Development and assessment of a multiepitope synthetic antigen for the diagnosis of Dengue virus infection.

Immunodiagnostic tests for detecting dengue virus infections encounter challenges related to cross-reactivity with other related flaviviruses. Our research focuses on the development of a synthetic multiepitope antigen tailored for dengue immunodiagnostics. Selected dengue epitopes involved structural linearity and dissimilarity from the proteomes of Zika and Yellow fever viruses which served for computationally modeling the three-dimensional protein structure, resulting in the design of two proteins: rDME-C and rDME-BR. Both proteins consist of seven epitopes, separated by the GPGPG linker, and a carboxy-terminal 6 × -histidine tag. The molecular weights of the final proteins rDME-C and rDME-BR are 16.83 kDa and 16.80 kDa, respectively, both with an isoelectric point of 6.35. The distinguishing factor between the two proteins lies in the origin of their epitope sequences, where rDME-C is based on the reference dengue proteome, while rDME-BR utilizes sequences from prevalent Dengue genotypes in Brazil from 2008 to 2019. PyMol analysis revealed exposure of epitopes in the secondary structure. Successful expression of the antigens was achieved in soluble form and fluorescence experiments indicated a disordered structure. In subsequent testing, rDME-BR and rDME-C antigens were assessed using an indirect Elisa protocol against Dengue infected serum, previously examined with a commercial diagnostic test. Optimal concentrations for antigens were determined at 10 µg/mL for rDME-BR and 30 µg/mL for rDME-C, with serum dilutions ranging from 1:50 to 1:100. Both antigens effectively detected IgM and IgG antibodies in Dengue fever patients, with rDME-BR exhibiting higher sensitivity. Our in-house test showed a sensitivity of 77.3 % and 82.6 % and a specificity of 89.4 % and 71.4 % for rDME-C and rDEM-BR antigens. No cross-reactivity was observed with serum from Zika-infected mice but with COVID-19 serum samples. Our findings underscore the utility of synthetic biology in crafting Dengue-specific multiepitope proteins and hold promise for precise clinical diagnosis and monitoring responses to emerging Dengue vaccines.

Accuracy of a raw saliva-based COVID-19 RT-LAMP diagnostic assay.

The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic demanded rapid diagnosis to isolate new COVID-19 cases and prevent disease transmission. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) rapidly became the gold standard for diagnosis. However, due to the high cost and delay of the results, other types of diagnosis were implemented, such as COVID-19 Ag Rapid Tests and Reverse Transcription Technique followed by Loop-Mediated isothermal Amplification (RT-LAMP). In this work, we validated the use of RT-LAMP in saliva samples rather than nasopharyngeal swabs, as the collection is more comfortable. First, we selected 5 primer sets based on the limit of detection for SARS-CoV-2 RNA, then validated their sensitivity and specificity in patient samples. A total of 117 samples were analyzed by fluorometric RT-LAMP and compared with qRT-PCR results. Our results show that the use of a high-sensitive primer ORF1-a, together with a low-sensitive primer set Gene E (time to threshold of 22.9 and 36.4 minutes, respectively, using 200 copies of viral RNA), achieved sensitivity in purified RNA from saliva samples of 95.2% (95% CI 76.1‒99.8) with 90.5% specificity (95% CI 69.6‒98.8) (n = 42).As RNA purification increases the turnaround time, we tested the outcome of RT-LAMP utilizing raw saliva samples without purification. The test achieved a sensitivity of 81.8% (95% CI 59.7‒94.8) and a specificity of 90.9% (95% CI 70.8‒98.8). As a result, the accuracy of 92.9% (95% CI 80.5‒98.5) in purified RNA-saliva samples was lowered to an acceptable level of 86.4% (95% CI 72.6‒94.8) in raw saliva. Although mass vaccination has been implemented, new strains and low vaccination progress helped to spread COVID-19. This study shows that it is feasible to track new COVID-19 cases in a large population with the use of raw saliva as sample in RT-LAMP assay which yields accurate results and offers a less invasive test.

Accuracy of a raw saliva-based COVID-19 RT-LAMP diagnostic assay

ABSTRACT The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic demanded rapid diagnosis to isolate new COVID-19 cases and prevent disease transmission. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) rapidly became the gold standard for diagnosis. However, due to the high cost and delay of the results, other types of diagnosis were implemented, such as COVID-19 Ag Rapid Tests and Reverse Transcription Technique followed by Loop-Mediated isothermal Amplification (RT-LAMP). In this work, we validated the use of RT-LAMP in saliva samples rather than nasopharyngeal swabs, as the collection is more comfortable. First, we selected 5 primer sets based on the limit of detection for SARS-CoV-2 RNA, then validated their sensitivity and specificity in patient samples. A total of 117 samples were analyzed by fluorometric RT-LAMP and compared with qRT-PCR results. Our results show that the use of a high-sensitive primer ORF1-a, together with a low-sensitive primer set Gene E (time to threshold of 22.9 and 36.4 minutes, respectively, using 200 copies of viral RNA), achieved sensitivity in purified RNA from saliva samples of 95.2% (95% CI 76.1-99.8) with 90.5% specificity (95% CI 69.6-98.8) (n = 42).As RNA purification increases the turnaround time, we tested the outcome of RT-LAMP utilizing raw saliva samples without purification. The test achieved a sensitivity of 81.8% (95% CI 59.7-94.8) and a specificity of 90.9% (95% CI 70.8-98.8). As a result, the accuracy of 92.9% (95% CI 80.5-98.5) in purified RNA-saliva samples was lowered to an acceptable level of 86.4% (95% CI 72.6-94.8) in raw saliva. Although mass vaccination has been implemented, new strains and low vaccination progress helped to spread COVID-19. This study shows that it is feasible to track new COVID-19 cases in a large population with the use of raw saliva as sample in RT-LAMP assay which yields accurate results and offers a less invasive test.

New Findings on LMO7 Transcripts, Proteins and Regulatory Regions in Human and Vertebrate Model Organisms and the Intracellular Distribution in Skeletal Muscle Cells.

LMO7 is a multifunctional PDZ-LIM protein that can interact with different molecular partners and is found in several intracellular locations. The aim of this work was to shed light on LMO7 evolution, alternative transcripts, protein structure and gene regulation through multiple in silico analyses. We also explored the intracellular distribution of the LMO7 protein in chicken and zebrafish embryonic skeletal muscle cells by means of confocal fluorescence microscopy. Our results revealed a single LMO7 gene in mammals, sauropsids, Xenopus and in the holostean fish spotted gar while two lmo7 genes (lmo7a and lmo7b) were identified in teleost fishes. In addition, several different transcripts were predicted for LMO7 in human and in major vertebrate model organisms (mouse, chicken, Xenopus and zebrafish). Bioinformatics tools revealed several structural features of the LMO7 protein including intrinsically disordered regions. We found the LMO7 protein in multiple intracellular compartments in chicken and zebrafish skeletal muscle cells, such as membrane adhesion sites and the perinuclear region. Curiously, the LMO7 protein was detected within the nuclei of muscle cells in chicken but not in zebrafish. Our data showed that a conserved regulatory element may be related to muscle-specific LMO7 expression. Our findings uncover new and important information about LMO7 and open new challenges to understanding how the diverse regulation, structure and distribution of this protein are integrated into highly complex vertebrate cellular milieux, such as skeletal muscle cells.

Three-Finger Toxins from Brazilian Coral Snakes: From Molecular Framework to Insights in Biological Function.

Studies on 3FTxs around the world are showing the amazing diversity in these proteins both in structure and function. In Brazil, we have not realized the broad variety of their amino acid sequences and probable diversified structures and targets. In this context, this work aims to conduct an in silico systematic study on available 3FTxs found in Micrurus species from Brazil. We elaborated a specific guideline for this toxin family. First, we grouped them according to their structural homologue predicted by HHPred server and further curated manually. For each group, we selected one sequence and constructed a representative structural model. By looking at conserved features and comparing with the information available in the literature for this toxin family, we managed to point to potential biological functions. In parallel, the phylogenetic relationship was estimated for our database by maximum likelihood analyses and a phylogenetic tree was constructed including the homologous 3FTx previously characterized. Our results highlighted an astonishing diversity inside this family of toxins, showing some groups with expected functional similarities to known 3FTxs, and pointing out others with potential novel roles and perhaps structures. Moreover, this classification guideline may be useful to aid future studies on these abundant toxins.

Properties of novel surfactin-derived biosurfactants obtained through solid-phase synthesis.

Eight molecules, four peptides (SPs) and four lipopeptides (LPs) derived by rational design from surfactin, a well-known secreted biosurfactant from Bacillus subtilis, were produced employing Fmoc-based solid-phase synthesis. These new peptides were tested to evaluate their potential biosurfactant and biological activities, aiming at possible applications in industrial, biological, pharmaceutical, and medical use. Five molecules (SP1, SP2, SP4, LP5, and LP8) presented potential for medical uses, mainly due to their drug delivery properties as suggested by their synergistic activity with the antibiotic vancomycin against Staphylococcus aureus. All synthetic peptides showed low toxicity against Vero cell cultures, in assays of hemolysis, and in different cytotoxicity assays. In addition, we found that three peptides (SP1, LP6, and LP7) had potential technological and industrial use because of their emulsifying capacity, low toxicity, and ability to physically stabilize solutions. These novel molecules retained some properties of the parental molecule (surfactin, which was originally obtained through nonribosomal synthesis in Bacillus subtilis) but have the advantage of being linear peptides, which can be produced at large scales through the use of conventional heterologous protein expression protocols.

Ligand-free method to produce the anti-angiogenic recombinant Galectin-3 carbohydrate recognition domain.

Galectin-3 (Gal3) is involved in many physiological processes related to tumor growth, such as promoting angiogenesis, cell migration/invasion, resistance to apoptosis and immune response modulation. Usually the overexpression of Gal3 is a poor prognostic marker for cancer patients. Recombinant Gal3 carbohydrate domain (Gal3C) has been proposed as a useful tool to inhibit angiogenesis. So far, all production protocols reported for Gal3C production have used proteolytic cleavage of full length Gal3 and/or affinity-based purification. This involves dialysis, a time consuming step used to eliminate the elution ligand, usually lactose. In this report, we describe an alternative method to produce human recombinant Gal3C in E. coli, purified with cationic exchange and size exclusion chromatography. The recombinant protein was characterized using circular dichroism and nuclear magnetic resonance, showing a beta sheet enriched well-folded globular structure. The average yield obtained was 26 mg/L of broth and the purity was above 99%. The anti-angiogenic activity was assessed in vitro and showed a reduction of 70% and 77% in endothelial cells tubule formation upon treatment with 10 and 20 µg/mL, respectively and also had no impact on cell viability. The method described here is more suitable for both laboratory and industrial production of the potential anti-tumor Gal3C.

Comprehensive structural analysis of designed incomplete polypeptide chains of the replicase nonstructural protein 1 from the severe acute respiratory syndrome coronavirus.

The cotranslational folding is recognized as a very cooperative process that occurs after the nearly completion of the polypeptide sequence of a domain. Here we investigated the challenges faced by polypeptide segments of a non-vectorial ß-barrel fold. Besides the biological interest behind the SARS coronavirus non-structural protein 1 (nsp1, 117 amino acids), this study model has two structural features that motivated its use in this work: 1- its recombinant production is dependent on the temperature, with greater solubility when expressed at low temperatures. This is an indication of the cotranslational guidance to the native protein conformation. 2- Conversely, nsp1 has a six-stranded, mixed parallel/antiparallel ß-barrel with intricate long-range interactions, indicating it will need the full-length protein to fold properly. We used non-denaturing purification conditions that allowed the characterization of polypeptide chains of different lengths, mimicking the landscape of the cotranslational fold of a ß-barrel, and avoiding the major technical hindrances of working with the nascent polypeptide bound to the ribosome. Our results showed partially folded states formed as soon as the amino acids of the second ß-strand were present (55 amino acids). These partially folded states are different based on the length of polypeptide chain. The native α-helix (amino acids 24-37) was identified as a transient structure (~20-30% propensity). We identified the presence of regular secondary structure after the fourth native ß-strand is present (89 amino acids), in parallel to the collapse to a non-native 3D structure. Interestingly the polypeptide sequences of the native strands ß2, ß3 and ß4 have characteristics of α-helices. Our comprehensive analyses support the idea that incomplete polypeptide chains, such as the ones of nascent proteins much earlier than the end of the translation, adopt an abundance of specific transient folds, instead of disordered conformations.

Heterologous expression and purification of a biologically active legume lectin from Cratylia mollis seeds (CRAMOLL 1).

CRAMOLL 1 is a mannose/glucose isolectin isolated from Cratylia mollis seeds. This lectin has 82% sequence identity with Con A and essentially the same quaternary structure. As with Con A, CRAMOLL 1 seems to undergo complex post-translational processing which makes it difficult to the use of traditional molecular cloning for heterologous expression. Here we report the expression and purification of functional recombinant CRAMOLL 1 (rCRAMOLL 1) in Escherichia coli. This was accomplished by introducing a chemically synthesized DNA encoding the mature CRAMOLL 1 amino acid sequence into a bacterial expression vector under T7 promoter control. Most of the recombinant lectin was found in insoluble aggregates (inclusion bodies), but we were able to recover reasonable amounts of soluble lectin in the active form by decreasing the protein induction temperature. The recombinant lectin was purified to homogeneity with one-step affinity chromatography. The plant CRAMOLL 1 (pCRAMOLL 1) and rCRAMOLL 1 share several physicochemical properties such as molecular mass, charge density and secondary and tertiary structures. However, pCRAMOLL 1 has a lower thermodynamic stability than rCRAMOLL 1 when probed by acidification, high temperature or high hydrostatic pressure, and this is probably caused by the presence of tetramers composed of fragmented monomers, which are formed in the plant cotyledon but absent from the recombinant protein. rCRAMOLL 1 behaves identically to its plant counterpart with respect to its specificity for monosaccharides, and to its agglutinating activities against rabbit erythrocytes and Trypanosoma cruzi epimastigote cells.