Graphene-Cu Nanocomposites Induce Tolerance against Fusarium oxysporum, Increase Antioxidant Activity, and Decrease Stress in Tomato Plants.

The tomato crop is susceptible to various types of stress, both biotic and abiotic, which affect the morphology, physiology, biochemistry, and genetic regulation of plants. Among the biotic factors, is the phytopathogen Fusarium oxysporum f. sp. lycopersici (Fol), which can cause losses of up to 100%. Graphene-Cu nanocomposites have emerged as a potential alternative for pathogen control, thanks to their antimicrobial activity and their ability to induce the activation of the antioxidant defense system in plants. In the present study, the effect of the Graphene-Cu nanocomposites and the functionalization of graphene in the tomato crop inoculated with Fol was evaluated, analyzing their impacts on the antioxidant defense system, the foliar water potential (Ψh), and the efficiency of photosystem II (PSII). The results demonstrated multiple positive effects; in particular, the Graphene-Cu nanocomposite managed to delay the incidence of the "vascular wilt" disease and reduce the severity by 29.0%. This translated into an increase in the content of photosynthetic pigments and an increase in fruit production compared with Fol. In addition, the antioxidant system of the plants was improved, increasing the content of glutathione, flavonoids, and anthocyanins, and the activity of the GPX, PAL, and CAT enzymes. Regarding the impact on the water potential and the efficiency of the PSII, the plants inoculated with Fol and treated with the Graphene-Cu nanocomposite responded better to biotic stress compared with Fol, reducing water potential by up to 31.7% and Fv/Fm levels by 32.0%.

The Banana MaWRKY18, MaWRKY45, MaWRKY60 and MaWRKY70 Genes Encode Functional Transcription Factors and Display Differential Expression in Response to Defense Phytohormones.

WRKY transcription factors (TFs) play key roles in plant defense responses through phytohormone signaling pathways. However, their functions in tropical fruit crops, especially in banana, remain largely unknown. Several WRKY genes from the model plants rice (OsWRKY45) and Arabidopsis (AtWRKY18, AtWRKY60, AtWRKY70) have shown to be attractive TFs for engineering disease resistance. In this study, we isolated four banana cDNAs (MaWRKY18, MaWRKY45, MaWRKY60, and MaWRKY70) with homology to these rice and ArabidopsisWRKY genes. The MaWRKY cDNAs were isolated from the wild banana Musa acuminata ssp. malaccensis, which is resistant to several diseases of this crop and is a progenitor of most banana cultivars. The deduced amino acid sequences of the four MaWRKY cDNAs revealed the presence of the conserved WRKY domain of ~60 amino acids and a zinc-finger motif at the N-terminus. Based on the number of WRKY repeats and the structure of the zinc-finger motif, MaWRKY18 and MaWRKY60 belong to group II of WRKY TFs, while MaWRKY45 and MaWRKY70 are members of group III. Their corresponding proteins were located in the nuclei of onion epidermal cells and were shown to be functional TFs in yeast cells. Moreover, expression analyses revealed that the majority of these MaWRKY genes were upregulated by salicylic acid (SA) or methyl jasmonate (MeJA) phytohormones, although the expression levels were relatively higher with MeJA treatment. The fact that most of these banana WRKY genes were upregulated by SA or MeJA, which are involved in systemic acquired resistance (SAR) or induced systemic resistance (ISR), respectively, make them interesting candidates for bioengineering broad-spectrum resistance in this crop.

The Entry Blocker Peptide Produced in Chlamydomonas reinhardtii Inhibits Influenza Viral Replication in vitro.

This year, a respiratory virus caused an emergency pandemic alert in health services around the world, showing the need for biotechnological approaches to fight these diseases. The influenza virus is one of the main viral agents that generate pandemic outbreaks. Currently, the majority of co-circulating influenza A virus (IAV) strains are adamantine- and oseltamivir-resistant strains, and the challenge is to find new antivirals for more efficient treatments. The antiviral entry blocker (EB) peptide is a promising candidate for blocking the virus entry into cells. The aim of this research was to express the EB peptide in the microalgae Chlamydomonas reinhardtii and test its antiviral activity against IAV in vitro. The EB peptide nucleotide sequence was introduced into the nuclear genome of microalgae using Agrobacterium tumefaciens transformation. The EB peptide amount produced in transformed microalgae was 4.99 ± 0.067% of the total soluble protein. In hemagglutination inhibition assays using influenza A/H1N1 pdm and influenza A H1N1/Virginia/ATCC/2009 strains, we reported that the EB peptide extract from the microalgae showed 100-fold higher efficiency than the EB synthetic peptide. In addition, both the EB peptide extract and synthetic peptide inhibited viral replication in MDCK cells (IC50 = 20.7 nM and IC50 = 754.4 nM, respectively); however, the EB peptide extract showed a 32-fold higher antiviral effectiveness than the synthetic peptide against influenza A/H1N1 pdm. Extracts from untransformed and transformed microalgae and synthetic peptide did not show cytotoxic effect on MDCK cell monolayers. Thus, C. reinhardtii may be a fast, safe, and effective expression platform for production of peptides with significant antiviral activity and can be used as a prophylactic treatment to reduce viral propagation.

Carbon Nanotubes Decrease the Negative Impact of Alternaria solani in Tomato Crop.

The diseases that attack the tomato crop are a limiting factor for its production and are difficult to control or eradicate. Stem and fruit rot and leaf blight caused by Alternaria solani causes severe damage and substantial yield losses. Carbon nanotubes (CNTs) could be an alternative for the control of pathogens since they have strong antimicrobial activity, in addition to inducing the activation of the antioxidant defense system in plants. In the present study, multi-walled carbon nanotubes were evaluated on the incidence and severity of A. solani. Moreover, to the impact they have on the antioxidant defense system and the photosynthetic capacity of the tomato crop. The results show that the application of CNTs had multiple positive effects on tomato crop. CNTs decreased the incidence and severity of A. solani. Furthermore, CNTs increased the fruit yield of tomato crop and dry shoot biomass. The antioxidant system was improved, since the content of ascorbic acid, flavonoids, and the activity of the glutathione peroxidase enzyme were increased. The net photosynthesis and water use efficiency were also increased by the application of CNTs. CNTs can be an option to control A. solani in tomato crop, and diminish the negative impact of this pathogen.

Impact of Carbon Nanomaterials on the Antioxidant System of Tomato Seedlings.

Tomato is one of the most economically important vegetables worldwide and is constantly threatened by various biotic and abiotic stress factors reducing the quality and quantity in the production of this crop. As an alternative to mitigate stress in plants, carbon nanomaterials (CNMs) have been used in agricultural areas. Therefore, the objective of the present work was to evaluate the antioxidant responses of tomato seedlings to the application via foliar and drench of carbon nanotubes (CNTs) and graphene (GP). Different doses (10, 50, 100, 250, 500, and 1000 mg L-1) and a control were evaluated. The results showed that the fresh and dry root weight increased with the application of CNMs. Regarding the antioxidant responses of tomato seedlings, the application of CNMs increased the content of phenols, flavonoids, ascorbic acid, glutathione, photosynthetic pigments, activity of the enzyme's ascorbate peroxidase, glutathione peroxidase, catalase, and phenylalanine ammonia lyase as well as the content of proteins. Therefore, the use of carbon-based nanomaterials could be a good alternative to induce tolerance to different stress in tomato crop.

Association of cytokines polymorphisms with chronic peridontitis and rheumatoid arthritis in a Mexican population.

OBJECTIVE: Historically, it has been shown that rheumatoid arthritis (RA) and periodontitis (PE) share pathophysiological similarities and possibly a genetic background. In order to elucidate the genetic background between both diseases, we evaluated the distributions of five SNPs genotypes and all the possible haplotypes composed in subjects with isolated RA, PE, combined diseases and healthy controls. MATERIALS AND METHODS: The study population consisted of 280 Mexican subjects. Genomic DNA was isolated from buccal epithelial cells collected by cheek scrapings and analyzed for the determination of the following SNPs: IL-1α + 4845 (rs17561), IL-1α -889 (rs1800587), IL-1ß + 3954 (rs1143634), IL-1ß -511(rs16944) and TNF-α -308 (rs1800629). RESULTS: After adjustment for age, sex and smoking status, multiple logistic regression analysis revealed a no significant association in the genotype frequencies of TNF-α -308 and IL-1α + 4845 SNPs. Otherwise a significant association was observed in IL-1ß + 3954 and IL-1ß -511 (p < 0.05) while IL-1α -889 was of borderline statistical significance (p = 0.054). Also, we found three negative associated haplotypes with PE: IL-1α + 4845 G/IL-1ß -511 A, IL-1ß + 3954 C/IL-1ß -511 A and interestingly IL-1α -889 C/IL-1ß -511 A also with a positive association with RA. CONCLUSIONS: Some genotypes and haplotypes are associated with the diseases. But it seems that the genetic background of the association between RA and PE needs to be explored deeper.

Effect of green and red light in lipid accumulation and transcriptional profile of genes implicated in lipid biosynthesis in Chlamydomonas reinhardtii.

Microalgae have the potential to accumulate triacylglycerols under different light spectra. In this work, Chlamydomonas reinhardtii was grown under white (400-700 nm), red (650 nm), and green (550 nm) lights. According to our results, red light (650 nm) has a positive effect in the microalgae growth and chlorophyll concentration. About the lipid content, the control culture (white light-illuminated) reached a 4.4% of dry cell weight (dcw), whereas the culture grown at 550 nm showed an increase of 1.35-fold in the lipids accumulation (5.96% dcw). Interestingly, the most significant accumulation was found in the culture grown at 650 nm (14.78% dcw) which means 3.36-fold higher with respect to the white light-illuminated culture. The most abundant fatty acids found in lipid extracts obtained from the cultures under different light wavelength were palmitic (C16: 0), oleic (C18: 1n9), stearidonic (C18: 4), and linoleic (C18: 2), which are useful in the biodiesel production. Changes in gene expression in response to different wavelength illuminations were assessed; however, an in-depth analysis of a larger number of genes involved in lipid biosynthesis is necessary to fully explain the highest accumulation of lipids in the culture grown under red light. This approach will be useful to find a sustainable source of lipids for biodiesel production. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1404-1411, 2016.

Expression of an Escherichia coli antigenic fusion protein comprising the heat labile toxin B subunit and the heat stable toxin, and its assembly as a functional oligomer in transplastomic tobacco plants.

Enterotoxigenic Escherichia coli (ETEC) strains are important pathogens in developing countries. Some vaccine formulations containing the heat labile toxin B subunit (LTB) have been used in clinical trials; however, the induction of neutralizing antibodies against the heat-stable toxin (ST), a poor immunogenic peptide, is necessary, as most ETEC strains can produce both toxins. In this study, a plant optimized synthetic gene encoding for the LTB-ST fusion protein has been introduced into plastids of tobacco leaf tissues, using biolistic microprojectile bombardment, in an effort to develop a single plant-based candidate vaccine against both toxins. Transplastomic tobacco plants carrying the LTB-ST transgene have been recovered. Transgene insertion into the plastid was confirmed by both PCR and Southern blot analysis. GM1-ELISA revealed that the LTB-ST fusion protein retained its oligomeric structure, and displayed antigenic determinants for both LTB and ST. Western blot analysis, using LTB antisera, confirmed the presence of a 17-KDa protein in transplastomic lines, with the correct antigenicity of the fusion protein. Expression levels of this fusion protein in different lines reached up to 2.3% total soluble protein. Oral immunization of mice with freeze-dried transplastomic tobacco leaves led to the induction of both serum and mucosal LTB-ST specific antibodies. Following cholera toxin challenge, a decrease of intestinal fluid accumulation was observed in mice immunized with LTB-ST-containing tobacco. These findings suggest that tobacco plants expressing LTB-ST could serve as a plant-based candidate vaccine model providing broad-spectrum protection against ETEC-induced diarrhoeal disease.

Distribution of Streptococcus mutans and Streptococcus sobrinus in saliva of Mexican preschool caries-free and caries-active children by microbial and molecular (PCR) assays.

The aim of the study was to investigate the distribution of mutans streptococci (MS) infection of caries-free (CF) and caries-active (CA) preschool Mexican children by microbial and molecular assays. Eighty preschool children were divided into two groups, 40 CF and 40 CA children. Saliva samples were inoculated onto MSB to identify CFU and DNA extractions were tested by PCR. Our results indicated that there was no statistical difference (p > 0.05) between groups either in age, weight, height or sex. S. sobrinus was detected by PCR twice as much in the CA group, the difference being statistically significant (p < 0.05). dmfs index was positive correlated with S. mutans (r = 0.2941, p = 0.0081), S. sobrinus (r = 0.3384, p = 0.0021) and S. mutans-S. sobrinus (r = 0.3978, p = 0.0003). ANCOVA revealed that dmfs index had a significant effect on the distribution of CFU of S. mutans (p = 0.0118) and S. sobrinus (p = 0.03). When MSB was compared with PCR to identify MS, there was no statistical difference (p > 0.05). We conclude that S. mutans and S. sobrinus were isolated in higher numbers from CA children and those harbouring both bacteria had higher dmfs scores. PCR is a useful tool in molecular epidemiology for dental caries studies; it was effective in detecting and identifying MS from saliva in children.

Ingestion of transgenic carrots expressing the Escherichia coli heat-labile enterotoxin B subunit protects mice against cholera toxin challenge.

Diarrheal diseases caused by Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) are worldwide health problems that might be prevented with vaccines based on edible plants expressing the B subunit from either the cholera toxin (CTB) or the E. coli heat labile toxin (LTB). In this work we analyzed the immunity induced in Balb/c mice by ingestion of three weekly doses of 10 mug of LTB derived from transgenic carrot material. Although the anti-LTB serum immunoglobulin G (IgG) and intestinal IgA antibody responses were higher with 10 mug-doses of pure bacterial recombinant LTB (rLTB), the transgenic carrot material also elicited significant serum and intestinal antibody responses. Serum anti-LTB IgG1 antibodies predominated over IgG2a antibodies, suggesting that mainly Th2 responses were induced. A decrease of intestinal fluid accumulation after cholera toxin challenge was observed in mice immunized with either rLTB or LTB-containing carrot material. These results demonstrate that ingestion of carrot-derived LTB induces antitoxin systemic and intestinal immunity in mice and suggest that transgenic carrots expressing LTB may be used as an effective edible vaccine against cholera and ETEC diarrhea in humans.

Transgenic tomatoes express an antigenic polypeptide containing epitopes of the diphtheria, pertussis and tetanus exotoxins, encoded by a synthetic gene.

A current priority of vaccinology is the development of multicomponent vaccines that protect against several pathogens. The diphtheria-pertussis-tetanus (DPT) vaccine prevents the symptoms of three serious and often fatal diseases due to the exotoxins produced by Corynebacterium diphteriae, Bordetella pertussis and Clostridium tetani. We are attempting to develop an edible DPT multicomponent vaccine in plants, based on the fusion of protective exotoxin epitopes encoded by synthetic genes. By means of Agrobacterium mediated transformation we generated transgenic tomatoes with a plant-optimised synthetic gene encoding a novel polypeptide containing two adjuvant and six DPT immunoprotective exotoxin epitopes joined by peptide linkers. In transformed tomato plants, integration of the synthetic DPT (sDPT) gene detected by PCR was confirmed by Southern blot, and specific transcripts of the expected molecular size were detected by RT-PCR. Expression of the putative polypeptide encoded by the sDPT gene was detected by immunoassay with specific antibodies to the diphtheria, pertussis and tetanus exotoxins. The sDPT gene is therefore integrated, transcribed and translated as the expected recombinant sDPT multiepitope polypeptide in transgenic tomatoes that constitute a potential edible vaccine.

Expression of Escherichia coli heat-labile enterotoxin b subunit (LTB) in carrot (Daucus carota L.).

We expressed the B subunit of enterotoxigenic Escherichia coli heat-labile enterotoxin (LTB) encoded by a synthetic codon-optimized gene in carrot. An Agrobacterium-mediated transformation method was used. Thirty independent transgenic lines were regenerated via somatic embryogenesis after 6 months in culture and were transferred to a greenhouse. GM1-ELISA assay was used to assess LTB protein content in mature taproots. Some transgenic lines expressed LTB up to 0.3% of the total soluble protein, which is tenfold higher than the expression levels reported earlier using the native bacterial gene in plants. Immunological assay confirmed proper assembly of the pentameric complex and in vitro activity of the recombinant LTB protein, suggesting that it can be functional in prevention of diarrhea.

Distribution of genotypes of Porphyromonas gingivalis in type 2 diabetic patients with periodontitis in Mexico.

OBJECTIVE: To determine and compare the distribution of Porphyromonas gingivalis fimA genotypes in type 2 diabetes mellitus (T2DM) patients affected by periodontitis, using non-diabetic subjects with and without periodontitis as control groups. MATERIAL AND METHODS: This study involved 75 subjects divided into three groups of 25 subjects each: Group 1 (non-T2DM without periodontitis), Group 2 (non-T2DM with periodontitis) and Group 3 (T2DM with periodontitis). The outcome variable was periodontitis, and explanatory variables were age, sex, T2DM and specific P. gingivalis fimA genotypes. RESULTS: In non-T2DM subjects with healthy periodontal tissues, type I fimA was the most frequently detected individually (40%) or in combinations (40%). In non-T2DM subjects with periodontitis, the most frequently detected type was Ib individually (20%) or in combinations (36%). In T2DM patients with periodontitis, the most frequently detected types were types I (20%) and III (20%), but there was no statistical difference (p>0.05) with non-T2DM periodontitis subjects. CONCLUSIONS: Type I genotype was more frequently detected in periodontally healthy sites from non-T2DM subjects than in periodontitis sites from either subjects with or without T2DM. However, in sites affected by periodontitis from T2DM subjects the predominating types were I and III, which are less virulent strains of P. gingivalis.