Yi-Shen-Hua-Shi regulates intestinal microbiota dysbiosis and protects against proteinuria in patients with chronic kidney disease: a randomized controlled study.

CONTEXT: Yi-Shen-Hua-Shi (YSHS) is a traditional Chinese medicine that treats chronic kidney disease (CKD). However, its efficacy in reducing proteinuria and underlying mechanisms is unknown. OBJECTIVE: This single-center randomized controlled trial explored whether YSHS could improve proteinuria and modulate the gut microbiota. MATERIALS AND METHODS: 120 CKD patients were enrolled and randomized to receive the renin-angiotensin-aldosterone system (RAAS) inhibitor plus YSHS (n = 56) or RAAS inhibitor (n = 47) alone for 4 months, and 103 patients completed the study. We collected baseline and follow-up fecal samples and clinical outcomes from participants. Total bacterial DNA was extracted, and the fecal microbiome was analyzed using bioinformatics. RESULTS: Patients in the intervention group had a significantly higher decrease in 24-h proteinuria. After 4 months of the YSHS intervention, the relative abundance of bacteria that have beneficial effects on the body, such as Faecalibacterium, Lachnospiraceae, Lachnoclostridium, and Sutterella increased significantly, while pathogenic bacteria such as the Eggerthella and Clostridium innocuum group decreased. However, we could not find these changes in the control group. Redundancy analysis showed that the decline in 24-h proteinuria during follow-up was significantly correlated with various taxa of gut bacteria, such as Lachnospiraceae and the Lachnoclostridium genus in the YSHS group. KEGG analysis also showed the potential role of YSHS in regulating glycan, lipid, and vitamin metabolism. DISCUSSION AND CONCLUSION: The YSHS granule reduced proteinuria associated with mitigating intestinal microbiota dysbiosis in CKD patients. The definite mechanisms of YSHS to improve proteinuria need to be further explored. TRIAL REGISTRATION: ChiCTR2300076136, retrospectively registered.

[Genetic analysis of a Chinese pedigree affected with Mucopolysaccharidosis type â ¢A].

OBJECTIVE: To explore the clinical and genetic characteristics of a patient with hypertrophic cardiomyopathy as the initial manifestation of Mucopolysaccharidosis type Ⅲ A (MPS Ⅲ A). METHODS: A female patient with MPS Ⅲ A who was admitted to the Affiliated Hospital of Jining Medical University in January 2022 and her family members (seven individuals from three generations) were selected as the study subjects. Clinical data of the proband were collected. Peripheral blood samples of the proband was collected and subjected to whole exome sequencing. Candidate variants were verified by Sanger sequencing. Heparan-N-sulfatase activity was determined for the disease associated with the variant site. RESULTS: The proband was a 49-year-old woman, for whom cardiac MRI has revealed significant thickening (up to 20 mm) of left ventricular wall and delayed gadolinium enhancement at the apical myocardium. Genetic testing revealed that she has harbored compound heterozygous variants in exon 17 of the SGSH gene, namely c.545G>A (p.Arg182His) and c.703G>A (p.Asp235Asn). Based on guidelines from the American College of Medical Genetics and Genomics (ACMG), both variants were predicted to be pathogenic (PM2_Supporting +PM3+PP1Strong+PP3+PP4; PS3+PM1+PM2_Supporting +PM3+PP3+PP4). Sanger sequencing confirmed that her mother was heterozygous for the c.545G>A (p.Arg182His) variant, whilst her father, sisters and her son were heterozygous for the c.703G>A (p.Asp235Asn) variant. Determination of blood leukocyte heparan-N-sulfatase activity suggested that the patient had a low level of 1.6 nmol/(g·h), whilst that of her father, elder and younger sisters and son were all in the normal range. CONCLUSION: The compound heterozygous variants of the SGSH gene probably underlay the MPS ⅢA in this patient, for which hypertrophic cardiomyopathy is an associated phenotype.

Molecular mechanisms of centipede toxin SsTx-4 inhibition of inwardly rectifying potassium channels.

Inwardly rectifying potassium channels (Kirs) are important drug targets, with antagonists for the Kir1.1, Kir4.1, and pancreatic Kir6.2/SUR1 channels being potential drug candidates for treating hypertension, depression, and diabetes, respectively. However, few peptide toxins acting on Kirs are identified and their interacting mechanisms remain largely elusive yet. Herein, we showed that the centipede toxin SsTx-4 potently inhibited the Kir1.1, Kir4.1, and Kir6.2/SUR1 channels with nanomolar to submicromolar affinities and intensively studied the molecular bases for toxin-channel interactions using patch-clamp analysis and site-directed mutations. Other Kirs including Kir2.1 to 2.4, Kir4.2, and Kir7.1 were resistant to SsTx-4 treatment. Moreover, SsTx-4 inhibited the inward and outward currents of Kirs with different potencies, possibly caused by a K+ "knock-off" effect, suggesting the toxin functions as an out pore blocker physically occluding the K+-conducting pathway. This conclusion was further supported by a mutation analysis showing that M137 located in the outer vestibule of the Kir6.2/ΔC26 channel was the key residue mediating interaction with SsTx-4. On the other hand, the molecular determinants within SsTx-4 for binding these Kir channels only partially overlapped, with K13 and F44 being the common key residues. Most importantly, K11A, P15A, and Y16A mutant toxins showed improved affinity and/or selectivity toward Kir6.2, while R12A mutant toxin had increased affinity for Kir4.1. To our knowledge, SsTx-4 is the first characterized peptide toxin with Kir4.1 inhibitory activity. This study provides useful insights for engineering a Kir6.2/SUR1 channel-specific antagonist based on the SsTx-4 template molecule and may be useful in developing new antidiabetic drugs.

Unusual Tetrahydropyridoindole-Containing Tetrapeptides with Human Nicotinic Acetylcholine Receptors Targeting Activity Discovered from Antarctica-Derived Psychrophilic Pseudogymnoascus sp. HDN17-933.

Chemical investigation of the psychrophilic fungus Pseudogymnoascus sp. HDN17-933 derived from Antarctica led to the discovery of six new tetrapeptides psegymamides A-F (1-6), whose planar structures were elucidated by extensive NMR and MS spectrometric analyses. Structurally, psegymamides D-F (4-6) possess unique backbones bearing a tetrahydropyridoindoles unit, which make them the first examples discovered in naturally occurring peptides. The absolute configurations of structures were unambiguously determined using solid-phase total synthesis assisted by Marfey's method, and all compounds were evaluated for their inhibition of human (h) nicotinic acetylcholine receptor subtypes. Compound 2 showed significant inhibitory activity. A preliminary structure-activity relationship investigation revealed that the tryptophan residue and the C-terminal with methoxy group were important to the inhibitory activity. Further, the high binding affinity of compound 2 to hα4ß2 was explained by molecular docking studies.

Secretome of Mesenchymal Stem Cells from Consecutive Hypoxic Cultures Promotes Resolution of Lung Inflammation by Reprogramming Anti-Inflammatory Macrophages.

The secretome from hypoxia-preconditioned mesenchymal stem cells (MSCs) has been shown to promote resolution of inflammation and alleviate acute lung injury (ALI) through its immunomodulatory function. However, the effects of consecutive hypoxic culture on immunomodulatory function of the MSCs secretome are largely unclarified. Here, we intend to investigate the effects of consecutive hypoxia on therapeutic efficacy of conditioned medium derived from MSCs (MSCs-CM) in alleviating ALI. Human umbilical cord-derived MSCs (UC-MSCs) were consecutively cultured in 21% O2 (Nor-MSCs) or in 1% O2 (Hypo-MSCs) from passage 0. Their conditioned medium (Nor-CM and Hypo-CM respectively) was collected and administered into ALI models. Our findings confirmed that Hypo-MSCs exhibited increased proliferation ability and decreased cell senescence compared with Nor-MSCs. Consecutive hypoxia promoted UC-MSCs to secrete immunomodulatory cytokines, such as insulin-like growth factor 1(IGF1), IL10, TNFα-stimulated gene 6(TSG6), TGFß, and prostaglandin E2 (PGE2). Both Nor-CM and Hypo-CM could effectively limit lung inflammation, promote efferocytosis and modulate anti-inflammatory polarization of lung macrophages in ALI models. Moreover, the effects of Hypo-CM were more potent than Nor-CM. Taken together, our findings indicate that consecutive hypoxic cultures could not only promote both proliferation and quality of UC-MSCs, but also enhance the therapeutic efficacy of their secretome in mitigating lung inflammation by promoting efferocytosis and anti-inflammatory polarization of macrophages.

Hypoxic Preconditioning Enhances the Efficacy of Mesenchymal Stem Cells-Derived Conditioned Medium in Switching Microglia toward Anti-inflammatory Polarization in Ischemia/Reperfusion.

Activation of pro-inflammatory microglia is an important mechanism of the cerebral ischemia-reperfusion (I/R)-induced neuronal injury and dysfunction. Mesenchymal stem cells (MSCs) together with their paracrine factors demonstrated curative potential in immune disorders and inflammatory diseases, as well as in ischemic diseases. However, it remains unclear whether conditioned medium from MSCs could effectively regulate the activation and polarization of microglia exposed to I/R stimulation. In this study, we investigated the effects of conditioned medium from bone marrow MSCs (BMSCs-CM) on I/R-stimulated microglia and the potential mechanism involved, as well as the way to obtain more effective BMSCs-CM. First, cell model of oxygen-glucose deprivation/reoxygenation (OGD/R) was established in microglia to mimic the I/R. BMSCs-CM from different culture conditions (normoxic: 21% O2; hypoxic: 1% O2; hypoxia preconditioning: preconditioning with 1% O2 for 24 h) was used to treat the microglia. Our results showed that BMSCs-CM effectively promoted the survival and alleviated the injury of microglia. Moreover, in microglia exposed to OGD/R, BMSCs-CM inhibited significantly the expression of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6), CD86 and inducible nitric oxide synthase, whereas upregulated the levels of anti-inflammatory cytokine (IL-10), CD206 and Arginase-1. These results suggested that BMSCs-CM promoted the polarization of anti-inflammatory microglia. In particular, BMSCs-CM from cultures with hypoxia preconditioning was more effective in alleviating cell injury and promoting anti-inflammatory microglia polarization than BMSCs-CM from normoxic cultures and from hypoxic cultures. Furthermore, inhibition of exosomes secretion could largely mitigate these effects of BMSCs-CM. In conclusion, our results suggested that hypoxia preconditioning of BMSCs could enhance the efficacy of BMSCs-CM in alleviating OGD/R-induced injury and in promoting the anti-inflammatory polarization of microglia, and these beneficial effects of BMSCs-CM owed substantially to exosomes.

Laboratory virulence of entomopathogenic nematodes to the sweetpotato whitefly, Bemisia tabaci.

The sweetpotato whitefly, Bemisia tabaci Middle East-Asia Minor 1 (MEAM1), is a major insect pest on vegetable crops worldwide. Enormous economic losses result from direct and indirect plant damage caused by MEAM1. Biological control using entomopathogenic nematodes (EPN) may be an effective alternative strategy against MEAM1 because this pest has developed resistance to most insecticides. First, nine EPN species (Heterorhabditis bacteriophora, H. indica, H. georgiana, H. floridensis, Steinernema feltiae, S. carpocapsae, S. riobrave, S. glaseri, and S. rarum) were investigated for virulence to MEAM1 third instar nymphs on snap bean leaves under laboratory conditions. The mortality of MEAM1 nymphs was evaluated at 3 days post-inoculation (dpi). Compared to the water control, the application of the nine EPN species except S. glaseri resulted in significantly higher mortality of MEAM1 nymphs, such as H. bacteriophora (66.31%), H. floridensis (56.38%), S. carpocapsae (54.54%), and S. rarum (57.80%). Subsequently, the four virulent EPN species, H. bacteriophora, H. floridensis, S. carpocapsae, and S. rarum were evaluated further for virulence to MEAM1 nymphs on snap bean and tomato leaves. The mortality of MEAM1 nymphs was assessed at 3 dpi and 7 dpi. There were no significant differences in MEAM1 nymphal mortality between tomato and snap bean at either 3 dpi or 7 dpi. The mortality of MEAM1 nymphs caused by the application of H. floridensis (99.25%) was significantly higher than the other three EPN species and the water control at 7dpi. The results indicate that H. floridensis is a very promising biocontrol agent for B. tabaci management.

IRF9 promotes apoptosis and innate immunity by inhibiting SIRT1-p53 axis in fish.

As a NAD+-dependent deacetylase, SIRT1 is widely involved in apoptosis and cellular inflammation via multiple pathways such as p53, NF-кB and STAT. More and more studies have shown that p53 is the first non-histone deacetylation target of SIRT1. SIRT1-p53 axis thus plays an important role in mammalian cells. IRF9 is an important member of interferon regulator factor family and performs an important role in innate immunity against foreign virus invasion. More importantly, human IRF9 can suppress the SIRT1-p53 axis. However, the functions and relationship between IRF9 and SIRT1-p53 axis are rarely studied in fish. To this end, we made a preliminary research on the functions of grass carp (Ctenopharyngodon idella) IRF9, SIRT1 and p53 in apoptosis and innate immunity. Firstly, we cloned and identified the ORF of SIRT1 (named CiSIRT1, MN125614) from C. idella and demonstrated that CiIRF9 promoted apoptosis, while CiSIRT1 inhibited apoptosis by flow cytometry and TUNEL experiments. Next, we found the interaction between CiSIRT1 and Cip53 in vivo by co-immunoprecipitation experiments. Moreover, the colocalization analysis also showed CiSIRT1 and Cip53 were mainly distributed in nucleus. Thirdly, we got a conclusion that CiIRF9 can repress the expression of CiSIRT1, implying that CiIRF9 regulates CiSIRT1-p53 axis. Finally, CiSIRT1 mRNA level was significantly up-regulated and the expression reached the highest level at 24 h post poly (I:C) stimulation in CIK cells. So, CiSIRT1 may exert an important function in innate immunity. Furthermore, we found CiSIRT1 down-regulated the expression of CiIFN1. In summary, CiIRF9 promotes apoptosis and innate immunity by inhibiting SIRT1-p53 axis. These findings will provide a new theoretical basis for the research on teleost innate immunity.

Influencing factors of vascular endothelial function in patients with non-obstructive coronary atherosclerosis: a 1-year observational study.

BACKGROUND: Endothelial dysfunction may play a key role in non-obstructive coronary artery atherosclerosis. Our study aimed to evaluate the vascular endothelial function and its influencing factors in patients with non-obstructive coronary artery atherosclerosis. METHODS: A total of 131 consecutive patients with non-obstructive coronary artery atherosclerosis were enrolled. Flow-mediated dilatation (FMD) was measured at baseline and 1-year follow-up. Endothelial progenitor cells (EPCs) were counted by staining the fasting venous blood with antibodies against CD34 and vascular endothelial growth factor receptor 2. RESULTS: Systolic blood pressure, pulse pressure and the levels of HbA1c in participants with baseline FMD < 6% (n = 65) were significantly higher than those with baseline FMD ≥ 6% (n = 66). Baseline FMD was negatively associated with EPC counts (r = - 0.199, P < 0.05) and systolic blood pressure (r = - 0.315, P < 0.01). The 1-year FMD was significantly increased compared to the baseline FMD [(9.31 ± 5.62) % vs (7.31 ± 5.26) %, P < 0.001]. Independent predictors of FMD improvement included elevated EPC counts (OR = 1.104, 95% CI: 1.047-1.165, P < 0.001) and decreased levels of serum creatinine (OR = 0.915, 95% CI: 0.843-0.993, P = 0.034). CONCLUSIONS: Family history of premature cardiovascular diseases, hypertension, elevated systolic pressure, and HbA1c > 6.5% are independent risk factors for endothelial dysfunction in non-obstructive atherosclerotic patients. Elevated peripheral blood EPC counts and decreased levels of serum creatinine are independent predictors of endothelial function improvement.

Interleukin-1ß Promotes Ox-LDL Uptake by Human Glomerular Mesangial Cells via LOX-1.

The aim of this study was to determine whether interleukin-1ß (IL-1ß) promotes oxidised low-density lipoprotein (Ox-LDL) uptake by human glomerular mesangial cells (HMCs) and its effect on the expression of lectin-like Ox-LDL receptor 1 (LOX-1) and to identify pathways through which IL-1ß affects lipid uptake. Confocal laser scanning microscopy and flow cytometry were used to observe the effect of IL-1ß on lipid uptake by HMCs and the pathway by which IL-1ß may mediate lipid uptake. Real-time polymerase chain reaction (PCR) and western blotting were used to evaluate the effect of IL-1ß on LOX-1 expression. Confocal laser scanning microscopy and flow cytometry revealed that IL-1ß promoted uptake of fluorescent Dil-labelled Ox-LDL(Dil-Ox-LDL) by HMCs and the enhanced uptake of Dil-Ox-LDL was partially inhibited by an anti-LOX-1 antibody evaluated by flow cytometry. Further, IL-1ß promoted LOX-1 mRNA and protein expression of HMCs in a dose- and time-dependent manner. Thus, Ox-LDL is ingested by HMCs under basic conditions. Inflammatory cytokine IL-1ß promotes Ox-LDL uptake by HMCs. Furthermore, IL-1ß promotes the mRNA and protein expression of LOX-1, a specific receptor of Ox-LDL, suggesting that the enhancement of Ox-LDL uptake may be mediated by LOX-1 pathway. Anti-LOX-1 therapy may be a promising option for treatment of glomerulosclerosis.

Characterization of Low Molecular Weight Sulfate Ulva Polysaccharide and its Protective Effect against IBD in Mice.

Inflammatory bowel disease (IBD) has been gradually considered a public health challenge worldwide. Sulfated polysaccharides, extracted from seaweed, have been shown to have an anti-inflammatory effect on the disease[...].

Bone marrow mesenchymal stem cells-derived conditioned medium protects cardiomyocytes from hypoxia/reoxygenation-induced injury through Notch2/mTOR/autophagy signaling.

Bone marrow mesenchymal stem cells (BMSC) can ameliorate ischemic injury of various tissues. However, the molecular mechanisms involved remain to be clarified. In this study, we intend to investigate the effects of BMSC-derived conditioned medium (BMSC-CM) on hypoxia/reoxygenation (H/R)-induced injury of H9c2 myocardial cells, and the potential mechanisms. Cell injury was determined through level of cell viability, lactate dehydrogenase (LDH) release, total intracellular reactive oxygen species (ROS), mitochondrial membrane potential (Δψm), and cell apoptosis. Autophagic activity of cells was detected through levels of the autophagy-associated proteins and autophagic flux. Results showed that BMSC-CM alleviated H/R-induced injury in H9c2 cells, as demonstrated by increased cell viability and Δψm, decreased ROS production, LDH release, and cell apoptosis. Furthermore, the H/R treatment induced a decrease in autophagic activity and an increase in Notch2 signaling activation in H9c2 cells. In the presence of BMSC-CM, the autophagic activity impaired by the H/R treatment was upregulated with decreased phosphorylation of mTOR, and the activation of Notch2 signaling was downregulated. These effects of BMSC-CM could be replicated by Notch signaling inhibitor. In contrast, inhibitors of cell autophagy including chloroquine (CQ) and 3-methyladenine, diminished the protective effects of BMSC-CM. Taken together results, our study showed that BMSC-CM could protect H9c2 cells from H/R-induced injury potentially through regulating Notch2/mTOR/autophagy signaling. These findings may provide a novel insight into the mechanisms of BMSC-CM in therapy of myocardial ischemia/reperfusion injury as well as other ischemic diseases.

Identification and functional characterization of IRAK-4 in grass carp (Ctenopharyngodon idellus).

IL-1R-associated kinase 4 (IRAK4), a central TIR signaling mediator in innate immunity, can initiate a cascade of signaling events and lead to induction of inflammatory target gene expression eventually. In the present study, we cloned and characterized an IRAK4 orthologue from grass carp (Ctenopharyngodon idella). The full length cDNA of CiIRAK4 was 2057 bp with an ORF of 1422 bp encoding a polypeptide of 472 amino acids. Multiple alignments showed that IRAK4s were highly conserved among different species. Phylogenetic tree analysis revealed that CiIRAK4 shared high homologous with zebra fish IRAK4. Expression analysis indicated that CiIRAK4 was widely expressed in all tested tissues. It was significantly up-regulated after treatment with poly I:C, especially obvious in liver and spleen. Also, CiIRAK4 could be induced by poly I:C and LPS in CIK cells. Fluorescence microscopy assays showed that CiIRAK4 localized in the cytoplasm. RNAi-mediated knockdown and overexpression assays indicated that CiIRAK4 might have little effect on NF-kappa B p65 translocation from cytoplasm to nucleus, indicating that CiIRAK4 was dispensable for activation of NF-kappa B p65. In addition, IRAK4 promoted IRF5 nuclear translocation, which has nothing to do with the interaction between IRAK4 and IRF5. It suggested that fish IRAK4 kinase regulated IRF5 activity through indirect ways.

The association of hepatitis c virus infection status with serum glucose levels.

BACKGROUND: Hepatitis C virus (HCV) infection is commonly associated with a disturbance of glucose metabolism. However, there have been conflicting reports on whether the clearance of the HCV may be followed by changes of serum blood glucose and insulin resistance. The aim of the present study was to evaluate the impact of HCV and antiviral treatment on serum glucose levels and other glucose metabolism parameters. METHODS: A retrospective analysis of 306 HCV-infected patients was performed. Fasting serum blood glucose (FBG) levels in these patients were compared with that of 325 healthy individuals. Serum parameters of glucose metabolism were measured in 183 patients with chronic hepatitis C at baseline, at the end of interferon α-2b plus ribavirin treatment, and at 24 weeks post-treatment. RESULTS: Patients with HCV infection had significantly higher FBG level than healthy controls (5.57 ± 0.74 vs. 5.11 ± 0.83 mmol/l, P < 0.001). After antiviral treatment, we found a significant reduction in FBG levels regardless of the outcome of treatment. However, after stopping treatment the serum FBG levels were significantly elevated in the sustained virological response (SVR) and non-responder groups, and maintained high level until week 24 post-treatment. In both groups, the levels of serum FBG after 24 weeks post-treatment were still lower than pre-treatment levels. In sustained responders, fasting insulin (P = 0.007), C-peptide (P < 0.001) and HOMA-IR (P < 0.001) significantly decreased, and the insulin sensitivity index (ISI) increased (P < 0.001) at the end of the treatment comparing with pre-treatment levels, while no significant difference was observed in non-responder group. HOMA-ß values were increased in both groups at the end of treatment (both P < 0.001). CONCLUSION: The total serum FBG level in HCV infected patients was higher than that in healthy controls. Clearance of HCV was associated with reduced glucose and improved insulin resistance.

Enzymatic rhamnosylation of anticancer drugs by an α-L-rhamnosidase from Alternaria sp. L1 for cancer-targeting and enzyme-activated prodrug therapy.

The synthesis of rhamnosylated compounds has gained great importance since these compounds have potential therapeutic applications. The enzymatic approaches for glycosylation of bioactive molecules have been well developed; however, the enzymatic rhamnosylation has been largely hindered by lacking of the glycosyl donor for rhamnosyltransferases. Here, we employed an α-L-rhamnosidase from Alternaria sp. L1 (RhaL1) to perform one-step rhamnosylation of anticancer drugs, including 2'-deoxy-5-fluorouridine (FUDR), cytosine arabinoside (Ara C), and hydroxyurea (Hydrea). The key synthesis conditions including substrate concentrations and reaction time were carefully optimized, and the maximum yields of each rhamnosylated drugs were 57.7 mmol for rhamnosylated Ara C, 68.6 mmol for rhamnosylated Hydrea, and 42.2 mmol for rhamnosylated FUDR. It is worth pointing out that these rhamnosylated drugs exhibit little cytotoxic effects on cancer cells, but could efficiently restore cytotoxic activity when incubated with exogenous α-L-rhamnosidase, suggesting their potential applications in the enzyme-activated prodrug system. To evaluate the cancer-targeting ability of rhamnose moiety, the rhamnose-conjugated fluorescence dye rhodamine B (Rha-RhB) was constructed. The fluorescence probe Rha-RhB displayed much higher cell affinity and cellular internalization rate of oral cancer cell KB and breast cancer cell MDA-MB-231 than that of the normal epithelial cells MCF 10A, suggesting that the rhamnose moiety could mediate the specific internalization of rhamnosylated compounds into cancer cells, which greatly facilitated their applications for cancer-targeting drug delivery.

Characterization and Hypoglycemic Activity of a Rhamnan-Type Sulfated Polysaccharide Derivative.

Polysaccharide chromium (III) derivatives are gaining increasing attention in improving type 2 diabetes. In this study, the sulfated polysaccharide from Enteromorpha prolifera (SPE) with 4.8 kDa was prepared by specific enzymatic hydrolysis. The obtained SPE was used to prepare a rhamnan-type sulfated polysaccharide derivative (SPED). Results indicated that O-H, C=O, and S=O were effectively involved in the chelation of SPED (chromium content 20.26%). Acute (half lethal dose > 2.38 g/kg) and sub-acute toxicity showed that SPED had no damaging effects on mice. Anti-diabetic experiment demonstrated that SPED improved glucose metabolism. Moreover, SPED promoted the PI3K/PKB/GSK-3ß signaling pathway by regulating mRNA expression of insulin receptors (IR), insulin receptor substrate 2 (IRS-2), phosphatidylinositol 3 kinase (PI3K), protein kinase B (PKB), and glycogen synthase kinase 3ß (GSK-3ß). In conclusion, the SPED might represent a novel marine-derived candidate against hyperglycemia, which may undergo further pharmaceutical development as a hypoglycemic agent.

Genetic Mapping and Molecular Characterization of a Broad-spectrum Phytophthora sojae Resistance Gene in Chinese Soybean.

Phytophthora root rot (PRR) causes serious annual soybean yield losses worldwide. The most effective method to prevent PRR involves growing cultivars that possess genes conferring resistance to Phytophthora sojae (Rps). In this study, QTL-sequencing combined with genetic mapping was used to identify RpsX in soybean cultivar Xiu94-11 resistance to all P. sojae isolates tested, exhibiting broad-spectrum PRR resistance. Subsequent analysis revealed RpsX was located in the 242-kb genomic region spanning the RpsQ locus. However, a phylogenetic investigation indicated Xiu94-11 carrying RpsX is distantly related to the cultivars containing RpsQ, implying RpsX and RpsQ have different origins. An examination of candidate genes revealed RpsX and RpsQ share common nonsynonymous SNP and a 144-bp insertion in the Glyma.03g027200 sequence encoding a leucine-rich repeat (LRR) region. Glyma.03g027200 was considered to be the likely candidate gene of RpsQ and RpsX. Sequence analyses confirmed that the 144-bp insertion caused by an unequal exchange resulted in two additional LRR-encoding fragments in the candidate gene. A marker developed based on the 144-bp insertion was used to analyze the genetic population and germplasm, and proved to be useful for identifying the RpsX and RpsQ alleles. This study implies that the number of LRR units in the LRR domain may be important for PRR resistance in soybean.

Next-generation sequencing to identify candidate genes and develop diagnostic markers for a novel Phytophthora resistance gene, RpsHC18, in soybean.

KEY MESSAGE: A novel Phytophthora sojae resistance gene RpsHC18 was identified and finely mapped on soybean chromosome 3. Two NBS-LRR candidate genes were identified and two diagnostic markers of RpsHC18 were developed. Phytophthora root rot caused by Phytophthora sojae is a destructive disease of soybean. The most effective disease-control strategy is to deploy resistant cultivars carrying Phytophthora-resistant Rps genes. The soybean cultivar Huachun 18 has a broad and distinct resistance spectrum to 12 P. sojae isolates. Quantitative trait loci sequencing (QTL-seq), based on the whole-genome resequencing (WGRS) of two extreme resistant and susceptible phenotype bulks from an F2:3 population, was performed, and one 767-kb genomic region with ΔSNP-index ≥ 0.9 on chromosome 3 was identified as the RpsHC18 candidate region in Huachun 18. The candidate region was reduced to a 146-kb region by fine mapping. Nonsynonymous SNP and haplotype analyses were carried out in the 146-kb region among ten soybean genotypes using WGRS. Four specific nonsynonymous SNPs were identified in two nucleotide-binding sites-leucine-rich repeat (NBS-LRR) genes, RpsHC18-NBL1 and RpsHC18-NBL2, which were considered to be the candidate genes. Finally, one specific SNP marker in each candidate gene was successfully developed using a tetra-primer ARMS-PCR assay, and the two markers were verified to be specific for RpsHC18 and to effectively distinguish other known Rps genes. In this study, we applied an integrated genomic-based strategy combining WGRS with traditional genetic mapping to identify RpsHC18 candidate genes and develop diagnostic markers. These results suggest that next-generation sequencing is a precise, rapid and cost-effective way to identify candidate genes and develop diagnostic markers, and it can accelerate Rps gene cloning and marker-assisted selection for breeding of P. sojae-resistant soybean cultivars.

Ctenopharyngodon idella TBK1 activates innate immune response via IRF7.

In mammals, IFN regulatory factor (IRF) 7 is a central regulator of IFN-α expression in response to variable pathogenic infections. There are several pathogenic sensors involved in monitoring pathogen intrusion in mammals. These sensors trigger IRF7-mediated responses through different pathways. TANK-binding kinase 1 (TBK1) is a critical mediator of IRF7 activation upon pathogen infection. In fish, there are many reports on TBK1, IRF3 and IRF7, especially on TBK1-IRF3 signaling pathway. However, it is not very clear how TBK1-IRF7 works in innate immune signaling pathway. In this study, we explored how TBK1 up-regulates IFN, ISG expression, and how TBK1 initiates innate immune response through IRF7 in fish under lipopolysaccharides (LPS) stimulation. After stimulation with LPS, grass carp IRF3 and IRF7 transcriptions were up-regulated, indicating they participate in TLR-mediated antiviral signaling pathway. It is interesting that the response time of grass carp IRF3 to LPS was earlier than that of IRF7. In addition, IRF7 rather than IRF3 acted as a stronger positive regulator of IFN and ISG transcription in Ctenopharyngodon idella kidney cells (CIKs). It is suggested the potential function differentiation between IRF3 and IRF7 upon LPS infection in fish. Dual luciferase assays also showed that overexpression of grass carp IRF7 and TBK1 up-regulated the transcription level of IFN and PKR. However, knockdown of IRF7 inhibits ISG expression, suggesting that grass carp TBK1 regulates the transcription via IRF7. Co-immunoprecipitation and GST pull-down assays proved the binding of grass carp IRF7 to TBK1. Furthermore, grass carp TBK1 can promote the nuclear translocation of IRF7. The results indicated that grass carp TBK1 can bind directly to and activate IRF7.

[Clinical Manifestations and Complications in Patients with Anti-NMDA Receptor Encephalitis].

OBJECTIVE: To investigate the clinical manifestations and complications in the patients of anti-N-methyl-D-aspartate (anti-NMDA) receptor encephalitis. METHODS: We retrospectively collected clinical data of 143 patients diagnosed with anti-NMDA receptor encephalitis. According to the complexity of clinical symptoms, the patients were divided into two groups: ≥4 clinical symptoms group (96 cases, 67.1%) and <4 group (47 cases, 32.9%). There were 109 patients with complications and 34 patients without any complicaitons, The related factors to symptom complexity and complications were analyzed by univariate and multivariate analysis method. RESULTS: In the 143 patients of anti-NMDA receptor encephalitis, 120 (83.9%) exhibited seizures, 130 (90.9%) presented with psychiatric symptoms. Pulmonary infections (58.7%) and gastrointestinal disorders (49.0%) were the most common complications. Univariate analysis showed that the complexity of clinical symptoms was related to abnormal electroencephalography (EEG), positive anti-NMDA receptor antibody in cerebrospinal fluid(CSF) and serum. There were more frequent seizures, disorders of consciousness, abnormal movements, central hypoventilation in the patients with complications than those in patients without complications, all the differences were signifcant (P<0.05). Multivariate analysis showed that abnormal EEG was an independent risk factor for symptom complexity 〔odds ratio(OR)=2.620, P<0.05〕. The abnormal movements and the activities of daily living (ADL) on admission were predictor factors for the incidence of complications (OR=4.338, 0.980, respectively, P<0.05). CONCLUSIONS: In the patients of anti-NMDA receptor encephalitis, abnormal EEG may related to more complex clinical symptoms. Abnormal movements and low ADL on admission seems to be independent risk factors related to the incidence of complications.