Cocoa (Theobroma cacao L) is one of the main agricultural products of Ecuador. However, it is susceptible to diseases and pests being those of fungal origin, the most common [1]. Black sheath caused by Phytophthora sp., the witch's broom caused by Moniliophthora perniciosa, and moniliasis caused by Moniliophthora roreri are the most reported conditions. Moniliasis is considered the most destructive than all diseases that affect this crop [2].

M.roreri manifests itself on the cob of the plant and presents two phases of infection: the biotrophic where it can be seen malformations in the fruit, and the necrotic where rot and death of the fruit occur [3]. The spore masses break off the fungus pseudostroma and move by different mechanisms (wind, humans, animals, and water) [4]. The climatic conditions and the amount of free spores are determinants in the life cycle of this pathogen [5]. Nevertheless, the fungus is present during year-round as a component of the air layer in growing regions, making eradication difficult. A better knowledge of the genetic diversity of this microorganism could lead to a greater understanding of its distribution and adaptation mechanisms.

Since this disease causes great economic losses, and there have been few studies of its molecular level, the objective of this investigation was the characterization of M. roreri isolates, to determinate its genetic diversity. To do this, 75 samples of the M. roreri fungus from three Ecuadorian provinces of high cocoa productivity were taken. Nucleotide diversity was determined through the amplification and sequencing of the ITS1 and ITS2 ribosomal regions of each sample.

It was found a low level of genetic differentiation between the samples analyzed. The results of the molecular variance analysis (AMOVA) revealed that most of the genetic variability is found within individuals (71.29%). To study phylogenetic relationships, an MCC algorithm tree was made using the sequences of the ITS regions from the samples in this study, together with other continental sequences previously reported in public databases (42 samples in total). Interestingly, it was found after performing the phylogenetic analyzes that Ecuador could be the center of origin of M. roreri, with multiple reintroductions of the pathogen occurring throughout Latin America. Data should be corroborated with future research.

Penco azul (Agave americana subsp. andina) is part of the landscape of the Ecuadorian Andean region, in the same way, of its culture and tradition. Moreover, it is interesting from the agricultural perspective, due to its type of asexual reproduction (of which the genus Agave or Agavaceae is characterized), since it does so through its rhizomes (underground stems that form the young or genetically identical replicas). Thus, it is common in Latin American countries, such as Mexico and Ecuador, that there are crops taking advantage of this property. However, the advantages are also observed from a biotechnological perspective, due to the possible separation of steroidal saponins (SE) such as hecogenin and tigogenin, from the rhizomes by enzymatic hydrolysis. Among the properties of these molecules, the one described by Santos, Fajardo, Romo & Uribe (2015), who concluded in their work with Agave Salmiana, that the anticancer effect of SE is verified by their first evaluation of the apoptotic potential of kammogenins and mangenins found in mead . Therefore, thanks to these SE, "programmed cell death" can be caused against colon cancer.
Steroidal saponins are glycosides, that is, they are compounds that by hydrolysis (acidic or diastatic) give one or more sugar molecules (glucones) and other non-glycidic substances or aglucones (for example, acids, phenols, alcohols or aldehydes). In samples of A. americana has been found that the content of hecogenin and tigogenin increases by a ratio of 12: 1 (respectively) during plant growth. For this reason, the author in this research proposes to separate steroidal saponins in the most adult specimens, whose approximate age is 12 years (determined empirically). This is due to the high content of hecogenin, the most active steroidal saponin of the genus Agave.
On the other hand, at the University of Manchester, Cambridge and Bahuguna Garhwal, special glycosides such as steroidal saponin were found in the structure of the rhizomes. However, the traditional method for extraction consists in the following treatment of the sample: it starts with drying at 40 ° C; macerate with ethanol and water (at room temperature and in the absence of light); the extract obtained is evaporated in vacuo; 10 g of this content are dissolved in water, and extracted again in separatory funnels with hexane and butanol; for hydrolysis, 5 g of the saponin extract was dissolved in 75 mL of ethanol and 75 mL of 2M hydrochloric acid was added; finally, it was extracted with ethyl acetate.
To conclude, hydrolysis is a water solvolysis, which allows a solvent (nucleophile) to break one or more bonds in the solute. The method proposed with the present study is through enzymatic hydrolysis. In this way, we seek to adapt a technique made by García et al. (2009), who experimented with potato dextrose agar. From this culture medium, the extract of the hydrolyzing enzyme was obtained, inoculating yeasts that ferment the water-honey of the Agave tequilana. In Ecuador, "penco azul" mead is known as mishki or chaguarmishki, so it is sought to replicate the experiment on a laboratory scale, with this native drink. As for the treatment of the sample, the traditional method will be used.

Blood disorders like B-thalassemia or sickle cell anaemia obligate patients to require constant blood transfusions. These, among others, make blood a highly demanded resource in the medical field. Blood has been obtained from in-vitro erythroid differentiation from embryonic stem cells or induced pluripotent stem cells, but this blood tends to produce embryonic haemoglobin.

In this project, we aim to use a CRISPR/Cas9 modified version, combined with an aptamer approach, to alter the expression of the β-globin locus upregulating the gamma globin gene to produce foetal haemoglobin.

Guide vectors were created targeting previously known sequences in the gamma haemoglobin gene. HEK cells were transfected with different combinations of CRISPR/Cas9 elements to test the amount of gene activation achieved. Gene expression analysis was performed through RTq/PCR.

It was found that HbG activation can be achieved to some extent, however, the levels of gamma haemoglobin attained are very low compared with the normal levels of HbG expressed in other cell lines like K562. From the combinations tested, the combination of dCas9ES + PP7/PCP + VPH presented the best performance.

Introduction

The genus Listeria is constituted by several species; however, Listeria monocytogenes is the most well-known species due to its impact as common foodborne pathogen and also is the leading cause for major food recalls. In recent years, several others Listeria species had been identified worldwide due to the availability of molecular tools for rapid characterization of putative Listeria isolates, such as L. innocua, L. seeligeri and L. welshimeri (2). Nowadays, several studies had been identifying Listeria monocytogenes with co-infection with Listeria innocua, on food plants surfaces, it is really worrying because L. innocua difficult the detection of L. monocytogenes due to its accelerated growth rate. Our previous results identified several Listeria sp. on fresh cheese from several farm markets, where L. innocua was also identified among the contaminated samples. The origin of the contamination of these processed foods is due to the persistence of Listeria in the environment.

L. monocytogenes has the ability to colonize surfaces in food processing plants and it is really difficult to eliminate because of its ability to form biofilms and survive at different temperatures(1). On the other hand, Listeria innocua biofilms have been detected on food processing plants surfaces although it usually coexists with Listeria monocytogenes, it is really important to mention that it was detected on surfaces at different temperatures including freezer areas. Little is still known about L. innocua and L. monocytogenes interactions and mixed biofilm formation. Therefore, it is important to understand the dynamic of biofilm formation of L. innocua to further analyze mixed biofilm with L. monocytogenes. Therefore, our main goal was to evaluate L. innocua ability on biofilm formation under a variability of temperatures, more exactly, at 4, 22 and 37°C.

Materials and Methods

The isolate of L. innocua was obtained from artisanal soft cheese used in this study. The biofilm assays were realized on a glass surface placed in a 6-well plate and growth in static conditions. Briefly, an initial inoculation of 1 – 2 Log 8 CFU/ml, through a standard growth curve, was prepared in each well and then incubated for 48h at different temperatures (4, 22 and 37°C). The medium was changed at 24 hours for sterile TBS and each assay had negative controls (coverslip with sterile medium). Each biofilm assay and samples were done in duplicate. The results were analyzed by crystal violet measurement (OD 630 nm) and CFU counting. This CFU counting allowed to evaluate the biofilm viability by culturing in TSA. Finally, each biofilm growth was also evaluated through microscopy analysis.

Results and Discussion

L. innocua demonstrated the ability to form biofilms in all temperatures (4, 22 and 37°C). The biofilm production index was higher at 37°C and gradually decreased to 4°C. Furthermore, biofilm viability showed the same pattern for 37°C (log 9), 22°C (log 8) and 4°C (log 7). Meanwhile, microscopy analysis confirmed biofilm formation in all temperatures, where at 4°C L. innocua showed less surface coverage in contrast to 37°C where the same strain was able to cover the entire glass surface.

Conclusions

To conclude, Listeria innocua had undoubtedly demonstrated its ability to form biofilms in a large range of temperatures, showing a great adaptation to the thermal conditions and comparable to L. monocytogenes ability to form biofilm, as previously described by other studies. To our best knowledge, this is the first study in Ecuador to evaluate L. innocua biofilms (3)

References

1. Orsi R, Wiedmann M. Characteristics and distribution of Listeria spp., including Listeria species newly described since 2009. Applied Microbiology and Biotechnology. 2016 Abril; 100.

2. Di Bonaventura G, Piccolomini R, Paludi D. Influence of temperature on biofilm formation by Listeria monocytogenes on various food-contact surfaces: relationship with motility and cell surface hydrophobicity. Journal of Applied Microbiology. 2008 Junio; 104(6).

3. Kubota H, Senda s, Nomura N, Tokuda H. Biofilm formation by lactic acid bacteria and resistance to environmental stress. Journal of Bioscience and Bioengineering. 2008 Octubre; 106(4).

Glycosylation mechanisms in saponins of Caryophyllaceae plant family were subjected to simulation by statistically exploring variability of 231 chemical structures belonging to four different aglycones: gypsogenin (Gyp), quillaic acid (QA), gypsogenic acid (GA), 16-OH-gypsogenic acid (16-OH-GA). Saponins based on different aglycones were initially characterized by relative glycosylation levels of different carbons. Simulation was initialized by combining the four saponin groups using Scheffé’s mixture design which provides a complete set of N gradual weightings of groups. Combined saponins were randomly and iteratively sampled from different groups by bootstrap technique. For a same combination, saponins were averaged leading to barycentric glycosylation profile. Iterations of the N barycentric profiles and averaging provided a final response matrix of N smoothed glycosylation profiles from which regulation mechanisms of carbons were highlighted in different aglycone-based saponins. Glucose (Glc) was revealed to be widely favored in GA and 16-OH-GA with more target aspect of 28-Glc in 16-OH-GA and relatively shared distribution between C28 (mainly) C3 and C23 in GA. Strong competition for galactose (Gal) was highlighted between C3 and C28 with target aspects to 28-Gal in GA and 3-Gal in (Gyp, QA). Gyp and QA showed higher regulations of pentoses (xylose, Xyl; arabinose, Ara) with more affinity of GA for (3-Ara, 28-Xyl) and 16-OH-GA for (3-Xyl, 28-Ara). These results call for further investments in simulations of glycosylation mechanisms helping for better understanding metabolic aspects of saponins, and encouraging future analytic experiments in the field.

Eukaryotic pathogens have developed the ability to colonize a variety of hosts, including animals and plants. In that sense, these organisms have developed proteins called effectors, which have the ability to manipulate innate immunity and host homeostasis (Whisson et al., 2007). For example, the Hopl1 effector of Pseudomonas syringae promotes bacterial virulence in hosts by suppressing plant defenses. One of the main characteristics of Hopl1 is the presence of a J domain (Jelenska et al., 2007). This domain characterizes the group of proteins called DnaJ (also called Hsp40). Members of this family are a heterogeneous group of multidomain proteins defined by the highly conserved J domain. They function as co-chaperones of the Hsp70 proteins and are involved in several essential cellular processes that include folding, degradation and protein refolding (Ito et al., 2016).

Although DnaJ effectors have been identified in phytopathogens of bacterial origin, so far this type of protein has not been reported in nematodes (one of the most important groups of plant parasites). For this reason, the present work aimed to explore the presence of DnaJ effectors in the parasitic nematode proteome of M. incognita plants through in silico analysis. In the first instance, a search was performed with PSI-BLAST (3 iterations), using the M. incognita proteome (PRJNA340324) as a database and the A0A2L0VY59_MELAR protein (annotated as DnaJ in the InterPro database) as a query (Figure 1). We identified 56 DnaJ proteins but only 47 with a J domain with the conserved HPD motif. We searched within this group of proteins for signal peptide containing sequences, using SignalP program. Additionally, the transmembrane regions were detected using the TMHMM program. Eight putative extracellular proteins were identified with DnaJ annotation, signal peptide and without transmembrane regions. Finally, nuclear localization signals were predicted using NLS mapper software.. Two of the eight proteins obtained high prediction values ​​for nuclear localization sites (g14296 and g10686).

Previous studies have identified effectors with similar characteristics to the candidates found in this work. The MiISE6 effector of Meloidogyne incognita has a signal peptide, an OGFr_N domain, and two NLS motifs to target the nucleus and facilitate parasitism in Arabidopsis (Shi et al., 2018). White, Potnis, Jones, & Koebnik (2009) determined that the conserved C-terminal portion of the TAL effectors of the genus Xanthomonas contains a nuclear localization signal (NLS) motifs which are essential for pathogen virulence and effects associated with the symptoms of the disease in the host. Likewise, Mueller et al. (2008) reported in Ustilago maydis secreted effector proteins, which also have nuclear localization signals (NLS). The functional analysis of the effector PsCRN63 from Phytophthora sojae showed that this is an inducer of cell death in the host which is secreted and has nuclear localization signals (Liu et al., 2011). These examples suggest that the presence of secretion signal and NLS characterize important effectors in different plant parasites.

In conclusion, 2 M. incognita proteins with characteristics frequently observed in effectors were identified. These proteins could be secreted from the nematode during infection and then transported to the host nucleus to alter their cellular processes and facilitate the infection. It should be noted that the effectors of the DnaJ family have not been previously reported in a parasitic plant nematodes. This work constitutes a basis for future in-plant studies and provides possible targets for nematode control.

The yellowfin tuna (Thunnus albacares) is a bony fish belonging to the Scombridae family. It is found on tropical and subtropical pelagic waters around the world. Thunnus albacares, particularly, is a species of high economic, ecologic and social value as it supports a relevant sector of the fishery industry [1]. Therefore, ensure an adequate management of this resource is necessary to maintain both sustainability and commercial trade [2][3]. Moreover, as an apex predator, the conservation of the yellowfin tuna is relevant for the preservation of healthy marine ecosystems and prevent the loss of this species [4]. However, to date, multiple mismatches between management units and biological populations have been reported in the case of the yellowfin tuna in different regions of the world. These mismatches put in danger the correct management of the marine resource [5][6]. Specifically, in the Eastern Pacific Ocean, previous studies have suggested genetic differences between northern and equatorial samples [7][8]. Thus, the objective established in the present study was to characterize the genetic diversity and population structure of Thunnus albacares in a section of the Eastern Pacific. We collected 630 samples from artisanal fisheries of Ecuador and Mexico in three distinct years (2015-2017). The samples were molecularly characterized using 18 microsatellite loci. The expected heterozygosity index (H_E = 0.85) together with an allelic richness per locus of 18.40 showed high genetic diversity in the sample analyzed. No population structure was found, although low genetic differences were found between samples from Mexico and Ecuador (F_ST < 0.02). The low genetic differentiation found was statistically significant (P < 0.05). Low levels of relatedness, as well as the detection of first generation migrants, suggest continuous gene flow in the section of the Eastern Pacific Ocean analyzed. The results support the concordance between one biological population and the actual management unit administrated by the Inter-American-Tropical-Tuna-Commission (IATTC). However, model-based clustering STRUCTURE results also show a slight difference in the composition of the genetic pools of northern and equatorial samples. Additionally, differences between the genetic pool of samples collected in the same region in different years suggest temporal changes in allelic frequencies or the dependence of the collection on fishery effort. It is recommended to continue the monitoring of T. albacares in the Eastern Pacific Ocean, especially taking into account the actual diminish of tuna populations worldwide [9]. Future studies should consider the inclusion of samples from higher latitudes where geographical barriers like the California and Humboldt [10] cold currents occur. In addition, new approaches such as second-generation sequencing analyze a greater proportion of the genome and they can provide new information that contributes to the development of better management plants for T. albacares in the fishery industry [11].

Graphical Abstract

Abstract

In continuation of study of 17 metal oxide nanoparticles (MNPs) cytotoxicity to E. coli after 2 hrs. exposure under dark conditions, we have evaluated cytotoxicity of additional 8 MNPs maintaining equivalent experiemental conditions following the literature (1). The overall cytotoxicity ranking of these additional MNPs according to LC₅₀ values in descending order is Er₂O₃, Gd₂O₃, CeO₂, Co₂O₃, Mn₂O₃, Co₃O₄, Fe₃O4/WO₃. The in vitro study clearly suggested that Er₂O₃ and Gd₂O₃ are the most toxic among all 25 MNPs under the mentioned experiemental conditions. The present experiemental data is one of the biggest available nano metal oxide cytotoxicity data for E. coli at present time performed under similar experimental conditions and same laboratory. Therefore, the obtained data is significant in terms of OECD principle 1 of defined endpoint to develop statistically acceptable and predictive in silico models. As two MNPs, WO₃ and Fe₃O₄ did not show toxicity even at 2000 ppm, the highest tested concentration in this study we can’t quantify the toxicity data for these two specific MNPs. On the other hand, Er₂O₃ and Gd₂O₃ showed significantly higher toxicity value compare to remaining metal oxides which make them influential data points for the in silico studies. In this background, we have considered a comprehensive approach that includes a total of seven classification and machine learning algorithms i.e. linear discriminant analysis (LDA), naïve bayes, multinomial logistic regression, Sequential minimal optimization (SMO), AdaBoost, J48 and random forest to model all 25 MNPs to understand and identify the major mechanism for such toxicities (2,3) without excluding any MNPs. To correlate the toxicity employing the in silico tools, we have employed 1^st (4) and 2^nd (5) generation periodic table descriptors developed by us which can be computed in no time without any sophisticated computing facilities. Among the seven models, the LDA based model emerged as the best model considering goodness-of-fit and prediction capability checked on training (N_Training=17) and test set (N_Test=8). The electronegativity count of oxygen and the core environment of metal defined by the ratio of the number of core electrons to the number of valence electrons showed positive contributions towards toxicity. The identification of these molecular descriptors may be beneficial in explaining the mechanisms of nanotoxicity and for predicting the environmental risk associated with release of the MNPs. The developed models can be resourcefully employed for environmental risk assessment tools for the E. coli for any new/untested MNPs along with the influence in the future design and manufacture of safe nanomaterials.

References

1. T. Puzyn, B. Rasulev, A. Gajewicz, X. Hu, T.P. Dasari, A. Michalkova, H.M. Hwang, A. Toropov, D. Leszczynska, J. Leszczynski, Using nano-QSAR to predict the cytotoxicity of metal oxide nanoparticles, Nat. Nanotechnol. 2011, 6, 175-178.
2. Weka 3: Machine Learning Software in Java (https://www.cs.waikato.ac.nz/ml/weka/)
3. DTC Lab Software Tools available at https://sites.google.com/site/dtclabdc/
4. S. Kar, A. Gajewicz, T. Puzyn, K. Roy, J. Leszczynski, Periodic table-based descriptors to encode cytotoxicity profile of metal oxide nanoparticles: A mechanistic QSTR approach, Ecotoxicol. Environ. Saf. 2014, 107, 162-169.
5. P. De, S. Kar, K. Roy, J. Leszczynski, Second Generation Periodic Table Based Descriptors to Encode Toxicity of Metal Oxide Nanoparticles to Multiple Species: QSTR Modeling for Exploration of Toxicity Mechanisms. Environ. Sci.: Nano, 2018, 5, 2742-2760.

Acknowledgement:

Authors are thankful to the National Science Foundation (NSF/CREST HRD-1547754 and NSF/RISE HRD-1547836) for financial support.

The use of Jamaican extract (Hibiscus sabdariffa) in different concentrations (2, 4 and 6%) as a natural antioxidant in the preparation of sausages allows us to offer healthy alternatives as a sausage preservative. Due to its composition, meat and fat present several alteration phenomena, among which the oxidation or rancidity produced by exposure to air stands out, which causes losses in trade and industrialization. The proper treatment of Hibiscus sabdariffa extract was determined by bromatological, sensory and evaluation of antioxidant activity over time. The sausage was made with pork meat and fat, mainly as raw materials to be ground and mixed into a thick paste along with the other ingredients. The results determined that the 4% addition of Hibiscus sabdariffa extract provides acceptable bromatological and sensory characteristics. The evaluation of antioxidant activity determined that the 4% incorporated product provides better conservation qualities, a result that corroborates that of the sensory evaluation in which the product presents stable appearance and acceptable coloring with the same treatment. The bromatological analysis presents variations in the concentrations of protein, fat, water, carbohydrates, minerals, when compared the results of day 1 versus day 30 showed that the time traveled directly influences the results with a reduction of each component by dehydration in the elapsed time.

Linear, quadratic, cubic, compound, exponential and logistic regression models were used to evaluate the temperature and pH dynamics of a solid-state fermented (SSF) cocoa shell (Theobroma cacao L.) treated with natural yogurt for use in animals. For this purpose, temperature and pH were checked in silage samples of 0, 1, 4, 8, 15 and 30 days. For the processing of the data, the statistical package SPSS version 22 was used. The behavior of the two variables were adjusted to a cubic model: temperature (R² = 0.88; Y = 23.8145630348868 + 0.188*X - 0.022*X² + 0.00054*X³) and pH (R² = 0.95; Y = 5.543420742606556 - 0.3620949736293026*X + 0.02686662258086579*X² - 0.0005405196051196668*X³). In conclusion, the temperature and pH behavior were adjusted to a non-linear cubic model, and were kept within the optimal ranges for the production of SSF of optimum quality for use in animals.