PCR-RFLP for Detection of Fusarium graminearum Genotypes with Resistance to Phenamacril.

Phenamacril is a cyanoacrylate fungicide that provides excellent control of Fusarium head blight (FHB) or wheat scab, which is caused predominantly by Fusarium graminearum and F. asiaticum. Previous studies revealed that codon mutations of the myosin-5 gene of Fusarium spp. conferred resistance to phenamacril in in vitro lab experiments. In this study, PCR restriction fragment length polymorphism (RFLP) was developed to detect three common mutations (A135T, GCC to ACC at codon 135; S217L, TCA to TTA at codon 217; and E420K, GAA to AAA at codon 420) in F. graminearum induced by fungicide domestication in vitro. PCR products of 841 bp (for mutation of A135T), 802 bp (for mutation of S217L), or 1,649 bp (for mutation of E420K) in the myosin-5 gene were amplified by appropriate primer pairs. Restriction enzyme KpnI, TasI, or DraI was used to distinguish phenamacril-sensitive and -resistant strains with mutation genotypes of A135T, S217L, and E420K, respectively. KpnI digested the 841-bp PCR products of phenamacril-resistant strains with codon mutation A135T into two fragments of 256 and 585 bp. In contrast, KpnI did not digest the PCR products of sensitive strains. TasI digested the 802-bp PCR products of phenamacril-resistant strains with codon mutation S217L into three fragments of 461, 287, and 54 bp. In contrast, TasI digestion of the 802-bp PCR products of phenamacril-sensitive strains resulted in only two fragments of 515 and 287 bp. DraI digested the 1,649-bp PCR products of phenamacril-resistant strains with codon mutation E420K into two fragments of 932 and 717 bp, while the PCR products of phenamacril-sensitive strains was not digested. The three genotypes of resistance mutations were determined by analyzing electrophoresis patterns of the digestion fragments of PCR products. The PCR-RFLP method was evaluated on 48 phenamacril-resistant strains induced by fungicide domestication in vitro and compared with the conventional method (mycelial growth on fungicide-amended agar). The accuracy of the PCR-RFLP method for detecting the three mutation genotypes of F. graminearum resistant to phenamacril was 95.12% compared with conventional method. Bioinformatics analysis revealed that the PCR-RFLP method could also be used to detect the codon mutations of A135T and E420K in F. asiaticum.

Monocyte mediated brain targeting delivery of macromolecular drug for the therapy of depression.

Leukocytes can cross intact blood-brain barrier under healthy conditions and in many neurological diseases, including psychiatric diseases. In present study, a cyclic RGD (cRGD) peptide with high affinity for integrin receptors of leukocytes was used to modify liposomes. The cRGD-modified liposomes (cRGDL) showed high affinity for monocytes in vitro and in vivo and co-migrated across in vitro BBB model with THP-1. The trefoil factor 3 (TFF3), a macromolecular drug, was rapidly and persistently delivered to brain for at least 12 h when loaded into cRGDL while 2.8-fold increase in drug concentration in basolateral amygdala regions related to depression was observed. A systemic administration of cRGDL-TFF3 mimicked antidepressant-like effect of direct intra-basolateral amygdala administration of TFF3 solution in rats subjected to chronic mild stress. The effective dual-brain targeting delivery resulting from the combination and co-migration of cRGDL with leukocyte cross BBB may be a promising strategy for targeted brain delivery. FROM THE CLINICAL EDITOR: In an effort to treat depression, brain targeted delivery via monocyte-cRGD liposome complexes capable of crossing the intact BBB was performed in this study in a murine model. Similar approaches may be helpful in the treatment of other neuropsychiatric conditions.

Resistance mechanism of Fusarium fujikuroi to phenamacril in the field.

BACKGROUND: Rice bakanae disease, mainly caused by Fusarium fujikuroi, is an important disease of rice. Phenamacril has been used to control the disease for a few years in China. In 2016, nine phenamacril-resistant strains were found in the field in Zhejiang Province. The aim of the study was to clarify the mechanism of resistance of F. fujikuroi to phenamacril and the fitness of resistant strains. RESULTS: The nine F. fujikuroi strains examined were highly resistant to phenamacril. Eight of them had the point mutation TCA (Ser) → CCA (Pro) at codon 219 in the Myosin-5 protein, while the other had the point mutation TCA (Ser) → TTA (Leu) at codon 219. Myosin-5 replacement between resistant and sensitive strains confirmed that the point mutation in Myosin-5 caused the resistance of F. fujikuroi to phenamacril. Docking of phenamacril into the modeled binding pocket of Myosin-5 showed that the affinity between phenamacril and Myosin-5 decreased and a hydrogen bond could not be formed between phenamacril and the amino acid at codon 219 after it changed to Pro or Leu. There was no cross-resistance between phenamacril and other fungicides. The eight resistant strains containing the point mutation S219P had almost the same fitness as the sensitive strains, while the one resistant strain containing the point mutation S219 L showed decreased mycelial growth, sporulation and pathogenicity. CONCLUSION: In the field, the point mutation S219P or S219 L in Myosin-5 conferred high resistance to phenamacril in F. fujikuroi. The point mutation S219P did not affect the fitness of F. fujikuroi, while the point mutation S219 L decreased its fitness. © 2017 Society of Chemical Industry.

Direct Macromolecular Drug Delivery to Cerebral Ischemia Area using Neutrophil-Mediated Nanoparticles.

Delivery of macromolecular drugs to the brain is impeded by the blood brain barrier. The recruitment of leukocytes to lesions in the brain, a typical feature of neuroinflammation response which occurs in cerebral ischemia, offers a unique opportunity to deliver drugs to inflammation sites in the brain. In the present study, cross-linked dendrigraft poly-L-lysine (DGL) nanoparticles containing cis-aconitic anhydride-modified catalase and modified with PGP, an endogenous tripeptide that acts as a ligand with high affinity to neutrophils, were developed to form the cl PGP-PEG-DGL/CAT-Aco system. Significant binding efficiency to neutrophils, efficient protection of catalase enzymatic activity from degradation and effective transport to receiver cells were revealed in the delivery system. Delivery of catalase to ischemic subregions and cerebral neurocytes in MCAO mice was significantly enhanced, which obviously reducing infarct volume in MCAO mice. Thus, the therapeutic outcome of cerebral ischemia was greatly improved. The underlying mechanism was found to be related to the inhibition of ROS-mediated apoptosis. Considering that neuroinflammation occurs in many neurological disorders, the strategy developed here is not only promising for treatment of cerebral ischemia but also an effective approach for various CNS diseases related to inflammation.

Enhanced brain targeting by synthesis of 3',5'-dioctanoyl-5-fluoro-2'-deoxyuridine and incorporation into solid lipid nanoparticles.

To overcome the limited access of the drug 5-fluoro-2'-deoxyuridine (FUdR) to the brain, 3',5'-dioctanoyl-5-fluoro-2'-deoxyuridine (DO-FUdR) was synthesized and incorporated into solid lipid nanoparticles (DO-FUdR-SLN). DO-FUdR-SLN were prepared by a thin-layer ultrasonication technique and a central composite design (CCD) was applied to optimize the formulation. The median particle size of DO-FUdR-SLN was 76 nm with drug loading of 29.02% and entrapment efficiency of 96.62%. The in vitro drug release was studied by a bulk-equilibrium reverse dialysis bag technique in phosphate-buffered saline (pH 7.4) containing 0.3% pancreatic enzyme at 37 degrees C. The concentrations of FUdR in various organs were determined by reversed-phase high-performance liquid chromatography after intravenous administration of DO-FUdR-SLN, DO-FUdR or FUdR. The brain area under the concentration-time curve of DO-FUdR-SLN and DO-FUdR were 10.97- and 5.32-fold higher than that of FUdR, respectively. These results indicated that DO-FUdR-SLN had a good brain targeting efficiency in vivo. SLN can improve the ability of the drug to penetrate through the blood-brain barrier and is a promising drug targeting system for the treatment of central nervous system disorders.

cRGD mediated liposomes enhanced antidepressant-like effects of edaravone in rats.

The delayed onset of therapeutic outcomes is a major drawback of the current antidepressants. The blood-brain barrier is the most important bottleneck impeding drug transport into the brain. Therefore, development of novel antidepressant medications with rapid onset and sustained activity is urgent. RGD liposomes showed an excellent effect of brain-targeting drug delivery and increased the entering rate to the brain. In the present study, we prepared cyclic RGD liposomes loaded with edaravone (cRGD-ERLs) and evaluated the potential antidepressant-like effects of this drug delivery system in rats. The results showed single injection of cRGD-ERLs produced significant antidepressant-like effects in both forced swim and novelty suppressed feeding test. Moreover, acute cRGD-ERLs increased the expression of c-fos in the medial prefrontal cortex, suggesting that cRGD-ERLs could activate the neuronal function. Furthermore, cRGD-ERLs reversed the increase of lipopolysaccharides (LPS)-induced plasma cytokine IL-1ß and IL-6, suggesting that normalization of cytokine level might be involved in the behavioral response of cRGD-ERLs. Finally, cRGD-ERLs prevented the increase of immobility induced by LPS in the forced swim test. Overall, the current data revealed a novel brain-target drug delivery system, which can be used to improve the therapeutic outcomes of antidepressants by increase of crossing rate to the brain.