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Serological tests are critical tools in the fight against infectious disease. They detect antibodies produced during an adaptive immune response against a pathogen with an immunological reagent, whose antibody binding characteristics define the specificity and sensitivity of the assay. While pathogen proteins have conveniently served as reagents, their performance is limited by the natural grouping of specific and non-specific antibody binding sites, epitopes. An attractive solution is to build synthetic proteins that only contains pathogen-specific epitopes, which could theoretically reach 100% specificity. However, the genesis of de novo proteins remains a challenge. To address the uncertainty of producing a synthetic protein, we have repurposed the beta barrel of fluorescent proteins into a receptacle that can receive several epitope sequences without compromising its ability to be expressed. Here, two versions of a multiepitope protein were built using the receptacle that differ by their grouping of epitopes specific to the parasite Trypanosoma cruzi, the causative agent for Chagas disease. An evaluation of their performance as the capture reagent in ELISAs showed near-complete agreement with recommended diagnostic protocols. The results suggest that a single assay could be developed for the diagnosis of Chagas disease and that this approach could be applied to other diseases.

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The gut microbiota has a great diversity of genera and species. The most recent studies show an effective participation of the microbiome not only in the modulation and production of virulence factors of bacteria present in the intestine, but also in the signaling and communication between the body's organs. The study models used so far have limitations and do not allow an effective study of identification and quantification of distinct populations of related species. The cellular assays used for the study of biofilm formation and bacterial adhesion are very effective for visualization of samples, but in the case of mixed cultures of species with similar characteristics it is not possible to distinguish. In this study, bacterial transformation was performed by inserting the plasmid containing the green fluorescent protein (GFP) gene, giving the samples the ability to emit green light. After confirmation of the transformation, the samples were used in biofilm formation and bacterial adhesion assays in in vitro epithelial tissues containing more than one bacterial population. It was possible to visualize and identify the different samples with the use of staining and fluorescence microscopy.

A microbiota intestinal possui grande diversidade de gêneros e espécies. Os estudos mais recentes evidenciam uma participação efetiva do microbioma não só na modulação e produção de fatores de virulência de bactérias presentes no intestino como na sinalização e comunicação entre os órgãos do corpo. Os modelos de estudo utilizados até o momento possuem limitações e não permitem um estudo eficaz de identificação e quantificação de populações distintas de espécies relacionadas. Os ensaios celulares utilizados para o estudo de formação de biofilme e adesão bacteriana são bastante eficazes para visualização de amostras, porém no caso de culturas mistas de espécies com características semelhantes não é possível a distinção. Neste estudo foi realizada a transformação bacteriana para a inserção do plasmídeo contendo o gene green fluorescent protein (GFP) conferindo às amostras a capacidade de emitir luz verde. Após a confirmação da transformação, as amostras foram utilizadas em ensaios de formação de biofilme e adesão bacteriana em tecidos epiteliais in vitro contendo mais de uma população bacteriana. Foi possível a visualização e identificação das diferentes amostras com a utilização de coloração e microscopia de fluorescência.

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A green fluorescent protein (GFP)-based whole-cell biosensor (WCB-GFP) for monitoring arsenic (As) was developed in Bacillus subtilis. To this end, we designed a reporter gene fusion carrying the gfpmut3a gene under the control of the promoter/operator region of the arsenic operon (Pars::gfpmut3a) in the extrachromosomal plasmid pAD123. This construct was transformed into B. subtilis 168, and the resultant strain was used as a whole-cell biosensor (BsWCB-GFP) for the detection of As. The BsWCB-GFP was specifically activated by inorganic As(III) and As(V), but not by dimethylarsinic acid [DMA(V)], and exhibited high tolerance to the noxious effects of arsenic. Accordingly, after 12 h exposure, B. subtilis cells carrying the Pars::gfpmut3a fusion exhibited 50 and 90% lethal doses (LD50 and LD90) to As(III) of 0.89 mM and As 1.71 mM, respectively. Notably, dormant spores from the BsWCB-GFP were able to report the presence of As(III) in a concentration range from 0.1 to 1,000 µM 4 h after the onset of germination. In summary, the specificity and high sensitivity for As, as well as its ability to proliferate under concentrations of the metal that are considered toxic in water and soil, makes the B. subtilis biosensor developed here a potentially important tool for monitoring environmental samples contaminated with this pollutant. IMPORTANCE Arsenic (As) contamination of groundwater is associated with serious worldwide health risks. Detection of this pollutant at concentrations that are established as permissible for water consumption by WHO is a matter of significant interest. Here, we report the generation of a whole-cell biosensor for As detection in the Gram-positive spore former B. subtilis. This biosensor reports the presence of inorganic As, activating the expression of the green fluorescent protein (GFP) under the control of the promoter/operator of the ars operon. The biosensor can proliferate under concentrations of As(III) that are considered toxic in water and soil and detect this ion at concentrations as low as 0.1 µM. Of note, spores of the Pars-GFP biosensor exhibited the ability to detect As(III) following germination and outgrowth. Therefore, this novel tool has the potential to be directly applied to monitor As contamination in environmental samples.

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In potato (Solanum tuberosum L.), protoplast techniques are limited to a few genotypes; thus, the use of regular regeneration procedures of multicellular explants causes us to face complexities associated to CRISPR/Cas9 gene editing efficiency and final identification of individuals. Geminivirus-based replicons contained in T-DNAs could provide an improvement to these procedures considering their cargo capability. We built a Bean yellow dwarf virus-derived replicon vector, pGEF-U, that expresses all the editing reagents under a multi-guide RNA condition, and the Green Fluorescent Protein (GFP) marker gene. Agrobacterium-mediated gene transfer experiments were carried out on 'Yagana-INIA', a relevant local variety with no previous regeneration protocol. Assays showed that pGEF-U had GFP transient expression for up to 10 days post-infiltration when leaf explants were used. A dedicated potato genome analysis tool allowed for the design of guide RNA pairs to induce double cuts of genes associated to enzymatic browning (StPPO1 and 2) and to cold-induced sweetening (StvacINV1 and StBAM1). Monitoring GFP at 7 days post-infiltration, explants led to vector validation as well as to selection for regeneration (34.3% of starting explants). Plant sets were evaluated for the targeted deletion, showing individuals edited for StPPO1 and StBAM1 genes (1 and 4 lines, respectively), although with a transgenic condition. While no targeted deletion was seen in StvacINV1 and StPPO2 plant sets, stable GFP-expressing calli were chosen for analysis; we observed different repair alternatives, ranging from the expected loss of large gene fragments to those showing punctual insertions/deletions at both cut sites or incomplete repairs along the target region. Results validate pGEF-U for gene editing coupled to regular regeneration protocols, and both targeted deletion and single site editings encourage further characterization of the set of plants already generated.

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Insect neuropeptides, play a central role in the control of many physiological processes. Based on an analysis of Nyssorhynchus albimanus brain transcriptome a neuropeptide precursor database of the mosquito was described. Also, we observed that adipokinetic hormone/corazonin-related peptide (ACP), hugin and corazonin encoding genes were differentially expressed during Plasmodium infection. Transcriptomic data from Ny. albimanus brain identified 29 pre-propeptides deduced from the sequences that allowed the prediction of at least 60 neuropeptides. The predicted peptides include isoforms of allatostatin C, orcokinin, corazonin, adipokinetic hormone (AKH), SIFamide, capa, hugin, pigment-dispersing factor, adipokinetic hormone/corazonin-related peptide (ACP), tachykinin-related peptide, trissin, neuropeptide F, diuretic hormone 31, bursicon, crustacean cardioactive peptide (CCAP), allatotropin, allatostatin A, ecdysis triggering hormone (ETH), diuretic hormone 44 (Dh44), insulin-like peptides (ILPs) and eclosion hormone (EH). The analysis of the genome of An. albimanus and the generated transcriptome, provided evidence for the identification of myosuppressin neuropeptide precursor. A quantitative analysis documented increased expression of precursors encoding ACP peptide, hugin and corazonin in the mosquito brain after Plasmodium berghei infection. This work represents an initial effort to characterize the neuropeptide precursors repertoire of Ny. albimanus and provides information for understanding neuroregulation of the mosquito response during Plasmodium infection.

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BACKGROUND: Maize is one of the most important crops worldwide and has been a target of nuclear-based transformation biotechnology to improve it and satisfy the food demand of the ever-growing global population. However, the maize plastid transformation has not been accomplished due to the recalcitrant condition of the crop. RESULTS: In this study, we constructed two different vectors with homologous recombination sequences from maize (Zea mays var. LPC13) and grass (Bouteloua gracilis var. ex Steud) (pZmcpGFP and pBgcpGFP, respectively). Both vectors were designed to integrate into rrn23S/rrn16S from an inverted repeat region in the chloroplast genome. Moreover, the vector had the mgfp5 gene driven by Prrn, a leader sequence of the atpB gene and a terminator sequence from the rbcL gene. Also, constructs have an hph gene as a selection marker gene driven by Prrn, a leader sequence from rbcL gene and a terminator sequence from the rbcL gene. Explants of maize, tobacco and Escherichia coli cells were transformed with both vectors to evaluate the transitory expression­an exhibition of green and red fluorescent light under epifluorescence microscopy. These results showed that both vectors were expressed; the reporter gene in all three organisms confirmed the capacity of the vectors to express genes in the cell compartments. CONCLUSIONS: This paper is the first report of transient expression of GFP in maize embryos and offers new information for genetically improving recalcitrant crops; it also opens new possibilities for the improvement in maize chloroplast transformation with these vectors.

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Solubility tags are commonly fused to target recombinant proteins to enhance their solubility and stability. In general, these protein tags must be removed to avoid misfolding of the partner protein and to allow for downstream applications. Nevertheless, in vitro tag removal increases process complexity and costs. Herein, we describe a synthetic biology-based strategy to permit in vivo removal of a solubility tag (EDA, KDPG aldolase), through co-expression of the fusion recombinant protein (EDA-EGFP) and the tag-cleaving protease (TEVp), in a controlled manner. Basically, the system uses three repressor proteins (LacI, cI434, and TetR) to regulate the expressions of EDA-EGFP and TEVp, in a regulatory cascade that culminates with the release of free soluble target protein (EGFP), following a single chemical induction by IPTG. The system worked consistently when all biological parts were cloned in a single plasmid, pSolubility(SOL)A (7.08 Kb, AmpR), and transformed in Escherichia coli Rosetta (DE3) or BL21(DE3) strains. Total soluble recombinant protein yield (EDA-EGFP + free EGFP) was ca. 272.0 ± 60.1 µg/mL of culture, following IMAC purification; free EGFP composed great part (average = 46.5%; maximum = 67.3%) of the total purified protein fraction and was easily separated from remaining fusion EDA-EGFP (53 KDa) through filtration using a 50 KDa cut-off centrifugal filter.

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Green fluorescent protein (GFP) is a globular protein used as biosensor and biomarker in medical and industrial fields. However, due to the expensive production costs of expressing proteins using high-cost inducers like isopropyl-ß-d-1-thiogalactopyranoside (IPTG), the number of GFP applications are still scarce. This work studied the production of enhanced GFP (EGFP) using Escherichia coli BL21 (DE3) [pLysS; pET28(a)], aiming to increase its yield and reduce costs. First, the influence of agitation rate, induction time, and concentration of IPTG in the production of EGFP was evaluated, but only the first two parameters were significant. Subsequently, aiming to reduce costs related to the use of inducer, the IPTG concentration (0.005, 0.010, and 0.025 mM) was decreased and, interestingly, the production levels were maintained or increased. These results show that a proper choice of production conditions, particularly through the decrease of inducer concentration, is effective to reduce the upstream production costs and guarantee high EGFP expression.

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Recombinant virus vectors represent a promising strategy for vaccine research. Among available viral vectors, members of the Poxviridae family-especially the modified Vaccinia virus Ankara (MVA)-stand out as immunogenic and safe vaccine platforms. Because MVA usually does not produce plaques in cell culture, visible selection markers such as the green fluorescent protein (GFP) are frequently incorporated into the constructions in order to facilitate the recognition of recombinants. However, these genetic markers have to be removed before any clinical trial. Here, we evaluated the acute responses generated in mice immunized with a MVA vector in which the GFP marker was not removed. We observed no differences in neutrophil, monocyte, or total leucocyte recruitment among animals inoculated with MVA or MVA-GFP. Likewise, there were no differences in neutrophil activation between mice groups. Hepatic functions were not altered in either MVA or MVA-GFP-inoculated mice, and we observed no histopathological alterations in different tissues from virus-inoculated animals. In conclusion, the presence of GFP is innocuous to immunized animals and do not alter acute physiopathological responses to the MVA vector. We suggest that keeping the GFP marker may be a good strategy for vaccine development, production, and evaluation.

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The fungal cell wall building processes are the ultimate determinants of hyphal shape. In Neurospora crassa the main cell wall components, ß-1,3-glucan and chitin, are synthesized by enzymes conveyed by specialized vesicles to the hyphal tip. These vesicles follow different secretory routes, which are delicately coordinated by cargo-specific Rab GTPases until their accumulation at the Spitzenkörper. From there, the exocyst mediates the docking of secretory vesicles to the plasma membrane, where they ultimately get fused. Although significant progress has been done on the cellular mechanisms that carry cell wall synthesizing enzymes from the endoplasmic reticulum to hyphal tips, a lot of information is still missing. Here, the current knowledge on N. crassa cell wall composition and biosynthesis is presented with an emphasis on the underlying molecular and cellular secretory processes.

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This study reports the introduction of egfp or mCherry markers to the Sp245, Sp7, and M40 wild-type strains of Azospirillum brasilense and the hhkB (encoding for a putative hybrid histidine kinase) minus mutant an isogenic strain of A. brasilense Sp245 to monitor colonization of wheat (Triticum aestivum). Two plasmids were constructed: (1) the pJMS-2 suicide plasmid derived from pSUP202 and harboring the mCherry gene expressed under the constitutive kanamycin resistance promoter to create a cis tag and (2) the broad-range plasmid pMP2449-5 that carries the mCherry gene under the lac promoter, which is derived from the plasmid pMP2444; to create the in trans tag. The stability of the plasmids encoding egfp and mCherry were confirmed in vitro for seven days of bacterial growth, and then, the A. brasilense strains harboring the plasmids were studied under nonselective conditions for adherence to seeds and, at seven or 14 days post-inoculation, for wheat root colonization. The utility of the labeled strains was proven by observation, using fluorescence microscopy and confocal laser scanning microscopy (CLSM) in wheat plants inoculated with the labeled strains and compared with the CFU g-1 for seed and wheat root. The method was suitable for observation of the in situ formation of mini-colonies, enabled visualization of bacterial colonization sites on large root fragments, and showed adherence to germinated seeds and root colonization of all strains by cell counts and direct microscopic examination. Thus, we are able to quantify the structures of the biofilms formed by each strain.

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Site-specific, genetic incorporation of unnatural amino acids (UAAs) into proteins in living cells using engineered orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs is a powerful tool for studying and manipulating protein structure and function. To date, UAA incorporation systems have been developed for several bacterial and eukaryotic model hosts. Due to the importance of Streptomyces as prolific producers of bioactive natural products and as model hosts for natural product biosynthesis and bioengineering studies, we have developed systems for the incorporation of the UAAs p-iodo-L-phenylalanine (pIPhe) and p-azido-L-phenylalanine (pAzPhe) into green fluorescent protein (GFP) in Streptomyces venezuelae ATCC 15439. Here, we describe the procedure for using this system to site-specifically incorporate pIPhe or pAzPhe into proteins of interest in S. venezuelae. The modular design of plasmids harboring UAA incorporation systems enables use of other aaRS or aaRS/tRNA pairs for the incorporation of other UAAs; and the vector backbone used allows the system to be transferred to diverse Streptomyces species via both protoplast transformation and conjugation.

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BACKGROUND Gene reporter-fluorescent cells have emerged as alternative method for drug screening. OBJECTIVE Achievement of constitutive expression of fluorescent protein GFP by Leishmania braziliensis as alternative method for drug screening. METHODS L. braziliensis-GFP was generated using Leishmania tarentolae pLEXSY-egfp for constitutive expression of GFP. Fluorescent cells were selected and subjected to standardisation tests of anti-promastigote and anti-intracellular amastigote assays. FINDINGS Our results showed that L. braziliensis-GFP method is faster and more sensitive than Allamar Blue-resazurin. MAIN CONCLUSION Transfected parasites maintained stable fluorescence after successive in vitro passages and pLEXSY system can be used to achieve non-L. tarentolae fluorescent cells.

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Leishmaniasis is a parasitic infection caused by several species of the genus Leishmania that is considered as a neglected disease. Drug development process requires a robust and updated high-throughput technology to the evaluation of candidate compounds that imply the manipulation of the pathogenic species of the parasite in the laboratory. Therefore, it is restricted to trained personal and level II biosafety environments. However, it has been established the utility of Leishmania tarentolae as a model for in vitro screening of antileishmanial agents without the necessity of level II biosafety setups. In parallel the transfection of Leishmania parasites with reporter genes as the eGFP using non-commercial integration vectors like the pIRmcs3(-) has proved to be a powerful tool for the implementation of semi automatized high-throughput platforms for the evaluation of antileishmanial compounds. Here we report the generation of a new L. tarentolae strain overexpressing the eGFP gene harboured by the non-commercial vector pIR3(-). We also demonstrate its utility for the semi-automatized screening of antileshmanial compounds in intracellular forms of the L. tarentolae parasite.

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Many Actinobacteria, most notably Streptomyces, produce structurally diverse bioactive natural products, including ribosomally synthesized peptides, by multistep enzymatic pathways. The use of site-specific genetic incorporation of unnatural amino acids to investigate and manipulate the functions of natural product biosynthetic enzymes, enzyme complexes, and ribosomally derived peptides in these organisms would have important implications for drug discovery and development efforts. Here, we have designed, constructed, and optimized unnatural amino acid systems capable of incorporating p-iodo-l-phenylalanine and p-azido-l-phenylalanine site-specifically into proteins in the model natural product producer Streptomyces venezuelae ATCC 15439. We observed notable differences in the fidelity and efficiency of these systems between S. venezuelae and previously used hosts. Our findings serve as a foundation for using an expanded genetic code in Streptomyces to address questions related to natural product biosynthesis and mechanism of action that are relevant to drug discovery and development.

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The experiment aimed to specifically monitor the passage of lactobacilli in vivo after oral administration. The green fluorescent protein (GFP) gene was cloned downstream from the constitutive p32 promoter from L. lactis subsp. cremoris Wg2. The recombinant expression vector, pLEM415-gfp-p32, was electroporated into Lactobacillus plantarum (L. plantarum) isolated from goat. Green fluorescent protein (GFP) was successfully expressed in L. plantarum. After 2 h post-administration, transformed Lactobacillus could be detectable in all luminal contents. In the rumen, bacteria concentration initially decreased, reached the minimum at 42 h post-oral administration and then increased. However, this concentration decreased constantly in the duodenum. This result indicated that L. plantarum could colonize in the rumen but not in the duodenum.

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The experiment aimed to specifically monitor the passage of lactobacilli in vivo after oral administration. The green fluorescent protein (GFP) gene was cloned downstream from the constitutive p32 promoter from L. lactis subsp. cremoris Wg2. The recombinant expression vector, pLEM415-gfp-p32, was electroporated into Lactobacillus plantarum (L. plantarum) isolated from goat. Green fluorescent protein (GFP) was successfully expressed in L. plantarum. After 2 h post-administration, transformed Lactobacillus could be detectable in all luminal contents. In the rumen, bacteria concentration initially decreased, reached the minimum at 42 h post-oral administration and then increased. However, this concentration decreased constantly in the duodenum. This result indicated that L. plantarum could colonize in the rumen but not in the duodenum..(AU)

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The experiment aimed to specifically monitor the passage of lactobacilli in vivo after oral administration. The green fluorescent protein (GFP) gene was cloned downstream from the constitutive p32 promoter from L. lactis subsp. cremoris Wg2. The recombinant expression vector, pLEM415-gfp-p32, was electroporated into Lactobacillus plantarum (L. plantarum) isolated from goat. Green fluorescent protein (GFP) was successfully expressed in L. plantarum. After 2 h post-administration, transformed Lactobacillus could be detectable in all luminal contents. In the rumen, bacteria concentration initially decreased, reached the minimum at 42 h post-oral administration and then increased. However, this concentration decreased constantly in the duodenum. This result indicated that L. plantarum could colonize in the rumen but not in the duodenum.

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Autophagy is a critical cellular homeostatic process that controls the turnover of damaged organelles and proteins. Impaired autophagic activity is involved in a number of diseases, including idiopathic pulmonary fibrosis suggesting that altered autophagy may contribute to fibrogenesis. However, the specific role of autophagy in lung fibrosis is still undefined. In this study, we show for the first time, how autophagy disruption contributes to bleomycin-induced lung fibrosis in vivo using an Atg4b-deficient mouse as a model. Atg4b-deficient mice displayed a significantly higher inflammatory response at 7 d after bleomycin treatment associated with increased neutrophilic infiltration and significant alterations in proinflammatory cytokines. Likewise, we found that Atg4b disruption resulted in augmented apoptosis affecting predominantly alveolar and bronchiolar epithelial cells. At 28 d post-bleomycin instillation Atg4b-deficient mice exhibited more extensive and severe fibrosis with increased collagen accumulation and deregulated extracellular matrix-related gene expression. Together, our findings indicate that the ATG4B protease and autophagy play a crucial role protecting epithelial cells against bleomycin-induced stress and apoptosis, and in the regulation of the inflammatory and fibrotic responses.

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Mesenchymal stem cells (MSC) are multipotent progenitor cells defined by their ability to self-renew and give rise to differentiated progeny. Since MSC from adult tissues represent a promising source of cells for a wide range of cellular therapies, there is high scientific interest in better understanding the potential for genetic modification and the mechanism underlying differentiation. The main objective of this study was to evaluate the potential for gene delivery using a GFP vector and lipofectamine, and to quantify the expression of epigenetic enzymes during foetal bMSC multilineage differentiation. Proportion of GFP-positive cells achieved (15.7% ± 3.5) indicated moderately low transfection efficiency. Analysis of DNA methyltransferase expression during MSC multilineage differentiation suggested no association with osteogenic and chondrogenic differentiation. However, up-regulation of KDM6B expression during osteogenic differentiation was associated with adoption of osteogenic lineage. Furthermore, increase in epigenetic enzyme expression suggested an intense epigenetic regulation during adipogenic differentiation.

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