Persistent Lipid Accumulation Leads to Persistent Exacerbation of Endoplasmic Reticulum Stress and Inflammation in Progressive NASH via the IRE1α/TRAF2 Complex.

Non-alcoholic steatohepatitis (NASH) is a metabolic disorder that often leads to other severe liver diseases, yet treatment options are limited. Endoplasmic reticulum (ER) stress is an important pathogenetic mechanism of NASH and plays a key role in tandem steatosis as well as liver inflammation. This study aims to develop a progressive NASH model through sustained lipid accumulation and to elucidate its molecular mechanism through IRE1α/TRAF2 complex. Male SD rats were fed a high-fat diet (HFD) for 4, 8, and 12 weeks to induce progressive NASH. MRNA sequencing and PPI analysis were used to screen core genes. Transmission electron microscopy, immunofluorescence staining, ELISA, qRT-PCR, and Western blotting were used at each time point to compare differences between each index of progressive NASH at 4, 8, and 12 weeks. Sustained lipid accumulation led to structural disruption of the ER, a reduction in ER number, and an increase of lipid droplet aggregation in hepatocytes. Persistent lipid accumulation led to a persistent increase in mRNA and protein expression of the IRE1α/TRAF2 complex, IKK/IκB/NF-κB signaling pathway and ASK1/JNK1 signaling pathway, and TNF-α, IL-1ß, and IL-6 also continued to increase. Persistent lipid accumulation led to a persistent exacerbation of ER stress and inflammation in progressive NASH via the IRE1α/TRAF2 complex.

Twenty years of paradigm-breaking studies of taxonomy and symbiotic nitrogen fixation by beta-rhizobia, and indication of Brazil as a hotspot of Paraburkholderia diversity.

Twenty years ago, the first members of the genus Burkholderia capable of nodulating and fixing N2 during symbiosis with leguminous plants were reported. The discovery that ß-proteobacteria could nodulate legumes represented a breakthrough event because, for over 100 years, it was thought that all rhizobia belonged exclusively to the α-Proteobacteria class. Over the past 20 years, efforts toward robust characterization of these bacteria with large-scale phylogenomic and taxonomic studies have led to the separation of clinically important and phytopathogenic members of Burkholderia from environmental ones, and the symbiotic nodulating species are now included in the genera Paraburkholderia and Trinickia. Paraburkholderia encompasses the vast majority of ß-rhizobia and has been mostly found in South America and South Africa, presenting greater symbiotic affinity with native members of the families Mimosoideae and Papilionoideae, respectively. Being the main center of Mimosa spp. diversity, Brazil is also known as the center of symbiotic Paraburkholderia diversity. Of the 21 symbiotic Paraburkholderia species described to date, 11 have been isolated in Brazil, and others first isolated in different countries have also been found in this country. Additionally, besides the symbiotic N2-fixation capacity of some of its members, Paraburkholderia is considered rich in other beneficial interactions with plants and can promote growth through several direct and indirect mechanisms. Therefore, these bacteria can be considered biological resources employed as environmentally friendly alternatives that could reduce the agricultural dependence on agrochemical inputs.

Physalin A attenuates inflammation through down-regulating c-Jun NH2 kinase phosphorylation/Activator Protein 1 activation and up-regulating the antioxidant activity.

Physalin A (PA), a withanolide, was isolated from Physalis angulata L. In this study, it is shown that PA can inhibit the production of inflammatory cytokines such as PGE2, NO, IL-1ß, IL-6, and TNF-α in LPS-induced RAW 264.7 cells. Furthermore, the results indicated that PA suppressed the IκB/NF-κB and JNK/ AP-1 inflammatory signaling pathways and inhibited the levels of pro-inflammatory factors iNOS and COX-2 in LPS-stimulated RAW 264.7 cells. In the carrageenan-induced mouse hind paw edema study, PA was shown to inhibit the production of inflammatory mediators such as NO, MDA, and TNF-α production. Conversely, the antioxidant factor levels of SOD, CAT, and GPx were all increased by the treated PA. According to the data, we are suggesting that the anti-inflammatory effects of PA may be through the suppressions of the JNK/AP-1 and IκB/NF-κB signaling pathways and up-regulation of the anti-oxidative activity.

Association of vitamin D status and the risk of cardiovascular disease as assessed by various cardiovascular risk scoring systems in patients with type 2 diabetes mellitus.

OBJECTIVE: The aim of the present study was to evaluate the relationship between vitamin D (25[OH]D) status and the risk of cardiovascular disease as assessed by various cardiovascular risk scoring systems such as QRISK2, BNF, ASSING, SCORE, and Framingham in patients with type 2 diabetes mellitus(T2DM). METHODS: The study included 108 patients with vitamin D insufficiency (25[OH]D ≥ 10-30 ng/mL) and 100 patients with vitamin D deficiency (25[OH]D < 10 ng/mL), who were admitted to the diabetes outpatient clinics due to T2DM and who were aged 45-65 years. QRISK2, BNF, ASSING, SCORE, and Framingham were calculated and compared between the two groups. RESULTS: HbA1c levels were significantly higher in patients with vitamin D deficiency. Patients with vitamin D deficiency had significantly higher Framingham risk score (p < .001) and significantly lower BNF score (p < .001), whereas other scores did not significantly differ between the groups. There was a moderate, statistically significant correlation between 25[OH]D levels and Framingham risk score in negative direction (r = 0.537) and a weak but statistically significant correlation between 25[OH]D levels and BNF score (r = 0.295). 25[OH]D levels were significantly higher and HbA1c levels were significantly lower in patients with Framingham cardiovascular risk score ≤10%. CONCLUSION: We found a close relationship with Framingham cardiovascular risk score in diabetic patients with very low serum vitamin D levels. Cardiovascular risk as assessed by the Framingham's scale increases with decreasing 25[OH]D levels. BNF score was negatively correlated with 25[OH]D levels.

Atorvastatin might resist tobacco smoking-induced endothelial inflammation through the inhibition of NF-κB signal pathway.

Endothelial inflammation caused by tobacco smoking is widely considered as a pathogenic factor in many vascular diseases. Drugs such as atorvastatin were found to be an effective treatment in smoking-dependent vascular diseases, but the underlying mechanism is still unclear. Here, we investigated the mechanism of atorvastatin resisting endothelial inflammation caused by tobacco smoking. Firstly, isolated human umbilical vein endothelial cells (HUVECs) were divided into normal control group, cigarette smoking extract (CSE) group, and atorvastatin (AS)+CSE group. Then the expressions of inflammatory factors (vascular cell adhesion molecule-1 (VCAM-1) and E-selectin) and nuclear transcription factor kappa (NF-κB) in HUVECs were detected by western blot after separate treatments. The results showed that the expressions of VCAM-1, E-selectin, and NF-κB in CSE group were significantly higher than the other two groups (P< 0.05). We also found that the expressions of VCAM-1, E-selectin, and NF-κB in CSE + atorvastatin group were a little higher than the normal control group (P< 0.05). Our results showed that atorvastatin might partly resist tobacco smoking-induced endothelial inflammation through the inhibition of NF-κB signal pathway.

Intracranial aneurysm growth: consistency of morphological changes.

OBJECTIVE: Previous studies have shown a relation between growth and rupture of intracranial aneurysms. Additionally, several morphological characteristics are frequently measured to estimate rupture risk. Little is known about how the rupture risk is associated with morphological characteristic changes during growth. The aim of this study was to provide insights into how morphological characteristics, associated with rupture, change during an aneurysm's growth. METHODS: The authors retrospectively identified patients with longitudinal MRA images of unruptured growing aneurysms. The MRA images had an in-plane resolution of 0.2-0.5 mm and a slice thickness of 0.2-0.75 mm. Therefore, growth was defined as an increase of at least 0.5 mm in two directions or 1 mm in one direction. Using the MRA images, the authors semiautomatically segmented the aneurysm and the perianeurysmal vasculature. Twelve morphological characteristics were automatically measured. These characteristics were related to size (diameter, height, width, neck diameter, volume, surface area, aspect ratio, height-width ratio, and bottleneck factor) and shape (ellipticity index, nonsphericity index, and undulation index) of the aneurysm. Morphological characteristics before and after growth were compared using the Wilcoxon signed-rank test. RESULTS: The authors included 31 patients with 38 growing aneurysms. The aneurysms' growth was detected after a mean of 218 weeks (range 23-567 weeks). A significant increase was seen in all size-related characteristics, and the bottleneck factor also significantly increased (from a median of 1.00 [IQR 0.85-1.04] to 1.03 [IQR 0.93-1.18]), while the ellipticity index decreased (from a median of 0.26 [IQR 0.25-0.28] to 0.25 [IQR 0.24-0.26]). The changes in size ratios and shape indices varied largely among patients. Larger aneurysms more often showed an increase in shape ratios. CONCLUSIONS: Although aneurysm growth, size-related characteristics, bottleneck factor, and ellipticity index changed significantly during growth, most size ratios and shape indices showed inconsistent changes among aneurysms. This suggests that, for an accurate rupture prediction, morphological parameters need to be reassessed after growth.

Dissipation of chlorantraniliprole in contrasting soils and its effect on soil microbes and enzymes.

An experiment was set up to determine the rate of dissipation of chlorantraniliprole (CTP) from two soils with contrasting properties. The other objective of the study was to find out the effect of CTP on soil microorganisms (population, microbial biomass carbon and soil enzymes) under controlled environment. CTP residues when applied at recommended dose ((RD) (at 40 g a.i./ha)) could not be recovered either from alluvial soil or red soil at 60 days post application of CTP in a microcosm study. Higher clay content led to higher half-life in alluvial soil compared to red soil. CTP could not be recovered from RD treatment at 30 days after pesticide application under controlled environment. Faster dissipation of CTP was observed in rice rhizosphere soil with 23.89 and 34.65 days dissipation half-lives for RD and double the recommended dose (DRD) treatments, respectively. Different doses of chlorantraniliprole did not have considerable negative effect on actinomycetes, fungi, biological nitrogen fixers and phospahte solubilising bacteria except the bacteria population. Among the treatments, DRD recorded the lowest activity of dehyrodeganse, fluoresein diacetate hydrolase, acid and alkaline phosphatases followed by RD treatment. Microbial biomass carbon, ß -glycosidase and urease did not vary significantly among the different doses of CTP. In general, RD did not have negative effcts on soil microbes. Hence, CTP can be recommeded in rice pest managment maintaining existing soil microbes and soil enzymes activity.

Site-directed mutagenesis to deactivate two nitrogenase isozymes of Kosakonia radicincitans DSM16656T.

Biological nitrogen fixation (BNF) is considered one of the key plant-growth-promoting (PGP) factors for diazotrophic organisms. Whether the iron and iron-molybdenum nitrogenases of Kosakonia radicincitans contribute to its PGP effect is yet to be proven. Hence, for the first time, we conducted site-directed mutagenesis in K. radicincitans to knock out anfH and (or) nifH as a mean to deactivate BNF in this strain. We used 15N2-labeled air to trace BNF activities in ΔanfH, ΔnifH, and ΔanfHΔnifH mutants. Assessing bacterial growth, nitrogen content, and 15N incorporation revealed that BNF is impaired in K. radicincitans DSM16656T ΔnifH and ΔanfHΔnifH. However, we detected no significant contribution of the Fe nitrogenase to biological dinitrogen assimilation under our pure bacterial culture experimental conditions. Such nondiazotrophic K. radicincitans DSM16656T mutants represent excellent tools for investigating nitrogen nutrition in K. radicincitans-inoculated plants.

[Effect of cadmium choride on oxidative damage and expression of NF-κB in brain tissue of mice].

OBJECTIVE: To investigate the oxidative damage induced by cadmium choride and the expression of nuclear factor κB( NF-κB), tumor necrosis factor-α( TNF-α) and intercellular cell adhesion molecule-1( ICAM-1) in brain tissue of mice. METHODS: A total of 32 adult ICR mice were divided into 4 groups randomly including CdCl_2 2. 0, 1. 0 and 0. 5 mg/kg and NS control group, all mice were through intraperitoneally injected administration for 14 days, after the last exposure, open-field test were used and all mice were sacrificed by cervical dislocation, the expression of reactive oxygen species( ROS)was measured and the expression of TNF-α, ICAM-1 were observed by ELISA, the level of NF-κB P65 in the brain were detected by immunohistochemistry and western blot, the slices of the brain were stained with HE for histopathological examination. RESULTS: Thebody weight gain in CdCl_22. 0 mg/kg was significant lower than others( P < 0. 05), the number of crossing lattice in CdCl_2 2. 0 mg/kg was higher than the NS control group in the open field test. The expression of ROS increased in CdCl_2 2. 0 and 1. 0 mg/kg groups( P < 0. 05), the expression of TNF-α increased in CdCl_2 2. 0 mg/kg group( P < 0. 05), no obvious change was found among every group of ICAM-1, the expression of NF-κB P65 in the brain increased in cadmium 2. 0 mg/kg group. The neuropathological test showed that the cerebral cortex and hippocampal cells were more loose in 2. 0 mg/kg group than the control. CONCLUSION: The level of ROS in mouse brain tissue increased, NF-κB pathway was activated by ROS and the expression of inflammatory cytokine TNF-α was increased.

Alternative NF-κB signaling regulates mTEC differentiation from podoplanin-expressing precursors in the cortico-medullary junction.

The thymic epithelium forms specialized niches to enable thymocyte differentiation. While the common epithelial progenitor of medullary and cortical thymic epithelial cells (mTECs and cTECs) is well defined, early stages of mTEC lineage specification have remained elusive. Here, we utilized in vivo targeting of mTECs to resolve their differentiation pathways and to determine whether mTEC progenitors participate in thymocyte education. We found that mTECs descend from a lineage committed, podoplanin (PDPN)-expressing progenitor located at the cortico-medullary junction. PDPN(+) junctional TECs (jTECs) represent a distinct TEC population that builds the thymic medulla, but only partially supports negative selection and thymocyte differentiation. Moreover, conditional gene targeting revealed that abrogation of alternative NF-κB pathway signaling in the jTEC stage completely blocked mTEC development. Taken together, this study identifies jTECs as lineage-committed mTEC progenitors and shows that NF-κB-dependent progression of jTECs to mTECs is critical to secure central tolerance.

Red blood membrane camouflaging Bismuth nanoflowers designed for radio-photothermal therapy in lung cancer.

Radio-photothermal therapy is an effective modality for cancer treatment. To overcome the radio-resistance in the hypoxic microenvironment and improve the sensitivity of radiotherapy, metal nanoparticles, and radio-photothermal therapy are widely used in the research of improving the curative effect and reducing the side effects of radiotherapy. Here, we developed red blood membrane camouflaging bismuth nanoflowers (RBCM-BNF) with outstanding physiological stability and biodegradability for lung tumours. In vitro data proved that the RBCM-BNF had the greatest cancer cell-killing ability combined with X-ray irradiation and photo-thermal treatment. Meanwhile, in vivo studies revealed that RBCM-BNF can alleviate the hypoxic microenvironment and promote tumour cell apoptosis by inhibiting HIF-1α expression and increasing caspase-3 expression. Therefore, RBCM-BNF had a good radio-sensitising effect and might be a promising biomimetic nanoplatform as a therapeutic target for cancer.

Yanghe Pingchuan granules mitigates oxidative stress and inflammation in a bronchial asthma rat model: role of the IKK/IκB/NF-κB signalling pathway.

Background: Bronchial asthma (BA) is a chronic inflammatory airway disease. Previous research has shown that Yanghe Pingchuan granules (YPG), among the granules formulated by the First Affiliated Hospital of the Anhui University of Chinese Medicine, exerts a precise therapeutic effect on BA. We previously showed that YPG improves airway inflammation in BA rats. Other studies have shown that the inhibitor of kappa-B kinase (IKK)/inhibitor of NF-κB (IκB)/nuclear factor kappa-B (NF-κB) signalling pathway plays a key role in inflammation mediation. Therefore, this study explored whether YPG could intervene in BA through the IKK/IκB/NF-κB signalling pathway. Methods: Ovalbumin-induced method was used to established BA rat model. After successful modelling, the authors used YPG to intervene the rats in BA rats. Hematoxylin-eosin (HE) staining was used to detect the bronchial pathological changes in BA rats, enzyme-linked immunosorbent assay (ELISA) was used to detect the changes of inflammatory factors (IL-1ß and IL-6) and oxidative stress indexes malondialdehyde (MDA), superoxide dismutase (SOD) and nitrogen monoxide (NO), Quantitative real-time polymerase chain reactionCR and western blot were used to detect the expression of IKK/IκB/NF-κB signalling pathway. Results: In BA model rats, YPG significantly improved the inflammatory response in bronchial tissues, reduced inflammatory factors IL-1ß and IL-6, alleviated oxidative stress, reduced MDA and NO, and increased SOD. Quantitative real-time polymerase chain reaction and western blot results showed that YPG could block the IKK/IκB/NF-κB signalling pathway. Conclusion: These findings showed that YPG had a definite therapeutic effect on BA, which may be related to blocking the IKK/IκB/NF-κB signalling pathway and improving inflammation and oxidative stress.

Novel phenanthrene/bibenzyl trimers from the tubers of Bletilla striata attenuate neuroinflammation via inhibition of NF-κB signaling pathway.

Five novel (9,10-dihydro) phenanthrene and bibenzyl trimers, as well as two previously identified biphenanthrenes and bibenzyls, were isolated from the tubers of Bletilla striata. Their structures were elucidated through comprehensive analyses of NMR and HRESIMS spectroscopic data. The absolute configurations of these compounds were determined by calculating rotational energy barriers and comparison of experimental and calculated ECD curves. Compounds 5b and 6 exhibited inhibitory effects on LPS-induced NO production in BV-2 cells, with IC50 values of 12.59 ± 0.40 and 15.59 ± 0.83 µmol·L-1, respectively. A mechanistic study suggested that these compounds may attenuate neuroinflammation by reducing the activation of the AKT/IκB/NF-κB signaling pathway. Additionally, compounds 3a, 6, and 7 demonstrated significant PTP1B inhibitory activities, with IC50 values of 1.52 ± 0.34, 1.39 ± 0.11, and 1.78 ± 0.01 µmol·L-1, respectively. Further investigation revealed that compound 3a might inhibit LPS-induced PTP1B overexpression and NF-κB activation, thereby mitigating the neuroinflammatory response in BV-2 cells.

Association of Barrett's esophagus with type II Diabetes Mellitus: results from a large population-based case-control study.

BACKGROUND & AIMS: Central obesity could increase the risk for Barrett's esophagus (BE) and esophageal adenocarcinoma by mechanical and/or metabolic mechanisms, such as hyperinsulinemia. We performed an epidemiologic study to determine whether prior type 2 diabetes mellitus (DM2) is associated with BE. METHODS: We performed a population-based case-control study using the General Practice Research Database, a UK primary care database that contains information on more than 8 million subjects, to identify cases of BE (using previously validated codes; n = 14,245) and matched controls without BE (by age, sex, enrollment date, duration of follow-up evaluation, and practice region by incidence density sampling; n = 70,361). We assessed the association of a prior diagnosis of DM2 with BE using conditional univariate and multivariable regression analysis. Confounders assessed included smoking, obesity measured by body mass index (BMI), and gastroesophageal reflux disease. RESULTS: BE cases were more likely than controls to have smoked (52.4% vs 49.9%), have a higher mean BMI (27.2 vs 26.9), and a higher prevalence of DM2 than controls (5.8% vs 5.3%). On multivariable analysis, DM2 was associated with a 49% increase in the risk of BE, independent of other known risk factors (odds ratio, 1.49; 95% confidence interval, 1.16-1.91). This association was stronger in women than men. Results remained stable with sensitivity analyses. CONCLUSIONS: In a large population-based case-control study, DM2 was a risk factor for BE, independent of obesity (as measured by BMI) and other risk factors (smoking and gastroesophageal reflux disease). These data suggest that metabolic pathways related to DM2 should be explored in BE pathogenesis and esophageal carcinogenesis.

Agronomic performance and quality of baby corn in response to the inoculation of seeds with Azospirillum brasilense and nitrogen fertilization in the summer harvest.

The association with Azospirillum brasilense promotes better growth and development in corn plants due to biological N fixation, the capacity to help in the synthesis of phytohormones and to improve the use of nutrients by crop plants. However, there aren't specific recommendations for the use of inoculation in baby corn crop. Thus, this study aimed to evaluate the effects of seed inoculation with A. brasilense, associated with nitrogen fertilization management, on the agronomic performance and chemical quality of baby corn grown in three summer growing seasons (2014/2015; 2015/2016 and 2016/2017). The evaluated treatments consisted of combination of five levels of seed inoculation (0.0, 50, 100, 150 and 200 mL 60,000 seeds-1) based on Azospirillum brasilense, two levels of nitrogen fertilization at sowing time (0.0 and 30.0 kg of N ha-1) and two levels of nitrogen in topdressing (0.0 and 110.0 kg of N ha-1), applied at the V4 stage of the popcorn hybrid IAC 125. The characteristics evaluated were: leaf area index (LAI), leaf nitrogen content (LNC), total husked spikelets yield (HSY) and commercial spikelets yield (CSY), and the chemical characteristics of the commercial spikelets: crude protein content (CPC), starch content (STC) and total sugar content (TSC). The inoculation, when combined with nitrogen fertilization, provided positive responses for LAI and provided an average increment of 6 kg ha-1 to CSY for every 10 mL 60,000 seeds-1 of inoculant added to the seeds. The LNC, CPC, STC and TSC weren't affected by seed inoculation. Nitrogen fertilization provided increments for all characteristics evaluated, except for TSC, which was negatively affected by nitrogen topdressing. The baby corn crop responded positively to seed inoculation with Azospirillum brasilense, combined with Nitrogen fertilization.

Dextran 40 hybrid biomimetic bismuth-nanoflower designed for NIR II-triggered hypoxic tumor thermoradiotherapy via macrophage escape.

At present, NIR-II-triggered photothermal biomedical applications are limited by complex synthesis reactions, mediocre photothermal conversion efficiency, and difficult degradation. Herein, we prepared biodegradable Bi flower-like nanoparticles (phospholipid-modified Bi nanoflowers, BNFs) with high photothermal conversion efficiency (∼33.52 %) in NIR-II by a simple method and then modified them with the red blood cell membrane and dextran 40 (DRBCM) to improve their in vitro stability, to escape macrophages clearance and to enhance tumor accumulation. Dextran coating onto the surface of particles as a dispersant shell stabilizes inorganic particles by maintaining the surface charges and creating steric repulsions upon compression of neighboring polymer chains. In vitro and in vivo experiments proved that combined thermoradiotherapy of DRBCM-BNFs exhibited significantly enhanced tumor inhibition efficacy than monotherapy with good biocompatibility and low toxicity due to its biodegradability. Furthermore, the mechanism studies demonstrated that DRBCM-BNFs could serve as a nano sensitizer to promote the thermoradiotherapy under NIR-II illumination and X-ray irradiation, by downregulating heat shock protein 70 (HSP70) and phosphorylated-p65 (p-p65) to reduce the thermal resistance and radioresistance of tumor cells and increasing the expression of apoptosis-related protein cleaved caspase-3. In conclusion, DRBCM-BNFs could be a promising green delivery platform for the sensitization of synergistic thermoradiotherapy.

YTHDF1 phase separation triggers the fate transition of spermatogonial stem cells by activating the IκB-NF-κB-CCND1 axis.

N6-methyladenosine (m6A) modification controls cell fate determination. Here, we show that liquid-liquid phase separation (LLPS) of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), a pivotal m6A "reader" protein, promotes the transdifferentiation of spermatogonial stem cells (SSCs) into neural stem cell-like cells by activating the IκB-nuclear factor κB (NF-κB)-CCND1 axis. The inhibition of IκBα/ß mRNA translation mediated by YTHDF1 LLPS is the key to the activation of the IκB-NF-κB-CCND1 axis. Disrupting either YTHDF1 LLPS or NF-κB activation inhibits transdifferentiation efficiency. Moreover, overexpression of the YTH domain of YTHDF1 inhibits the activation of the IκB-NF-κB-CCND1 axis by promoting IκBα/ß mRNA translation. Overexpression of the tau-YTH fusion protein reactivates IκB-NF-κB-CCND1 axis by inhibiting the translation of IκBα/ß mRNAs, and tau LLPS is observed, which can restore transdifferentiation efficiency. Our findings demonstrate that the protein-RNA LLPS plays essential roles in cell fate transition and provide insights into translational medicine and the therapy of neurological diseases.

Genetic diversity of Rhizobia isolates from Amazon soils using cowpea (Vigna unguiculata) as trap plant.

The aim of this work was to characterize rhizobia isolated from the root nodules of cowpea (Vigna unguiculata) plants cultivated in Amazon soils samples by means of ARDRA (Amplified rDNA Restriction Analysis) and sequencing analysis, to know their phylogenetic relationships. The 16S rRNA gene of rhizobia was amplified by PCR (polymerase chain reaction) using universal primers Y1 and Y3. The amplification products were analyzed by the restriction enzymes HinfI, MspI and DdeI and also sequenced with Y1, Y3 and six intermediate primers. The clustering analysis based on ARDRA profiles separated the Amazon isolates in three subgroups, which formed a group apart from the reference isolates of Bradyrhizobium japonicum and Bradyrhizobium elkanii. The clustering analysis of 16S rRNA gene sequences showed that the fast-growing isolates had similarity with Enterobacter, Rhizobium, Klebsiella and Bradyrhizobium and all the slow-growing clustered close to Bradyrhizobium.

[Anti-inflammation effect of moxibustion for rats with diarrhea-predominant irritable bowel syndrome based on multiple miRNAs regulating NF-κB signal pathway].

OBJECTIVE: To observe the effect of moxibustion on the regulation of nuclear factor-kappa B (NF-κB) and inflammatory factors by multiple microRNAs (miRNAs) in rats with diarrhea-predominant irritable bowel syndrome (IBS-D), and to explore the anti-inflammatory mechanism of moxibustion on IBS-D. METHODS: Twelve of 52 newborn rats were randomly selected into a normal group. The remaining rats were made into IBS-D model. A total of 36 rats with successful model were randomly divided into a model group, a medication group and a moxibustion group, 12 rats in each group. The rats in the medication group were intraperitoneally injected with pyrrolidine dithiocarbamate (PDTC). The rats in the moxibustion group were treated with moxibustion at "Tianshu" (ST 25) and "Shangjuxu" (ST 37) for 20 min each time. All the intervention was given once a day for 7 days. Before and after modeling as well as after intervention, the body mass, loose stool rate and the minimum volume threshold of abdominal withdrawal reflex (AWR) were measured. After intervention, the contents of serum tumor necrosis factor α (TNF-α), interleukin (IL)-1ß and IL-8 were detected by ELISA method; the morphology of colon tissues was observed by HE staining, and the expressions of miR-155, miR-125b, miR-29b, miR-31, miR-18a and NF-κB p65 mRNA in colon tissues were detected by real-time PCR. The expressions of NF-κB p65, TNF-α, IL-1ß and IL-8 protein in colon tissues were detected by immunofluorescence. RESULTS: After modeling, the body mass and the minimum volume threshold of AWR in the model group were lower than those in the normal group (P<0.01); the rates of loose stool in the model group were higher than those in the normal group (P<0.01); after intervention, in the model group, the inflammatory infiltration of colon tissues was obvious, and the serum levels of TNF-α, IL-1 ß, IL-8 were higher than those in the normal group (P<0.05); the expression of miR-155, miR-125b, miR-29b, miR-31, miR-18a and NF-κB p65 mRNA in colon tissues was higher than that in the normal group (P<0.05); the protein expression of NF-κB p65, TNF-α, IL-1ß, IL-8 was also higher than that in the normal group (P<0.01). After intervention, the body mass and the minimum volume threshold of AWR in the medication group and the moxibustion group were both higher than those in the model group (P<0.05); the loose stool rate in the medication group and the moxibustion group were lower than those in model group (P<0.05); the inflammatory cells infiltration in the colon tissues was less, the serum levels of TNF-α, IL-1ß and IL-8 as well as the protein expression of NF-κB p65, TNF-α, IL-1ß and IL-8 in the colon tissues in the medication group and the moxibustion group were lower than those in the model group (P<0.05, P<0.01). The expression of miR-125b, miR-31, miR-18a and NF-κB p65 mRNA in the medication group were lower than those in the model group (P<0.05). The expression of miR-155, miR-125b, miR-29b, miR-31, miR-18a and NF-κB p65 mRNA in the moxibustion group were lower than those in the model group (P<0.05). The miR-155, miR-125b, miR-29b, miR-31, miR-18a were positively correlated with NF-κB p65 mRNA (0

A Nodule-Localized Small Heat Shock Protein GmHSP17.1 Confers Nodule Development and Nitrogen Fixation in Soybean.

Small heat shock proteins (sHSPs) are ubiquitous proteins present in all organisms. The sHSPs are not only upregulated under heat shock as well as other stresses but also are expressed in unstressed cells, indicating quite diverse functions of sHSPs. However, there is little known about the role of sHSPs in nodulation and nitrogen fixation in soybean. In this study, we cloned a candidate protein of sHSP, GmHSP17.1, from proteome of nodule and analyzed its function in soybean nodulation. We found that GmHSP17.1 was a cytosolic protein and preferentially expressed during nodule development. An overexpression of GmHSP17.1 in composite transgenic plants showed increases in nodule number, fresh weight, nodule size, area of infection cells, and nitrogenase activity, and subsequently promoted the content of nitrogen and growth of soybean plants. While GmHSP17.1 RNA interference (RNAi) lines showed significantly impaired nodule development and nitrogen fixation efficiency. Through liquid chromatography-tandem mass spectrometry (LC-MS/MS), GmRIP1 was identified as the first potential target of GmHSP17.1, and was shown to be specifically expressed in soybean nodules. The interaction between GmHSP17.1 and GmRIP1 was further confirmed by yeast-two hybrid (Y2H), bimolecular fluorescence complementation (BiFC) in vivo and pull-down assay in vitro. Furthermore, peroxidase activity was markedly increased in GmHSP17.1 overexpressed nodules and decreased in RNAi lines. As a result, the reactive oxygen species (ROS) content greatly decreased in GmHSP17.1 overexpression lines and increased in suppression lines. Taken together, we conclude that GmHSP17.1 plays an important role in soybean nodulation through interacting with GmRIP1. Our results provide foundation for studying the mechanism of nitrogen fixation and for the genetics improvement of legume plants.