Exploring causal effects of gut microbiota and metabolites on body fat percentage using two-sample Mendelian randomization.

AIM: The relationship between the gut microbiota, metabolites and body fat percentage (BFP) remains unexplored. We systematically assessed the causal relationships between gut microbiota, metabolites and BFP using Mendelian randomization analysis. MATERIALS AND METHODS: Single nucleotide polymorphisms associated with gut microbiota, blood metabolites and BFP were screened via a genome-wide association study enrolling individuals of European descent. Summary data from genome-wide association studies were extracted from the MiBioGen consortium and the UK Biobank. The inverse variance-weighted model was the primary method used to estimate these causal relationships. Sensitivity analyses were performed using pleiotropy, Mendelian randomization-Egger regression, heterogeneity tests and leave-one-out tests. RESULTS: In the aspect of phyla, classes, orders, families and genera, we observed that o_Bifidobacteriales [ß = -0.05; 95% confidence interval (CI): -0.07 to -0.03; false discovery rate (FDR) = 2.76 × 10-3], f_Bifidobacteriaceae (ß = -0.05; 95% CI: -0.07 to -0.07; FDR = 2.76 × 10-3), p_Actinobacteria (ß = -0.06; 95% CI: -0.09 to -0.03; FDR = 6.36 × 10-3), c_Actinobacteria (ß = -0.05; 95% CI: -0.08 to -0.02; FDR = 1.06 × 10-2), g_Bifidobacterium (ß = -0.05; 95% CI: -0.07 to -0.02; FDR = 1.85 × 10-2), g_Ruminiclostridium9 (ß = -0.03; 95% CI: -0.06 to -0.01; FDR = 4.81 × 10-2) were negatively associated with BFP. G_Olsenella (ß = 0.02; 95% CI: 0.01-0.03; FDR = 2.16 × 10-2) was positively associated with BFP. Among the gut microbiotas, f_Bifidobacteriales, o_Bifidobacteriales, c_Actinobacteria and p_Actinobacteria were shown to be significantly associated with BFP in the validated dataset. In the aspect of metabolites, we only observed that valine (ß = 0.77; 95% CI: 0.5-1.04; FDR = 8.65 × 10-6) was associated with BFP. CONCLUSIONS: Multiple gut microbiota and metabolites were strongly associated with an increased BFP. Further studies are required to elucidate the mechanisms underlying this putative causality. In addition, BFP, a key indicator of obesity, suggests that obesity-related interventions can be developed from gut microbiota and metabolite perspectives.

Microbial community and transcriptional responses to V. coralliilyticus stress in coral Favites halicora and Pocillopora damicornis holobiont.

Variability in coral hosts susceptibility to Vibrio coralliilyticus is well-documented; however, the comprehensive understanding of tolerance of response to pathogen among coral species is lacked. Herein, we investigated the microbial communities and transcriptome dynamics of two corals in response to Vibrio coralliilyticus. Favites halicora displayed greater resistance to Vibrio coralliilyticus challenge than Pocillopora damicornis. Furthermore, the relative abundances of Flavobacteriaceae, Vibrionacea, Rhodobacteraceae, and Roseobacteraceae increased significantly in Favites halicora following pathogen stress, whereas that of Akkermansiaceae increased significantly in Pocillopora damicornis, leading to bacterial community imbalance. In contrast to the previous results, pathogen infection did not have much effect on the community structures of Symbiodiniaceae and fungi, but led to a decrease in the density of Symbiodiniaceae. Transcriptome analysis indicated that Vibrio infection triggered a coral immune response, resulting in higher expression of immune-related genes, which appeared to have higher transcriptional plasticity in Favites halicora than in Pocillopora damicornis. Specifically, the upregulated genes of Favites halicora were predominantly involved in the apoptosis pathway, whereas Pocillopora damicornis were significantly enriched in the nucleotide excision repair and base excision repair pathways. These findings suggest that coral holobionts activate different mechanisms across species in response to pathogens through shifts in microbial communities and transcriptomes, which provides novel insight into assessing the future coral assemblages suffering from disease outbreaks.

Ammonia removal mitigates white plague type II in the coral Pocillopora damicornis.

White Plague Type II (WPL II) is a disease increasingly affecting scleractinian coral species and progresses rapidly. However, the etiological pathogen and remedy remain elusive. In this study, transmission experiments demonstrated that Aureimonas altamirensis and Aurantimonas coralicida, representing the WPL II pathogens, could infect Pocillopora damicorni. The infection produced selected pathological symptoms, including bleaching, tissue loss, and decolorization. Furthermore, ammonia degradation significantly reduced the severity of infection by these pathogens, indicating that ammonia may be a virulence factor for WPL II. Coral microbiome analysis suggested that ammonia degradation mediates the anti-white plague effect by maintaining the density of Symbiodiniaceae and stabilizing the core and symbiotic bacteria. Aureimonas altamirensis and Aurantimonas coralicida have been shown to cause diseases of P. damicornis, with ammonia acting as a virulence factor, and ammoniac degradation may be a promising and innovative approach to mitigate coral mortality suffering from increasing diseases.

Tolerability and effectiveness of albuvirtide combined with dolutegravir for hospitalized people living with HIV/AIDS.

Treatment options for hospitalized people living with HIV/AIDS (PLWHA) with opportunistic infections and comorbidities are limited in China. Albuvirtide (ABT), a new peptide drug, is a long-acting HIV fusion inhibitor with limited drug-drug interactions and fast onset time. This single-center, retrospective cohort study investigated the effectiveness and safety of ABT plus dolutegravir (DTG) therapy in a real-world setting. We performed a chart review on the electronic patient records for hospitalized PLWHA using ABT plus DTG between April and December 2020. The clinical outcomes were retrospectively analyzed. Among 151 PLWHA (mean age 47.6 ± 15.9 years), 140 (93%) had at least 1 episode of bacterial and/or fungal infections and 64 (42%) had other comorbidities including syphilis, hepatitis B, and/or hypertension. ABT plus DTG was given to 87 treatment-naïve (TN) and 64 treatment-experienced (TE) PLWHA. Regardless of treatment history, mean HIV-1 RNA levels significantly decreased from 4.32 log10copies/mL to 2.24 log10copies/mL, 2.10 log10copies/mL and 1.89 log10copies/mL after 2, 4 and 8 weeks of treatment, respectively (P < .0001). Compared with baseline mean CD4 + T-cell counts of 122.72 cells/µL, it increased to 207.87 cells/µL (P = .0067) and 218.69 cells/µL (P = .0812) after 4 and 8 weeks of treatment. Except for limited laboratory abnormalities such as hyperuricemia, increased creatinine level, and hyperglycemia observed after treatment, no other clinical adverse events were considered related to ABT plus DTG. Data suggests that ABT plus DTG is safe and effective for critically-ill hospitalized PLWHA. In view of the rapid viral load suppression and restoration of CD4 + count within 8 weeks of treatment, its clinical application warrants further investigation.

Significant Shifts in Microbial Communities Associated with Scleractinian Corals in Response to Algae Overgrowth.

Microbes play a key role in reef dynamics, mediating the competition between scleractinian corals and benthic algae; however, major shifts in bacterial communities among coral species in response to increases in the abundance of algae are not well understood. We investigated the taxonomic composition of coral-associated microbial communities under algae-overgrowth conditions using 16S rRNA gene sequencing. The results showed that non-algal (i.e., healthy) tissue (HH) had lower bacterial abundance and diversity than tissue collected from the coral-algae interface boundary (HA) and areas of algae growth (AA). Specifically, the HA and AA samples had higher relative abundances of Saprospiraceae, Rhodobacteraceae, and Alteromonadaceae. Compared with Platygyra sp. and Montipora sp., the physiological response of Pocillopora sp. was more intense under algae-induced stress based on microbial gene function prediction. Our results indicate that algal pressure can significantly alter the microbial community structure and function of coral ecosystems. Our data thus provide new insight into the relationship between corals and their microbiome under environmental stress.

Clostridium, Bacteroides and Prevotella associates with increased fecal metabolites Trans-4-Hydroxy-L-proline and Genistein in active pulmonary tuberculosis patients during anti-tuberculosis chemotherapy with isoniazid-rifampin-pyrazinamide-ethambutol (HRZE).

Purpose: To investigated the changes of gut microbiome and fecal metabolome during anti-tuberculosis chemotherapy with isoniazid (H)-rifampin (R)-pyrazinamide (Z)-ethambutol (E). Patients and methods: (1) In this study, we recruited 168 stool specimens from 49 healthy volunteers without M. tuberculosis (Mtb), 30 healthy volunteers with latently infected by Mtb, 41 patients with active tuberculosis (ATB), 28 patients with 2-month HRZE treatment and 20 patients with 2-month HRZE followed by 4-month HR treatment. (2) We used 16S rRNA sequencing and an untargeted Liquid Chromatograph Mass Spectrometer-based metabolomics to investigate the changes of gut microbiome and the alteration of fecal metabolome, respectively, during anti-TB chemotherapy. Results: Mtb infection can reduce the diversity of intestinal flora of ATB patients and change their taxonomic composition, while the diversity of intestinal flora of ATB patients were restored during anti-TB chemotherapy. Especially, family Veillonellacea and Bateroidaceae and their genera Veillonella and Bacteroides significantly increased in the gut microbiota during anti-TB chemotherapy. Additionally, Mtb infection dynamically regulates fecal metabolism in ATB patients during anti-TB chemotherapy. Interestingly, the altered abundance of fecal metabolites correlated with the altered gut microbiota, especially the change of gut Clostridium, Bacteroides and Prevotella was closely related to the change of fecal metabolites such as Trans-4-Hydroxy-L-proline and Genistein caused by Mtb infection or anti-TB chemotherapy. Conclusion: Anti-TB chemotherapy with HRZE can disrupt both gut microbiotas and metabolome in ATB patients. Some specific genera and metabolites are depleted or enriched during anti-TB chemotherapy. Therefore, revealing potential relevance between gut microbiota and anti-TB chemotherapy will provide potential biomarkers for evaluating the therapeutic efficacy in ATB patients. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01003-2.

Use of a purified ß-glucosidase from coral-associated microorganisms to enhance wine aroma.

BACKGROUND: ß-Glucosidases (3.2.1.21) play essential roles in the removal of nonreducing terminal glucosyl residues from saccharides and glycosides. However, the full potential and different applications of recombinant high-yield microbial ß-glucosidase-producing systems remain to be tackled. RESULTS: A ß-glucosidase gene designated as Mg132 was isolated from a coral microorganism by high-throughput sequencing and functional screening. The deduced amino acid sequences of Mg132 showed a highest identity of 97% with ß-glucosidase predicted in the GenBank database. This gene was cloned and overexpressed in Escherichia coli BL21 (DE3) for the first time. The optimal pH and temperature of purified recombinant Mg132 were 8.0 and 50 °C respectively. It exhibited a high level of stability at high concentration of glucose and ethanol, and glucose concentrations below 300 mmol L-1 distinctly stimulated p-nitrophenyl-ß-d-glucopyranoside hydrolysis, reaching 200% at 15% ethanol. The Km and Vmax values were 0.293 mmol L-1 and 320 µmol min-1  mg-1 respectively while using p-nitrophenyl-ß-d-glucopyranoside as a substrate. Wine treated with Mg132 had an obvious positive catalytic specificity for glycosides, which give a pleasant flavor of temperate fruity and floral aromas. The total concentration of fermentative volatiles was 201.42 ± 10.22 µg L-1 following Mg132 treatment and 99.21 ± 7.72 µg L-1 in control samples. CONCLUSION: Good tolerance of winemaking and aroma fermentative properties suggest that Mg132 has potential application in aroma enhancement in wine and warrants further study. © 2021 Society of Chemical Industry.

Control of chronic obstructive pulmonary disease in urban populations: findings from a cross-sectional prevalence survey in Shenzhen, China.

The prevalence of chronic obstructive pulmonary disease (COPD) among urban populations is generally lower than rural residents, but the disease burden is still high. We conducted a cross-sectional prevalence survey of COPD among residents aged ≥40 years in an emerging city Shenzhen, China from September 2018 to June 2019. Through multi-stage stratified random sampling, a total of 4157 eligible participants were invited to complete a questionnaire and to take the spirometry test; 3591 with available data were enrolled in the final analysis. Individuals were diagnosed with COPD if the post-bronchodilator FEV1/FVC ratio was less than 0.7. The estimated standardized prevalence of COPD among residents over 40 years old in Shenzhen was 5.92% (95% confidential intervals [CI] 4.05-8.34). Risk factors for COPD included elder age (adjusted odds ratio 1.206, 95% CI 1.120-1.299 per 10-year increase), smoking over 20 pack-years (1.968, 1.367-2.832), history of chronic bronchitis (1.733, 1.036-2.900) or asthma (4.920, 2.425-9.982), and exposure to higher annual minimum concentrations of ambient SO2 (1.156, 1.053-1.270 per 1-µg/m3 increase). Among 280 spirometry-diagnosed patients, most (221, 78.93%) patients were classified as mild COPD (GOLD stage I). This survey found that the prevalence of COPD in Shenzhen is low and most patients had mild symptoms, thus recommended screening using spirometry in primary health care to detect early-stage COPD. Increased risk from the exposure to air pollutants also indicated the urgent need for environmental improvement in city settings.

A Novel Neutral and Mesophilic ß-Glucosidase from Coral Microorganisms for Efficient Preparation of Gentiooligosaccharides.

ß-glucosidases can produce gentiooligosaccharides that are lucrative and promising for the prebiotic and alternative food industries. However, the commercial production of gentiooligosaccharides using ß-glucosidase is challenging, as this process is limited by the need for high thermal energy and increasing demand for the enzyme. Here, a putative ß-glucosidase gene, selected from the coral microbial metagenome, was expressed in Escherichia coli. Reverse hydrolysis of glucose by Blg163 at pH 7.0 and 40 °C achieved a gentiooligosaccharide yield of 43.02 ± 3.20 g·L-1 at a conversion rate of 5.38 ± 0.40%. Transglycosylation of mixed substrates, glucose and cellobiose, by Blg163 consumed 21.6 U/0.5 g glucose/g cellobiose, achieving a gentiooligosaccharide yield of 70.34 ± 2.20 g·L-1 at a conversion rate of 15.63%, which is close to the highest yield reported in previous findings. Blg163-mediated synthesis of gentiooligosaccharides is the mildest reaction and the lowest ß-glucosidase consumption reported to date.

High Diversity of ß-Glucosidase-Producing Bacteria and Their Genes Associated with Scleractinian Corals.

ß-Glucosidase is a microbial cellulose multienzyme that plays an important role in the regulation of the entire cellulose hydrolysis process, which is the rate-limiting step in bacterial carbon cycling in marine environments. Despite its importance in coral reefs, the diversity of ß-glucosidase-producing bacteria, their genes, and enzymatic characteristics are poorly understood. In this study, 87 ß-glucosidase-producing cultivable bacteria were screened from 6 genera of corals. The isolates were assigned to 21 genera, distributed among three groups: Proteobacteria, Firmicutes, and Actinobacteria. In addition, metagenomics was used to explore the genetic diversity of bacterial ß-glucosidase enzymes associated with scleractinian corals, which revealed that these enzymes mainly belong to the glycosidase hydrolase family 3 (GH3). Finally, a novel recombinant ß-glucosidase, referred to as Mg9373, encompassing 670 amino acids and a molecular mass of 75.2 kDa, was classified as a member of the GH3 family and successfully expressed and characterized. Mg9373 exhibited excellent tolerance to ethanol, NaCl, and glucose. Collectively, these results suggest that the diversity of ß-glucosidase-producing bacteria and genes associated with scleractinian corals is high and novel, indicating great potential for applications in the food industry and agriculture.

Vitamin D receptor and Alzheimer's disease: a genetic and functional study.

Genetic studies on late-onset Alzheimer's disease (AD) have repeatedly mapped susceptibility loci onto chromosome 12q13, encompassing the vitamin D receptor (VDR) gene. Epidemiology studies have indicated vitamin D insufficiency as a risk factor for AD. Given that VDR is the major mediator for vitamin D's actions, we sought to clarify the role of VDR in late-onset AD. We conducted an association study in 492 late-onset AD cases and 496 controls with 80 tagging single nucleotide polymorphisms (SNPs). The strongest association was found at a promoter SNP rs11568820 (P = 9.1 × 10(-6), odds ratio (OR) = 1.69), which resides within the transcription factor Cdx-2 binding site and the SNP has been also known as CDX2. The risk-allele at rs11568820 is associated with lower VDR promoter activity (p < 10(-11)). The overexpression of VDR or vitamin D treatment suppressed amyloid precursor protein (APP) transcription in neuroblastoma cells (p < 0.001). We provide both statistical evidence and functional data suggesting VDR confers genetic risk for AD. Our findings are consistent with epidemiology studies suggesting that vitamin D insufficiency increases the risk of developing AD.

Presynaptic low- and high-affinity kainate receptors in nociceptive spinal afferents.

Presynaptic ionotropic glutamate receptors are increasingly attributed a role in the modulation of sensory input at the first synapse of dorsal root ganglion (DRG) neurons in the spinal dorsal horn. Central terminals of DRG neurons express AMPA and NMDA receptors whose activation modulates the release of glutamate, the main transmitter at these synapses. Previous work, with an antibody that recognizes all low-affinity kainate receptor subunits (GluR5, 6, 7), provided microscopic evidence of presynaptic kainate receptors in unidentified primary afferent terminals in superficial laminae of the spinal dorsal horn (Hwang SJ, Pagliardini S, Rustioni A, Valtschanoff JG. Presynaptic kainate receptors in primary afferents to the superficial laminae of the rat spinal cord. J Comp Neurol 2001; 436: pp. 275-289). We show here that, although all such subunits may be expressed in these terminals, GluR5 is the subunit most readily detectable at presynaptic sites in sections processed for immunocytochemistry. We also show that the high-affinity kainate receptor subunits KA1 and KA2 are expressed in central terminals of DRG neurons and are co-expressed with low-affinity receptor subunits in the same terminals. Quantitative data show that kainate-expressing DRG neurons are about six times more likely to express the P2X(3) subunit of the purinergic receptor than to express substance P. Thus, nociceptive afferents that express presynaptic kainate receptors are predominantly non-peptidergic, suggesting a role for these receptors in the modulation of neuropathic rather than inflammatory pain.

Ionotropic glutamate receptors are expressed in GABAergic terminals in the rat superficial dorsal horn.

Ionotropic glutamate receptors (IGR), including NMDA, AMPA, and kainate receptors, are expressed in terminals with varied morphology in the superficial laminae (I-III) of the dorsal horn of the spinal cord. Some of these terminals can be identified as endings of primary afferents, whereas others establish symmetric synapses, suggesting that they may be gamma-aminobutyric acid (GABA)-ergic. In the present study, we used confocal and electron microscopy of double immunostaining for GAD65, a marker for GABAergic terminals, and for subunits of IGRs to test directly whether IGRs are expressed in GABAergic terminals in laminae I-III of the dorsal horn. Although colocalization is hard to detect with confocal microscopy, electron microscopy reveals a substantial number of terminals immunoreactive for GAD65 also stained for IGRs. Among all GAD65-immunoreactive terminals counted, 37% express the NMDA receptor subunit NR1; 28% are immunopositive using an antibody for the GluR2/4 subunits of the AMPA receptor; and 20-35% are immunopositive using antibodies for the kainate receptor subunits GluR5, GluR6/7, KA1, or KA2. Terminals immunoreactive for IGR subunits and GAD65 establish symmetric synapses onto dendrites and perikarya and can be presynaptic to primary afferent terminals within both type 1 and type 2 synaptic glomeruli. Activation of presynaptic IGR may reduce neurotransmitter release. As autoreceptors in terminals of Adelta and C afferent fibers in laminae I-III, presynaptic IGRs may play a role in inhibiting nociception. As heteroreceptors in GABAergic terminals in the same laminae, on the other hand, presynaptic IGRs may have an opposite role and even contribute to central sensitization and hyperalgesia.

Primary afferent terminals that express presynaptic NR1 in rats are mainly from myelinated, mechanosensitive fibers.

Presynaptic N-methyl-D-aspartate (NMDA) receptors in terminals of primary afferents to spinal cord of rats were first reported by Liu et al. (1994; Proc. Natl. Acad. Sci. USA 91:8383-8387) and were proposed to modulate nociceptive input (Liu et al. [1997] Nature 386:721-724). We previously demonstrated kainate and AMPA receptors in numerous primary afferent terminals in the spinal cord fixed with diluted paraformaldehyde and no glutaraldehyde. Therefore, we reinvestigated the occurrence of presynaptic NMDAR1 (NR1) with this fixation protocol. With confocal microscopy, numerous immunofluorescent puncta were double-stained for NR1 and the presynaptic marker synaptophysin throughout the spinal gray. NR1-immunostained puncta costained more frequently with a tracer that labels myelinated afferents (cholera toxin subunit B; CTB) than with a tracer that labels non-peptidergic unmyelinated afferents (Griffonia simplicifolia isolectin B4; IB4). Virtually no double staining was found for NR1 and calcitonin gene-related peptide (CGRP), which labels somatic peptidergic primary afferents. In the gracile nucleus, virtually all puncta labeled for CTB appeared immunopositive for NR1. At the electron microscopic level, most immunopositive terminals in spinal cord and gracile nucleus displayed morphological characteristics of endings of myelinated primary afferents. NR1 was presynaptic in 60-65% of all synapses in which it was expressed pre- or postsynaptically, or both, in spinal laminae I-IV. Estimates for the gracile nucleus were higher (80%). No presynaptic NR1 was found in the ventroposterior thalamus. Because of the relative sparsity of presynaptic NR1 in terminals in laminae I and IIo and in terminals of peptidergic unmyelinated afferents, it is suggested that presynaptic NMDA receptors play a more significant role in modulation of mechanosensitive, innocuous input than in nociception.

Primary afferent terminals in spinal cord express presynaptic AMPA receptors.

Larger dorsal root ganglion neurons are stained by an antibody for the C terminus of glutamate receptor subunit 2 (GluR2) and GluR3 (GluR2/3) rather than by an antibody for GluR4. In dorsal roots, anti-GluR2/3 stains predominantly myelinated fibers; anti-GluR4 or anti-GluR2/4 stains predominantly unmyelinated fibers. In the dorsal horn, puncta immunopositive for synaptophysin and GluR2/3 are predominantly in laminas III and IV, whereas puncta immunopositive for synaptophysin and GluR4 or GluR2/4 are predominantly in laminas I and II. Puncta immunopositive for GluR2/3 costain with the B subunit of cholera toxin, whereas puncta immunopositive for GluR2/4 costain with isolectin B4 after injections of these tracers in the sciatic nerve. No puncta costain with calcitonin gene-related peptide and AMPA receptor subunits. Electron microscopy indicates that AMPA receptor-immunopositive terminals are more numerous than suggested by confocal microscopy. Of all synapses in which immunostaining is presynaptic, postsynaptic, or both, the percentage of presynaptic immunostain is approximately 70% with anti-GluR4 or anti-GluR2/4 (in laminas I-III), 25-30% with anti-GluR2/3 (in laminas III and IV), and 5% with anti-GluR2 (in laminas I-III). Because of fixation constraints, the types of immunostained terminals could be identified only on the basis of morphological characteristics. Many terminals immunostained for GluR2/3, GluR4, or GluR2/4 have morphological features of endings of primary afferents. Terminals with morphological characteristics of presumed GABAergic terminals are also immunostained with anti-GluR2/4 and anti-GluR4 in laminas I and II and with anti-GluR2/3 in laminas III and IV. The conspicuous and selective expression of presynaptic AMPA receptor subunits may contribute to the characteristic physiological profile of different classes of primary afferents and suggests an important mechanism for the modulation of transmitter release by terminals of both myelinated and unmyelinated primary afferents.

Two major distinct subpopulations of neurokinin-3 receptor-expressing neurons in the superficial dorsal horn of the rat spinal cord.

This study was designed to provide evidence for elucidating the mechanisms of neurokinin-3 receptor (NK3) in spinal pain modulation. First, colocalization of NK3 with the micro -opioid receptor (MOR1) was studied in the spinal dorsal horn of the rat. Confocal microscopy showed that about 44% of NK3-expressing neurons in laminae I and II were immunoreactive for MOR1, which corresponded to about 93% of the total population of MOR1-containing neurons in these laminae. Second, the relationship between NK3/MOR1-coexpressing neurons and those that express nitric oxide synthase (NOS) was examined by using a triple immunofluorescent staining method. About 37% of NK3-immunoreactive neurons were also NOS-immunoreactive, which constituted about 82% of NOS-immunoreacitve neurons in the superficial laminae. However, no triple-labelled neurons were detected. The present results indicate that there are two major distinct subpopulations of NK3-expressing neurons in the superficial dorsal horn, which suggests that the involvement of NK3 receptor in spinal nociception could be mediated by two distinct mechanisms, i.e. opioid and nitric oxide.