Differential Impacts on Bacterial Composition and Abundance in Rhizosphere Compartments between Al-Tolerant and Al-Sensitive Soybean Genotypes in Acidic Soil.

In this study, two soybean genotypes i.e. aluminum-tolerant Baxi 10 (BX10) and aluminum-sensitive Bendi 2 (BD2) were used as plant materials and the acidic red soil was used as growth medium. The soil layers from the inside to the outside of the root are: rhizospheric soil after washing (WRH), rhizospheric soil after brushing (BRH) and rhizospheric soil at two sides (SRH), respectively. The rhizosphere bacterial communities were analyzed by high-throughput sequencing of V4 hypervariable regions of 16S rRNA gene (16S rDNA) amplicons via Illumina MiSeq. The results of alpha diversity showed that the BRH and SRH of BX10 were significantly lower on community richness than that of BD2, while the WRH existed no significant difference between BX10 and BD2. Among the three sampling compartments of the same soybean genotype, WRH had the lowest community richness and diversity while existed the highest coverage. Beta diversity analysis results displayed no significant difference for any compartment between the two genotypes, or among the three different sampling compartments for any same soybean genotype. However, the relative abundance of major bacterial taxa specifically nitrogen-fixating and/or aluminum-tolerant bacteria was significantly different in the compartments of the BRH and/or SRH at phylum and genus levels depicting genotype dependent variations in rhizosphere bacterial community. Strikingly, as compared with BRH and SRH, the WRH within the same genotype (BX10 or BD2) always had an enrichment effect on rhizosphere bacteria associated with nitrogen-fixation.

A novel epigenetic signature to predict recurrence-free survival in patients with colon cancer.

BACKGROUND: DNA methylation plays an important role in the initiation and progression of colon cancer. The aim of the present study was to perform a comprehensive analysis of DNA methylation and gene expression profiles in order to develop a signature to predict recurrence-free survival (RFS) of colon cancer. METHODS: DNA methylation and mRNA expression data were obtained from TCGA database, and were analyzed using an R package MethylMix. Functional enrichment analysis was performed on statistically significant genes identified by MethylMix criteria. The epigenetic signature and nomogram associated with the RFS of colon cancer were established by the Least Absolute Shrinkage and Selection Operator (LASSO) Cox model. Additionally, a joint survival analysis of gene expression and methylation was performed to identify potential prognostic factors for patients with colon cancer. RESULTS: A total of 179 differentially methylated genes were obtained using MethylMix algorithm. An epigenetic signature for RFS was developed using LASSO. Patients with high-risk had significantly worse RFS than those with low-risk. The signature is independent of clinicopathological variables and indicated better predictive power than other clinicopathological variables in patients with colon cancer. Moreover, joint survival analysis of gene expression and methylation revealed that seven methylated genes could be independent prognostic factors for RFS in colon cancer. CONCLUSIONS: Our proposed epigenetic signature presents potential prognostic significance in assessing recurrence risk stratification for patients with colon cancer.

Histone deacetylase 2 is involved in µ­opioid receptor suppression in the spinal dorsal horn in a rat model of chronic pancreatitis pain.

Chronic pain occurs in ~85-90% of chronic pancreatitis (CP) patients. However, as the pathogenesis of CP pain remains to be fully understood, the current therapies for CP pain remain inadequate. Emerging evidence has suggested that the epigenetic modulations of genes are involved in chronic pain. In the present study, intrapancreatic trinitrobenzene sulfonic acid infusions were used to establish a CP model in rats. Mechanical allodynia was measured with von Frey filaments. Immunofluorescent staining analysis was used to observe the expression changes of histone deacetylase 2 (HDAC2) and µ­opioid receptor (MOR), and intrathecal administration of the selective HDAC2 inhibitor AR­42 was used to assess the underlying mechanisms. The expression levels of c­Jun N­terminal kinase (JNK) in the thoracic spinal cord were detected by western blotting, and the mRNA expression levels of interleukin (IL)1­ß, IL­6 and tumor necrosis factor (TNF)­α were detected by reverse transcription­quantitative polymerase chain reaction. The results demonstrated that HDAC2 expression was upregulated during the course of CP induction, while MOR activity in the thoracic spinal dorsal horn was significantly suppressed. Intrathecal infusion of AR­42 significantly attenuated CP­induced mechanical allodynia, with rescued MOR activity. Additionally, HDAC2 facilitated the release of inflammatory cytokines, including IL­1ß, IL­6 and TNF­α. These results suggested that the underlying mechanisms of HDAC2 regulating MOR activity under CP induction may occur via promoting the release of inflammatory cytokines, thus activating the JNK signaling pathway. The present study suggested that the epigenetic­regulated disturbance of MOR is dependent on the endogenous analgesia system in CP, which may a provide novel therapeutic strategy for treating pain in CP.

Astrocytic NDRG2 is involved in glucocorticoid-mediated diabetic mechanical allodynia.

AIMS: The present study aims to test whether astrocytes contribute to glucocorticoid-mediated diabetic mechanical allodynia. METHODS: Streptozotocin (STZ)-induced diabetic rats were used in our study. The intrathecal operation was performed 21 days after the onset of diabetes. Diabetic mechanical allodynia was present 28 d after the onset of diabetes, and the mechanical threshold was tested using von Frey filaments. Immunohistochemistry, including immunofluorescent histochemical staining, was performed to observe the morphology of the spinal dorsal horn (SDH). Western blot analysis was employed as a semi-quantitative assay of the expression levels of GFAP and NDRG2 associated with diabetic mechanical allodynia. RESULTS: Diabetic rats displayed mechanical allodynia and activated astrocytes in the SDH 28 days after the onset of diabetes. This allodynia was attenuated by intrathecal administration of the astrocyte-specific inhibitor l-α-aminoadipate. In parallel, intrathecal injection of RU486, a glucocorticoid receptor antagonist, inhibited the activation of astrocytes in the SDH, alleviating the diabetes-induced mechanical allodynia. Furthermore, we found that dorsal horn astrocytes express abundant N-myc downstream-regulated gene 2 (NDRG2), which contributes to astrocyte reactivity. NDRG2 was over-expressed in activated astrocytes in diabetic rats with mechanical allodynia. Intrathecal injection of RU486 prevented the over-expression of NDRG2, which reversed the astrocyte reactivity and diabetic tactile allodynia. CONCLUSIONS: These results suggest that glucocorticoid-mediated over-expression of NDRG2 may contribute to the activation of dorsal horn astrocytes, which play a crucial role in diabetic mechanical allodynia. Thus, inhibiting glucocorticoid receptors and/or astrocyte reactivity in the SDH may be a therapeutic strategy for treating diabetic tactile allodynia.

Puerarin alleviates noise-induced hearing loss via affecting PKCγ and GABAB receptor expression.

Noise-induced hearing loss (NIHL) often results from prolonged exposure to high levels of noise. Our previous study revealed that during the development of NIHL, the expression of protein kinase C γ subunit (PKCγ) and GABAB receptor (GABABR) was changed within the cochlear nuclear complex (CNC), suggesting that these molecules might be the potential targets for the treatment of NIHL. As an extending study, here we focused on puerarin, a major isoflavonoid extracted from Pueraria lobota, which has been used in the treatment of cardiovascular and cerebrovascular diseases, and investigated whether it could protect against NIHL by acting on PKCγ and GABABR. Transgenic GAD67-GFP knock-in mice were subjected to the NIHL model and their auditory functions were evaluated by the auditory brainstem response thresholds and distortion product oto-acoustic emission signals. Our results showed that 200mg/kg puerarin treatment ameliorated the thresholds of auditory brainstem response of NIHL mice significantly. Triple immunofluorescence staining and electron microscopy results revealed that GFP-positive neurons in the superficial layers of CNC expressed both PKCγ and GABABR1, and GAD67-positive terminals contacted PKCγ- or GABABR1-positive neurons. Immunoblotting and RT-PCR results showed that NIHL increased the expression of PKCγ but decreased that of GABABR1 and GABABR2 at both protein and mRNA levels in the CNC. Puerarin significantly attenuated the increased expression of PKCγ but elevated the reduced expression of GABABR1 and GABABR2 after noise exposure. Thus, we provided the first evidence that puerarin ameliorated the auditory functions of NIHL mice, and this effect may be due to its ability to regulate the expression of PKCγ and GABABR.

Postsynaptic insertion of AMPA receptor onto cortical pyramidal neurons in the anterior cingulate cortex after peripheral nerve injury.

Long-term potentiation (LTP) is the key cellular mechanism for physiological learning and pathological chronic pain. Postsynaptic accumulation of AMPA receptor (AMPAR) GluA1 plays an important role for injury-related cortical LTP. However, there is no direct evidence for postsynaptic GluA1 insertion or accumulation after peripheral injury. Here we report nerve injury increased the postsynaptic expression of AMPAR GluA1 in pyramidal neurons in the layer V of the anterior cingulate cortex (ACC), including the corticospinal projecting neurons. Electrophysiological recordings show that potentiation of postsynaptic responses was reversed by Ca2+ permeable AMPAR antagonist NASPM. Finally, behavioral studies show that microinjection of NASPM into the ACC inhibited behavioral sensitization caused by nerve injury. Our findings provide direct evidence that peripheral nerve injury induces postsynaptic GluA1 accumulation in cingulate cortical neurons, and inhibits postsynaptic GluA1 accumulation which may serve as a novel target for treating neuropathic pain.

Alterations in the neural circuits from peripheral afferents to the spinal cord: possible implications for diabetic polyneuropathy in streptozotocin-induced type 1 diabetic rats.

Diabetic polyneuropathy (DPN) presents as a wide variety of sensorimotor symptoms and affects approximately 50% of diabetic patients. Changes in the neural circuits may occur in the early stages in diabetes and are implicated in the development of DPN. Therefore, we aimed to detect changes in the expression of isolectin B4 (IB4, the marker for nonpeptidergic unmyelinated fibers and their cell bodies) and calcitonin gene-related peptide (CGRP, the marker for peptidergic fibers and their cell bodies) in the dorsal root ganglion (DRG) and spinal cord of streptozotocin (STZ)-induced type 1 diabetic rats showing alterations in sensory and motor function. We also used cholera toxin B subunit (CTB) to show the morphological changes of the myelinated fibers and motor neurons. STZ-induced diabetic rats exhibited hyperglycemia, decreased body weight gain, mechanical allodynia and impaired locomotor activity. In the DRG and spinal dorsal horn, IB4-labeled structures decreased, but both CGRP immunostaining and CTB labeling increased from day 14 to day 28 in diabetic rats. In spinal ventral horn, CTB labeling decreased in motor neurons in diabetic rats. Treatment with intrathecal injection of insulin at the early stages of DPN could alleviate mechanical allodynia and impaired locomotor activity in diabetic rats. The results suggest that the alterations of the neural circuits between spinal nerve and spinal cord via the DRG and ventral root might be involved in DPN.

Neurochemical phenotype and function of endomorphin 2-immunopositive neurons in the myenteric plexus of the rat colon.

The distribution and activity of endomorphins (EMs), which are endogenous µ-opioid receptor (MOR) ligands in the gastrointestinal tract (GI), are yet to be elucidated. The current study aimed to shed light on this topic. EM2 was expressed in the enteric neurons in the myenteric plexus of the mid-colon. Of the EM2-immunoreactive (EM2-IR) neurons, 53 ± 4.6%, 26 ± 4.5%, 26 ± 2.8% and 49 ± 4.2% displayed immunopositive staining for choline acetyl transferase (ChAT), substance P (SP), vasoactive intestinal peptide (VIP) and nitric oxide synthetase (NOS), respectively. A bath application of EM2 (2 µM) enhanced spontaneous contractile amplitude and tension, which were reversed by ß-FNA (an antagonist of MOR) but not NG-nitro-L-arginine methyl ether (L-NAME, a non-selective inhibitor of NOS) or VIP6-28 (an antagonist of the VIP receptor) in the colonic strips. EM2 significantly suppressed inhibitory junction potentials (IJPs) in 14 of the 17 examined circular muscle cells, and this effect was not antagonized by preincubation in L-NAME. EM2 was widely expressed in interneurons and motor neurons in the myenteric plexus and presynaptically inhibited fast IJPs, thereby enhancing spontaneous contraction and tension in the colonic smooth muscle.

Comprehensive application of modern technologies in precise liver resection.

BACKGROUND: Liver surgery has gone through the phases of wedge liver resection, regular resection of hepatic lobes, irregular and local resection, extracorporeal hepatectomy, hemi-extracorporeal hepatectomy and Da Vinci surgical system-assisted hepatectomy. Taking advantage of modern technologies, liver surgery is stepping into an age of precise liver resection. This review aimed to analyze the comprehensive application of modern technologies in precise liver resection. DATA SOURCE: PubMed search was carried out for English-language articles relevant to precise liver resection, liver anatomy, hepatic blood inflow blockage, parenchyma transection, and down-staging treatment. RESULTS: The 3D image system can imitate the liver operation procedures, conduct risk assessment, help to identify the operation feasibility and confirm the operation scheme. In addition, some techniques including puncture and injection of methylene blue into the target Glisson sheath help to precisely determine the resection. Alternative methods such as Pringle maneuver are helpful for hepatic blood inflow blockage in precise liver resection. Moreover, the use of exquisite equipment for liver parenchyma transection, such as cavitron ultrasonic surgical aspirator, ultrasonic scalpel, Ligasure and Tissue Link is also helpful to reduce hemorrhage in liver resection, or even operate exsanguinous liver resection without blocking hepatic blood flow. Furthermore, various down-staging therapies including transcatheter arterial chemoembolization and radio-frequency ablation were appropriate for unresectable cancer, which reverse the advanced tumor back to early phase by local or systemic treatment so that hepatectomy or liver transplantation is possible. CONCLUSIONS: Modern technologies mentioned in this paper are the key tool for achieving precise liver resection and can effectively lead to maximum preservation of anatomical structural integrity and functions of the remnant liver. In addition, large randomized trials are needed to evaluate the usefulness of these technologies in patients with hepatocellular carcinoma who have undergone precise liver resection.

Synaptic connections between endomorphin 2-immunoreactive terminals and µ-opioid receptor-expressing neurons in the sacral parasympathetic nucleus of the rat.

The urinary bladder is innervated by parasympathetic preganglionic neurons (PPNs) that express µ-opioid receptors (MOR) in the sacral parasympathetic nucleus (SPN) at lumbosacral segments L6-S1. The SPN also contains endomorphin 2 (EM2)-immunoreactive (IR) fibers and terminals. EM2 is the endogenous ligand of MOR. In the present study, retrograde tract-tracing with cholera toxin subunit b (CTb) or wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) via the pelvic nerve combined with immunohistochemical staining for EM2 and MOR to identify PPNs within the SPN as well as synaptic connections between the EM2-IR terminals and MOR-expressing PPNs in the SPN of the rat. After CTb was injected into the pelvic nerve, CTb retrogradely labeled neurons were almost exclusively located in the lateral part of the intermediolateral gray matter at L6-S1 of the lumbosacral spinal cord. All of the them also expressed MOR. EM2-IR terminals formed symmetric synapses with MOR-IR, WGA-HRP-labeled and WGA-HRP/MOR double-labeled neuronal cell bodies and dendrites within the SPN. These results provided morphological evidence that EM2-containing axon terminals formed symmetric synapses with MOR-expressing PPNs in the SPN. The present results also show that EM2 and MOR might be involved in both the homeostatic control and information transmission of micturition.

Down-regulation of insulin signaling is involved in painful diabetic neuropathy in type 2 diabetes.

BACKGROUND: Previous theories considered that the main cause of painful diabetic neuropathy (PDN) was due to hyperglycemia. However, recent evidence indicated that hyperinsulinemia plays a greater role in type 2 diabetic metabolisms (T2DM). OBJECTIVES: Our aim was to explore insulin signaling to determine the molecular mechanism involved in the pathogenesis of PDN in T2DM. STUDY DESIGN: A randomized, double blind, controlled animal trial. METHODS: We observed the localization of insulin receptor (IR) and phosphorylated insulin receptor substrate 1 (IRS-1) in the spinal cord using in situ hybridization and immunohistochemistry. Then we investigated the alternations of IR and pIRS-1 and the activity of the JAK2/STAT3 pathway by immunohistochemistry, Western Blotting, and cell culture. Finally, we detected the influence of intrathecal JAK2/STAT3 inhibitor (AG490) on nociceptive behavior and insulin signaling in ob/ob mice using Western Blotting. RESULTS: We found that IR and pIRS-1 are mainly located in neurons in the superficial layer of the spinal dorsal horn. The expressions of IR and pIRS-1 decreased and the JAK2/STAT3 pathway activated in the spinal dorsal horn in ob/ob mice with mechanical hyperalgesia. Next, our in vitro RESULTS indicated that hyperinsulinemia and hyperglycemia impaired insulin signaling along with the activated JAK2/STAT3 pathway in differentiated human neuronal cells (SH-SY5Y). Treatment through intrathecal injection of AG490, an inhibitor of the JAK2/STAT3 pathway, alleviated mechanical hyperalgesia in ob/ob mice and prevented impaired insulin signaling in the spinal cord. LIMITATIONS: The activation of the JAK2/STAT3 pathway could not explain the mechanism of PDN in T1DM.   CONCLUSIONS: We demonstrate that insulin signaling impairment in the spinal dorsal horn is associated with the activated JAK2/STAT3 pathway, which contributes to the progressive PDN in T2DM.

Beneficial effects of splenectomy on liver regeneration in a rat model of massive hepatectomy.

BACKGROUND: Small-for-size syndrome is a widely recognized clinical complication after living donor liver transplantation or extended hepatectomy due to inadequate liver mass. The purpose of this study was to investigate the role of splenectomy in rats after massive hepatectomy, a surrogate model of small-for-size graft. METHODS: Rats were divided into eight groups, each with 20 animals: 50% hepatectomy (50% Hx), 50% hepatectomy+splenectomy (50% Hx+Sp), 60% Hx, 60% Hx+Sp, 70% Hx, 70% Hx+Sp, 90% Hx and 90% Hx+Sp. The following parameters were evaluated: liver function tests (ALT, AST and TBIL), liver regeneration ratio, DNA synthesis, proliferation cell nuclear antigen, hepatic oxygen delivery (HDO2) and hepatic oxygen consumption (HVO2). RESULTS: The liver regeneration ratio was enhanced in the Hx+Sp groups (P<0.05). In addition, compared with the Hx groups, the Hx+Sp groups had better liver functions (P<0.05). DNA synthesis and proliferation cell nuclear antigen were also increased in the Hx+Sp groups compared with the Hx groups (P<0.05). Furthermore, in the Hx+Sp groups, HDO2 and HVO2 were increased over those in the Hx groups (P<0.05), and were positively correlated with the liver regeneration ratio. CONCLUSIONS: Splenectomy significantly improved liver function, and enhanced DNA synthesis and proliferation cell nuclear antigen after massive hepatectomy in rats. This operation could be mediated through increased HDO2 and HVO2, which facilitate liver regeneration.

Sodium channel Nav1.6 is up-regulated in the dorsal root ganglia in a mouse model of type 2 diabetes.

Neuropathic pain is one of the most common chronic complications of diabetes, of which the underlying mechanisms are unclear. Expression changes of voltage-gated sodium channels in dorsal root ganglia (DRG) are involved in the production of ectopic spontaneous activity. In the present study, we examined the changes of DRG Nav1.6 expression in a mouse model of type 2 diabetes (db/db mice). Db/db mice developed significant and persistent mechanical allodynia from postnatal 2 months compared to the heterozygous littermates (db/+) and C57 mice. Immunofluorescent staining showed that Nav1.6 was highly expressed in the normal DRG (approximately 31.3±5.2% of total DRG neurons), especially in the large-diameter neurons. In postnatal 5 months in db/db mice, percentage of Nav1.6 positive cells (62.9±5.5%) was significantly higher than that in C57 and db/+ mice. Western blot showed that from 2 to 5 months, Nav1.6 was increased by 1.67±0.16, 2.12±0.23, 1.89±0.32, and 2.01±0.35 folds of C57 mice, which were significantly higher than that of the C57 and db/+ mice. Real-time PCR showed that in postnatal 1 month of db/db mice, mRNA level of Nav1.6 was increased by 1.72±0.22 fold, which was significantly higher than that of C57 and db/+ mice. Nav1.6 mRNA was increased thereafter and maintained at high levels throughout the observed period. Our results provide direct evidence that type 2 diabetes induces significant and persistent increase of Nav1.6 expression in the DRG, which may participate in the diabetic neuropathic pain.

Spinal toll like receptor 3 is involved in chronic pancreatitis-induced mechanical allodynia of rat.

BACKGROUND: Mechanisms underlying pain in chronic pancreatitis (CP) are incompletely understood. Our previous data showed that astrocytes were actively involved. However, it was unclear how astrocytic activation was induced in CP conditions. In the present study, we hypothesized that toll-like receptors (TLRs) were involved in astrocytic activation and pain behavior in CP-induced pain. RESULTS: To test our hypothesis, we first investigated the changes of TLR2-4 in the rat CP model induced by intrapancreatic infusion of trinitrobenzene sulfonic acid (TNBS). Western blot showed that after TNBS infusion, TLR3, but not TLR2 or TLR4, was increased gradually and maintained at a very high level for up to 5 w, which correlated with the changing course of mechanical allodynia. Double immunostaining suggested that TLR3 was highly expressed on astrocytes. Infusion with TLR3 antisense oligodeoxynucleotide (ASO) dose-dependently attenuated CP-induced allodynia. CP-induced astrocytic activation in the spinal cord was also significantly suppressed by TLR3 ASO. Furthermore, real-time PCR showed that IL-1ß, TNF-α, IL-6 and monocyte chemotactic protein-1 (MCP-1) were significantly increased in spinal cord of pancreatic rats. In addition, TLR3 ASO significantly attenuated CP-induced up-regulation of IL-1ß and MCP-1. CONCLUSIONS: These results suggest a probable "TLR3-astrocytes-IL-1ß/MCP-1" pathway as a positive feedback loop in the spinal dorsal horn in CP conditions. TLR3-mediated neuroimmune interactions could be new targets for treating persistent pain in CP patients.

Spinal astrocytic activation contributes to mechanical allodynia in a mouse model of type 2 diabetes.

Diabetic neuropathic pain (DNP) plays a major role in decreased life quality of type 2 diabetes patients, however, the molecular mechanisms underlying DNP remain unclear. Emerging research implicates the participation of spinal glial cells in some neuropathic pain models. However, it remains unknown whether spinal glial cells are activated under type 2 diabetic conditions and whether they contribute to diabetes-induced neuropathic pain. In the present study, using a db/db type 2 diabetes mouse model that displayed obvious mechanical allodynia, we found that spinal astrocyte but not microglia was dramatically activated. The mechanical allodynia was significantly attenuated by intrathecally administrated l-α-aminoadipate (astrocytic specific inhibitor) whereas minocycline (microglial specific inhibitor) did not have any effect on mechanical allodynia, which indicated that spinal astrocytic activation contributed to allodynia in db/db mice. Further study aimed to identify the detailed mechanism of astrocyte-induced allodynia in db/db mice. Results showed that spinal activated astrocytes dramatically increased interleukin (IL)-1ß expression which may induce N-methyl-D-aspartic acid receptor (NMDAR) phosphorylation in spinal dorsal horn neurons to enhance pain transmission. Together, these results suggest that spinal activated astrocytes may be a crucial component of mechanical allodynia in type 2 diabetes and "Astrocyte-IL-1ß-NMDAR-Neuron" pathway may be the detailed mechanism of astrocyte-induced allodynia. Thus, inhibiting astrocytic activation in the spinal dorsal horn may represent a novel therapeutic strategy for treating DNP.