Characterization of a Molecularly Engineered Banlec-Type Lectin (rBTL).

Lectins are proteins that reversibly bind to carbohydrates and are commonly found across many species. The Banana Lectin (BanLec) is a member of the Jacalin-related Lectins, heavily studied for its immunomodulatory, antiproliferative, and antiviral activity. In this study, a novel sequence was generated in silico considering the native BanLec amino acid sequence and 9 other lectins belonging to JRL. Based on multiple alignment of these proteins, 11 amino acids of the BanLec sequence were modified because of their potential for interference in active binding site properties resulting in a new lectin named recombinant BanLec-type Lectin (rBTL). rBTL was expressed in E. coli and was able to keep biological activity in hemagglutination assay (rat erythrocytes), maintaining similar structure with the native lectin. Antiproliferative activity was demonstrated on human melanoma lineage (A375), evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT). rBTL was able to inhibit cellular growth in a concentration-dependent manner, in an 8-h incubation, 12 µg/mL of rBTL led to a 28.94% of cell survival compared to cell control with 100%. Through a nonlinear fit out log-concentration versus biological response, an IC50% of 3.649 µg/mL of rBTL was determined. In conclusion, it is possible to state that the changes made to the rBTL sequence maintained the structure of the carbohydrate-binding site without changing specificity. The new lectin is biologically active, with an improved carbohydrate recognition spectrum compared to nBanLec, and can also be considered cytotoxic for A375 cells.

Intranasal administration of interleukin-4 ameliorates depression-like behavior and biochemical alterations in mouse submitted to the chronic unpredictable mild stress: modulation of neuroinflammation and oxidative stress.

Physical and psychological stress modulates the hypothalamic pituitary adrenal (HPA) axis, and the redox and inflammatory systems. Impairments in these systems have been extensively reported in major depression (MD) patients. Therefore, our study aimed to investigate the effects of the intranasal administration of interleukin-4 (IL-4) in mice with depressive-like behavior induced by chronic unpredictable mild stress (CUMS) for 28 days. On the 28th day, mice received IL-4 intranasally (1 ng/mouse) or vehicle (sterile saline), and after 30 min, they were submitted to behavioral tests or euthanasia for blood collection and removal of the adrenal glands, axillary lymph nodes, spleen, thymus, prefrontal cortices (PFC), and hippocampi (HC). A single administration of IL-4 reversed CUMS-induced depression-like behavior in the tail suspension test and splash test, without evoking locomotor changes. IL-4 administration reduced the plasma levels of corticosterone and the increased weight of suprarenal glands in stressed mice. Moreover, IL-4 restored the expression of nuclear factor erythroid 2-related factor 2 (NRF2), nuclear factor kappa B (NF-kB), interleukin 1 beta (IL-1ß), IL-4, brain derived neurotrophic factor (BDNF), and indoleamine 2,3-dioxygenase (IDO) in the PFC and HC and modulated oxidative stress markers in these brain structures in stressed mice. Our results showed for the first time the antidepressant-like effect of IL-4 through the modulation of neuroinflammation and oxidative stress. The potential effect of IL-4 administered intranasally arises as an innovative strategy for MD treatment.

In silico analyses and design of chimeric proteins containing epitopes of Bartonella henselae antigens for the control of cat scratch disease.

Bartonella henselae is a Gram-negative bacterium that causes cat scratch disease (CSD), as well as bacteremia, endocarditis, and other clinical presentations. CSD remains one of the most common infections caused by bacteria in the genus Bartonella, and it is transmitted to humans through a scratch or cat bite. Vaccination and more efficient diagnostic methods would represent a promising and sustainable alternative measure for CSD control in humans and animals. Here, we described the in silico analyses and design of three recombinant chimeric proteins (rC1, rC2, and rC3), for use in the control of CSD. The chimeras were constructed with epitopes identified from the sequences of the GroEL, 17 kDa, P26, BadA, Pap31, OMP 89, and OMP 43, previously described as the most important B. henselae antigens. The rC1, rC2, and rC3 were expressed and purified using a heterologous system based on Escherichia coli and reacted with antibodies present in the sera of humans naturally infected. The chimeric proteins were used to immunize mice using Freund adjuvant, and the humoral immune response was evaluated. Animals immunized with rC1 and rC3 showed a significant IgG antibodies response from the 28th day (P < 0.05), and the animals immunized with the rC2 from the 35th day (P < 0.05) remained until the 56th day of experimentation, with a titer of 1:3200 (P < 0.05), 1:1600 (P < 0.05) and 1:1600 (P < 0.05) from rC1, rC2, and rC3, respectively. Significant production of IgA and IgG1 isotype was detected in animals immunized with rC1 and rC2 proteins. Additionally, analysis using 13 serum samples from naturally infected patients showed that the proteins are recognized by antibodies present in sera, reinforcing the possibility of using these chimeras for CSD control. KEY POINTS: • The recombinant chimeras were expressed in Escherichia coli with 37 kDa (rC1), 35 kDa (rC2), and 38 kDa (rC3). • Animals immunized with rC1, rC2, and rC3 showed significant antibody response. • The chimeras were recognized by the sera of naturally infected patients.

DNA microarray for forensic intelligence purposes: High-density SNP profiles obtained directly from casework-like samples with and without a DNA purification step.

SNP analyses from a forensic intelligence perspective have proven to be an important tool to restrict the number of suspected offenders and find missing persons. DNA microarray assays have been demonstrated as a potential feature in forensic analysis, like such as forensic genetic genealogy. The objective of this study was to describe the results from DNA microarray assay from saliva samples deposited on a glass surface collected from by a double swab technique, commonly applied in crime scenes. Eighteen samples from the same person were subjected to Infinium® Global Screening Array-24 v1.0 (~642.824 SNP markers) in two different protocols - with or without the DNA purification procedure. The measured genotype was compared with a Consensus Genotype, obtained from standard control samples, and the parameters such as Call Rate and GenCall Scores were evaluated. Results showed that the Call Rate parameter is enough to estimate the probability of obtaining a correct genotype in the SNP assay. Reliable genotypes with a confidence level of more than 90% (at least 90.15%) were observed in Call Rates above 69.41%, regardless of the experimental condition. Our data demonstrate that DNA Microarray from samples collected under conditions such as those found at crime scenes can generate high-density SNP genetic profiles with a confidence level greater than 90%. Enzymatic adjustments and protocol changes may enable DNA microarray assays for crime analysis and investigation purposes eliminating the purification step in the future. Our data suggest that DNA microarray can support criminal investigation teams from a forensic intelligence perspective.

In silico analyses and design of a chimeric protein containing epitopes of SpaC, PknG, NanH, and SodC proteins for the control of caseous lymphadenitis.

Caseous lymphadenitis (CLA) is an infectious disease that affects goats and sheep causing drastic impacts on milk and meat production and is caused by Corynebacterium pseudotuberculosis. The disease can be prevented through vaccination but currently, vaccines demonstrate limited efficacy consequently leading to a need for the development of new ones. Here, we described the in silico development of a new chimeric protein constructed with epitopes identified from the sequences of the genes nanH, pknG, spaC, and sodC, previously described as potential vaccinal targets against C. pseudotuberculosis. The chimera was expressed, purified, and its immunogenicity was evaluated using sera of immunized mice. Results indicate the chimeric protein was able to stimulate antibody production. Additionally, analysis using serum from naturally infected goats showed that the protein is recognized by sera from these animals, indicating the possibility for using this chimera in new diagnostic methods. KEY POINTS: • The chimera was expressed with 52 kDa and a yield of 7 mg/L after purification. • The chimera was recognized by the sera of animals immunized with this formulation. • Chimera reacted with the serum of goats naturally infected with C. pseudotuberculosis.

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.

Genome sequence of Xanthomonas fuscans subsp. fuscans strain Xff49: a new isolate obtained from common beans in Southern Brazil.

The genus Xanthomonas comprises Gram-negative bacteria, many of which are phytopathogens. Xanthomonas fuscans subsp. fuscans is one of the most devastating pathogens affecting the bean plant, resulting in the common bacterial blight of bean (CBB). The disease is mainly foliar and affects a wide variety of bean species, thus acting as the yield-limiting factor for the bean crop. Here, we report the whole-genome sequencing of a new strain of X. fuscans subsp. fuscans, named Xff49, isolated from the infected and symptomatic beans from Capão do Leão, Southern Brazil. The genetic analysis demonstrated the presence of single-nucleotide variants (SNVs) in this strain, potentially affecting the mobilome, cell mobility, and inorganic ion metabolism. In addition, the analysis resulted in the identification of a new plasmid similar to the pAX22 derived from Achromobacter denitrificans, which was named plX, along with plA and plC, previously reported in other strains of X. fuscans subsp. fuscans. Xff49 represents the first Brazilian genome of X. fuscans subsp. fuscans and might provide useful information applicable to the studies of phylogenetics, evolution, and pathogenomics, thereby allowing a better understanding of the genomic features present in the Brazilian strains.

Heterologous expression and characterization of a new galactose-binding lectin from Bauhinia forficata with antiproliferative activity.

In this study, we describe the characterization of a new lectin, BfL-II, purified from the seeds of Bauhinia forficata, which is distinct, at sequence-level, from the previously reported lectin from the same specie (BfL). In addition, the gene for this lectin was cloned and expressed in Escherichia coli and its antiproliferative activity was evaluated against human breast and colorectal cancer cells (MCF-7 and HT-29, respectively). The treatment with 100 µg/µL of either native or recombinant BfL (nBfL or rBfL) significantly reduced the proliferation of both cancer cell lines (p < 0.01). The inhibition of HT-29 cell proliferation was as high as 82.5% and 93.6% with 100 µg/µL of rBfL-I and nBfL after 24 h, respectively. Therefore, BfL-II presents a promising antiproliferative activity, which may be applied in the development of new anticancer treatments.

Whole-genome sequencing of Leptospira interrogans from southern Brazil: genetic features of a highly virulent strain

BACKGROUND Leptospirosis is the most widespread zoonotic disease. It is caused by infection with pathogenic Leptospira species, of which over 300 serovars have been described. The accurate identification of the causative Leptospira spp. is required to ascertain the pathogenic status of the local isolates. OBJECTIVES This study aimed to obtain the complete genome sequence of a virulent Leptospira interrogans strain isolated from southern Brazil and to describe its genetic features. METHODS The whole genome was sequenced by next-generation sequencing (Ion Torrent). The genome was assembled, scaffolded, annotated, and manually reviewed. Mutations were identified based on a variant calling analysis using the genome of L. interrogans strain Fiocruz L1-130 as a reference. FINDINGS The entire genome had an average GC content of 35%. The variant calling analysis identified 119 single nucleotide polymorphisms (SNPs), from which 30 led to a missense mutation. The structural analyses identified potential evidence of genomic inversions, translocations, and deletions in both the chromosomes. MAIN CONCLUSIONS The genome properties provide comprehensive information about the local isolates of Leptospira spp., and thereby, could facilitate the identification of new targets for the development of diagnostic kits and vaccines.

Whole-genome sequencing of Leptospira interrogans from southern Brazil: genetic features of a highly virulent strain.

BACKGROUND Leptospirosis is the most widespread zoonotic disease. It is caused by infection with pathogenic Leptospira species, of which over 300 serovars have been described. The accurate identification of the causative Leptospira spp. is required to ascertain the pathogenic status of the local isolates. OBJECTIVES This study aimed to obtain the complete genome sequence of a virulent Leptospira interrogans strain isolated from southern Brazil and to describe its genetic features. METHODS The whole genome was sequenced by next-generation sequencing (Ion Torrent). The genome was assembled, scaffolded, annotated, and manually reviewed. Mutations were identified based on a variant calling analysis using the genome of L. interrogans strain Fiocruz L1-130 as a reference. FINDINGS The entire genome had an average GC content of 35%. The variant calling analysis identified 119 single nucleotide polymorphisms (SNPs), from which 30 led to a missense mutation. The structural analyses identified potential evidence of genomic inversions, translocations, and deletions in both the chromosomes. MAIN CONCLUSIONS The genome properties provide comprehensive information about the local isolates of Leptospira spp., and thereby, could facilitate the identification of new targets for the development of diagnostic kits and vaccines.

Draft Genome Sequences of Leptospira santarosai Strains U160, U164, and U233, Isolated from Asymptomatic Cattle.

In the present work, we announce the draft genomes for three new strains (U160, U164, and U233) of Leptospira santarosai, isolated from urine samples from asymptomatic cattle in Rio de Janeiro, Brazil.