TLR9-independent activation of B lymphocytes by bacterial DNA

The intracellular Toll-like receptor 9 (TLR9) is unique in its ability to recognize single-stranded DNA unmethylated at CpG motifs. Work from this laboratory showed that plasmid DNA is spontaneously internalized in B lymphocytes. This event is followed by the upregulation of costimulatory molecules and the acquisition of antigen presenting function by these cells. However, it is not known whether this phenomenon depends on TLR9. Because of the relevant role played by DNA-based drugs in immunotherapy and vaccination, and the central role of TLR9 signaling by CpG motifs, we decided to investigate whether signaling through TLR9 is a prerequisite for spontaneous transgenesis of lymphocytes. Here we found that transgene expression and upregulation of CD40 and CD86 costimulatory molecules was not inhibited by chloroquine treatment. Spontaneous transgenesis also occurred in B lymphocytes from TLR9-/- mice, and the injection of TLR9-/- transgenic B lymphocytes in C57Bl/6 mice induced both CD4 and CD8 T cell responses comparable to those induced by wild-type B lymphocytes. Collectively, these results suggest that plasmid DNA activates mammalian B lymphocytes through a TLR9 independent pathway.     

CyDisCo production of functional recombinant SARS‐CoV‐2 spike receptor binding domain

The COVID‐19 pandemic caused by SARS‐CoV‐2 has applied significant pressure on overtaxed healthcare around the world, underscoring the urgent need for rapid diagnosis and treatment. We have developed a bacterial strategy for the expression and purification of a SARS‐CoV‐2 spike protein receptor binding domain (RBD) that includes the SD1 domain. Bacterial cytoplasm is a reductive environment, which is problematic when the recombinant protein of interest requires complicated folding and/or processing. The use of the CyDisCo system (cytoplasmic disulfide bond formation in E. coli) bypasses this issue by pre‐expressing a sulfhydryl oxidase and a disulfide isomerase, allowing the recombinant protein to be correctly folded with disulfide bonds for protein integrity and functionality. We show that it is possible to quickly and inexpensively produce an active RBD in bacteria that is capable of recognizing and binding to the ACE2 (angiotensin‐converting enzyme) receptor as well as antibodies in COVID‐19 patient sera.     

A single prion protein peptide can elicit a panel of isoform specific monoclonal antibodies

The main step in the pathogenesis of transmissible spongiform encephalopathies (TSE) is the conformational change of the normal cellular prion protein (PrP(C)) into the abnormal isoform, named prion (PrP(Sc)). Since PrP is a highly conserved protein, the production of monoclonal antibodies (mAbs) of high specificity and affinity to PrP is a difficult task. In the present study we show that it is possible to overcome the unresponsiveness of the immune system by immunizing wild-type BALB/c mice with a 13 amino acid PrP peptide from the C-terminal part of PrP, bound to the keyhole limpet hemocyanin (KLH). Immunization induced predominantly anti-PrP(Sc) humoral immune response. Furthermore, we were able to obtain a panel of mAbs of IgG class specific for different non-self-conformations of PrP, with anti-PrP(Sc)-specific mAbs being the most abundant.     

Biomphalaria tenagophila: dynamics of populations of resistant and susceptible strains to Schistosoma mansoni, with or without pressure of the parasite

Resistant (Taim, RS) and susceptible albino (Joinville, SC) Biomphalaria tenagophila populations were kept together, at different proportions, throughout a 18-month-period. Some of the snail groups were submitted to Schistosoma mansoni infection. The targets of this study were (a) to analyze the populational dynamics among resistant and susceptible individuals to S. mansoni; (b) to study the resistance phenotype in descendants of cross-breeding; (c) to observe whether the parasite could exert any kind of selection in those snail populations. Throughout the experiment it could be observed that the susceptible B. tenagophila strain (Joinville) underwent a selective pressure of the parasite that was negative, since the individuals showed a high mortality rate. Although B. tenagophila (Taim) population presented a higher mortality rate without pressure of the parasite, this event was compensated by a reproductive capacity. B. tenagophila Taim was more fecund than B. tenagophila Joinville and was able to transmit the resistance character to their descendants. F1 generation obtained by cross-breeding between resistant and susceptible lineages was completely resistant to S. mansoni infection, irrespective of the Taim proportion. Moreover, less than 5% of F2 progeny were susceptible to S. mansoni infection.     

The kinetics of Theileria parva infection and lymphocyte transformation in vitro

Theileriaparva is an intracellular protozoan parasite that causes a fatal lymphoproliferative disease of cattle known as East Coast Fever. The parasite infects host lymphocytes causing their transformation and uncontrolled proliferation. Infiltration of major organs with parasitized lymphoblasts results in most cases in death within 3 weeks. Although both T and B lymphocytes are susceptible to infection, the majority of cell lines arising from infection of peripheral blood mononuclear cells in vitro are of T cell lineage. To explore the basis of this phenotypic bias we have followed the very early stages of parasite development in vitro at the single cell level. Peripheral blood mononuclear cells were infected and stained for both surface phenotype and intracellular parasite antigen and analysed by flow cytometry. Although the parasite antigen was detected intracellularly as early as 6h p.i., our data indicate that parasite infection does not lead to cell transformation in all instances. Rather, specific cell types appear to undergo selection very early after infection and expansion of particular cell subsets results in survival and growth of only a small proportion of the cells originally parasitized.     

Antibiotic inducibility of the MexXY multidrug efflux system of Pseudomonas aeruginosa: involvement of the antibiotic-inducible PA5471 gene product

The MexXY components of the MexXY-OprM multidrug efflux system of Pseudomonas aeruginosa are encoded by a MexZ repressor-regulated operon that is inducible by antibiotics that target the ribosome. Mutant strains disrupted in a gene, PA5471, were shown to be compromised for drug-inducible mexXY expression and, therefore, MexXY-OprM-mediated antimicrobial resistance. The PA5471 gene was inducible by the same ribosome-targeting agents that induce mexXY expression. Moreover, vector-driven expression of cloned PA5471 was sufficient to promote mexXY expression and MexXY-mediated resistance in the absence of antibiotic exposure, consistent with PA5471 directly or indirectly activating mexXY expression following its own upregulation in response to antibiotics. The requirement for PA5471 for mexXY expression and antimicrobial resistance was, however, obviated in mutants lacking the MexZ repressor of mexXY expression, suggesting that PA5471 directly or indirectly modulates MexZ activity in effecting mexXY expression. While the recruitment of PA5471 and MexXY in response to ribosome disruption by antimicrobials is consistent with their genes playing a role in protecting cells from the adverse consequences of disrupting the translation process, reminiscent of trans-translation, these genes appear to operate independently in their contribution to resistance: mutants defective in trans-translation showed a much more modest (twofold) decrease in resistance to ribosome-targeting agents than those lacking PA5471 or MexXY, and this decrease was observed whether functional PA5471/MexXY was present or not.     

Banding pattern of A and B chromosomes of Prochilodus lineatus (Characiformes, Prochilodontidae), with comments on B chromosomes evolution

B chromosomes in Prochilodus lineatus, a migratory neotropical fish, were analyzed in a comparative study among populations from the Dourada lagoon (State of Paraná, Brazil) and from Mogi-Guaçu river (State of São Paulo, Brazil). The data on C-banding and fluorescent in situ hybridization with a satellite DNA probe (SATH1), indicate that the small metacentric B chromosome might correspond to an isochromosome. On the other hand, both populations presented a distinct set of B chromosomes, differentiated either by their number and by the presence of variant B types in the population from Mogi-Guaçu river. The present results indicate that the B chromosomes of P. lineatus should have an ancient origin, and have undergone a differential evolutionary pathway among distinct populations.     

Evolutionary chromosomal differentiation among four species of Conoderus Eschscholtz, 1829 (Coleoptera, Elateridae, Agrypninae, Conoderini) detected by standard staining, C-banding, silver nitrate impregnation, and CMA3/DA/DAPI staining

The speciose Brazilian Elateridae fauna is characterized by high karyotypic diversity, including one species (Chalcolepidius zonatus Eschscholtz, 1829) with the lowest diploid number within any Coleoptera order. Cytogenetic analysis of Conoderus dimidiatus Germar, 1839, C. scalaris (Germar, 1824,) C. ternarius Germar, 1839, and C. stigmosus Germar, 1839 by standard and differential staining was performed with the aim of establishing mechanisms of karyotypic differentiation in these species. Conoderus dimidiatus, C. scalaris, and C. ternarius have diploid numbers of 2n(♂) = 17 and 2n(♀) = 18, and a X0/XX sex determination system, similar to that encountered in the majority of Conoderini species. The karyotype of C. stigmosus was characterized by a diploid number of 2n=16 and a neoXY/neoXX sex determination system that was highly differentiated from other species of the genus. Some features of the mitotic and meiotic chromosomes suggest an autosome/ancestral X chromosome fusion as the cause of the neoXY system origin in C. stigmosus. C-banding and silver impregnation techniques showed that the four Conoderus species possess similar chromosomal characteristics to those registered in most Polyphaga species, including pericentromeric C band and autosomal NORs. Triple staining techniques including CMA₃/DA/DAPI also provided useful information for differentiating these Conoderus species. These techniques revealed unique GC-rich heterochromatin associated with NORs in C. scalaris and C. stigmosus and CMA₃-heteromorphism in C. scalaris and C. ternarius.