First report of Fusarium proliferatum causing Root and Bulb Rot on tiller onion (Allium cepa L. var. Agrogatum Don) in China.

Tiller onion is a biennial herb and a fascicular variety of onion. Tiller onion has strong tillering ability and can produce up to ten bulbs per plant. It is widely cultivated due to nutrition and special flavor. In July 2020, we observed a disease that seriously affected the normal growth of tiller onion in Halahai Town, Nongan County, Jilin Province, China. At least 70% of tiller onions in the field were affected by this disease. Aboveground parts of the symptomatic plants showed stunted growth, wilting and drying. Underground parts of infected plants were shown that onion increase tiller number but did not grow and expand. Root appeared red lesions and rot in severe cases. The bulb disc appeared brown to dark brown rot. Symptomatic roots were cut into 0.5 cm pieces and surface-sterilized by dipping in 75% ethanol for 60 s, 3% NaOCl for 3 min, and rinsing three times with sterile distilled water. Pieces were placed on potato dextrose agar (PDA) plates and incubated at 25±1℃ for 4 days. Fifteen isolates were obtained and pure-cultured through single-sporing. On PDA plates, the colonies initially had white aerial mycelia that then turned pale purple. The color of the colonies on the back of the plates was purple. Macroconidia were hyaline, falcate and 14.4 to 38.7 × 1.2 to 3.0 µm. Microconidia were hyaline, reniform or elliptic, unicellular or bicellular and were 7.62 to 19.61 µm in length, and 3.23 to 8.41 µm in width. Based on these morphological and culture characteristics, the causal agent was tentatively identified as F. proliferatum. To confirm the pathogen identity, segments of the internal transcribed spacer region of the rRNA gene ( ITS, primers ITS4 and ITS5, White et al., 1990), ß-tubulin gene (TUB2, primers T1 and T2, O'Donnell and Cigelnik, 1997), and translation elongation factor 1-alpha gene (TEF-1α, primers EF1 and EF2 from O'Donnell et al., 1998) were amplified by PCR. Per the BLASTN search, TEF-1α (Accession No. OL355013), TUB2 (Accession No. OL355012), and ITS (Accession No. OL355011) queries showed 99.26%, 100%, and 99.82% homology to F. proliferatum GenBank accessions KU872098, MH398224, and MH997878, respectively. Pathogenicity of fifteen isolates of F. proliferatum from tiller onion was confirmed by inoculating healthy tiller onion roots and bulb disc with a spore suspension (1 × 106 spores/ml) produced on PDA. For each treatment, five plants were injected with 5 ml of spore suspension. Control plants (n=5) were injected with sterilized water. All plants were enclosed in plastic bags for 48 h in a greenhouse at 28℃ and 12 h/d light cycle. After 10 days, inoculated plants showed similar symptoms to those on the original diseased plants, while control plants remained symptomless. F. proliferatum was successfully re-isolated from symptomatic plants to fulfill Koch's postulates. Diseases caused by F. proliferatum are only reported in A. cepa. To our knowledge, this is the first report of F. proliferatum in Allium cepa L. var. agrogatum Don in China. Our findings are important for informed surveillance of the disease in China as F. proliferatum infection can not only reduce the quality and yield of tiller onion but also can contaminate the bulbs with harmful mycotoxins.

[Application of long term video electroencephalogram and resting-state functional magnetic resonance imaging in detection of cognition in patients with benign epilepsy of childhood with centrotemporal spikes].

Objective: To study the relationship between the changes of brain network and cognition in patients with benign epilepsy of childhood with centrotemporal spikes (BECTS) by using long term video electroencephalogram (VEEG) and resting-state functional magnetic resonance imaging (RS-fMRI) technology. Methods: Eleven patients with right-handed were recruited (from April 2015 to September 2016) from epilepsy specialist outpatients and functional department of neurosurgery of Tianjin Medical University General Hospital. They all underwent the long term VEEG monitoring (one sleep cycle was included at least). According to the spike-wave index (SWI) during slow ware sleep, they were divided into two groups: SWI<50% (5 cases) and SWI≥50% (6 cases). All the patients were assessed with cognitional test including language, execution, memory and attention. They also underwent the head MRI, RS-fMRI examinations. Then the results were comparatively analysed. Results: (1)There were no statisticaly significance in sex, age, age of onset, disease course, total number of seizures, years of education (P>0.05). The Full Intelligence Quotient (FIQ) (87±18), Verbal Intelligence Quotient (VIQ) (88±15) and Performance Intelligence Quotient (PIQ) (89±20) of SWI≥50% group were lower than SWI<50% group(118±8, 114±11, 119±5) and the differences were statistically significant(P<0.05). (2)There was a negative correlation between the FIQ (P=0.002), VIQ (P=0.006), PIQ (P=0.001) and SWI. The FIQ, VIQ and PIQ had no correlation with the sex, age, age of onset, disease course, total number of seizures, years of education (P>0.05). (3)Compared with SWI<50% group, SWI≥50% group showed increased regional homogeneity (ReHo) in the bilateral precentral gyrus, premotor area and the subcortical structure, the right temporal lobe and the bilateral insular lobe(P<0.05); while they showed decreased ReHo in the posterior cingulate gyrus, right posterior inferior temporal lobe and right occipital lobe(P<0.05). Conclusion: The change of the brain network which is caused by the paradoxical and constant discharge during slow ware sleep in patients with BECTS may affect the development of cognition.

Dysregulation of TFDP1 and of the cell cycle pathway in high-grade glioblastoma multiforme: a bioinformatic analysis.

Despite extensive research, the prognosis of high-grade glioblastoma multiforme (GBM) has improved only slightly because of the limited response to standard treatments. Recent advances (discoveries of molecular biomarkers) provide new opportunities for the treatment of GBM. The aim of the present study was to identify diagnostic biomarkers of high-grade GBM. First, we combined 3 microarray expression datasets to screen them for genes differentially expressed in patients with high-grade GBM relative to healthy subjects. Next, the target network was constructed via the empirical Bayesian coexpression approach, and centrality analysis and a molecular complex detection (MCODE) algorithm were performed to explore hub genes and functional modules. Finally, a validation test was conducted to verify the bioinformatic results. A total of 277 differentially expressed genes were identified according to the criteria P < 0.05 and |log2(fold change)| ≥ 1.5. These genes were most significantly enriched in the cell cycle pathway. Centrality analysis uncovered 9 hub genes; among them, TFDP1 showed the highest degree of connectivity (43) and is a known participant in the cell cycle pathway; this finding pointed to the important role of TFDP1 in the progression of high-grade GBM. Experimental validation mostly supported the bioinformatic results. According to our study results, the gene TFDP1 and the cell cycle pathway are strongly associated with high-grade GBM; this result may provide new insights into the pathogenesis of GBM.

[Resting-state functional magnetic resonance study of the brain's network of the temporal lobe epilepsy patients with depression].

OBJECTIVE: To study brain networks of temporal lobe epilepsy (TLE) to investigate whether TLE brain dysfunction have an impact on depression, using resting state functional magnetic resonance (RS-fMRI) detection technology. METHODS: A total of 18 patients with TLE were included in this study. According to Beck Depression Inventory (BDI) and Hamilton's Depression Scale (HAMD)-17 score, we divided them into two groups: depression group 9 cases, non-depression group 9 cases. All patients underwent 3.0T MRI , RS-fMRI and magnetic resonance spectroscopy (MRS) examinations and then the results were analyzed. RESULTS: Disease course of depression group was longer than non-depression group and the difference was statistically significant (P<0.05). RS-fMRI examination showed that depression group had more active brain areas and more extral temporal active areas than non-depression group (P<0.05). By compared with the non-depression group, we found more strong active brain areas including thalamus, and the default-mode network which involved in prefrontal cortex, precuneus, ventral anterior cingulate and hippocampus. We found the NAA and NAA/Cho+ Cr of the hippocampus which were ipsilateral to the advantage discharge side were decreased in 5/9 cases with depression in MRS and 3/5 cases had hippocampal atrophy, while the non-depression group had no obvious abnormalities. CONCLUSION: The brain default-mode network activity in TLE patients with depression is increased and there is more extral temporal activation than the non-depression group; furthermore abnormal hippocampus structure is more common in depression group, which suggests that epileptic brain dysfunction may affect the development of depression.

[Stereotactic brain biopsy guided by iMRI co-registration combined with PET/CT].

OBJECTIVE: To explore the clinical value of intraoperative magnetic resonance imaging (iMRI) coregistration combined with position emission tomography/computed tomography (PET/CT) in stereotactic brain biopsy. METHODS: Forty nine patients with intracranial lesions were operated by stereotactic biopsy from June 2010 to June 2015 in Tianjin Medical University General Hospital. Seventeen patient's operation was guided by iMRI only (group A), thirty two patients' operation was guided by iMRI and PET/CT (group B). The diagnosis success rate and operation related complications were compared between the two groups. RESULTS: PET/CT and iMRI were integrated successfully in all cases of group B. Fourteen patients (82.4%) of group A and all 32 patients (100%) of group B had final diagnosis confirmed by histopathological and immunohistochemical observation. The diagnosis success rate of group B was higher than group A (P<0.05). There were 5 patients in total who had postoperative complication, 2 (11.8%) in group A and 3 (9.3%) in group B, but the difference was not statistically significant. CONCLUSIONS: PET/CT based metabolic imaging can be automatically integrated with standard MRI guided stereotactic biopsy. Compared with iMRI only, the combined treatment improves diagnosis success rate without increasing complications; it's safe, and has high clinical efficacy.

A new inducible expression system in a transformed green alga, Chlorella vulgaris.

Genetic transformation is useful for basic research and applied biotechnology. However, genetic transformation of microalgae is usually quite difficult due to the technical limitations of existing methods. We cloned the promoter and terminator of the nitrate reductase gene from the microalga Phaeodactylum tricornutum and used them for optimization of a transformation system of the microalga Chlorella vulgaris. This species has been used for food production and is a promising candidate as a bioreactor for large-scale production of value-added proteins. A construct was made containing the CAT (chloramphenicol acetyltransferase) reporter gene driven by the nitrate reductase promoter. This construct was transferred into the C. vulgaris genome by electroporation. Expression of CAT in transgenic Chlorella conferred resistance to the antibiotic chloramphenicol and enabled growth in selective media. Overall efficiency for the transformation was estimated to be approximately 0.03%, which is relatively high compared with other available Chlorella transformation systems. Expression of CAT was induced in the presence of nitrate and inhibited in the presence of ammonium as a sole nitrogen source. This study presented an inducible recombinant gene expression system, also providing more gene regulation elements with potential for biotechnological applications.

Perlecan domain I promotes fibroblast growth factor 2 delivery in collagen I fibril scaffolds.

Perlecan, a heparan sulfate proteoglycan, is widely distributed in developing and adult tissues and plays multiple, important physiological roles. Studies with knockout mouse models indicate that expression of perlecan and heparan sulfate is critical for proper skeletal morphogenesis. Heparan sulfate chains bind and potentiate the activities of various growth factors such as fibroblast growth factor 2 (FGF-2). Previous studies indicate that important biological activities are associated with the heparan sulfate-bearing domain I of perlecan (PlnDI; French et al. J. Bone Miner. Res. 17 , 48, 2002). In the present study, we have used recombinant, glycosaminoglycan-bearing PlnDI to reconstitute three-dimensional scaffolds of collagen I. Collagen I fibrils bound PlnDI much better than native collagen I monomers or heat-denatured collagen I preparations. Heparitinase digestion demonstrated that recombinant PlnDI was substituted with heparan sulfate and that these heparan sulfate chains were critically important not only for efficient integration of PlnDI into scaffolds, but also for FGF-2 binding and retention. PlnDI-containing collagen I scaffolds to which FGF-2 was bound sustained growth of both MG63, an osteoblastic cell line, and human bone marrow stromal cells (hBMSCs) significantly better than scaffolds lacking either PlnDI or FGF-2. Collectively, these studies demonstrate the utility of PlnDI in creating scaffolds that better mimic natural extracellular matrices and better support key biological activities.

Effects of N-methyl hexanoylhydroxamic acid (NMHH) and myoglobin on endothelial damage by hydrogen peroxide.

OBJECTIVE: The aim was to investigate the interaction of the novel antioxidant N-methyl hexanoylhydroxamic acid (NMHH) with myoglobin in protecting endothelial cells against H2O2 mediated damage. METHODS: Cultured bovine aortic endothelial cells were exposed to 50-100 microM H2O2 for 10-60 min with and without NMHH and/or myoglobin, and immediate or delayed damage was assessed by lactate dehydrogenase release, 3H adenine uptake, a tetrazolium reduction assay, and microscopy. RESULTS: Brief exposure to low concentrations of H2O2 caused cell damage, for which the tetrazolium reduction assay was the most sensitive assay, and inhibited subsequent cell division. NMHH in concentrations from 50 to 200 microM protected against damage provided it was present at the time of adding H2O2, and the effect was markedly potentiated by 10 microM oxymyoglobin, which had little protective effect alone. CONCLUSIONS: NMHH is an effective antioxidant which is markedly potentiated by low concentrations of oxymyoglobin. Oxymyoglobin may potentiate NMHH by scavenging H2O2 through the rapid formation of ferrylmyoglobin, which is then reduced by NMHH. This synergism may be particularly relevant to the protection of myoglobin-rich cells such as myocytes.

Exposure to low concentrations of hydrogen peroxide causes delayed endothelial cell death and inhibits proliferation of surviving cells.

Cultured bovine aortic endothelial cells were briefly exposed to low concentrations of hydrogen peroxide (0.005 to 0.05 mmol/l). At these concentrations cells do not suffer immediate lysis, but a high proportion underwent delayed cell death over the next 24 h, with changes in nuclear morphology, the appearance of nucleosomal fragments in extracted nuclear DNA, and the appearance of numerous DNA strand breaks demonstrated by 3'OH in situ end-labelling compatible with apoptotic cell death. At the same time, there was marked inhibition of [3H]thymidine uptake, and inhibition of the incorporation of the thymidine analogue 5-bromo-2'deoxy-uridine into nuclear DNA. Cells which survived apoptosis showed inhibition of cell division on subsequent culture for up to 15 days, and there were striking morphological changes, with the formation of uninucleate or multinucleate giant cells. These effects may be relevant to the mechanisms by which brief exposure to oxidative stress causes progressive vessel wall damage.

The role of nitric oxide in the cardiovascular system.

Nitric oxide (NO) exerts various pathophysiological effects on the cardiovascular system; inhibition of platelet aggregation or leukocyte adhesion on endothelium and vasorelaxation including lethal hypotension in endotoxic shock. In spite of these significant roles of NO, its direct action on individual cardiovascular cells remains unclarified. Therefore, we have investigated the function of NO on cells which constitute vascular wall and heart, and have found this new evidence. 1) ATP increased intracellular ([Ca2+]i) in vascular endothelial cells (ECs) and decreased [Ca2+]i of adjacently cocultured vascular smooth muscle cells (VSMCs), as detected by 2-D fura-2 image analysis. 2) The [Ca2+]i reduction in cocultured VSMCs with ECs by ATP was attenuated by pretreatment of several types of NO inhibitor, whereas the NO inhibitor potentiated the [Ca2+]i elevation in ECs, suggesting that NO affects VSMCs in a paracrine manner while ECs in an autocrine fashion. 3) Physiological concentration of lysophosphatidylcholine, which is an atherogenic constituent of oxidized LDL, but not native phosphatidylcholine, acted on ECs and VSMCs like a NO inhibitor, indicating that this material attenuates NO effect and disturbs vessel relaxation in the short term. 4) Highly efficient transfection of the ecNOS gene in rat heart showed a toxic effect on individual cardiomyocytes in vivo. In conclusion, NO may exert both beneficial and harmful effects on the cardiovascular system.

The effect of myoglobin on the stability of the hydroxyl-radical adducts of 5,5 dimethyl-1-pyrolline-N-oxide (DMPO), 3,3,5,5 tetramethyl-1-pyrolline-N-oxide (TMPO) and 1-alpha-phenyl-tert-butyl nitrone (PBN) in the presence of hydrogen peroxide.

The hydroxyl radical adducts of 5,5 dimethyl-1-pyrolline-N-oxide (DMPO) and 3,3,5,5 tetramethyl-1-pyrolline-N-oxide (TMPO) formed in the presence of hydrogen peroxide and FeII are normally quite stable, but in the presence of 5-20 micromolar myoglobin their ESR signals decay rapidly. This decay probably reflects further oxidation of the adduct to nonparamagnetic products. The ESR signal of the hydroxyl radical adduct of 1-alpha-phenyl-tert-butyl nitrone (PBN) formed under similar conditions is subject to non-heme dependent attenuation, possibly via hydroxyl radical scavenging, but not to heme dependent decay. Hydrogen peroxide readily converts myoglobin to its ferryl (FeIV) derivative, and this centre may be responsible for the oxidation of the DMPO and TMPO adducts. The different behaviour of PBN may be due to differences in susceptibility to ferrylmyoglobin mediated oxidation, or to steric differences controlling access to the heme pocket of myoglobin, and is relevant to the choice of spin trap for biological experiments aimed at detecting hydroxyl radicals in the presence of myoglobin or other heme proteins.

Differentiation between fresh beef and thawed frozen beef.

In order to prevent meat retailers offering thawed, imported frozen beef as fresh domestic beef, the method of Gottesmann and Hamm for differentiating between fresh and frozen/thawed meat is recommended. The principle of this method is based on the fact that freezing and thawing of meat results in a release of the enzyme ß-hydroxyacyl CoA-dehydrogenase (HADH) from the mitochondrion into the sarcoplasm; an elevated HADH activity in the muscle press-juice indicates freezing and thawing of tissue. The HADH colour test of Gottesmann and Hamm was modified by replacing the electron-transmitter meldolablue by resazurin which results in a much higher colour stability after reaction with fresh meat extracts.

[Spectroscopic study on the high voltage fast pulsed discharge of nitrogen, ammonia or their mixture].

The emission spectra from the pulsed discharge plasma of nitrogen, ammonia or their mixture were measured. In the discharge of pure nitrogen gas, as the pressure increased, the discharge volume decreased and more dissociation of nitrogen molecules occurred due to the higher energy density. In the discharge of ammonia, N,N+ and NH+ were observed, but no NH2 and NH3 were detected, indicating that ammonia, which has the lower dissociation and ionization energies as compared to nitrogen, was highly dissociated. The discharge of the mixture of N2 and NH3 was also studied. The dependence of the dissociation of nitrogen on the ratio of nitrogen to ammonia was investigated by emission spectra. The optimal ratio for nitrogen dissociation was obtained. The advantage of using the mixture of nitrogen and ammonia in the synthesis of nitrides was discussed.

[Direct detection of oxygen free radicals produced by myocardial reperfusion using electron spin resonance spectroscopy].

There is a growing evidence for the role of oxygen free radicals (OFR) in mediating myocardial tissue injury during myocardial ischemia and particularly during reperfusion. But almost all of the evidence was indirect, using electron spin resonance (ESR) spectroscopy, we have directly measured OFR generated in ischemic and reperfused isolated rabbit hearts. 17 hearts were rapidly frozen in liquid nitrogen after their arrest by cardioplegic solution and sampled after 150 min of sustained hypothermic global ischemia or after reperfusion. The ESR spectra obtained from experiment have directly demonstrated that OFR is produced in significant amounts in the isolated rabbit hearts during early stage of reperfusion but only small amount during ischemia. The mitochondrial electron transport chain appeared to be the main source of OFR. We found that superoxide dismutase scavenged OFR generated during reperfusion efficiently, but catalase did not. We believe that superoxide anion, not hydroxyl radical, is the main OFR which is responsible for myocardial reperfusion injury. We also found that Salvia, a traditional Chinese medicine, a very efficient OFR scavenger, had the similar effect as superoxide dismutase.

The effects of myoglobin and apomyoglobin on the formation and stability of the hydroxyl radical adduct of 5,5'-dimethyl-1-pyrroline-N-oxide.

When aqueous solutions of the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) are treated with hydrogen peroxide in the presence of either FeII or light, the hydroxyl radical adduct DMPO-OH is formed, with a characteristic 4 line ESR spectrum. When oxy- or metmyoglobin is added to such a system the initial yield and the halife of DMPO-OH are reduced, and at high myoglobin concentrations (about 0.1 mmol dm-3) DMPO-OH becomes undetectable. Using the stable nitroxide 2,2,6,6-tetramethyl-1-piperidinyloxy-N-oxyl (TMPO) for comparison it was found that neither hydrogen peroxide nor myoglobin alone caused a loss of signal, but together a marked loss of signal was induced. From the evidence of these and other experiments it was concluded that the DMPO-OH adduct reacts with hydrogen peroxide and myoglobin to give non-paramagnetic products, and hence that the use of the DMPO spin trap to detect hydroxyl or other active radicals in systems containing physiological concentrations of myoglobin may give misleading results.

[Fusion and expression of the gene encoding human Mn-SOD to anti-CEA single-chain antibody in Escherichia coli].

The gene encoding human manganese-superoxide dismutase (Mn-SOD) was fused to anti-carcinoembryonic antigen single-chain antibody gene to construct the fusion gene, then was ligated into prokaryotic expression vector pET-22b(+), The fusion gene was expressed in E. coli at high level, accounting for 24% of the total bacteria soluble protein; and was characterized by SDS-PAGE and Western-blot analysis; the expression product had the CEA-binding ability in RIA, and also had the SOD activity by pyrogallol autoxidation assay. So, the Mn-SOD moiety retains substantial enzymatic activity, where the ScFv moiety can deliver the fusion protein to tumor, Mn-SOD is a potential tumor-suppressor gene, maybe the fusion protein can provide a new pathway to tumor therapy.

A study of injectable tissue-engineered autologous cartilage.

OBJECTIVE: To test the effectiveness of the new techniques of tissue-engineered cartilage. METHODS: Chondrocytes were harvested through type II collagenase digestion from the auricle of New Zealand rabbits. The cells were mixed with alginate to generate chondrocytes/alginate composites with final cellular density of 50 x 10(6) per mL. Calcium chloride was used as the cross-linking agent to gel the aqueous alginate solution. The chondrocytes/alginate composites were injected into the dorsal subcutaneous tissue of New Zealand rabbits through autologous cells grafts. The specimens were observed during cartilage formation at 4, 8, and 12 weeks after injection. RESULTS: Prior to harvesting, chondrocytes/alginate composites were easily visualized under the dorsal skin of animals. The appearance of experimental specimens was similar to that of native cartilage in gross morphology. Using a standard hematoxylin and eosin stain, the histologic features of all experimental specimens demonstrated new cartilage formation. With a Masson's trichrome and safranin O stain, the presence of collagen and glycosaminoglycan (GAG) was observed at 8 and 12 weeks. CONCLUSION: This study demonstrated that polymerization of alginate hydrogel can be controlled to allow injection of chondrocytes that produce new autologous cartilage at subcutaneous dorsal site of rabbits. Injectable tissue-engineered autologous cartilage is promising for potential use in oral and maxillofacial surgery.

Autocrine and paracrine effects of endothelium-derived relaxing factor on intracellular Ca2+ of endothelial cells and vascular smooth muscle cells. Identification by two-dimensional image analysis in coculture.

To elucidate the effects of endothelium-derived relaxing factor (EDRF) released from vascular endothelial cells (ECs) on handling of intracellular calcium ion (Ca2+i) in ECs themselves and vascular smooth muscle cells (VSMCs), we measured the Ca2+i by two-dimensional digital image analysis of fura-2-loaded ECs and VSMCs in tissue culture. In isoculture of one cell type, adenosine triphosphate (ATP, 1 microM) transiently increased the Ca2+i of both ECs and VSMCs. High-K+ depolarization or angiotensin II also elevated the Ca2+i of VSMCs, whereas neither stimulants changed the Ca2+i of ECs. In coculture of ECs with VSMCs, the same dose of ATP rapidly increased the Ca2+i of ECs and then transiently decreased the Ca2+i of VSMCs to below the resting level. The maximal Ca2+i-modulating effects of ATP on both cell types were reproducible after the second application of ATP. Three kinds of EDRF blockers (L-NG-monomethylarginine, methemoglobin, or methylene blue) potentiated the ATP-induced Ca2+i rise in ECs and attenuated the Ca2+i reduction in VSMCs, suggesting the autocrine and paracrine effects of EDRF on ECs and VSMCs, respectively. However, neither indomethacin, superoxide dismutase, nor neutralizing monoclonal antibody to endothelin-1 altered the second responses. Thus, two-dimensional Ca2+i image analysis of ECs and VSMCs in coculture enabled direct visualization of the EDRF actions in ECs and VSMCs and their modifications.