Enzymatic modules of the SAGA chromatin-modifying complex play distinct roles in Drosophila gene expression and development.

The Spt-Ada-Gcn5-acetyltransferase (SAGA) chromatin-modifying complex is a transcriptional coactivator that contains four different modules of subunits. The intact SAGA complex has been well characterized for its function in transcription regulation and development. However, little is known about the roles of individual modules within SAGA and whether they have any SAGA-independent functions. Here we demonstrate that the two enzymatic modules of Drosophila SAGA are differently required in oogenesis. Loss of the histone acetyltransferase (HAT) activity blocks oogenesis, while loss of the H2B deubiquitinase (DUB) activity does not. However, the DUB module regulates a subset of genes in early embryogenesis, and loss of the DUB subunits causes defects in embryogenesis. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analysis revealed that both the DUB and HAT modules bind most SAGA target genes even though many of these targets do not require the DUB module for expression. Furthermore, we found that the DUB module can bind to chromatin and regulate transcription independently of the HAT module. Our results suggest that the DUB module has functions within SAGA and independent functions.

The SAGA core module is critical during Drosophila oogenesis and is broadly recruited to promoters.

The Spt/Ada-Gcn5 Acetyltransferase (SAGA) coactivator complex has multiple modules with different enzymatic and non-enzymatic functions. How each module contributes to gene expression is not well understood. During Drosophila oogenesis, the enzymatic functions are not equally required, which may indicate that different genes require different enzymatic functions. An analogy for this phenomenon is the handyman principle: while a handyman has many tools, which tool he uses depends on what requires maintenance. Here we analyzed the role of the non-enzymatic core module during Drosophila oogenesis, which interacts with TBP. We show that depletion of SAGA-specific core subunits blocked egg chamber development at earlier stages than depletion of enzymatic subunits. These results, as well as additional genetic analyses, point to an interaction with TBP and suggest a differential role of SAGA modules at different promoter types. However, SAGA subunits co-occupied all promoter types of active genes in ChIP-seq and ChIP-nexus experiments, and the complex was not specifically associated with distinct promoter types in the ovary. The high-resolution genomic binding profiles were congruent with SAGA recruitment by activators upstream of the start site, and retention on chromatin by interactions with modified histones downstream of the start site. Our data illustrate that a distinct genetic requirement for specific components may conceal the fact that the entire complex is physically present and suggests that the biological context defines which module functions are critical.

Transport model simulation prediction and environmental risk assessment of pyrethroid insecticides in urban artificial lakes caused by rainfall: A case study of Cloud Mountain Park in subhumid climate zone.

Pyrethroid insecticides are among urban parks' most widely used and harmful insecticides. The advanced prediction method is the key to studying the pollution and diffusion risk of plant conservation insecticides in parks. A two-dimensional advection-dispersion model was established for the North Lake of Cloud Mountain Park in the subhumid area of Hebei Province. The temporal and spatial distribution of lambda-cyhalothrin pollution required by plant growth in artificial lakes under different rainfall intensities and the time of water renewal after rainfall was simulated and predicted. According to the model efficiency (E: 0.98), mean absolute error (MAE: 0.016-0.064 cm), and root mean square error (RMSE: 0.014-0.041 cm), the prediction results showed that the model fits well. The results showed that the concentration of lambda-cyhalothrin in the artificial lake was positively correlated with the increase in rainfall intensity. Under the three scenarios of moderate rain, heavy rain, and rainstorm, the variation of total pollutants into the lake over time conformed to the first-order dynamic equation (R2＞0.97), and the cumulative rates were 0.013 min-1, 0.019 min-1 and 0.022 min-1, respectively. Under light rain, the accumulation rate of lambda-cyhalothrin showed a double-linear relationship, which was in accordance with the second-order kinetic equation (R2＞0.97). The rapid accumulation rate of early-stage rainfall was 0.0024 min-1, and the slow accumulation rate of late-stage rainfall was 0.0019 min-1. The human health risk assessment predicted by the simulation was lower than the hazard value (Rtgn(a-1): 9.65 E-11-1.12 E-10 a-1). However, the potential risk value to aquatic species was higher (RQ: 0.33-23.05). In addition, the increase in rainfall intensity has no significant effect on the acceleration of water renewal time. The two-dimensional dispersion model of pollutants driven by water dynamics provided relevant examples for evaluating the impact of runoff on pesticide scour in parks and supplied scientific support for improving the management of artificial lakes in urban parks.

Characterization of a metazoan ADA acetyltransferase complex.

The Gcn5 acetyltransferase functions in multiple acetyltransferase complexes in yeast and metazoans. Yeast Gcn5 is part of the large SAGA (Spt-Ada-Gcn5 acetyltransferase) complex and a smaller ADA acetyltransferase complex. In flies and mammals, Gcn5 (and its homolog pCAF) is part of various versions of the SAGA complex and another large acetyltransferase complex, ATAC (Ada2A containing acetyltransferase complex). However, a complex analogous to the small ADA complex in yeast has never been described in metazoans. Previous studies in Drosophila hinted at the existence of a small complex which contains Ada2b, a partner of Gcn5 in the SAGA complex. Here we have purified and characterized the composition of this complex and show that it is composed of Gcn5, Ada2b, Ada3 and Sgf29. Hence, we have named it the metazoan 'ADA complex'. We demonstrate that the fly ADA complex has histone acetylation activity on histones and nucleosome substrates. Moreover, ChIP-Sequencing experiments identified Ada2b peaks that overlap with another SAGA subunit, Spt3, as well as Ada2b peaks that do not overlap with Spt3 suggesting that the ADA complex binds chromosomal sites independent of the larger SAGA complex.

Signal multi-amplified electrochemical biosensor for voltammetric determination of tau-441 protein in biological samples using carbon nanomaterials and gold nanoparticles to hint dementia.

A signal multi-amplified electrochemical biosensor was fabricated for tau-441 protein, a dementia biomarker. It utilizes a carbon nanocomposite film modified gold electrode. The carbon nanocomposite film was composed of multi-walled carbon nanotubes (MWCNTs), reduced graphene oxide (rGO), and chitosan (CS). For the nanocomposite film, rGO improved the dispersibility of MWCNTs, and the effective surface area of MWCNTs was increased. On the other hand, MWCNTs also increased the interlayer spacing of rGO, resulting in a thinner rGO layer. MWCNTs-rGO had a better conductivity than that of MWCNTs and rGO due to the synergy effect. Biocompatible CS was employed for immobilization of the specific antibody. Tau-441 protein was modified with gold nanoparticles (AuNPs) for signal amplification again. The response of the electrochemical biosensor is linear in the range 0.5-80 fM (0.5, 1.5, 5, 10, 40, 80 fM) with a limit of detection (LOD) of 0.46 fM, using differential pulse voltammetry (DPV) in a potential range of - 100-500 mV. The biosensor was successfully applied to the analysis of serum samples of 14 normal people, 14 mild cognitive impairment (MCI) patients, and 14 dementia patients. Graphical abstract Schematic representation of signal multi-amplified electrochemical biosensor for determination of tau-441 protein in human serum.

Follicular T-helper cell recruitment governed by bystander B cells and ICOS-driven motility.

Germinal centres support antibody affinity maturation and memory formation. Follicular T-helper cells promote proliferation and differentiation of antigen-specific B cells inside the follicle. A genetic deficiency in the inducible co-stimulator (ICOS), a classic CD28 family co-stimulatory molecule highly expressed by follicular T-helper cells, causes profound germinal centre defects, leading to the view that ICOS specifically co-stimulates the follicular T-helper cell differentiation program. Here we show that ICOS directly controls follicular recruitment of activated T-helper cells in mice. This effect is independent from ICOS ligand (ICOSL)-mediated co-stimulation provided by antigen-presenting dendritic cells or cognate B cells, and does not rely on Bcl6-mediated programming as an intermediate step. Instead, it requires ICOSL expression by follicular bystander B cells, which do not present cognate antigen to T-helper cells but collectively form an ICOS-engaging field. Dynamic imaging reveals ICOS engagement drives coordinated pseudopod formation and promotes persistent T-cell migration at the border between the T-cell zone and the B-cell follicle in vivo. When follicular bystander B cells cannot express ICOSL, otherwise competent T-helper cells fail to develop into follicular T-helper cells normally, and fail to promote optimal germinal centre responses. These results demonstrate a co-stimulation-independent function of ICOS, uncover a key role for bystander B cells in promoting the development of follicular T-helper cells, and reveal unsuspected sophistication in dynamic T-cell positioning in vivo.

High-Frequency Repetitive Transcranial Magnetic Stimulation Reduces Pain in Postherpetic Neuralgia.

OBJECTIVES: Postherpetic neuralgia (PHN) is one of the most intractable pain disorders, especially in elderly patients. There is evidence that repetitive transcranial magnetic stimulation (rTMS) reduces neuropathic pain; however, its effectiveness for PHN is unknown. This study investigated the efficacy of high-frequency rTMS in patients with PHN. DESIGN: A total of 40 patients were randomly assigned to receive 10 sessions of real or sham rTMS of the primary motor cortex. Each stimulation session consisted of a series of 300 five-second pulses with a frequency of 10 Hz and an interval of 3 seconds between each train, giving a total of 1500 pulses per session. The primary outcome was pain intensity measured before stimulation from first intervention (T0) to the final stimulation (T10), and 1 and 3 months after final stimulation (T11 and T12). Other outcomes measured included scores on the short form McGill pain questionnaire, self-rating depression scale, quality of life (QOL), sleep quality, the patient global impression of change, medication regulation, and reported adverse events. RESULTS: The real rTMS group demonstrated greater reduction of visual analogue scale (VAS) than the sham group at each time point except for T0 (P = 0.399) and T1 (P = 0.091). Mean VAS reduction in the real rTMS group was 16.89% for duration of disease longer than 6 months. These analgesic effects were associated with long-term improvement in rating-scale items related to QOL. CONCLUSION: The results suggest that rTMS is an effective and safe therapy in patients with PHN.

Masticatory Muscles Dysfunction after CT-guided Percutaneous Trigeminal Radiofrequency Thermocoagulation for Trigeminal Neuralgia: A Detailed Analysis.

OBJECTIVE: The aim of this study was to investigate the severity and the natural course of masticatory muscles weakness that developed after CT-guided percutaneous trigeminal radiofrequency thermocoagulation (PT-RFT) for the treatment of idiopathic trigeminal neuralgia (ITN). METHODS: Twenty-seven patients with ITN were treated by CT-guided percutaneous trigeminal radiofrequency thermocoagulation. Each patients' occlusal function and surface electromyographic (sEMG) activity of the ipsilateral anterior temporalis (TA) and masseter muscles (MM) at mandibular postural position (MPP), and during a fast maximum voluntary clenching (MVC) from MPP to intercuspal position (ICP), were simultaneously recorded by the T-Scan III system and Bio-pak sEMG III system before (baseline), 3 days, 3 months, and 12 months after procedure. The incidence, degree, and prognosis of masticatory muscles dysfunction related to trigeminal nerve motor-branch injury were analyzed. RESULTS: Three days and 3 months after procedure, both the occlusal symmetry and the sEMG activity of ipsilateral TA and MM became significantly decreased compared to the baseline (P < 0.05). However, they demonstrated a gradual improvement toward preoperative values in follow-up, returning to complete in 23 patients at 12 months after procedure. None reported permanent masticatory paralysis. Pain relief was most significant on the third day after procedure. At the final clinical visit, a pain-free status was observed in 25 patients (92.6%). Meanwhile, the intensity of facial dysesthesia was mildest, whereas there were statistic differences compared with baseline. CONCLUSION: CT-guided PT-RFT for ITN remains an effective and safe surgical procedure, but there is a high rate of temporary masticatory dysfunction during a short time after procedure, appearing to be reversible in a period of 12 months.

Repeated CT-guided percutaneous radiofrequency thermocoagulation for recurrent trigeminal neuralgia.

AIMS: To investigate the long-term outcomes of repeated percutaneous radiofrequency thermocoagulation (PRT) for recurrent trigeminal neuralgia (TN) patients. METHODS: Between 2002 and 2012, 33 patients with recurrent TN following an initial PRT procedure were retrospectively studied and underwent 43 repeated PRT procedures. RESULTS: The mean length of follow-up after repeated PRT was 34 months. Pain relief was immediate in 30 patients (90.9%), and no pain relief occurred in 3 patients (9.1%) following a second PRT procedure. The percentage of patients who remained in an 'excellent' and 'good' pain relief condition (pain intensity ≤BIN grade III) after the second PRT procedure was 75% at 1 year, 68% at 2 years and 68% at 5 years, and 22 of these patients (54.5%) remained satisfied with their pain relief during the follow-up period. Nine patients underwent PRT three times and 1 patient four times. The total number of patients who benefited from repeated PRT was 28 (84.8%). Postprocedure complications including masseter weakness were present in 3 patients and limited mouth opening affected 1 patient. No mortalities were observed during or after repeated PRT procedures. CONCLUSION: Repeated PRT provides long-term pain relief benefits to patients with recurrent TN and should be considered as an alternative treatment for recurrent TN.

Long-term outcome of computed tomography-guided percutaneous radiofrequency thermocoagulation for classic trigeminal neuralgia patients older than 70 years.

The incidence of trigeminal neuralgia (TN) in elderly patients is higher. However, for those with poor fitness, the optimal surgical treatment for those refractory to medical treatment is controversial. The aim of current study was to investigate the long-term outcome of computed tomography (CT)-guided percutaneous radiofrequency thermocoagulation (PRT) for 304 TN patients 70 years or older. We conducted a retrospective study of 304 elderly patients with TN who were treated with CT-guided PRT between 2002 and 2012. Follow-up was censored at the time of last contact, additional surgery, or death. Sixty-seven patients (22.1%) were of more than American Society of Anesthesiologists classification system physical status II. Excellent pain relief was 100% at discharge, 85% at 1 year, 75% at 3 years, 71% at 5 years, and 49% at 10 years. Pain relief outcomes were correlated with facial numbness. Lower temperature group (≤75°C) can attain the same long-term pain relief as higher temperature group (≥80°C); however, the incidence of painful dysesthesia rate of higher temperature group was higher than lower temperature group. Postoperative morbidity included facial numbness, masseter weakness, corneitis, hearing loss, dropping eyelid, and limited mouth opening. There were no mortalities observed during or after PRT. Our result showed CT-guided PRT is safe and effective for classic TN patients 70 years or older, including poor-fitness patients (American Society of Anesthesiologists classification system physical status >II). Lower temperature (≤75°C) is recommended for PRT in the treatment of TN.

The role of the environmental subsystem in sustainable urban development: Evidence from megacities in China.

Conflicts in urban subsystems have severely hindered the realization of sustainable development, among which the most serious is the conflict between the environmental subsystem and urban development. Differing from studies considering individual environmental elements, this paper innovatively investigates the quantitative relationship between overall environmental performance and other development dimensions to understand the quantitative role of the environmental subsystem in sustainable urban development. Taking the nine megacities in China as an example, this paper first develops the performance variables of four urban subsystems, including the environment, by entropy method and analyzes the conflict or coordination level between the environment and other subsystems through the coupling coordination degree model (CCDM). Then, the interaction mechanism is further analyzed by the fully modified ordinary least square (FMOLS) and vector error correction model (VECM). This paper tries to provide a new reference for management and decision-making by focusing on the whole environmental subsystem rather than separate elements, which is of theoretical and practical significance. The main conclusions are as follows: (1) The coordination level between the environment and other urban subsystems is low; (2) 1 % rise in the economic and resource performance can respectively lead to 0.2014 % and 0.1388 % declines in the environmental performance; (3) 1 % increase in social performance can bring a 0.3738 % rise in environmental performance; (4) Improving environmental and resource subsystems' performance is the priority; (5) Coordinating urban subsystems is the key to long-run sustainable development. Despite the case studies on megacities in China, we hope to provide a new reference for cities worldwide with concentrated populations, rapid growth, and complex development contradictions.

Effects of Low-Impact Development Facilities (Water Systems of the Park) on Stormwater Runoff in Shallow Mountainous Areas Based on Dual-Model (SWMM and MIKE21) Simulations.

Rapid urbanization has triggered more serious urban flood risks. Many studies have focused on intra-urban flooding, but less attention has been paid to rainfall and flood risks at the urban fringe. Nowadays, China is vigorously promoting the construction of sponge cities in the whole area. It is important to study the construction of sponge cities in shallow mountainous areas, which are an important barrier between cities and mountains. The purpose of this paper is to investigate the performance of Low-Impact Development (LID) facilities under different rainfall scenarios in developed shallow mountainous areas. The second garden and flower exposition ("the Expo Park") in Hebei Province is used as an example. The SWMM and MIKE21 models were used to simulate the hydrological processes before and after the construction of "the Expo Park", and the models were calibrated with the measured data. Peak flow rate, outflow volume, rainfall-outflow ratio, runoff velocity, and water feature area of the water system were used as indicators to evaluate their effectiveness. The results showed that the placement of LID facilities had a positive impact on the construction of the shallow mountain area. Specifically, (1) LID facilities can reduce the peak flow rate, delayed peak flow time, outflow volume, and rainfall outflow ratio of stormwater runoff in mountainous areas; and (2) they can effectively collect rainwater and become a supplement to the landscape water system of the site. These findings provide a scientific basis for the construction of LID facilities in shallow mountainous areas, which is important for the development and flood management of shallow mountainous areas.

Corrigendum: Distinct Age-Related Epigenetic Signatures in CD4 and CD8 T Cells.

[This corrects the article DOI: 10.3389/fimmu.2020.585168.].

Reduced chromatin accessibility to CD4 T cell super-enhancers encompassing susceptibility loci of rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is an inflammatory disease that manifests as a preclinical stage of systemic autoimmunity followed by chronic progressive synovitis. Disease-associated genetic SNP variants predominantly map to non-coding, regulatory regions of functional importance in CD4 T cells, implicating these cells as key regulators. A better understanding of the epigenome of CD4 T cells holds the promise of providing information on the interaction between genetic susceptibility and exogenous factors. METHODS: We mapped regions of chromatin accessibility using ATAC-seq in peripheral CD4 T cell subsets of patients with RA (n=18) and compared them to T cells from patients with psoriatic arthritis (n=11) and age-matched healthy controls (n=10). Transcripts of selected genes were quantified using qPCR. FINDINGS: RA-associated epigenetic signatures were identified that in part overlapped between central and effector memory CD4 T cells and that were to a lesser extent already present in naïve cells. Sites more accessible in RA were highly enriched for the motif of the transcription factor (TF) CTCF suggesting differences in the three-dimensional chromatin structure. Unexpectedly, sites with reduced chromatin accessibility were enriched for motifs of TFs pertinent for T cell function. Most strikingly, super-enhancers encompassing RA-associated SNPs were less accessible. Analysis of selected transcripts and published DNA methylation patterns were consistent with this finding. The preferential loss in accessibility at these super-enhancers was seen in patients with high and low disease activity and on a variety of immunosuppressive treatment modalities. INTERPRETATION: Disease-associated genes are epigenetically less poised to respond in CD4 T cells from patients with established RA. FUNDING: This work was supported by I01 BX001669 from the Veterans Administration.

Activation of mTORC1 at late endosomes misdirects T cell fate decision in older individuals.

The nutrient-sensing mammalian target of rapamycin (mTOR) is integral to cell fate decisions after T cell activation. Sustained mTORC1 activity favors the generation of terminally differentiated effector T cells instead of follicular helper and memory T cells. This is particularly pertinent for T cell responses of older adults who have sustained mTORC1 activation despite dysfunctional lysosomes. Here, we show that lysosome-deficient T cells rely on late endosomes rather than lysosomes as an mTORC1 activation platform, where mTORC1 is activated by sensing cytosolic amino acids. T cells from older adults have an increased expression of the plasma membrane leucine transporter SLC7A5 to provide a cytosolic amino acid source. Hence, SLC7A5 and VPS39 deficiency (a member of the HOPS complex promoting early to late endosome conversion) substantially reduced mTORC1 activities in T cells from older but not young individuals. Late endosomal mTORC1 is independent of the negative-feedback loop involving mTORC1-induced inactivation of the transcription factor TFEB that controls expression of lysosomal genes. The resulting sustained mTORC1 activation impaired lysosome function and prevented lysosomal degradation of PD-1 in CD4+ T cells from older adults, thereby inhibiting their proliferative responses. VPS39 silencing of human T cells improved their expansion to pertussis and to SARS-CoV-2 peptides in vitro. Furthermore, adoptive transfer of CD4+ Vps39-deficient LCMV-specific SMARTA cells improved germinal center responses, CD8+ memory T cell generation, and recall responses to infection. Thus, curtailing late endosomal mTORC1 activity is a promising strategy to enhance T cell immunity.

Ecto-NTPDase CD39 is a negative checkpoint that inhibits follicular helper cell generation.

Vaccination is a mainstay in preventive medicine, reducing morbidity and mortality from infection, largely by generating pathogen-specific neutralizing antibodies. However, standard immunization strategies are insufficient with increasing age due to immunological impediments, including defects in T follicular helper (Tfh) cells. Here, we found that Tfh generation is inversely linked to the expression of the ecto-NTPDase CD39 that modifies purinergic signaling. The lineage-determining transcription factor BCL6 inhibited CD39 expression, while increased Tfh frequencies were found in individuals with a germline polymorphism preventing transcription of ENTPD1, encoding CD39. In in vitro human and in vivo mouse studies, Tfh generation and germinal center responses were enhanced by reducing CD39 expression through the inhibition of the cAMP/PKA/p-CREB pathway, or by blocking adenosine signaling downstream of CD39 using the selective adenosine A2a receptor antagonist istradefylline. Thus, purinergic signaling in differentiating T cells can be targeted to improve vaccine responses, in particular in older individuals who have increased CD39 expression.

FOXO1 deficiency impairs proteostasis in aged T cells.

T cell differentiation involves the dynamic regulation of FOXO1 expression, which rapidly declines after activation and is subsequently restored. Reexpression is impaired in naïve CD4+ T cell responses from older individuals. Here, we show that FOXO1 promotes lysosome function through the induction of the key transcription factor for lysosomal proteins, TFEB. Subdued FOXO1 reexpression in activated CD4+ T cells impairs lysosomal activity, causing an expansion of multivesicular bodies (MVBs). Expansion of the MVB compartment induces the sequestration of glycogen synthase kinase 3ß (GSK3ß), thereby suppressing protein turnover and enhancing glycolytic activity. As a consequence, older activated CD4+ T cells develop features reminiscent of senescent cells. They acquire an increased cell mass, preferentially differentiate into short-lived effector T cells, and secrete exosomes that harm cells in the local environment through the release of granzyme B.

Distinct Age-Related Epigenetic Signatures in CD4 and CD8 T Cells.

Healthy immune aging is in part determined by how well the sizes of naïve T cell compartments are being maintained with advancing age. Throughout adult life, replenishment largely derives from homeostatic proliferation of existing naïve and memory T cell populations. However, while the subpopulation composition of CD4 T cells is relatively stable, the CD8 T cell compartment undergoes more drastic changes with loss of naïve CD8 T cells and accumulation of effector T cells, suggesting that CD4 T cells are more resilient to resist age-associated changes. To determine the epigenetic basis for these differences in behaviors, we compared chromatin accessibility maps of CD4 and CD8 T cell subsets from young and old individuals and related the results to the expressed transcriptome. The dominant age-associated signatures resembled hallmarks of differentiation, which were more pronounced for CD8 naïve and memory than the corresponding CD4 T cell subsets, indicating that CD8 T cells are less able to keep cellular quiescence upon homeostatic proliferation. In parallel, CD8 T cells from old adults, irrespective of their differentiation state, displayed greater reduced accessibility to genes of basic cell biological function, including genes encoding ribosomal proteins. One possible mechanism is the reduced expression of the transcription factors YY1 and NRF1. Our data suggest that chromatin accessibility signatures can be identified that distinguish CD4 and CD8 T cells from old adults and that may confer the higher resilience of CD4 T cells to aging.

[Effect of fentanyl on expression of mu-receptor and beta-arrestin 2 in periaqueductal gray of rats tolerant to morphine].

OBJECTIVE: To investigate the effect of fentanyl upon the expression of mu-receptor and beta-arrestin 2 in peri-aqueductal gray of morphine-tolerant rats. METHODS: Forty male SD rats weighing (230 +/- 20) g were randomly divided into 5 groups of eight animals each: group NS, group M, group MF1, group MF2 and group MF3. Rats in group NS received only subcutaneous normal saline 1 ml/kg twice a day for 9 consecutive days; group M received subcutaneous morphine 10 mg/kg followed by NS 1 ml/kg twice a day for 9 consecutive days; In groups MF1, MF2 and MF3, morphine 10 mg x kg(-1) was injected subcutaneously followed by fentanyl 3, 6, 12 microg/kg respectively. All animals were sacrificed at Day 9 after measurement of pain threshold. Periaqueductal gray was removed for determination of the expression of mRNA (RT-PCR) and protein (Western-blot) of mu-receptor and beta-arrestin 2. RESULTS: Compared with group NS, TFL of group M was significantly elevated after the first morphine injection (P < 0.01). But TFL of group M returned to the baseline value after chronic morphine treatment. Compared with group M, TFL increased in groups MF2 and MF3 at Days 7 and 9 (P < 0.05 or 0.01). However, TFL of group MF1 was negative (P > 0.05). The expression of mu-receptor mRNA and protein was significantly lower in group M than in group NS (P < 0.01). Compared with group M, the expressions of mu-receptor mRNA and protein were significantly elevated in group MF2 and MF3 (P < 0.05 or 0.01) but there was no significant change in group MF1 (P > 0.05). The expression of beta-arrestin 2 mRNA and protein significantly decreased in group M as compared with group NS (P < 0.01). Compared with group M, the expressions of beta-arrestin 2 mRNA and protein were significantly elevated in group MF2 and MF3 (P < 0.05 or 0.01), but there was no significant change in group MF1 (P > 0.05). CONCLUSION: Fentanyl at 6 and 12 microg/kg can partly inhibit morphine tolerance through an increased expression of mu-receptor and beta-arrestin 2 in periaqueductal gray of morphine-tolerant rats.

Biosynthesis of gold nanoparticles using fungus Trichoderma sp. WL-Go and their catalysis in degradation of aromatic pollutants.

An efficient green method of gold nanoparticles (AuNPs) biosynthesis was achieved by cell-free extracts of fungus Trichoderma sp. WL-Go. Based on UV-Vis spectra, AuNPs biosynthesised by cell-free extracts with 90 mg/l protein exhibited a characteristic absorption band at 556 nm and was stable for 7 days. Transmission electron microscopy images revealed that the as-synthesised AuNPs were spherical and pseudo-spherical, and the average size was calculated to be 9.8 nm with a size range of 1-24 nm. The AuNPs illustrated their good catalytic activities for reduction of nitro-aromatics (2-nitrophenol, 3-nitrophenol, 4-nitrophenol, 2-nitroaniline, 3-nitroaniline) with catalytic rate constants of 7.4 × 10-3 s-1, 10.3 × 10-3 s-1, 4.9 × 10-3 s-1, 5.8 × 10-3 s-1, 15.0 × 10-3 s-1, respectively. Meanwhile, the AuNPs also showed excellent catalytic performance in decolourisation of azo dyes with decolourisation efficiency from 82.2 to 97.5%. This study provided a green gentle method for AuNPs synthesis as well as exhibiting efficient catalytic capability for degradation of aromatic pollutants.