Cloning, Characterization, and Functional Expression of Phospholipase D α

Phospholipase D (PLD) plays a key role in adaptive responses of postharvest fruits. A cDNA clone of banana (Musa acuminate L.) PLDα (MaPLDα) was obtained by RT-PCR in this study. The MaPLDα gene contains a complete open reading frame (ORF) encoding a 92-kDa protein composed of 832 amino acid residues and possesses a characteristic C2 domain and two catalytic H×K×××D (abbr. HKD) motifs. The two HKD motifs are separated by 341 amino acid residues in the primary structure. Relatively higher PLD activity and expression of MaPLDα mRNA were detected in developing tissues compared to senescent or mature tissues in individual leaves, flower, stem, and fruit organs, respectively. The expression profile of PLDα mRNA in postharvest banana fruits at different temperatures was determined, and the MaPLDα mRNA reached the highest expression peak on day 5 at 25°C and on day 7 at 12°C. The results provide useful information for maintaining postharvest quality and extending the storage life of banana fruit

Role of phospholipase C in banana in response to anthracnose infection

Phospholipase C (PLC) plays an important role in plant immunity, and anthracnose caused by the Colletotrichum species is a common postharvest disease of the banana fruit. This study aims to evaluate the role of PLC in anthrax resistance in banana. The experimental group of banana samples was treated with a banana anthracnose conidia suspension, and the control group was treated with distilled water. After inoculation, the groups were sprayed with ethephon, and indicators, such as hardness and conductivity changes; PLC activity, 1,2-diacylglycerol (DAG) and phosphatidic acid (PA)content; and MaPLC-1and MaPLC-2 expression levels, were assessed at 0, 3, 6, 9, 12, and 15 days. Moreover, the expression levels of MaPLC-1 and MaPLC-2 were detected in various tissues. The hardness of banana fruits in the experimental group decreased faster than that in the control group. Furthermore, the conductivity was higher in the experimental group than in the control group. Regarding PLC activity, DAG, and PA content, bananas in the experimental group showed higher activities than those in the control group. Moreover, relatively higher expression of PLC mRNA was detected in anthracnose-inoculated tissues. The evaluation of MaPLC-1 and MaPLC-2 expression levels showed that the mature peel had the highest MaPLC-1 expression level. However, the MaPLC-2 gene was expressed at relatively low levels in the fruit and at relatively high levels in the flower organs. PLC might play a role in fruit ripening in response to anthracnose resistance

Transcriptome profiling helps to elucidate the mechanisms of ripening and epidermal senescence in passion fruit (Passiflora edulia Sims).

Passion fruit (Passiflora edulia Sims), an important tropical and subtropical species, is classified as a respiration climacteric fruit, and its quality deteriorates rapidly after harvest. To elucidate the mechanisms involved in ripening and rapid fruit senescence, phytochemical characteristic analysis and RNA sequencing were performed in purple passion fruit with different treatments, that is, 1-methylcyclopropene (1-MCP) and preservative film (PF). Comprehensive functional annotation and KEGG enrichment analysis showed that starch and sucrose metabolism, plant hormone signal transduction, phenylpropanoid biosynthesis, flavonoid biosynthesis, and carotenoid biosynthesis were involved in fruit ripening. Treatment with PF and 1-MCP significantly affected the transcription levels of passion fruit during postharvest storage. A large number of differentially expressed unigenes (DEGs) were identified as significantly enriched in starch and sucrose metabolism, plant hormone signal transduction and phenylpropanoid biosynthesis at the postharvest stage. The PF and 1-MCP treatments increased superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) gene expression levels and enzyme activities, accelerated lignin accumulation, and decreased β-galactosidase (β-Gal), polygalacturonase (PG) and cellulose activities and gene expression levels to delay cell wall degradation during fruit senescence. The RNA sequencing data for cell wall metabolism and hormone signal transduction pathway-related unigenes were verified by RT-qPCR. The results of this study indicate that the cell wall metabolism and hormone signaling pathways are closely related to passion fruit ripening. PF and 1-MCP treatment might inhibit ethylene signaling and regulate cell wall metabolism pathways to inhibit cell wall degradation. Our results demonstrate the involvement of ripening- and senescence-related networks in passion fruit ripening and may establish a foundation for future research investigating the effects of PF and 1-MCP treatment on fruit ripening

The Ion Source of Nitrogen Direct Analysis in Real-Time Mass Spectrometry as a Highly Efficient Reactor: Generation of Reactive Oxygen Species

Abstract An innovative strategy for sustainably active oxygen capture using nitrogen (N2) instead of helium (He) as direct analysis in real-time (DART) gas is demonstrated in this work. DART MS was carried out to analyze different polarity compounds including organophosphorus pesticides, amino acids, hormones, and poly brominated diphenyl ethers by using He and N2 as DART gas, respectively. The unexpectedly characteristic ionization reactions, including replacement reaction where the sulfur atom of P=S group, were replaced by oxygen atom, oxidation ([M + nO + H]+ or [M + nO-H]− (n = 1, 2, 3, 4, 5)), and hydrogen loss (loss of two hydrogens) rapidly occurred in situ in the presence of N2 under ambient conditions without any additives. The reaction mechanisms were proposed and further confirmed by high-resolution tandem mass spectrometry. Our study under high temperature and high voltage provides a powerful tool for generating unique ionic species that may be difficult to form by other means, which also creates favorable conditions for the future study of the mechanism of DART MS. Graphical Abstrac

Antibacterial Activity and Mechanism of Action of Bovine Lactoferricin Derivatives with Symmetrical Amino Acid Sequences

In recent years, the overuse of antibiotics has become very serious. Many pathogenic bacteria have become resistant to them, with serious potential health consequences. Thus, it is urgent that we develop new antibiotic drugs. Antimicrobial peptides (AMPs) are important endogenous antibacterial molecules that contribute to immunity. Most have spectral antibacterial properties and do not confer drug resistance. In this paper, an 11-residue peptide (LFcinB18–28) with a sequence of KCRRWQWRMKK was modified by amino acid substitution to form a symmetrical amino acid sequence. The antibacterial activities and mechanisms of action of engineered peptides including KW-WK (KWRRWQWRRWK), FP-PF (FPRRWQWRRPF), FW-WF (FWRRWQWRRWF), and KK-KK (KKRRWQWRRKK) were investigated. The four engineered peptides could more effectively inhibit bacteria than the original peptide, LFcinB18–28. This suggested that a symmetrical amino acid sequence might enhance the antibacterial activity of AMPs. However, only peptides KW-WK, FP-PF, and KK-KK were safe; FW-WF displayed hemolytic activity. The engineered peptides shared cationic and amphipathic characteristics that facilitated interactions with the anionic microbial membranes, leading to disruption of membrane integrity and permeabilizing microbial membranes, resulting in cell death. Therefore, a symmetrical amino acid sequence and related structural parameters offer an alternative approach to the design of AMPs. This will provide a scientific basis for the design and synthesis of new AMPs

Reproducibility and Validity of a Semi-Quantitative Food Frequency Questionnaire for Assessing Dietary Intake of Vegetarians and Omnivores in Harbin, China

This study aims to evaluate the reproducibility and validity of a semi-quantitative food frequency questionnaire (SQFFQ) developed for vegetarians and omnivores in Harbin, China. Participants (36 vegetarians and 64 omnivores) administered SQFFQ at baseline (SQFFQ1) and six months later (SQFFQ2) to assess the reproducibility. The 24 h recalls (24 HRs) for three consecutive days were completed between the administrations of two SQFFQs to determine the validity. For reproducibility, Pearson correlation coefficients between SQFFQ1 and SQFFQ2 for vegetarians and omnivores were 0.45~0.88 and 0.44~0.84, respectively. For validity, unadjusted Pearson correlation coefficients were 0.46~0.83 with an average of 0.63 and 0.43~0.86 with an average of 0.61, respectively; energy-adjusted Pearson correlation coefficients were 0.43~0.82 with an average of 0.61 and 0.40~0.85 with an average of 0.59, respectively. Majority of the correlation coefficients for food groups and macronutrients decreased or remained unchanged after energy adjustment. Furthermore, all correlations were statistically significant (p < 0.05). Bland–Altman plots also showed reasonably acceptable agreement between the two methods. In conclusion, the SQFFQ developed in this study has reasonably acceptable reproducibility and validity

Molecular Characterization Of Pathogenic Salmonella Spp From Raw Beef In Karachi, Pakistan

The aim of the present study was to estimate the prevalence of Salmonella and investigate the dominant serovars distribution in raw beef and to screen the isolated serovars for the prescense of beta-lactamases and virulence genes. A total of 150 samples of raw beef sold at butcher shops (n = 75) and supermarkets (n = 75) in Karachi city were collected (50 samples each from muscles, lymph nodes, and minced beef). The samples were cultured according to the ISO-6579-1guidlines. The overall prevalence of Salmonella strains was found to be 21.34%. A total of 56 isolates of Salmonella belonging to four serogroups (Salmonella Pullorum, Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Choleraesuis) were isolated from beef muscles (12%), lymph nodes (24%) and minced beef (28%) samples collected from butcher shops (av. 21.34%). No Salmonella was detected in beef samples collected from supermarkets. S. Enteritidis contamination was highest (37.5%), followed by S. Choleraesuis (30.4%), S. Pullorum (19.6%) and S. Typhimurium (12.5 %). Antibiotic susceptibility testing revealed that Salmonella isolates were highly resistant to Oxytetracycline (90%), Ampicillin (90.5%), Amoxicillin (81.1%), Tetracycline (76%), Neomycin, (79.8%) and Ciprofloxacin (61.4%). The Salmonella isolates examined were more susceptible to the Cephalosporin antibiotics such as Cefixime (43.2%), Cefepime (48.2) and Cefoxitin (49.8%). PCR based screening of blaTEM, blaCTX-M and blaSHV revealed that blaCTX-M and blaTEM were the dominant resistant genes in S. Enteritidis and S. Typhimurium followed by S. Pullorum and S. Choleraesuis whereas blaSHV was the least detected beta-lactamase in Salmonella isolates. Virulence genes screening revealed that at least five genes were present in all the serovars, highest being present in S. Enteritidis (12/17) and S. Typhimurium (12/17). S. Cholerasuis (5/17) carried the least number of virulence genes followed by S. Pullorum (6/17). The present data suggest that beef samples from butcher shops of Karachi city are heavily contaminated with MDR Salmonella. The presence of resistance and virulence genes in MDR strains of Salmonella may play a significant role in transmission and development of Salmonella infection in humans

Characterization and Expression of Phospholipase D Putatively Involved in <i>Colletotrichum</i><i>musae</i> Disease Development of Postharvest Banana Fruit

Phospholipase D (PLD) in plants plays an important role in growth, development, and stress response. The effect of hexanal on PLD in banana fruit responding to Colletotrichum musae infection remains poorly understood. In this study, four putative PLD genes, named as MaPLD1, MaPLD2, MaPLD3, and MaPLD4 were identified from banana fruit. The four MaPLDs can be classified into three of the seven known PLD families according to sequence characterization. Their deduced amino acid sequences displayed homology of PLDs from other plant species. Furthermore, the specific expression analysis of PLD genes in banana fruit in response to infection in C. musae was studied and the response relationship between PLD family members and banana fruit under anthracnose stress was clarified. Changes in both the activity of PLD and PLC, and the connection between hexanal and phospholipases in the banana fruit C. musae infection were compared. The results showed that the incidence of disease in banana inoculated with C. musae was dramatically increased after 6 days of storage, the activation of PLD and PLC in infected anthracnose fruit before disease development, and that this activation was inhibited by hexanal treatment, which suggested that both enzymes play a protective role in banana fruit to cope with C. musae infection and the participation of hexanal in their regulation. Of the four MaPLD genes, the anthracnose had a stronger effect on MaPLD1 and MaPLD4. These data demonstrated that hexanal treatment could enhance fruit disease resistance to C. musae, and that PLD could take part in the disease defensive system of harvested banana fruit to C. musae by modulating the metabolism of cell membrane lipids, and thus suppress disease development in C. musae -inoculated banana during storage