Application of the solid-state fermentation process and its variations in PHA production: a review.

Solid-state fermentation (SSF) is a type of fermentation process with potential to use agro-industrial by-products as a carbon source. Nonetheless, there are few studies evaluating SSF compared to submerged fermentation (SmF) to produce polyhydroxyalkanoates (PHAs). Different methodologies are available associating the two processes. In general, the studies employ a 1st step by SSF to hydrolyze the agro-industrial by-products used as a carbon source, and a 2nd step to produce PHA that can be carried out by SmF or SSF. This paper reviewed and compared the different methodologies described in the literature to assess their potential for use in PHA production. The studies evaluated showed that highest PHA yields (86.2% and 82.3%) were achieved by associating SSF and SmF by Cupriavidus necator. Meanwhile, in methodologies using only SSF, Bacillus produced the highest yields (62% and 56.8%). Since PHA (%) does not necessarily represent a higher production by biomass, the productivity parameter was also compared between studies. We observed that the highest productivity results did not necessarily represent the highest PHA (%). C. necator presented the highest PHA yields associating SSF and SmF, however, is not the most suitable microorganism for PHA production by SSF. Concomitant use of C. necator and Bacillus is suggested for future studies in SSF. Also, it discusses the lack of studies on the association of the two fermentation methodologies, and on the scaling of SSF process for PHA production. In addition to demonstrating the need for standardization of results, for comparison between different methodologies.

The addition of residue from pruning of yerba mate (Ilex paraguariensis) in laying hens modulates fatty acid profile and incorporates chlorogenic acid in the egg.

This study aimed to determine whether the addition of Ilex paraguariensis residue to the basal diet of laying hens improves bird health, production, fatty acids, egg quality, and transfer of chlorogenic acids to the egg. One hundred twenty birds of the Hy-line lineage were used, divided into five groups with six replicates and four birds per replicate, as follows: T0 (control group, without IPPR supplementation), T0.5 (basal diet plus 0.5% of IPPR), T1 (basal diet plus 1% IPPR), T1.5 (basal diet plus 1.5% IPPR), and T2 (basal diet plus 2% IPPR), for two consecutive cycles of 21 days each. The productive performance of the birds, physicochemical composition of eggs, fatty acid profile, the concentration of chlorogenic acids in eggs, leukocyte count, serum biochemistry, oxidant and antioxidant status, total bacterial count in eggs, and feces were evaluated at 21 and 42 days of the experiment. The addition of IPPR to the birds' diet no-change eggs production and feed conversion. In the egg yolks of birds that consumed IPPR, there were chlorogenic acids. There was a reduction in the total bacterial count in the feces and eggshells of the birds that received 2% of the IPPR. IPPR intake also reflected a reduction in total saturated fatty acid levels and increased monounsaturated fatty acids. These findings suggest that IPPR is a compelling alternative for laying hens; however, its ingestion also has negative effects that are discussed in below. Chlorogenic acids in the egg are desirable, as these phenolics have nutraceutical effects in humans.

Development of ELISA Using Recombinant Proteins for the Diagnosis of Mycoplasma hyopneumoniae Infection.

In order to develop a more sensitive and reliable method for detection of serum antibodies against Mycoplasma hyopneumoniae infection in pigs, six recombinant proteins of M. hyopneumoniae (P102, P95, P46, P97 like, Lppt, and hypothetical P987) were used for the standardization of an indirect enzyme-linked immunosorbent assay (ELISA). The proteins were evaluated against 50 sera of the specific pathogen-free and 50 sera of pigs with lesions suggestive of infection. The sensitivity was 88%, 86%, 78%, 74%, 66%, and 60% for the proteins P102, P95, P46, P97 like, Lppt, and hypothetical protein P987, respectively. Moreover, the proteins were used to establish the seroprevalence in two different commercial herds (254 sera pigs from farm considered free of M. hyopneumoniae and 246 from farm with clinical signs of enzootic pneumonia and positive serology for M. hyopneumoniae) and the positive rate was 65.2% for P95, 54.6% for P102, 40.2% for P46, 37.2% for P97 like, 17.4% for the hypothetical P987, and 14% for Lppt protein. In addition, the ELISA with six recombinant proteins was compared to commercial HerdCheck kit using 118 random pig sera samples and the results showed that ELISA with recombinant proteins were more sensitive than the commercial test. These data show that the recombinant proteins P95 and P102 are potential targets to be used in diagnostic tests to detect antibodies against M. hyopneumoniae. Although more studies are necessary, this study provides insights that these recombinant proteins can be useful in epidemiological investigations and as potential biomarkers in differentiating infected animals from those vaccinated.

Multivariate optimization results in an edible extract from Ilex paraguariensis unexplored residues with a high amount of phenolic compounds.

Ilex paraguariensis A. St. Hil. plants are used for the preparation of food and drinks which are widely consumed worldwide. During the harvest season of these plants, 2-5 ton hec-1 of agricultural residue is generated, which remains underutilized. Therefore, this study aimed to obtain an edible extract with high content of bioactive compounds and antimicrobial properties from the agricultural residue of I. paraguariensis for industrial use in food applications. The extraction conditions were optimized through a multivariate experimental design using ethanol:water. The extracted compounds were characterized by HPLC-ESY-QTOF-MS. In the optimal extraction conditions, 55 compounds were extracted, including 8 compounds that were not previously reported in I. paraguariensis. The method proved to be simple, fast, economical and environmentally friendly, with the use of green solvents. This optimization allowed for the extraction of 15.07 g of phenolic compounds per 100 g of residue. The extract showed high antioxidant activity and the capacity to inhibit Staphylococcus aureus. Results indicate that it is possible to obtain an edible extract with a high content of bioactive compounds, particularly phenolic compounds, from the I. paraguariensis residue, which has high prospects for the valorization of unexplored natural resources.

Lipid oxidation and sensory characterization of Omega-3 rich buffalo burgers enriched with chlorogenic acids from the mate (Ilex paraguariensis) tree harvesting residues.

A freeze-dried extract from the bark of mate branches (BMBE) containing high chlorogenic acids (CGA) content (30 g 100 g-1) was produced. Then, chia oil was mixed with 7.5% BMBE and sonicated for 0, 10, and 20 min. Chia oil with or without the addition of BMBE was hydrogelled and used to produce buffalo burgers with 50% reduction in animal fat. CGA levels and the nutritional, oxidative, and sensory properties of the burgers were analyzed. A reduction of ~30% fat and an increase above 60% PUFA/SFA ratio was observed for the reformulated raw and cooked burgers. In addition, the Omega-6/Omega-3 PUFA ratio of the burgers decreased from 20.8 (raw) and 31.9 (cooked) to values lower than 2. The addition of BMBE enriched the burgers with CGA, preventing an increase in lipid oxidation caused by chia oil. The addition of BMBE-enriched hydrogelled chia oil not subjected to sonication did not affect the sensory properties of the burgers.

Characterization and quantification of bioactive compounds from Ilex paraguariensis residue by HPLC-ESI-QTOF-MS from plants cultivated under different cultivation systems.

Ilex paraguariensis is a perennial plant used in the production of mate tea, "chimarrão" and "tererê," cosmetics, and other food products. Its leaves are harvested every 12 or 18 months. Approximately 2 to 5 tons of residue are generated per hectare during the harvest. The bioactive composition of this residue has not been characterized to date. Therefore, this paper presents for the first time, the simultaneous characterization of the bioactive compounds of the leaves, thin branches, and thick branches (residue) from I. paraguariensis grown under two cultivation systems: "full sun" and "shaded." The identification and quantification of the compounds was performed using high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometer (HPLC-ESI-QTOF-MS). Consequently, 35 compounds were identified. The average dry weight of phenolic compounds in the residue was 4.1 g/100 g, whereas that in the bark of the residue was 12.9 g/100 g, which was similar to the content found in leaves. The same compounds were identified in the two cultivation systems but with a difference in their contents. While the "full sun" cultivation had a higher content of phenolic acids, the "shaded" cultivation had a higher content of flavonoids and saponins. It was found that the I. paraguariensis residue, particularly the bark, is rich in bioactive compounds, such as quinic, 3-caffeoylquinic, 5-cafefoylquinic, 3,5-dicaffeoylquinic, and 4,5-dicaffeoylquinic acids as well as rutin, and their contents vary according to the cultivation system. Therefore, this residue is an underutilized natural resource with a potential for industrial applications. PRACTICAL APPLICATION: Yerba mate producers will be able to choose the best cultivation system ("full sun" or "shaded") to increase the content of bioactive compounds. New products may be developed with yerba mate residue due to its high concentration of compounds that are beneficial to the human health. New destinations may be applied to yerba mate residue from the harvest pruning, adding commercial value to this unexplored natural resource.

The CYP707A Gene Family in Strawberry (Fragaria × ananassa)

Abstract Abscisic acid (ABA) is a plant hormone that plays several roles in plant development. The de novo synthesis and the reversible inactivation of ABA have been largely described in the literature; however, the degradation of ABA, promoted by the enzymes Abscisic Acid 8'-Hydroxylase, encoded by the CYP707A gene family, is still poorly elucidated. Strawberry (Fragaria x ananassa) has been used as a model to study the ABA-dependent maturation process of non-climacteric fruits, and the ABA-dependent response to abiotic stress. However, the CYP707A genes from this species have not been fully described and characterized. In this perspective, FaCYP707A sequences were identified from strawberry fruit transcriptome and several structural and comparative genomic analyzes were performed. Moreover, the expression of the FaCYP707A sequences identified was investigated in fruits under salt stress and ABA application. Four putative FaCYP707A were identified and the structural analysis confirmed the identity of three of them. The phylogenetic analysis allowed to determine their homologous in other plant species and to predict their evolutionary history; and the expression profile of the FaCYP707As demonstrated that FaCYP707A3 seems to be involved in the response against salt stress in an ABA-dependent manner. Moreover, the interaction network analysis pointed out proteins involved in the ABA metabolism, heavy metal homeostasis and detoxification, and cell wall dissemble. This study characterized for the first time the CYP707A gene family in F. ananassa; this information will guide future studies in order to develop biofortified fruits and stress tolerant plants.

Genome-wide identification, and characterization of the CDPK gene family reveal their involvement in abiotic stress response in Fragaria x ananassa.

Calcium-dependent protein kinases (CDPKs) are encoded by a large gene family and play important roles against biotic and abiotic stresses and in plant growth and development. To date, little is known about the CDPK genes in strawberry (Fragaria x ananassa). In this study, analysis of Fragaria x ananassa CDPK gene family was performed, including gene structures, phylogeny, interactome and expression profiles. Nine new CDPK genes in Fragaria x ananassa were identified based on RNA-seq data. These identified strawberry FaCDPK genes were classified into four main groups, based on the phylogenetic analysis and structural features. FaCDPK genes were differentially expressed during fruit development and ripening, as well as in response to abiotic stress (salt and drought), and hormone (abscisic acid) treatment. In addition, the interaction network analysis pointed out proteins involved in the ABA-dependent response to plant stress via Ca2+ signaling, especially RBOHs. To our knowledge, this is the first report on CDPK families in Fragaria x ananassa, and it will provide valuable information for development of biofortified fruits and stress tolerant plants.

Transcriptome Analysis Identifies Candidate Target Genes Involved in Glyphosate-Resistance Mechanism in Lolium multiflorum.

Italian ryegrass (Lolium multiflorum; LOLMU) is one of the most troublesome weeds in temperate regions in the world. This weed species interfere with wheat, corn, rye, and oat, causing significant crop yield losses. This species has evolved glyphosate resistance, making it difficult to control. The mechanisms of glyphosate resistance are still unknown, and an understanding thereof will favor the development of new strategies of management. The present study is the first transcriptome study in LOLMU using glyphosate-resistant and -sensitive biotypes, aiming to identify and to provide a list of the candidate target genes related to glyphosate resistance mechanism. The transcriptome was assembled de novo, producing 87,433 contigs with an N50 of 740 bp and an average length of 575 bp. There were 92 and 54 up- and down-regulated genes, respectively, in the resistant biotype, while a total of 1683 were differentially expressed in the sensitive biotype in response to glyphosate treatment. We selected 14 highly induced genes and seven with repressed expression in the resistant biotype in response to glyphosate. Of these genes, a significant proportion were related to the plasma membrane, indicating that there is a barrier making it difficult for glyphosate to enter the cell.

The postharvest ripening of strawberry fruits induced by abscisic acid and sucrose differs from their in vivo ripening.

In this study, we compared the chemical composition of strawberry (Fragaria × ananassa) fruits that were ripened in vivo (attached to plant) to those ripened during postharvest storage. The effects of the application of abscisic acid (ABA) and sucrose on the postharvest ripening were also evaluated. The results suggested that the postharvest ripening process was dependent on the signal triggered by ABA and differed from in vivo ripening, resulting in fruits with altered chemical composition and firmness. The application of sucrose in unripe strawberries resulted in the induction of ripening, which is dependent on ABA and its derivatives. This induction was more pronounced during the first days of storage and associated with the application of mannitol rather than water, suggesting that mannitol negatively regulated the postharvest strawberry ripening. These results provide further insights into the role of ABA and sucrose in the regulation of postharvest ripening of strawberry.

Untargeted metabolomics of strawberry (Fragaria x ananassa 'Camarosa') fruit from plants grown under osmotic stress conditions.

BACKGROUND: Plants activate defense mechanisms to cope with adverse environmental conditions, leading to the accumulation and / or depletion of general and specialized metabolites. In this study, a multiplatform untargeted metabolomics strategy was employed to evaluate metabolic changes in strawberry fruit of cv. Camarosa grown under osmotic stress conditions. Liquid chromatography-mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) data from strawberries grown under two water-deficit conditions, irrigated at 95% crop evapotranspiration (ETc) and 85% ETc, and one excess salt condition with a 80 mmol L-1 NaCl solution, were analyzed to determine treatment effects on fruit metabolism. RESULTS: Multivariate principal component analysis, orthogonal projections to latent structures - discriminant analysis (OPLS-DA), and univariate statistical analyses were applied to the data set. While multivariate analyses showed group separation by treatment, T-tests and fold change revealed 12 metabolites differentially accumulated in strawberries from different treatments - among them phenolic compounds, glycerophospholipids, phytosterols, carbohydrates, and an aromatic amino acid. CONCLUSION: Untargeted metabolomic analysis allowed for the annotation of compounds differentially accumulated in strawberry fruit from plants grown under osmotic stress and non-stressed plants. The metabolic disturbance in plants under stress involved metabolites associated with the inhibition of reactive oxygen species and cell-wall and membrane lipid biosynthesis, which might serve as osmotic stress biomarkers. © 2019 Society of Chemical Industry.

Transcriptomic Analysis Identifies New Non-Target Site Glyphosate-Resistance Genes in Conyza bonariensis.

Conyza bonariensis (hairy fleabane) is one of the most problematic and widespread glyphosate-resistant weeds in the world. This highly competitive weed species significantly interferes with crop growth and substantially decreases crop yield. Despite its agricultural importance, the molecular mechanisms of glyphosate resistance are still unknown. The present RNA-Seq study was performed with the goal of identifying differentially expressed candidate transcripts (genes) related to metabolism-based non-target site glyphosate resistance in C. bonariensis. The whole-transcriptome was de novo assembled from glyphosate-resistant and -sensitive biotypes of C. bonariensis from Southern Brazil. The RNA was extracted from untreated and glyphosate-treated plants at several timepoints up to 288 h after treatment in both biotypes. The transcriptome assembly produced 90,124 contigs with an average length of 777 bp and N50 of 1118 bp. In response to glyphosate treatment, differential gene expression analysis was performed on glyphosate-resistant and -sensitive biotypes. A total of 9622 genes were differentially expressed as a response to glyphosate treatment in both biotypes, 4297 (44.6%) being up- and 5325 (55.4%) down-regulated. The resistant biotype presented 1770 up- and 2333 down-regulated genes while the sensitive biotype had 2335 and 2800 up- and down-regulated genes, respectively. Among them, 974 up- and 1290 down-regulated genes were co-expressed in both biotypes. In the present work, we identified 41 new candidate target genes from five families related to herbicide transport and metabolism: 19 ABC transporters, 10 CYP450s, one glutathione S-transferase (GST), five glycosyltransferases (GT), and six genes related to antioxidant enzyme catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD). The candidate genes may participate in metabolic-based glyphosate resistance via oxidation, conjugation, transport, and degradation, plus antioxidation. One or more of these genes might 'rescue' resistant plants from irreversible damage after glyphosate treatment. The 41 target genes we report in the present study may inform further functional genomics studies, including gene editing approaches to elucidate glyphosate-resistance mechanisms in C. bonariensis.

Carotid-artery cochlear dehiscence

Changes in course of the internal carotid artery (ICA) are uncommon, and dehiscence of the carotid canal with cochlea may occur. A 48-year-old female individual with pulsatile tinnitus. No other otologic symptoms observed. Otolaryngologic examination and audiometric test with normal results. Computed tomography (CT) scan of the mastoid bones showed dehiscence of cochlea with ICA on the right side. An option for monitored observation was made after analysis of the risks and undefined results of surgery. Patient maintained clinical and audiometric profile. Carotid-artery cochlear dehiscence is a condition that must be known, remembered and investigated, because it may mimic other otologic pathologies. Knowledge about it prevents serious complications that can be difficult to reverse.

Presence of genes associated with adhesion, invasion, and toxin production in Campylobacter jejuni isolates and effect of temperature on their expression.

The aims of this study were to evaluate the presence of genes associated with adhesion (cadF), invasion (ciaB), and cytotoxin production (cdtA, cdtB, and cdtC) among Campylobacter jejuni isolates from a poultry slaughterhouse and to investigate the effect of different temperatures on the expression of these virulence-associated genes. A total of 88 C. jejuni isolates from cecum, liver, chicken carcasses, chilled water, and scalding water were submitted to PCR assay for detection of virulence genes. Representative isolates were selected for gene expression evaluation at 37 and 42 °C, according to their virulence gene profile and genotypic typing. All C. jejuni isolates carried the five virulence-associated genes, which play an important role in the infectious process. Differential gene expression by RT-qPCR was observed among C. jejuni isolates at 37 and 42 °C. The expression levels at 37 °C showed upregulation of the ciaB, cdtA, cdtB, and cdtC genes in five isolates, with the exception of ciaB for isolate 4. At 42 °C, upregulation was observed for ciaB and cdtC, cdtA and cdtB, and cadF in four, three, and two isolates, respectively. The C. jejuni isolates expressed the virulence genes evaluated, and the expression is gene- and isolate-dependent and varied according the temperature.

Transcriptome analysis of strawberry (Fragaria × ananassa) fruits under osmotic stresses and identification of genes related to ascorbic acid pathway.

Strawberry (Fragaria ananassa Duch.) is an economically important fruit with a high demand owing to its good taste and medicinal properties. However, its cultivation is affected by various biotic and abiotic stresses. Plants exhibit several intrinsic mechanisms to deal with stresses. In the case of strawberry, the mechanisms highlighting the response against these stresses remain to be elucidated, which has hampered the efforts to develop and cultivate strawberry plants with high yield and quality. Although a virtual reference genome of F. ananassa has recently been published, there is still a lack of information on the expression of genes in response to various stresses. Therefore, to provide molecular information for further studies with strawberry plants, we present the reference transcriptome dataset of F. ananassa, assembled and annotated from deep RNA-Seq data of fruits cultivated under salinity and drought stresses. We also systematically arranged a series of transcripts differentially expressed during these stresses, with an emphasis on genes related to the accumulation of ascorbic acid (AsA). Ascorbic acid is the most potent antioxidant present in these fruits and highly considered during biofortification. A comparison of the expression profile of these genes by RT-qPCR with the content of AsA in the fruits verified a tight regulation and balance between the expression of genes, from biosynthesis, degradation and recycling pathways, resulting in the reduced content of AsA in fruits under these stresses. These results provide a useful repertoire of genes for metabolic engineering, thereby improving the tolerance to stresses.

ABA-dependent salt and drought stress improve strawberry fruit quality.

Strawberry crop is very sensitive to osmotic stress conditions. We investigated the effect of the stress induced by mild drought (DS) and salt (SS) stresses, on molecular, physiological, and metabolic processes in the strawberry crop (Fragaria ananassa), cv. Camarosa. The results showed that the fruit yield was not affected. Mild DS and SS also resulted in an increased content of phenolics, anthocyanins, and l-ascorbic acid, and an increased antioxidant activity. These effects were accompanied by increased levels of ABA and its derivatives (phaseic and dehydrophasic acids), alongside the upregulation of several genes involved on their synthesis. Therefore, the results obtained in this study suggest that mild DS and SS improve the functional quality of strawberry fruits through and ABA-dependent mechanism.

Validation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in strawberry fruits using different cultivars and osmotic stresses.

The increasing demand of strawberry (Fragaria×ananassa Duch) fruits is associated mainly with their sensorial characteristics and the content of antioxidant compounds. Nevertheless, the strawberry production has been hampered due to its sensitivity to abiotic stresses. Therefore, to understand the molecular mechanisms highlighting stress response is of great importance to enable genetic engineering approaches aiming to improve strawberry tolerance. However, the study of expression of genes in strawberry requires the use of suitable reference genes. In the present study, seven traditional and novel candidate reference genes were evaluated for transcript normalization in fruits of ten strawberry cultivars and two abiotic stresses, using RefFinder, which integrates the four major currently available software programs: geNorm, NormFinder, BestKeeper and the comparative delta-Ct method. The results indicate that the expression stability is dependent on the experimental conditions. The candidate reference gene DBP (DNA binding protein) was considered the most suitable to normalize expression data in samples of strawberry cultivars and under drought stress condition, and the candidate reference gene HISTH4 (histone H4) was the most stable under osmotic stresses and salt stress. The traditional genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 18S (18S ribosomal RNA) were considered the most unstable genes in all conditions. The expression of phenylalanine ammonia lyase (PAL) and 9-cis epoxycarotenoid dioxygenase (NCED1) genes were used to further confirm the validated candidate reference genes, showing that the use of an inappropriate reference gene may induce erroneous results. This study is the first survey on the stability of reference genes in strawberry cultivars and osmotic stresses and provides guidelines to obtain more accurate RT-qPCR results for future breeding efforts.

Micronutrient and functional compounds biofortification of maize grains.

Maize, in addition to being the main staple food in many countries, is used in the production of hundreds of products. It is rich in compounds with potential benefits to health, such as carotenoids, phenolic compounds, vitamin E, and minerals that act as cofactors for antioxidant enzymes. Many of these compounds have been neglected thus far in the scientific literature. Nevertheless, deficiencies in the precursors of vitamin A and some minerals, such as iron and zinc, in maize, in association with the great genetic variability in its cultivars and our genomic, transcriptomic, and metabolomic knowledge of this species make targeted biofortification strategies for maize promising. This review discusses the potential of the main microconstituents found in maize with a focus on studies aimed at biofortification.