Effective antimicrobial activity of Cbf‐K16 and Cbf‐A7A13 against NDM‐1‐carrying Escherichia coli by DNA binding after penetrating the cytoplasmic membrane in vitro

New Delhi metallo‐beta‐lactamase‐1(NDM‐1)‐carrying isolates, which are resistant to most clinical used antibiotics except for tigecycline and colistin, have been found worldwide. Cathelicidin‐BF (BF‐30) is found in the venom of the snake Bungarus fasciatus and exhibits broad antimicrobial activity. Cbf‐K₁₆ and Cbf‐A₇A₁₃ were obtained by mutating Lys¹⁶, Ala⁷, and Ala¹³ of BF‐30, respectively. To investigate their antimicrobial activities against NDM‐1 carrying bacteria, recombinant Escherichia coli BL21 (DE3)‐NDM‐1 with high NDM‐1 activity was constructed by inserting the Klebsiella pneumoniae NDM‐1 gene (GenBank accession no. HQ328085) into a pET28a vector and transforming it into E. coli BL21 (DE3). The peptides showed effective antimicrobial activities against NDM‐1‐carrying E. coli, and the minimum inhibitory concentrations of Cbf‐K₁₆ and Cbf‐A₇A₁₃ were only 4 and 8 µg/ml, whereas those of minimum bactericidal concentrations were 8 and 16 µg/ml, respectively. A time course experiment showed that colony forming unit counts rapidly decreased, and bacteria were thoroughly eliminated within 3 and 6 h by the Cbf‐K₁₆ and Cbf‐A₇A₁₃ treatments, respectively. The peptides penetrated the bacterial cell membrane and enabled β‐galactosidase leakage, and caused the cytoplasmic membrane to become permeable, and finally bound to the DNA. The genomic DNA of E. coli was completely unable to migrate on an agarose gel after Cbf‐K₁₆ treatment (8 µg/ml). These data demonstrated that Cbf‐K₁₆ and Cbf‐A₇A₁₃ possess effective antimicrobial activity against drug‐resistant strains, including NDM‐1 carrying E. coli BL21 (DE3)‐NDM‐1, by binding to DNA after penetrating the cytoplasmic membrane in vitro, which may have potential therapeutic value for the treatment of NDM‐1‐carrying bacterial infections. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Genetic mapping reveals a candidate gene (<Emphasis Type="Italic">ClFS1</Emphasis>) for fruit shape in watermelon (<Emphasis Type="Italic">Citrullus lanatus</Emphasis> L.)

Key message

A 159 bp deletion in ClFS1 gene encoding IQD protein is responsible for fruit shape in watermelon.

Abstract

Watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] is known for its rich diversity in fruit size and shape. Fruit shape has been one of the major objectives of watermelon breeding. However, the candidate genes and the underlying genetic mechanism for such an important trait in watermelon are unknown. In this study, we identified a locus on chromosome 3 of watermelon genome controlling fruit shape. Segregation analysis in F₂ and BC₁ populations derived from a cross between two inbred lines “Duan125” (elongate fruit) and “Zhengzhouzigua” (spherical fruit) suggests that fruit shape of watermelon is controlled by a single locus and elongate fruit (OO) is incompletely dominant to spherical fruit (oo) with the heterozygote (Oo) being oval fruit. GWAS profiles among 315 accessions identified a major locus designated on watermelon chromosome 3, which was confirmed by BSA-seq mapping in the F₂ population. The candidate gene was mapped to a region 46 kb on chromosome 3. There were only four genes present in the corresponding region in the reference genome. Four candidate genes were sequenced in this region, revealing that the CDS of Cla011257 had a 159 bp deletion which resulted in the omission of 53 amino acids in elongate watermelon. An indel marker was derived from the 159 bp deletion to test the F₂ population and 105 watermelon accessions. The results showed that Cla011257 cosegregated with watermelon fruit shape. In addition, the Cla011257 expression was the highest at ovary formation stage. The predicted protein of the Cla011257 gene fitted in IQD protein family which was reported to have association with cell arrays and Ca²⁺-CaM signaling modules. Clear understanding of the genes facilitating the fruit shape along with marker association selection will be an effective way to develop new cultivars.

     

Tillage effect on seasonal nitrogen availability in corn supplied with legume green manures

Colonization of sorghum by Macrophomina phaseolina in field plots was determined at nitrogen fertilization rates of 0, 56, 112, and 168 kg ha^-1 in 1988 and 0, 84, 168, and 256 kg ha^-1 in 1989. Above ground plant tissue and roots were sampled monthly to determine total nitrogen and percent colonization of root segments by natural inoculum. Root infection was not affected by nitrogen treatment, but was affected by growth stage and environment. High root infection occurred before reproductive development (growth stage 3) in 1988 and was associated with hot, dry weather early in the growing season. In 1989, when the weather was cool and wet, root infection began after reproductive development (growth stage 4). The effect of nitrogen treatments on lesion length was determined in sorghum stalks artificially inoculated with M. phaseolina. Lesion lengths were significantly affected by both nitrogen treatments and growth stage. Lesions were significantly longer with all nitrogen treatments at growth stage 9 than with the no-nitrogen treatment, and lesions tended to increase with increased levels of nitrogen fertilization. Significant increases in lesion length occurred between growth stages 5, 7, and 9 in 1988 and between 7 and 9 in 1989. This study demonstrates that nitrogen fertilization affects colonization of sorghum stalks but not root infection by M. phaseolina.     

Does the γ subunit move to an abortive position for ATP hydrolysis when the F1·ADP·Mg complex isomerizes to the inactive F1*·ADP·Mg complex?

F₁-ATPases transiently entrap inhibitory MgADP in a catalytic site during turnover when noncatalytic sites are not saturated with ATP. An initial burst of ATP hydrolysis rapidly decelerates to a slow intermediate rate that gradually accelerates to a final steady-state rate. Transition from the intermediate to the final rate is caused by slow binding of ATP to noncatalytic sites which promotes dissociation of inhibitory MgADP from the affected catalytic site. Evidence from several laboratories suggests that the γ subunit rotates with respect to α/β subunit pairs of F₁-ATPases during ATP hydrolysis. The α₃β₃ and α₃β₃δ subcomplexes of the TF₁-ATPase do not entrap inhibitory MgADP in a catalytic site during turnover, suggesting involvement of the γ subunit in the entrapment process. From these observations, it is proposed that the γ subunit moves into an abortive position for ATP hydrolysis when inhibitory MgADP is entrapped in a catalytic site during ATP hydrolysis.     

Enhanced‐efficiency fertilizers are not a panacea for resolving the nitrogen problem

Improving nitrogen (N) management for greater agricultural output while minimizing unintended environmental consequences is critical in the endeavor of feeding the growing population sustainably amid climate change. Enhanced‐efficiency fertilizers (EEFs) have been developed to better synchronize fertilizer N release with crop uptake, offering the potential for enhanced N use efficiency (NUE) and reduced losses. Can EEFs play a significant role in helping address the N management challenge? Here we present a comprehensive analysis of worldwide studies published in 1980–2016 evaluating four major types of EEFs (polymer‐coated fertilizers PCF, nitrification inhibitors NI, urease inhibitors UI, and double inhibitors DI, i.e. urease and nitrification inhibitors combined) regarding their effectiveness in increasing yield and NUE and reducing N losses. Overall productivity and environmental efficacy depended on the combination of EEF type and cropping systems, further affected by biophysical conditions. Best scenarios include: (i) DI used in grassland (n = 133), averaging 11% yield increase, 33% NUE improvement, and 47% decrease in aggregated N loss (sum of NO₃^‐, NH₃, and N₂O, totaling 84 kg N/ha); (ii) UI in rice‐paddy systems (n = 100), with 9% yield increase, 29% NUE improvement, and 41% N‐loss reduction (16 kg N/ha). EEF efficacies in wheat and maize systems were more complicated and generally less effective. In‐depth analysis indicated that the potential benefits of EEFs might be best achieved when a need is created, for example, by downward adjusting N application from conventional rate. We conclude that EEFs can play a significant role in sustainable agricultural production but their prudent use requires firstly eliminating any fertilizer mismanagement plus the implementation of knowledge‐based N management practices.     

SNRPA enhances tumour cell growth in gastric cancer through modulating NGF expression

Objectives

SNRPA is a protein component of U1 small nuclear ribonucleoprotein (U1 snRNP) complex, which takes part in the splicing of pre‐mRNAs. Its expression and function in tumour remain unknown. Herein, we elucidated the functional contribution of SNRPA to the progression of gastric cancer (GC).

Materials and methods

SNRPA expression was investigated in a GC tissue microarray by immunohistochemical staining. Cell proliferation was evaluated by CCK‐8, colony formation and EdU incorporation assays. A mouse xenograft model was used to detect the tumourigenicity. Gene expression profiling was performed and then the potential target genes were verified by quantitative real‐time PCR and western blot analyses. The functional relevance between SNRPA and its target gene was examined by cell growth assays.

Results

SNRPA expression was higher in tumour tissues than in matched normal gastric mucosa tissues, and it was positively correlated with the tumour size and progression. High SNRPA expression indicated poor prognosis of GC patients. Silencing SNRPA in GC cells markedly inhibited cell proliferation in vitro and tumour growth in a xenograft model, while overexpressing SNRPA exhibited opposite results. Moreover, we identified NGF (Nerve growth factor) as a downstream effector of SNRPA and further proved that NGF was crucial for SNRPA‐mediated GC cell growth.

Conclusions

These findings suggested that SNRPA may contribute to GC progression via NGF and could be a prognostic biomarker for GC.

     

Discovery of new GSK-3β inhibitors through structure-based virtual screening

Glycogen synthase kinase-3β (GSK-3β) is an attractive therapeutic target for human diseases, such as diabetes, cancer, neurodegenerative diseases, and inflammation. Thus, structure-based virtual screening was performed to identify novel scaffolds of GSK-3β inhibitors, and we observed that conserved water molecules of GSK-3β were suitable for virtual screening. We found 14 hits and D1 (IC₅₀ of 0.71?μM) were identified. Furthermore, the neuroprotection activity of D1–D3 was validated on a cellular level. 2D similarity searches were used to find derivatives of high inhibitory compounds and an enriched structure–activity relationship suggested that these skeletons were worthy of study as potent GSK-3β inhibitors.