Characterization of Aspergillus species on Brazil nut from the Brazilian Amazonian region and development of a PCR assay for identification at the genus level.

BACKGROUND: Brazil nut is a protein-rich extractivist tree crop in the Amazon region. Fungal contamination of shells and kernel material frequently includes the presence of aflatoxigenic Aspergillus species from the section Flavi. Aflatoxins are polyketide secondary metabolites, which are hepatotoxic carcinogens in mammals. The objectives of this study were to identify Aspergillus species occurring on Brazil nut grown in different states in the Brazilian Amazon region and develop a specific PCR method for collective identification of member species of the genus Aspergillus. RESULTS: Polyphasic identification of 137 Aspergillus strains isolated from Brazil nut shell material from cooperatives across the Brazilian Amazon states of Acre, Amapá and Amazonas revealed five species, with Aspergillus section Flavi species A. nomius and A. flavus the most abundant. PCR primers ASP_GEN_MTSSU_F1 and ASP_GEN_MTSSU_R1 were designed for the genus Aspergillus, targeting a portion of the mitochondrial small subunit ribosomal RNA gene. Primer specificity was validated through both electronic PCR against target gene sequences at Genbank and in PCR reactions against DNA from Aspergillus species and other fungal genera common on Brazil nut. Collective differentiation of the observed section Flavi species A. flavus, A. nomius and A. tamarii from other Aspergillus species was possible on the basis of RFLP polymorphism. CONCLUSIONS: Given the abundance of Aspergillus section Flavi species A. nomius and A. flavus observed on Brazil nut, and associated risk of mycotoxin accumulation, simple identification methods for such mycotoxigenic species are of importance for Hazard Analysis Critical Control Point system implementation. The assay for the genus Aspergillus represents progress towards specific PCR identification and detection of mycotoxigenic species.

Minimal Bacterial Cell JCVI-syn3B as a Chassis to Investigate Interactions between Bacteria and Mammalian Cells.

Mycoplasmas are atypical bacteria with small genomes that necessitate colonization of their respective animal or plant hosts as obligate parasites, whether as pathogens, or commensals. Some can grow axenically in specialized complex media yet show only host-cell-dependent growth in cell culture, where they can survive chronically and often through interactions involving surface colonization or internalization. To develop a mycoplasma-based system to identify genes mediating such interactions, we exploited genetically tractable strains of the goat pathogen Mycoplasma mycoides (Mmc) with synthetic designer genomes representing the complete natural organism (minus virulence factors; JCVI-syn1.0) or its reduced counterpart (JCVI-syn3B) containing only those genes supporting axenic growth. By measuring growth of surviving organisms, physical association with cultured human cells (HEK-293T, HeLa), and induction of phagocytosis by human myeloid cells (dHL-60), we determined that JCVI-syn1.0 contained a set of eight genes (MMSYN1-0179 to MMSYN1-0186, dispensable for axenic growth) conferring survival, attachment, and phagocytosis phenotypes. JCVI-syn3B lacked these phenotypes, but insertion of these genes restored cell attachment and phagocytosis, although not survival. These results indicate that JCVI-syn3B may be a powerful living platform to analyze the role of specific gene sets, from any organism, on the interaction with diverse mammalian cells in culture.

Protocol for the establishment of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells.

Serine integrases (Ints) are a family of site-specific recombinases (SSRs) encoded by some bacteriophages to integrate their genetic material into the genome of a host. Their ability to rearrange DNA sequences in different ways including inversion, excision, or insertion with no help from endogenous molecular machinery, confers important biotechnological value as genetic editing tools with high host plasticity. Despite advances in their use in prokaryotic cells, only a few Ints are currently used as gene editors in eukaryotes, partly due to the functional loss and cytotoxicity presented by some candidates in more complex organisms. To help expand the number of Ints available for the assembly of more complex multifunctional circuits in eukaryotic cells, this protocol describes a platform for the assembly and functional screening of serine-integrase-based genetic switches designed to control gene expression by directional inversions of DNA sequence orientation. The system consists of two sets of plasmids, an effector module and a reporter module, both sets assembled with regulatory components (as promoter and terminator regions) appropriate for expression in mammals, including humans, and plants. The complete method involves plasmid design, DNA delivery, testing and both molecular and phenotypical assessment of results. This platform presents a suitable workflow for the identification and functional validation of new tools for the genetic regulation and reprogramming of organisms with importance in different fields, from medical applications to crop enhancement, as shown by the initial results obtained. This protocol can be completed in 4 weeks for mammalian cells or up to 8 weeks for plant cells, considering cell culture or plant growth time.

Exploring the Proteomic Profile of Soybean Bran: Unlocking the Potential for Improving Protein Quality and Quantity.

Soybean is a rich source of vegetal protein for both animal and human consumption. Despite the high levels of protein in soybean seeds, industrial processing to obtain soybean bran significantly decreases the final protein content of the byproducts. To overcome this problem, cultivars with higher protein contents must be developed. However, selecting the target proteins is difficult because of the lack of information on the proteome profile of soybean bran. Therefore, this study obtained the comparative proteomic profiles of both natural coatless seeds and defatted bran from an elite tropical-soybean cultivar. Thus, their extracts were characterized using LC-MS/MS and a total of 550 proteins were identified. Among these, 526 proteins were detected in coatless seeds and 319 proteins in defatted bran. Moreover, a total of 139 proteins were identified as presenting different levels of content in coatless seeds and defatted bran. Among them, only 46 were retained after the seed processing. These proteins were clustered in several important metabolic pathways, such as amino-acid biosynthesis, sugar biosynthesis, and antioxidant activity, meaning that they could act as targets for bioactive products or genome editing to improve protein quality and quantity in soybean grains. These findings can enhance our understanding regarding protein robustness for both soybean crops and the commercial bran improvement because target proteins must remain intact after processing and must be bioactive when overexpressed. Overall, the soybean bran proteomic profile was explored for the first time, providing a valuable catalogue of target proteins that can tolerate the industrial process.

Bioengineering of spider silks for the production of biomedical materials.

Spider silks are well known for their extraordinary mechanical properties. This characteristic is a result of the interplay of composition, structure and self-assembly of spider silk proteins (spidroins). Advances in synthetic biology have enabled the design and production of spidroins with the aim of biomimicking the structure-property-function relationships of spider silks. Although in nature only fibers are formed from spidroins, in vitro, scientists can explore non-natural morphologies including nanofibrils, particles, capsules, hydrogels, films or foams. The versatility of spidroins, along with their biocompatible and biodegradable nature, also placed them as leading-edge biological macromolecules for improved drug delivery and various biomedical applications. Accordingly, in this review, we highlight the relationship between the molecular structure of spider silk and its mechanical properties and aims to provide a critical summary of recent progress in research employing recombinantly produced bioengineered spidroins for the production of innovative bio-derived structural materials.

Traditional protocols and optimization methods lead to absent expression in a mycoplasma cell-free gene expression platform.

Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a Mycoplasma bacterium-based CFE system using lysates of the genome-minimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative Mycoplasma capricolum (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of Escherichia coli. Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of in vitro transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease (RNase) activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNase activity yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned E. coli cell-free extracts by degrading ribosomal RNAs, indicating that the mycoplasma cells, even the minimal cell, have a surface-associated RNase activity. However, it is not clear which gene encodes the RNase. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field. Graphical Abstract.

Molecular Dynamics of Synthetic Flagelliform Silk Fiber Assembly.

In order to better understand the relationship between Flagelliform (Flag) spider silk molecular structural organization and the mechanisms of fiber assembly, it was designed and produced the Nephilengys cruentata Flag spidroin analogue rNcFlag2222. The recombinant proteins are composed by the elastic repetitive glycine-rich motifs (GPGGX/GGX) and the spacer region, rich in hydrophilic charged amino acids, present at the native silk spidroin. Using different approaches for nanomolecular protein analysis, the structural data of rNcFlag2222 recombinant proteins were compared in its fibrillar and in its fully solvated states. Based on the results was possible to identify the molecular structural dynamics of NcFlag2222 prior to and after fiber formation. Overal rNcFlag2222 shows a mixture of semiflexible and rigid conformations, characterized mostly by the presence of PPII, ß-turn and ß-sheet. These results agree with previous studies and bring insights about the molecular mechanisms that might driven Flag silk fibers assembly and elastomeric behavior.

Piriform spider silk sequences reveal unique repetitive elements.

Orb-weaving spider silk fibers are assembled from very large, highly repetitive proteins. The repeated segments contain, in turn, short, simple, and repetitive amino acid motifs that account for the physical and mechanical properties of the assembled fiber. Of the six orb-weaver silk fibroins, the piriform silk that makes the attachment discs, which lashes the joints of the web and attaches dragline silk to surfaces, has not been previously characterized. Piriform silk protein cDNAs were isolated from phage libraries of three species: A. trifasciata , N. clavipes , and N. cruentata . The deduced amino acid sequences from these genes revealed two new repetitive motifs: an alternating proline motif, where every other amino acid is proline, and a glutamine-rich motif of 6-8 amino acids. Similar to other spider silk proteins, the repeated segments are large (>200 amino acids) and highly homogenized within a species. There is also substantial sequence similarity across the genes from the three species, with particular conservation of the repetitive motifs. Northern blot analysis revealed that the mRNA is larger than 11 kb and is expressed exclusively in the piriform glands of the spider. Phylogenetic analysis of the C-terminal regions of the new proteins with published spidroins robustly shows that the piriform sequences form an ortholog group.

Spinning gland transcriptomics from two main clades of spiders (order: Araneae)--insights on their molecular, anatomical and behavioral evolution.

Characterized by distinctive evolutionary adaptations, spiders provide a comprehensive system for evolutionary and developmental studies of anatomical organs, including silk and venom production. Here we performed cDNA sequencing using massively parallel sequencers (454 GS-FLX Titanium) to generate ∼80,000 reads from the spinning gland of Actinopus spp. (infraorder: Mygalomorphae) and Gasteracantha cancriformis (infraorder: Araneomorphae, Orbiculariae clade). Actinopus spp. retains primitive characteristics on web usage and presents a single undifferentiated spinning gland while the orbiculariae spiders have seven differentiated spinning glands and complex patterns of web usage. MIRA, Celera Assembler and CAP3 software were used to cluster NGS reads for each spider. CAP3 unigenes passed through a pipeline for automatic annotation, classification by biological function, and comparative transcriptomics. Genes related to spider silks were manually curated and analyzed. Although a single spidroin gene family was found in Actinopus spp., a vast repertoire of specialized spider silk proteins was encountered in orbiculariae. Astacin-like metalloproteases (meprin subfamily) were shown to be some of the most sampled unigenes and duplicated gene families in G. cancriformis since its evolutionary split from mygalomorphs. Our results confirm that the evolution of the molecular repertoire of silk proteins was accompanied by the (i) anatomical differentiation of spinning glands and (ii) behavioral complexification in the web usage. Finally, a phylogenetic tree was constructed to cluster most of the known spidroins in gene clades. This is the first large-scale, multi-organism transcriptome for spider spinning glands and a first step into a broad understanding of spider web systems biology and evolution.

A MaSp2-like gene found in the Amazon mygalomorph spider Avicularia juruensis.

Two unique spidroins are present in the silk of the Amazon mygalomorph spider - Avicularia juruensis (Theraphosidae), and for the first time the presence and expression of a major ampullate spidroin 2-like in Mygalomorphae are demonstrated. Molecular analysis showed the presence of (GA)(n,) poly-A and GPGXX motifs in the amino acid sequence of Spidroin 2, the last being a motif described so far only in MaSp2 and Flag spidroins. Phylogenetic analysis confirmed the previously known orthologous silk gene clusters, and placed this gene firmly within the orbicularian MaSp2 clade. Gene tree-species tree reconciliations show a pattern of multiple gene duplication throughout spider silk evolution, and pinpoint the oldest speciation in which MaSps must have been present in spiders on the mygalomorph-araneomorph split, 240 MYA. Therefore, while not refuting orb weaver monophyly, MaSp2s, and major ampullate silks in general cannot be classified as orbicularian synapomorphies, but have to be considered plesiomorphic for Opisthothelae. The evidence presented here challenges the simplified notion that mygalomorphs spin only one kind of silk, and adds to the suite of information suggesting a pattern of early niche diversification between Araneomorphae and Mygalomorphae. Additionally, mygalomorph MaSp2-like might accommodate mechanical demands arising from the arboreal habitat preference of Avicularia.

A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning.

The extreme strength and elasticity of spider silks originate from the modular nature of their repetitive proteins. To exploit such materials and mimic spider silks, comprehensive strategies to produce and spin recombinant fibrous proteins are necessary. This protocol describes silk gene design and cloning, protein expression in bacteria, recombinant protein purification and fiber formation. With an improved gene construction and cloning scheme, this technique is adaptable for the production of any repetitive fibrous proteins, and ensures the exact reproduction of native repeat sequences, analogs or chimeric versions. The proteins are solubilized in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 25-30% (wt/vol) for extrusion into fibers. This protocol, routinely used to spin single micrometer-size fibers from several recombinant silk-like proteins from different spider species, is a powerful tool to generate protein libraries with corresponding fibers for structure-function relationship investigations in protein-based biomaterials. This protocol may be completed in 40 d.

Prevalência da mastite bovina causada por Prototheca zopfii em rebanhos leiteiros, na regiäo norte do Paraná / Prevalence of bovine mastitis due to Prototheca zopfii in dairy cattle in north Paraná, Brazil

Algas do gênero Prototheca têm sido relacionadas como agente etiológico na ocorrência de mastite bovina, entretanto, nenhum caso foi relatado no estado do Paraná. O presente trabalho objetivou determinar a prevalência desta alga, enquanto agente etiológico nas mastites bovinas, em rebanhos leiteiros, na regiäo norte do Paraná. Foram visitadas 20 propriedades produtoras de leite e realizados exames clínicos e teste do CMT (California Mastitis Test) em todos os animais em lactaçäo. A alga Prototheca zopfii foi isolada de dois animais de uma das propriedades. Todas as cepas isoladas de Prototheca zopfii mostraram-se resistentes aos antimicrobianos testados.