Luis G C Pacheco

Luis G C Pacheco
Universidade Federal da Bahia · Department of Biotechnology

PhD

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83
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Publications

Publications (83)
Article
The appropriate choice of reference genes is essential for accurate normalization of gene expression data obtained by the method of reverse transcription quantitative real-time PCR (RT-qPCR). In 2009, a guideline called the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) highlighted the importance of the selecti...
Article
The recent availability of highly adaptable engineered nucleic acid binding proteins has brought about a revolution in the genome-editing field. Now, several studies have harnessed these Zinc-Finger-based, Transcription Activator-like Effector (TALE)-based, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based programmable DN...
Article
Full-text available
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 bi...
Article
Deep-learning (DL)-based image processing has potential to revolutionize the use of smartphones in mobile health (mHealth) diagnostics of infectious diseases. However, the high variability in cellphone image data acquisition and the common need for large amounts of specialist-annotated images for traditional DL model training may preclude generaliz...
Preprint
Corynebacterium striatum , a common constituent of the human skin microbiome, is now considered an emerging multidrug-resistant pathogen of immunocompromised and chronically ill patients. However, little is known about the molecular mechanisms in the transition from colonization to the multidrug-resistant (MDR) invasive phenotype in clinical isolat...
Article
Background: Allergic diseases figure among the most common immune-mediated diseases worldwide, affecting more than 25% of the world's population. Allergic reactions can be triggered by house dust mite (HDM) allergens, of which the so-called group 21 of allergens is considered as clinically relevant. Methods: Herein, we used a structural bioinfor...
Article
Full-text available
Blomia tropicalis and Dermatophagoides pteronyssinus play an important role in triggering allergy. Glycycometus malaysiensis causes IgE reaction in sensitive people, but is rarely reported in domestic dust, because it is morphologically similar to B. tropicalis making the identification of these species difficult. The identification of mites is mos...
Article
Recombinant proteins are generally fused with solubility enhancer tags to improve the folding and solubility of the target protein of interest. However, the fusion protein strategy usually requires expensive proteases to perform in vitro proteolysis and additional chromatographic steps to obtain tag-free recombinant proteins. Expression systems bas...
Article
CRISPR (Clustered regularly interspaced short palindromic repeats)-based diagnostic technologies have emerged as a promising alternative to accelerate delivery of SARS-CoV-2 molecular detection at the point of need. However, efficient translation of CRISPR-diagnostic technologies to field application is still hampered by dependence on target amplif...
Data
Correlations of sIgE binding assays. a Spearman’s correlation of BtE ELISA results and sIgE from ImmunoCAP assay (n=30). b Spearman’s correlation of rBlo t 5 ELISA results and sIgE from ImmunoCAP assay (n=25).
Data
PASTA 2.0 and AGGRESCAN3D analyses [19,20]. a Energy scores of the aggregation pairings retrieved from PASTA 2.0 server. b Aggregation free energy profile. c Aggregation helix profile from PASTA 2.0. d Ribbon representation of the aggregation-prone region, with the penultimate coil and β-sheet of Blo t 12. e Modelled 3D 10 structure retrieved from...
Data
Heterologous expressions and solubility test. Expression of rBlo t 12 in 5 mL of LB medium using BL21(DE3) (a), BL21(DE3)pLys S and TOP 10 F’ strains (b). c Western blot for confirmation of expression, using anti-HIS IgG antibodies. The sample added is indicated above each lane. MW: molecular weight. d 18% SDS-PAGE for the solubility test. e Wester...
Article
Full-text available
Successful research in the wide-ranging field of allergy is usually achieved by definition not only of physicochemical and immunological properties of natural, but also recombinant allergens. Blomia tropicalis mite is a well-known source for various groups of hypersensitivity-causing proteins. The goal of the present work was to produce, purify and...
Article
Full-text available
Aim Molecular sensitization profile analyses of allergic individuals to the house dust mites (HDM) Blomia tropicalis and Dermatophagoides pteronyssinus from Brazil and Austria, in the attempt to comprehend the individual contribution of the molecular components in the diagnosis of HDM allergy. Methodology These analyses were made using a new in vi...
Preprint
Full-text available
Recombinant proteins are generally fused with solubility enhancer tags to improve target protein folding and solubility. However, the fusion protein strategy usually requires the use of expensive proteases to perform in vitro proteolysis and additional chromatography steps to obtain tag-free recombinant proteins. Expression systems based on intrace...
Article
Full-text available
Background There is growing concern about individuals reported to suffer repeat COVID-19 disease episodes, these in a small number of cases characterised as de novo infections with distinct sequences, indicative of insufficient protective immunity even in the short term. Methods Observational case series and case-control studies reporting 33 cases...
Article
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Corynebacterium spp. are Gram-positive rods that are recognized to cause opportunistic diseases under certain predisposing clinical conditions. Some species have been described in urinary tract infections. In this report we document a new episode of urinary tract infection caused by Corynebacterium phoceense and describe the whole-genome sequencing...
Article
The prevalence of allergic diseases in Brazil is one of the biggest in the world. Among these pathologies, we highlight asthma as one of the most importance. Asthma is characterized as a chronic inflammatory disease of airways, associated with hyperresponsiveness. Many environmental factors can trigger asthma symptoms, among them house dust mites c...
Article
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Objectives Corynebacterium urealyticum is a non-diphtherial urease-producing clinically relevant corynebacterial associated with urinary tract infections. Most of the C. urealyticum clinical isolates are multidrug-resistant. Whole genome sequencing of C. urealyticum VH4248, isolated from a urine sample at the Hospital Universitario Marqués de Valde...
Article
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Arboviruses, including the Zika virus, have recently emerged as one of the most important threats to human health. The use of metagenomics-based approaches has already proven valuable to aid surveillance of arboviral infections, and the ability to reconstruct complete viral genomes from metatranscriptomics data is key to the development of new cont...
Article
Full-text available
Corynebacterium urealyticum is a non-diphtherial urease-producing clinically relevant corynebacterial, most frequently involved in urinary tract infections. Most of the C. urealyticum clinical isolates are frequently resistant to several antibiotics. We investigated the susceptibility of 40 C. urealyticum isolated in our institution during the peri...
Article
Toxocariasis, a natural helminth infection of dogs and cats caused by Toxocara canis and T. cati, respectively, that are transmitted to mammals, including humans. Infection control is based currently on periodic antihelmintic treatment and there is a need for the development of vaccines to prevent this infection. Materials and methods: Eight pote...
Data
Online Supporting Information of the Manuscript number: CEA-2020-0050. This file details the methods of the experiments, which were performed to verify the potential of BTH2 as an allergy vaccine.
Data
Figure S3. Pattern of BTH1-derived peptide clusters using the degradome assay. Peptides sequenced by tandem mass spectrometry after 48 h of in vitro digestion are shown. Grey bars indicate T-cell epitopes of Blo t 5 and Blo t 21. The immunogenic region of Blo t 5, which comprise the amino acid residues IIRELDVVCAMIEGAQGAL were previously identified...
Data
Figure S4. Pattern of rBlo t 5-derived peptide clusters using the degradome assay. Peptides sequenced by tandem mass spectrometry after 48 h of in vitro digestion are shown. Grey bars indicate T-cell epitopes of Blo t 5, as previously published. Positions of alpha-helical motifs are shown on the top. The predominant peptide clusters were around the...
Data
Figure S1. Sequence and model of BTH1 and BTH2; expression, purification, and structural features of rBlo t 5 and rBlo t 21. A: The hybrid protein BTH1 consists of fragments of Blo t 5 (green) and Blo t 21 (orange). The sequence regions highlighted in grey were subjected to a mutation scan to generate the hypoallergenic variant BTH2. The four resul...
Data
Figure S6. IgE reactivity profile of Blomia tropicalis parental and hybrid recombinant allergens. A: Specific IgE reactivity determined by ELISA using sera from donors with allergic rhinitis (n=31); B: Specific IgE reactivity determined by ELISA using sera from asthmatic donors with Blomia allergy (n=35). The cut-offs are represented by horizontal...
Data
Figure S8. Stimulation of PBMC with parental allergens and hybrid proteins. Titration curves were performed during PBMC culture as shown for rBlo t 5 (A), rBlo t 21 (B), BTH1 (C), and BTH2 (D). Forty-eight (for IL-10 detection) and 120 hours (for IL-5 and IFN-γ detection) after culture, PBMC proliferation was evaluated by the MTT assay. Data were e...
Data
Figure S2. Primary and secondary structure of Blomia tropicalis wild-type and hybrid recombinant allergens. Experimental determination of amino acid residues numbers in BTH1 (A) and BTH2 (B). Circular dichroism spectra of BTH1 (C) and BTH2 (D) recorded at 20 °C for 48h. E: FTIR 2nd derivative spectra are presented as second derivatives of Amide I a...
Data
Figure S5. Pattern of rBlo t 21-derived peptide clusters using the degradome assay. Peptides sequenced by tandem mass spectrometry after 48 h of in vitro digestion are shown. Grey bar indicates T-cell epitopes of Blo t 21, as predicted in the present work using IEDB analysis resource Consensus tool. Positions of alpha-helical motifs are shown on th...
Data
Figure S9. Optimum concentration of recombinant proteins as stimuli for PBMC culture. Concentration curves were performed during PBMC culture as shown for rBlo t 5 (A), rBlo t 21 (B), BTH1 (C), and BTH2 (D). Forty-eight (for IL-10 detection) and 120 hours (for IL-5 and IFN-γ detection) after culture, supernatants were collected. The amounts of cyto...
Data
Figure S7. The allergenic activity of Blomia tropicalis parental and hybrid recombinant allergens. Mediator release assays were used to determine the capacity of the antigens to trigger the release of an inflammatory mediator. Typical antigen-titration curves are shown. Donors’ numbers are indicated in the top of each curve.
Article
Herein, we describe the detection of a SARS-CoV-2 genome through metatranscriptome next-generation sequencing directly from the nasopharyngeal swab of a suspected case of local transmission of Covid-19, in Brazil. Depletion of human ribosomal RNA and use of an optimized in-house developed bioinformatics strategy contributed to successful detection...
Article
Full-text available
Introduction: Allergen-specific immunotherapy (AIT) represents a curative approach for treating allergies. In the tropical and subtropical regions of the world, Blomia tropicalis (Blo t 5 and Blo t 21) is the likely dominant source of indoor allergens. Aim: To generate a hypoallergenic Blo t 5/Blo t 21 hybrid molecule that can treat allergies ca...
Article
Introduction: Allergic illnesses are one of the most prevalent immunological disorders worldwide and house dust mites are important triggers of these diseases. Allergen-specific immunotherapy (AIT) is an alternative treatment to pharmacotherapy and among its technologies, recombinant hypoallergenic derivatives have shown promising features, turn th...
Chapter
Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) using fluorescent DNA-binding dyes is now a gold-standard methodology to study bacterial gene expression through relative quantitation of target mRNAs under specific experimental conditions, and recent developments in the technology allow for gene expression analysis i...
Article
Background: The dawn of the "omics" technologies has changed allergy research, increasing the knowledge and identification of new allergens. However, these studies have been almost restricted to Dermatophagoides spp. Although Blomia tropicalis has long been established as a clinically important source of allergens, a thorough proteomic characteriz...
Article
Full-text available
Biochemical tests are traditionally used for bacterial identification at the species level in clinical microbiology laboratories. While biochemical profiles are generally efficient for the identification of the most important corynebacterial pathogen Corynebacterium diphtheriae, their ability to differentiate between biovars of this bacterium is st...
Article
Full-text available
Multidrug-resistant (MDR) Corynebacterium striatum has been cited with increased frequency as pathogen of nosocomial infections. In this study, we report the draft genome of a C. striatum isolated from a patient with bloodstream infection in a hospital of Rio de Janeiro, Brazil. The isolate presented susceptibility only to tetracycline, vancomycin...
Article
Toxocariasis is a widespread helminth infection of dogs and cats, caused by Toxocara canis and Toxocara cati larvae, respectively. Toxocara spp. can cause zoonotic infections in humans by invading tissues and organs causing pathology. Toxocara spp. larvae release excretory-secretory molecules (TES) into the body of their host that are fundamental t...
Article
A multiplex-PCR (mPCR) assay was designed with species-specific primers which generate amplicons of 226bp, 434bp and 106bp for differentiating the species C. striatum, C. amycolatum, and C. xerosis, respectively. mPCR results were 100% in agreement with identifications achieved by 16S rRNA and rpoB gene sequencing and by VITEK-MS.
Article
The inverse relationship between helminth infections and the development of immune-mediated diseases is a cornerstone of the hygiene hypothesis and studies were carried out to elucidate the mechanisms by which helminth-derived molecules can suppress immunological disorders. These studies have fostered the idea that parasitic worms may be used as a...
Chapter
Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis, an infectious disease that affects sheep and goats worldwide. Even though the pathogenic process involved in this disease is well known, only a few virulence factors of C. pseudotuberculosis have been described so far, making it difficult to develop vaccines and the...
Article
Introduction: Allergies are among the most prevalent chronic diseases worldwide. Allergen-specific immunotherapy is used as an alternative treatment to pharmacotherapy. These immunotherapies are performed with crude extracts, which have disadvantages when compared to the new approaches, among them are recombinant proteins and hypoallergens. This r...
Article
Infection with helminthic parasites, including the soil-transmitted helminth Trichuris trichiura (human whipworm), has been shown to modulate host immune responses and, consequently, to have an impact on the development and manifestation of chronic human inflammatory diseases. De novo derivation of helminth proteomes from sequencing of transcriptom...
Article
Full-text available
Corynebacterium striatum commonly colonizes the normal skin and nasopharyngeal tract of humans; however, this potentially pathogenic bacterium has been identified as the causative agent of several nosocomial infections. The current study describes the draft genome of strain 1961 BR-RJ/09, isolated from the urine of a hospitalized patient from Brazi...
Article
Full-text available
Non-diphtheriae Corynebacterium species have been increasingly recognized as the causative agents of infections in humans. Differential identification of these bacteria in the clinical microbiology laboratory by the most commonly used biochemical tests is challenging, and normally requires additional molecular methods. Herein, we present the annota...
Article
Full-text available
Reference genes presenting stable expression profiles over a wide variety of conditions are required in relative expression studies of specific bacterial genes by quantitative reverse transcription PCR (RT-qPCR). High-throughput sequencing of bacterial transcriptomes using the RNA-seq methodology now provides a wealth of data that may be searched f...
Article
Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis a chronic infectious disease affecting small ruminants. The 2D-DIGE technique was used to compare the exoproteomes of two C. pseudotuberculosis biovar ovis strains isolated from goat (strain 1002) and sheep (strain C231). Seventeen proteins differentially produced we...
Article
This study involves the comparison between the exoproteomes of two different strains of Corynebacterium pseudotuberculosis, the etiologic agent of caseous lymphadenitis in small ruminants. In a previous study, based on a gel-free system (TPP-LC/MS(E)), 70 exoproteins for the strain 1002 and 67 for the strain C231, totaling 93 different extracellula...
Article
Full-text available
Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis constitute a group of potentially toxigenic microorganisms that are related to different infectious processes in animal and human hosts. Currently, there is a lack of information on the prevalence of disease caused by these pathogens, which is partially due...
Article
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Corynebacterium pseudotuberculosis equi is a Gram-positive pathogenic bacterium which affects a variety of hosts. Besides the great economic losses it causes to horse-breeders, this organism is also known to be an important infectious agent to cattle and buffaloes. As an outcome of the efforts in characterizing the molecular basis of its virulence,...
Article
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Corynebacterium pseudotuberculosis is a pathogen of great veterinary and economic importance, since it affects livestock, mainly sheep and goats, worldwide, together with reports of its presence in camels in several Arabic, Asiatic, and East and West African countries, as well as Australia. In this article, we report the genome sequence of Coryneba...
Article
Full-text available
Pathogenic intracellular bacteria can respond to antimicrobial mechanisms of the host cell through transient activation of stress-responsive genes by alternative sigma (σ) factors of the RNA polymerase. We evaluated the contribution of the extracytoplasmic function sigma factor σ(E) for Corynebacterium pseudotuberculosis resistance to stress condit...
Article
Caseous lymphadenitis (CLA) is a chronic disease affecting small ruminants caused by Corynebacterium pseudotuberculosis and is responsible for significant economic losses. Previous studies have reported that a set of C. pseudotuberculosis secreted proteins react with sera from infected goats. Mapping of the secretome will shed light on the pathogen...
Article
Full-text available
Despite the existence of a vaccine against diphtheria, this disease remains endemic and is reemerging in several regions due to many factors, including variations in genes coding for virulence factors. One common feature of virulence factors is their high concentration in pathogenicity islands (PAIs), very unstable regions acquired via horizontal g...
Article
Invasive infections caused by Corynebacterium diphtheriae in vaccinated and non-vaccinated individuals have been reported increasingly. In this study we used multilocus sequence typing (MLST) to study genetic relationships between six invasive strains of this bacterium isolated solely in the urban area of Rio de Janeiro, Brazil, during a 10-year pe...
Article
Full-text available
Corynebacterium pseudotuberculosis, a gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economi...
Article
Full-text available
Background Bacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA) in sheep and goats. Results An optimized pro...
Article
Full-text available
Corynebacterium pseudotuberculosis, a Gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economi...
Article
Full-text available
Background: Bacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA) in sheep and goats. Results: An optimized pr...
Article
Corynebacterium ulcerans was isolated from nares of one asymptomatic dog kept in an animal shelter in the metropolitan area of Rio de Janeiro, Brazil. The RNA polymerase beta subunit-encoding gene was sequenced to confirm the species identity. C. ulcerans strains producing phospholipase D, but not diphtheria toxin, are able to cause severe disease...