The autotetraploid potato genome provides insights into highly heterozygous species.

Potato (Solanum tuberosum L.) originated in the Andes and evolved its vegetative propagation strategy through short day-dependent tuber development. Herein, we present a high-quality, chromosome-scale reference genome sequence of a tetraploid potato cultivar. The total length of this genome assembly was 2.67 Gb, with scaffold N50 and contig N50 sizes of 46.24 and 2.19 Mb, respectively. In total, 1.69 Gb repetitive sequences were obtained through de novo annotation, and long terminal repeats were the main transposable elements. A total of 126 070 protein-coding genes were annotated, of which 125 077 (99.21%) were located on chromosomes. The 48 chromosomes were classified into four haplotypes. We annotated 31 506 homologous genes, including 5913 (18.77%) genes with four homologues, 11 103 (35.24%) with three homologues, 12 177 (38.65%) with two homologues and 2313 (7.34%) with one homologue. MLH3, MSH6/7 and RFC3, which are the genes involved in the mismatch repair pathway, were found to be significantly expanded in the tetraploid potato genome relative to the diploid potato genome. Genome-wide association analysis revealed that cytochrome P450, flavonoid synthesis, chalcone enzyme, glycosyl hydrolase and glycosyl transferase genes were significantly correlated with the flesh colours of potato tuber in 150 tetraploid potatoes. This study provides valuable insights into the highly heterozygous autotetraploid potato genome and may facilitate the development of tools for potato cultivar breeding and further studies on autotetraploid crops.

Age-Related Conservation in Plant-Soil Feedback Accompanied by Ectomycorrhizal Domination in Temperate Forests in Northeast China.

This study investigates the effects of forest aging on ectomycorrhizal (EcM) fungal community and foraging behavior and their interactions with plant-soil attributes. We explored EcM fungal communities and hyphal exploration types via rDNA sequencing and investigated their associations with plant-soil traits by comparing younger (~120 years) and older (~250 years) temperate forest stands in Northeast China. The results revealed increases in the EcM fungal richness and abundance with forest aging, paralleled by plant-soil feedback shifting from explorative to conservative nutrient use strategies. In the younger stands, Tomentella species were prevalent and showed positive correlations with nutrient availability in both the soil and leaves, alongside rapid increases in woody productivity. However, the older stands were marked by the dominance of the genera Inocybe, Hymenogaster, and Otidea which were significantly and positively correlated with soil nutrient contents and plant structural attributes such as the community-weighted mean height and standing biomass. Notably, the ratios of longer-to-shorter distance EcM fungal exploration types tended to decrease along with forest aging. Our findings underscore the integral role of EcM fungi in the aging processes of temperate forests, highlighting the EcM symbiont-mediated mechanisms adapting to nutrient scarcity and promoting sustainability in plant-soil consortia.

Gene Co-Expression Analysis Reveals the Transcriptome Changes and Hub Genes of Fructan Metabolism in Garlic under Drought Stress.

Drought has become a serious environmental factor that affects the growth and yield of plants. Fructan, as an important storage compound in garlic, plays an important role in drought tolerance. Genomic changes in plants under drought stress clarify the molecular mechanism of plants' responses to stress. Therefore, we used RNA-seq to determine the transcriptomic changes in garlic under drought stress and identified the key module related to fructan metabolism by weighted gene co-expression network analysis. We conducted a comprehensive analysis of the garlic transcriptome under drought stress over a time course (0, 3, 6, 9, 12, 15 d). Drought significantly induces changes in gene expression. The number of specifically expressed genes were 1430 (3 d), 399 (6 d), 313 (9 d), 351 (12 d), and 1882 (15 d), and only 114 genes responded at each time point. The number of upregulated DEGs was higher than the number of downregulated DEGs. Gene ontology and a Kyoto Encyclopedia of Genes and Genomes analysis showed that garlic was more likely to cause changes in carbohydrate metabolism pathways under drought stress. Fructan content measurements showed that drought stress significantly induced fructan accumulation in garlic. To determine whether there were modules involved in the transcriptional regulation of fructan content in garlic, we further analyzed the genes related to fructan metabolism using WGCNA. They were enriched in two modules, with F-box protein and GADPH as hub genes, which are involved in garlic fructan metabolism in response to drought stress. These results provide important insights for the future research and cultivation of drought-tolerant garlic varieties.

Effect of the structural characterization of the fungal polysaccharides on their immunomodulatory activity.

The immunomodulatory effects of the four extracellular polysaccharides, namely WPA, WPB, AP2A, and TP1A, which were isolated from the fermented broth of Aspergillus aculeatus, A. terreus and Trichoderma sp. KK19L1, were investigated in vitro. WPA, WPB, AP2A, and TP1A were not toxic to RAW264.7 cells. These polysaccharides enhanced cell viability. WPA, WPB, AP2A, and TP1A showed increased immunomodulatory effect by strengthening the phagocytic activity and enhancing the release of NO, TNF-α and IL-6 from RAW264.7 cells. WPA, WPB, AP2A, and TP1A exhibited different immunomodulatory activity in vitro due to their different structural characterizations, and their immunoregulatory effects decreased successively in the following order: WPA, WPB, AP2A, and TP1A. The extracellular polysaccharides WPA, WPB, AP2A, and TP1A had potent immunomodulatory effects and could be used as potential immunomodulatory agents in the fields of functional food and medicine.

Structure and anticoagulant activity of a sulfated fucan from the sea cucumber Acaudina leucoprocta.

A sulfated fucan was extracted, purified, and characterized from Acaudina leucoprocta (a low value sea cucumber) to better understand and utilize this species. The structure of the sulfated fucan was elucidated using chemical and modern spectroscopic analyses including HPGPC, IR, AFM, GC-MS, and NMR, and its bioactivity was investigated. Our results showed that the sulfated fucan was mainly composed of → 3)-α-L-Fucp-(1→ linkage, and that the sulfate groups were substituted at the O-2 and/or O-4 positions of the fucose ring. In detail, the sulfated fucan consisted of Fuc0S (40%), Fuc2S4S (24%), Fuc2S (24%), and Fuc4S (12%). On average, there were seven sulfate groups on every eight fucose residues. Assay for anticoagulant activity indicated that the sulfated fucan displayed intrinsic anticoagulant activity and specific anti-thrombin activity through heparin cofactor II. Our results showed that this bioactive sulfated fucan could enable the high-value utilization of this low-value sea cucumber.

Chemical characteristic and anticoagulant activity of the sulfated polysaccharide isolated from Monostroma latissimum (Chlorophyta).

A polysaccharide was isolated from marine green algae Monostroma latissimum, and its chemical characteristic and anticoagulant activity were investigated. The results demonstrated that the polysaccharide was high rhamnose-containing sulfated polysaccharide, and was mainly composed of 1,2-linked l-rhamnose residues with sulfate groups substituted at positions C-3 and/or C-4. The sulfated polysaccharide exhibited high anticoagulant activities by assays of the activated partial thromboplastin time (APTT) and thrombin time (TT). The anticoagulant property of the sulfated polysaccharide was mainly attributed to powerful potentiation thrombin by heparin cofactor II.

Antagonist of thromboxane A2 receptor by SQ29548 lowers DOCA-induced hypertension in diabetic rats.

Diabetes is one of high risk factors for cardiovascular diseases, including atherosclerosis and hypertension. This study was conducted to elucidate whether and how thromboxane receptor (TPr) activation contributes to hypertension in diabetes. Human umbilical vein endothelial cells (HUVECs) were cultured. The phosphorylated levels of endothelial nitric oxide synthase (eNOS) and Akt were monitored by western blot. Endothelial function was determined by organ bath. High glucose (HG) or thromboxane A2 mimetic U46619 significantly reduced the levels of p-eNOS and p-Akt in cultured HUVECs, which were reversed by inhibition of TPr. HG/U46619-induced reductions of p-eNOS and p-Akt were accompanied with increases of total and phosphorylated tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN). PTEN siRNA restored Akt-eNOS signaling in cells treated with HG. In rats, streptozotocin-induced hyperglycemia was associated with aortic PTEN upregulation and reductions of p-Akt and p-eNOS. TPr antagonist SQ29548 ablated these alterations and reduced blood pressure in rats with DOCA-induced hypertensive. In conclusion, hyperglycemia activates thromboxane A2 receptor to augment DOCA-induced high blood pressure in rats via the PTEN-Akt-eNOS signaling.

miR-218 inhibits acute promyelocytic leukemia cell growth by targeting BMI-1.

Acute promyelocytic leukemia (APL) is a subtype of acute myelocytic leukemia. Previous studies have reported a number of functions and therapeutic roles of microRNAs (miRs) in APL, and have suggested that miR-218 acts as a tumor suppressor in a number of types of human cancer; however, its role in APL remains unclear. In the present study, the expression of miR-218 and its effects on the viability and proliferation of HL-60 cells was investigated. Reverse transcription-quantitative polymerase chain reaction analysis demonstrated that miR-218 was frequently downregulated in APL marrow tissues compared with normal marrow tissues. Overexpression of miR-218 significantly inhibited cell proliferation, arrested the cell cycle in the G0/G1 phase and induced apoptosis. In addition, B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) mRNA expression was negatively associated with miR-218 expression; BMI-1 mRNA and protein expression were downregulated following transfection with miR-218 mimic. These results indicate that miR-218 functions as tumor suppressor in APL, and the miR-218/BMI-1 signaling axis may be a potential novel diagnostic marker and therapeutic target for the treatment of APL.

[Diagnostic value of citrulline and intestinal fatty acid binding protein on acute gastrointestinal injury in critical patients: a prospective study of 530 patients].

OBJECTIVE: To observe the incidence of acute gastrointestinal injury (AGI) in intensive care unit (ICU) patients, and to approach the value of serum citrulline and intestinal fatty acid binding protein (IFABP) on diagnosis of AGI in critical patients. METHODS: A prospective study was conducted. 576 critical patients admitted to ICU of Yantai Yuhuangding Hospital from February 2016 to February 2017 were enrolled. According to the AGI classification proposed by European Society of Intensive Care Medicine (ESICM) in 2012, the AGI and severity of the patients were observed. The general data, severity and prognosis of patients with different AGI grades were recorded. According to the random number table, 20 patients with normal kidney function from AGI I to IV were selected. The femoral artery blood was collected within 12 hours of ICU admission, and serum citrulline level was detected by high performance liquid chromatography (HPLC). Serum IFABP level was determined by enzyme-linked immunosorbent assay (ELISA). Twenty healthy subjects were selected as controls. The receiver operating characteristic curve (ROC) was drawn, and the predictive values of citrulline and IFABP for AGI diagnosis were evaluated. RESULTS: (1) 576 patients were enrolled in the analysis. Of which 530 patients (92.0%) had AGI, and 289 patients with grade I (54.5%), 154 with grade II (29.1%), 64 with grade III (12.1%), and 23 with grade IV (4.3%). With the increase in AGI classification, acute physiology and chronic health evaluation system II (APACHE II) score, sequential organ failure score (SOFA), the length of ICU stay and 28-day mortality were gradually increased. (2) Compared with health control group, the levels of serum citrulline in patients with different AGI grades were significantly decreased, and IFABP was significantly increased. With the increase in AGI classification, the citrulline level was gradually decreased, and IFABP level was gradually increased [citrulline levels (µmol/L) in AGI I,II,III,IV groups were 14.1±3.6, 12.7±3.1, 8.3±2.7, and 5.6±3.4, F = 3.287, P = 0.027, and IFABP levels (ng/L) were 526.7±204.9, 698.4±273.8, 894.7±455.9, and 1 062.8±532.2, F = 2.903, P = 0.043]. ROC curve analysis showed that citrulline had a higher predictive value for AGI diagnosis. The area under the ROC curve (AUC) was 0.927. When the cut-off value of citrulline was 9.7 µmol/L, the sensitivity and specificity were 87.5% and 87.5%, respectively. The AUC of IFABP was 0.043, which has no predictive value for the diagnosis of AGI. CONCLUSIONS: The AGI is extremely common in ICU. The higher the AGI grade is, the worse the prognosis is. Citrulline has high diagnostic value for AGI in critical patients, but IFABP has no predictive value on the diagnosis of AGI.

Polyacrylamide Gel Polymerization: Ascorbic Acid-ferrous Sulfate-ammonium Persulfate Initiator for Acid System.

A new initiator system for polyacrylamide gel polymerization at low pH is developed. The system consists of ascorbic acid, ferrous sulfate and ammonium persulfate (AP). It is effective under acid conditions even if the concentration of the initiators is very low. To get high incorporation efficiency of acrylamide, the ratio of the AP, ascorbic acid and ferrous sulfate must be in a suitable range. The conversion efficiency has been assessed as a function of the pH of the gelling solution, in pH2.0--6.58 range. New system is able to guarantee>98% conversion in the pH 2.0--5.0 range. In contrast to the hydrogen peroxide-ascorbic acid-ferrous sulfate system widely used under acid conditions, new system gives a much higher incorporation efficiency of acrylamide, lower gel pore size at the same concentration and cross linkage and better gel mechanical property that made the gel easy handle for general purpose. The recommended concentrations of the initiators are: AP, 2.2-3.3 mmol/L, ascorbic acid, 0.91-1.82mmol/L, FeSO(4), 0.046-0.092 mmol/L.

[Establishment of a sandwich ELISA for testing serum sB7-H3 and detection of sB7-H3 levels in liver disease].

AIM: To establish quantitative ELISA for soluble sB7-H3 and evaluate its clinical application. METHODS: Two mAbs of mouse anti-human sB7-H3(4H7 and 2E6)established by our lab were used. The mAb 4H7 was used as coating antibody and the 2E6 as a sandwich antibody, which recognized a different epitope and was labeled by biotin. The serum sB7-H3 levels of healthy volunteers and patients were measured by this method. RESULTS: Sandwich ELISA was established with the linear range covering from 8.192 ng/L to 2 000 ng/L. The outcomes of repeatability test, recovery test, stability test and specificity test were quite good. The increased levels of sB7-H3 were found in schistosomiasis cirrhosis, the reduced levels in severe hepatitis and the difference in both situations showed significance. CONCLUSION: A specific and sensitive sandwich ELISA is developed, the detection in liver disease indicates that the ELISA kit provides a potential tool in the clinical application.

Structural characterization of extracellular polysaccharides produced by the marine fungus Epicoccum nigrum JJY-40 and their antioxidant activities.

Two extracellular polysaccharides, ENP1 and ENP2, were isolated from the fermentation liquid of the marine fungus Epicoccum nigrum JJY-40 by anion-exchange chromatography and gel-filtration chromatography, and their structures were investigated using chemical and spectroscopic methods including methylation analysis and NMR spectroscopy. The results demonstrated that ENP1 was composed of mannose, glucose, and galactose in the molar ratio of 5.0:2.1:1.0, and the main chain of the polysaccharide consisted of (1 → 2)-linked mannose, (1 → 3)-linked mannose, terminal mannose, (1 → 6)-linked glucose, (1 → 4)-linked glucose, and (1 → 4)-linked galactose. ENP2 was composed of mannose, galactose, glucose, and glucuronic acid in a molar ratio of 12.4:11.2:8.3:1.0, and its glycosidic linkage patterns included terminal mannose, (1 → 6)-linked glucose, (1 → 4)-linked galactose, and (1 → 3)-linked mannose. The two polysaccharides had a partially branched structure with branch point located at C-3 position of (1 → 6)-linked glucose residue. The molecular weights of ENP1 and ENP2 were 19.2 kDa and 32.7 kDa, respectively. Antioxidant properties of the two polysaccharides were evaluated with hydroxyl, superoxide, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities and lipid peroxidation inhibition in vitro, and results showed that ENP2 and ENP1 had good antioxidant activities, especially ENP2. ENP2 could be effective as a potential antioxidant.

Solid-state NMR fermi contact and dipolar shifts in organometallic complexes and metalloporphyrins.

We have used density functional theory methods to investigate the solid-state "magic-angle" spinning (MAS) NMR and single-crystal NMR/ENDOR spectra of paramagnetic organometallic complexes and metalloporphyrins. The solid-state MAS NMR chemical shifts (including both diamagnetic and hyperfine contributions) are predicted with a slope of 1.007 and an R2 = 0.967, corresponding to a 28 ppm (or 6.3%) error over the entire 442 ppm range. Single-crystal ENDOR hyperfine values, including both isotropic Fermi contact and dipolar couplings, are predicted with a slope of 1.009 and an R2 = 0.998, corresponding to a 0.93 MHz (or 1.2%) error over the entire 78.37 MHz range. In addition, single-crystal NMR shifts (including both hyperfine terms) are predicted with an R2 = 0.961. The ability to compute solid-state MAS NMR and single-crystal NMR/ENDOR data should facilitate the use of these techniques in investigating paramagnetic metal complexes and should be of particular use in studying paramagnetic metalloproteins, where structures are less accurately known.

Solid-state NMR and quantum chemical investigations of 13Calpha shielding tensor magnitudes and orientations in peptides: determining phi and psi torsion angles.

We report the experimental determination of the (13)C(alpha) chemical shift tensors of Ala, Leu, Val, Phe, and Met in a number of polycrystalline peptides with known X-ray or de novo solid-state NMR structures. The 700 Hz dipolar coupling between (13)C(alpha) and its directly bonded (14)N permits extraction of both the magnitude and the orientation of the shielding tensor with respect to the C(alpha)-N bond vector. The chemical shift anisotropy (CSA) is recoupled under magic-angle spinning using the SUPER technique (Liu et al., J. Magn. Reson. 2002, 155, 15-28) to yield quasi-static chemical shift powder patterns. The tensor orientation is extracted from the (13)C-(14)N dipolar modulation of the powder line shapes. The magnitudes and orientations of the experimental (13)C(alpha) chemical shift tensors are found to be in good accord with those predicted from quantum chemical calculations. Using these principal values and orientations, supplemented with previously measured tensor orientations from (13)C-(15)N and (13)C-(1)H dipolar experiments, we are able to predict the (phi, psi, chi(1)) angles of Ala and Val within 5.8 degrees of the crystallographic values. This opens up a route to accurate determination of torsion angles in proteins based on shielding tensor magnitude and orientation information using labeled compounds, as well as the structure elucidation of noncrystalline organic compounds using natural abundance (13)C NMR techniques.

A solid state 13C NMR, crystallographic, and quantum chemical investigation of chemical shifts and hydrogen bonding in histidine dipeptides.

We report the first solid-state NMR, crystallographic, and quantum chemical investigation of the origins of the 13C NMR chemical shifts of the imidazole group in histidine-containing dipeptides. The chemical shift ranges for Cgamma and Cdelta2 seen in eight crystalline dipeptides were very large (12.7-13.8 ppm); the shifts were highly correlated (R2= 0.90) and were dominated by ring tautomer effects and intermolecular interactions. A similar correlation was found in proteins, but only for buried residues. The imidazole 13C NMR chemical shifts were predicted with an overall rms error of 1.6-1.9 ppm over a 26 ppm range, by using quantum chemical methods. Incorporation of hydrogen bond partner molecules was found to be essential in order to reproduce the chemical shifts seen experimentally. Using AIM (atoms in molecules) theory we found that essentially all interactions were of a closed shell nature and the hydrogen bond critical point properties were highly correlated with the N...H...O (average R2= 0.93) and Nepsilon2...H...N (average R2= 0.98) hydrogen bond lengths. For Cepsilon1, the 13C chemical shifts were also highly correlated with each of these properties (at the Nepsilon2 site), indicating the dominance of intermolecular interactions for Cepsilon1. These results open up the way to analyzing 13C NMR chemical shifts, tautomer states (from Cdelta2, Cepsilon1 shifts), and hydrogen bond properties (from Cepsilon1 shifts) of histidine residue in proteins and should be applicable to imidazole-containing drug molecules bound to proteins, as well.

A cyanopropyl reversed phase chromatography enables resolution of huperzine A and B on analytical and preparative scales.

Huperzine A (HupA) and huperzine B (HupB) are two medically important components of Huperzia serrata. It is difficult to obtain high yields of the separation from the plant using conventional liquid extraction and chromatography. However, this study has found that RP chromatography with cyanopropyl (CN) medium was able to separate these two analogs simultaneously from the plant extract with higher resolution. Comparison between a CN medium and a popular C18 medium demonstrated the superiority of the CN over the C18 in resolution for both analytical and preparative separation of HupA and HupB. A preparative process was developed for simultaneous purification of HupA and HupB from H. serrata. The yields on the basis of the mass of the herbal powder for HupA and HupB were 0.019 and 0.008% respectively, which were about 1.9 and 10 times of those reported in the literature.

Tryptophan chemical shift in peptides and proteins: a solid state carbon-13 nuclear magnetic resonance spectroscopic and quantum chemical investigation.

We have obtained the carbon-13 nuclear magnetic resonance spectra of a series of tryptophan-containing peptides and model systems, together with their X-ray crystallographic structures, and used quantum chemical methods to predict the (13)C NMR shifts (or shieldings) of all nonprotonated aromatic carbons (C(gamma), C(delta 2) and C(epsilon 2). Overall, there is generally good accord between theory and experiment. The chemical shifts of Trp C(gamma) in several proteins, hen egg white lysozyme, horse myoglobin, horse heart cytochrome c, and four carbonmonoxyhemoglobins, are also well predicted. The overall Trp C(gamma) shift range seen in the peptides and proteins is 11.4 ppm, and individual shifts (or shieldings) are predicted with an rms error of approximately 1.4 ppm (R value = 0.86). Unlike C(alpha) and N(H) chemical shifts, which are primarily a function of the backbone phi,psi torsion angles, the Trp C(gamma) shifts are shown to be correlated with the side-chain torsion angles chi(1) and chi(2) and appear to arise, at least in part, from gamma-gauche interactions with the backbone C' and N(H) atoms. This work helps solve the problem of the chemical shift nonequivalences of nonprotonated aromatic carbons in proteins first identified over 30 years ago and opens up the possibility of using aromatic carbon chemical shift information in structure determination.

Carbon-13 NMR shielding in the twenty common amino acids: comparisons with experimental results in proteins.

We have used ab initio quantum chemical techniques to compute the (13)C(alpha) and (13)C(beta) shielding surfaces for the 14 amino acids not previously investigated (R. H. Havlin et al., J. Am. Chem. Soc. 1997, 119, 11951-11958) in their most popular conformations. The spans (Omega = sigma(33) - sigma(11)) of all the tensors reported here are large ( approximately 34 ppm) and there are only very minor differences between helical and sheet residues. This is in contrast to the previous report in which Val, Ile and Thr were reported to have large ( approximately 12 ppm) differences in Omega between helical and sheet geometries. Apparently, only the beta-branched (beta-disubstituted) amino acids have such large CSA span (Omega) differences; however, there are uniformly large differences in the solution-NMR-determined CSA (Deltasigma = sigma(orth) - sigma(par)) between helices and sheets in all amino acids considered. This effect is overwhelmingly due to a change in shielding tensor orientation. With the aid of such shielding tensor orientation information, we computed Deltasigma values for all of the amino acids in calmodulin/M13 and ubiquitin. For ubiquitin, we find only a 2.7 ppm rmsd between theory and experiment for Deltasigma over an approximately 45 ppm range, a 0.96 slope, and an R(2) = 0.94 value when using an average solution NMR structure. We also report C(beta) shielding tensor results for these same amino acids, which reflect the small isotropic chemical shift differences seen experimentally, together with similar C(beta) shielding tensor magnitudes and orientations. In addition, we describe the results of calculations of C(alpha), C(beta), C(gamma)1, C(gamma)2, and C(delta) shifts in the two isoleucine residues in bovine pancreatic trypsin inhibitor and the four isoleucines in a cytochrome c and demonstrate that the side chain chemical shifts are strongly influenced by chi(2) torsion angle effects. There is very good agreement between theory and experiment using either X-ray or average solution NMR structures. Overall, these results show that both C(alpha) backbone chemical shift anisotropy results as well as backbone and side chain (13)C isotropic shifts can now be predicted with good accuracy by using quantum chemical methods, which should facilitate solution structure determination/refinement using such shielding tensor surface information.