, we used two consecutive cell-sorting steps by magnetic activated cell sorting (MACS). After centrifugation, the cells were resuspended and seeded in a 24-well culture plate. The primary microglia were cultured with complete medium (CM) or TIC medium (a serum-free medium with TGF-ß, IL-34 and cholesterol as the main nutritional components) for 4 days, and then were used for further experiments. The results showed that: (1) The cell viability was (56.03 ± 2.10)% by manual dissociation of hippocampus; (2) Compared with immunopanning, two-step MACS sorting allowed for efficient enrichment of microglia with higher purity of (86.20 ± 0.68)%; (3) After being incubated in TIC medium for 4 d, microglia exhibited branching, quiescent morphology; (4) The results from qRT-PCR assay showed that the levels of TNF-α, IL-1ß and CCL2 mRNA in TIC cultured-microglia were similar to freshly isolated microglia, while those were much higher in CM cultured-microglia after incubation for 4 d and 7 d (P < 0.05). Taken together, compared to the conventional approaches, this modified protocol of mouse hippocampal primary microglia culture by using MACS and TIC medium enables the increased yield and purity of microglia in the quiescent state, which is similar to normal ramified microglia in healthy brain in vivo.

Chemokine CCL2-CCR2 Signaling Induces Neuronal Cell Death via STAT3 Activation and IL-1ß Production after Status Epilepticus.

Elevated levels of chemokine C-C motif ligand 2 (CCL2) and its receptor CCR2 have been reported in patients with temporal lobe epilepsy and in experimental seizures. However, the functional significance and molecular mechanism underlying CCL2-CCR2 signaling in epileptic brain remains largely unknown. In this study, we found that the upregulated CCL2 was mainly expressed in hippocampal neurons and activated microglia from mice 1 d after kainic acid (KA)-induced seizures. Taking advantage of CX3CR1GFP/+:CCR2RFP/+ double-transgenic mice, we demonstrated that CCL2-CCR2 signaling has a role in resident microglial activation and blood-derived monocyte infiltration. Moreover, seizure-induced degeneration of neurons in the hippocampal CA3 region was attenuated in mice lacking CCL2 or CCR2. We further showed that CCR2 activation induced STAT3 (signal transducer and activator of transcription 3) phosphorylation and IL-1ß production, which are critical for promoting neuronal cell death after status epilepticus. Consistently, pharmacological inhibition of STAT3 by WP1066 reduced seizure-induced IL-1ß production and subsequent neuronal death. Two weeks after KA-induced seizures, CCR2 deficiency not only reduced neuronal loss, but also attenuated seizure-induced behavioral impairments, including anxiety, memory decline, and recurrent seizure severity. Together, we demonstrated that CCL2-CCR2 signaling contributes to neurodegeneration via STAT3 activation and IL-1ß production after status epilepticus, providing potential therapeutic targets for the treatment of epilepsy.SIGNIFICANCE STATEMENT Epilepsy is a global concern and epileptic seizures occur in many neurological conditions. Neuroinflammation associated with microglial activation and monocyte infiltration are characteristic of epileptic brains. However, molecular mechanisms underlying neuroinflammation in neuronal death following epilepsy remain to be elucidated. Here we demonstrate that CCL2-CCR2 signaling is required for monocyte infiltration, which in turn contributes to kainic acid (KA)-induced neuronal cell death. The downstream of CCR2 activation involves STAT3 (signal transducer and activator of transcription 3) phosphorylation and IL-1ß production. Two weeks after KA-induced seizures, CCR2 deficiency not only reduced neuronal loss, but also attenuated seizure-induced behavioral impairments, including anxiety, memory decline, and recurrent seizure severity. The current study provides a novel insight on the function and mechanisms of CCL2-CCR2 signaling in KA-induced neurodegeneration and behavioral deficits.

TNF-α Differentially Regulates Synaptic Plasticity in the Hippocampus and Spinal Cord by Microglia-Dependent Mechanisms after Peripheral Nerve Injury.

Clinical studies show that chronic pain is accompanied by memory deficits and reduction in hippocampal volume. Experimental studies show that spared nerve injury (SNI) of the sciatic nerve induces long-term potentiation (LTP) at C-fiber synapses in spinal dorsal horn, but impairs LTP in the hippocampus. The opposite changes may contribute to neuropathic pain and memory deficits, respectively. However, the cellular and molecular mechanisms underlying the functional synaptic changes are unclear. Here, we show that the dendrite lengths and spine densities are reduced significantly in hippocampal CA1 pyramidal neurons, but increased in spinal neurokinin-1-positive neurons in mice after SNI, indicating that the excitatory synaptic connectivity is reduced in hippocampus but enhanced in spinal dorsal horn in this neuropathic pain model. Mechanistically, tumor necrosis factor-alpha (TNF-α) is upregulated in bilateral hippocampus and in ipsilateral spinal dorsal horn, whereas brain-derived neurotrophic factor (BDNF) is decreased in the hippocampus but increased in the ipsilateral spinal dorsal horn after SNI. Importantly, the SNI-induced opposite changes in synaptic connectivity and BDNF expression are prevented by genetic deletion of TNF receptor 1 in vivo and are mimicked by TNF-α in cultured slices. Furthermore, SNI activated microglia in both spinal dorsal horn and hippocampus; pharmacological inhibition or genetic ablation of microglia prevented the region-dependent synaptic changes, neuropathic pain, and memory deficits induced by SNI. The data suggest that neuropathic pain involves different structural synaptic alterations in spinal and hippocampal neurons that are mediated by overproduction of TNF-α and microglial activation and may underlie chronic pain and memory deficits. SIGNIFICANCE STATEMENT: Chronic pain is often accompanied by memory deficits. Previous studies have shown that peripheral nerve injury produces both neuropathic pain and memory deficits and induces long-term potentiation (LTP) at C-fiber synapses in spinal dorsal horn (SDH) but inhibits LTP in hippocampus. The opposite changes in synaptic plasticity may contribute to chronic pain and memory deficits, respectively. However, the structural and molecular bases of these alterations of synaptic plasticity are unclear. Here, we show that the complexity of excitatory synaptic connectivity and brain-derived neurotrophic factor (BDNF) expression are enhanced in SDH but reduced in the hippocampus in neuropathic pain and the opposite changes depend on tumor necrosis factor-alpha/tumor necrosis factor receptor 1 signaling and microglial activation. The region-dependent synaptic alterations may underlie chronic neuropathic pain and memory deficits induced by peripheral nerve injury.

Heterosynaptic long-term potentiation from the anterior cingulate cortex to spinal cord in adult rats.

Spinal nociceptive transmission receives biphasic modulation from supraspinal structures. Recent studies demonstrate that the anterior cingulate cortex facilitates spinal excitatory synaptic transmission and nociceptive reflex. However, whether the top-down descending facilitation can cause long-term synaptic changes in spinal cord remains unclear. In the present study, we recorded C-fiber-evoked field potentials in spinal dorsal horn and found that the anterior cingulate cortex stimulation caused enhancement of C-fiber-mediated responses. The enhancement lasted for more than a few hours. Spinal application of N-methyl-D-aspartate (NMDA) receptor antagonist D-AP5 abolished this enhancement, suggesting that the activation of the NMDA receptor is required. Furthermore, spinal application of methysergide, a serotonin receptor antagonist, also blocked the anterior cingulate cortex-induced spinal long-term potentiation. Our results suggest that the anterior cingulate cortex stimulation can produce heterosynaptic form of long-term potentiation at the spinal cord dorsal horn, and this novel form of long-term potentiation may contribute to top-down long-term facilitation in chronic pain conditions.

Ammonia Monooxygenase-Mediated Cometabolic Biotransformation and Hydroxylamine-Mediated Abiotic Transformation of Micropollutants in an AOB/NOB Coculture.

Biotransformation of various micropollutants (MPs) has been found to be positively correlated with nitrification in activated sludge communities. To further elucidate the roles played by ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), we investigated the biotransformation capabilities of an NOB pure culture ( Nitrobacter sp.) and an AOB ( Nitrosomonas europaea)/NOB ( Nitrobacter sp.) coculture for 15 MPs, whose biotransformation was reported previously to be associated with nitrification. The NOB pure culture did not biotransform any investigated MP, whereas the AOB/NOB coculture was capable of biotransforming six MPs (i.e., asulam, bezafibrate, fenhexamid, furosemide, indomethacin, and rufinamide). Transformation products (TPs) were identified, and tentative structures were proposed. Inhibition studies with octyne, an ammonia monooxygenase (AMO) inhibitor, suggested that AMO was the responsible enzyme for MP transformation that occurred cometabolically. For the first time, hydroxylamine, a key intermediate of all aerobic ammonia oxidizers, was found to react with several MPs at concentrations typically occurring in AOB batch cultures. All of these MPs were also biotransformed by the AOB/NOB coculture. Moreover, the same asulam TPs were detected in both biotransformation and hydroxylamine-treated abiotic transformation experiments, whereas rufinamide TPs formed from biological transformation were not detected during hydroxylamine-mediated abiotic transformation, which was consistent with the inability of rufinamide abiotic transformation by hydroxylamine. Thus, in addition to cometabolism likely carried out by AMO, an abiotic transformation route indirectly mediated by AMO might also contribute to MP biotransformation by AOB.

Oral Application of Magnesium-L-Threonate Attenuates Vincristine-induced Allodynia and Hyperalgesia by Normalization of Tumor Necrosis Factor-α/Nuclear Factor-κB Signaling.

BACKGROUND: Antineoplastic agents, including vincristine, often induce neuropathic pain and magnesium deficiency clinically, but the causal link between them has not been determined. No drug is available for treating this form of neuropathic pain. METHODS: Injection of vincristine (0.1 mg · kg · day, intraperitoneally, for 10 days) was used to induce nociceptive sensitization, which was accessed with von Frey hairs and the plantar tester in adult male Sprague-Dawley rats. Magnesium-L- threonate was administered through drinking water (604 mg · kg · day). Extracellular and intracellular free Mg were measured by Calmagite chromometry and flow cytometry. Molecular biologic and electrophysiologic experiments were performed to expose the underlying mechanisms. RESULTS: Vincristine injection induced allodynia and hyperalgesia (n = 12), activated tumor necrosis factor-α/nuclear factor-κB signaling, and reduced free Mg in cerebrospinal fluid by 21.7 ± 6.3% (mean ± SD; n = 13) and in dorsal root ganglion neurons by 27 ± 6% (n = 11). Reducing Mg activated tumor necrosis factor-α/nuclear factor-κB signaling in cultured dorsal root ganglion neurons. Oral application of magnesium-L-threonate prevented magnesium deficiency and attenuated both activation of tumor necrosis factor-α/nuclear factor-κB signaling and nociceptive sensitization (n = 12). Mechanistically, vincristine induced long-term potentiation at C-fiber synapses, up-regulated N-methyl-D-aspartate receptor type 2B subunit of N-methyl-D-aspartate receptor, and led to peptidergic C-fiber sprouting in spinal dorsal horn (n = 6 each). The vincristine-induced pathologic plasticity was blocked by intrathecal injection of nuclear factor-κB inhibitor (n = 6), mimicked by tumor necrosis factor-α, and substantially prevented by oral magnesium-L-threonate (n = 5). CONCLUSIONS: Vincristine may activate tumor necrosis factor-α/nuclear factor-κB pathway by reduction of intracellular magnesium, leading to spinal pathologic plasticity and nociceptive sensitization. Oral magnesium-L-threonate that prevents the magnesium deficiency is a novel approach to prevent neuropathic pain induced by chemotherapy.

Interleukin-1ß overproduction is a common cause for neuropathic pain, memory deficit, and depression following peripheral nerve injury in rodents.

BACKGROUND: Chronic pain is often accompanied by short-term memory deficit and depression. Currently, it is believed that short-term memory deficit and depression are consequences of chronic pain. Here, we test the hypothesis that the symptoms might be caused by overproduction of interleukin-1beta (IL-1ß) in the injured nerve independent of neuropathic pain following spared nerve injury in rats and mice. RESULTS: Mechanical allodynia, a behavioral sign of neuropathic pain, was not correlated with short-term memory deficit and depressive behavior in spared nerve injury rats. Spared nerve injury upregulated IL-1ß in the injured sciatic nerve, plasma, and the regions in central nervous system closely associated with pain, memory and emotion, including spinal dorsal horn, hippocampus, prefrontal cortex, nucleus accumbens, and amygdala. Importantly, the spared nerve injury-induced memory deficits, depressive, and pain behaviors were substantially prevented by peri-sciatic administration of IL-1ß neutralizing antibody in rats or deletion of IL-1 receptor type 1 in mice. Furthermore, the behavioral abnormalities induced by spared nerve injury were mimicked in naïve rats by repetitive intravenous injection of re combinant rat IL-1ß (rrIL-1ß) at a pathological concentration as determined from spared nerve injury rats. In addition, microglia were activated by both spared nerve injury and intravenous injection of rrIL-1ß and the effect of spared nerve injury was substantially reversed by peri-sciatic administration of anti-IL-1ß. CONCLUSIONS: Neuropathic pain was not necessary for the development of cognitive and emotional disorders, while the overproduction of IL-1ß in the injured sciatic nerve following peripheral nerve injury may be a common mechanism underlying the generation of neuropathic pain, memory deficit, and depression.

Neuropathic Pain: Sensory Nerve Injury or Motor Nerve Injury?

Peripheral nerve injury often induces chronic neuropathic pain. Peripheral nerve is consisted of sensory fibers and motor fibers, it is questioned injury to which type of fibers is responsible for generation of neuropathic pain? Because neuropathic pain is sensory disorder, it is generally believed that the disease should be induced by injury to sensory fibers. In recent years, however, emergent evidence shows that motor fiber injury but not sensory fiber injury is necessary and sufficient for induction of neuropathic pain. Motor fiber injury leads to neuropathic pain by upregulating pro-inflammatory cytokines and brain-derived neurotrophic factor in pain pathway.

Antifungal Activity of Eucalyptus Oil against Rice Blast Fungi and the Possible Mechanism of Gene Expression Pattern.

Eucalyptus oil possesses a wide spectrum of biological activity, including anti-microbial, fungicidal, herbicidal, acaricidal and nematicidal properties. We studied anti-fungal activities of the leaf oil extracted from Eucalyptus. grandis × E. urophylla. Eleven plant pathogenic fungi were tested based on the mycelium growth rates with negative control. The results showed that Eucalyptus oil has broad-spectrum inhibitory effects toward these fungi. Remarkable morphological and structural alterations of hypha have been observed for Magnaporthe grisea after the treatment. The mRNA genome array of M. grisea was used to detect genes that were differentially expressed in the test strains treated by the Eucalyptus oil than the normal strains. The results showed 1919 genes were significantly affected, among which 1109 were down-regulated and 810 were up-regulated (p < 0.05, absolute fold change >2). According to gene ontology annotation analysis, these differentially expressed genes may cause abnormal structures and physiological function disorders, which may reduce the fungus growth. These results show the oil has potential for use in the biological control of plant disease as a green biopesticide.

Multi-Residue Analysis of Pesticide Residues in Crude Pollens by UPLC-MS/MS.

A multi-residue method for the determination of 54 pesticide residues in pollens has been developed and validated. The proposed method was applied to the analysis of 48 crude pollen samples collected from eight provinces of China. The recovery of analytes ranged from 60% to 136% with relative standard deviations (RSDs) below 30%. Of the 54 targeted compounds, 19 pesticides were detected. The major detection rates of each compound were 77.1% for carbendazim, 58.3% for fenpropathrin, 56.3% for chlorpyrifos, 50.0% for fluvalinate, 31.3% for chlorbenzuron, and 29.2% for triadimefon in crude pollen samples. The maximum values of each pesticide were 4516 ng/g for carbendazim, 162.8 ng/g for fenpropathrin, 176.6 ng/g for chlorpyrifos, 316.2 ng/g for fluvalinate, 437.2 ng/g for chlorbenzuron, 79.00 ng/g for triadimefon, and so on. This study provides basis for the research on the risks to honeybee health.

Calpain-2 contributes to neuropathic pain following motor nerve injury via up-regulating interleukin-6 in DRG neurons.

Motor nerve injury by L5 ventral root transection (L5-VRT) initiates interleukin-6 (IL-6) up-regulation in primary afferent system contributing to neuropathic pain. However, the early upstream regulatory mechanisms of IL-6 after L5-VRT are still unknown. Here, we monitored both the activity of calpain, a calcium-dependent protease suggested as one of the earliest mediators for cytokine regulation, and the expression of IL-6 in bilateral L4-L6 dorsal root ganglias (DRGs) soon after L5-VRT. We found that the protein level of calpain-2 in DRGs, but not calpain-1 was increased transiently in the first 10 min(-1)h ipsilaterally and 20 min(-1)h contralaterally after L5-VRT, long before mechanical allodynia was initiated (5-15 h ipsilaterally and 15 h(-1)d contralaterally). The early activation of calpain evaluated by the generation of spectrin breakdown products (SBDP) correlated well with IL-6 up-regulation in bilateral DRGs. Double immunofluorescence staining revealed that almost all the calpain-2 positive neurons expressed IL-6, indicating an association between calpain-2 and IL-6. Inhibition of calpain by pre-treatment with MDL28170 (25mg/kg, i.p.) attenuated the rat mechanical allodynia and prevented the early up-regulation of IL-6 following L5-VRT. Addition of exogenous calpain-2 onto the surface of left L5 DRG triggered a temporal allodynia and increased IL-6 in bilateral DRGs simultaneously. Taken together, the early increase of calpain-2 in L5-VRT rats might be responsible for the induction of allodynia via up-regulating IL-6 in DRG neurons.