Involvement of GPR43 Receptor in Effect of Lacticaseibacillus rhamnosus on Murine Steroid Resistant Chronic Obstructive Pulmonary Disease: Relevance to Pro-Inflammatory Mediators and Oxidative Stress in Human Macrophages.

BACKGROUND: Cytokine storm and oxidative stress are present in chronic obstructive pulmonary disease (COPD). Individuals with COPD present high levels of NF-κB-associated cytokines and pro-oxidant agents as well as low levels of Nrf2-associated antioxidants. This condition creates a steroid-resistant inflammatory microenvironment. Lacticaseibacillus rhamnosus (Lr) is a known anti-cytokine in lung diseases; however, the effect of Lr on lung inflammation and oxidative stress in steroid-resistant COPD mice remains unknown. OBJECTIVE: Thus, we investigated the Lr effect on lung inflammation and oxidative stress in mice and macrophages exposed to cigarette smoke extract (CSE) and unresponsive to steroids. METHODS: Mice and macrophages received dexamethasone or GLPG-094 (a GPR43 inhibitor), and only the macrophages received butyrate (but), all treatments being given before CSE. Lung inflammation was evaluated from the leukocyte population, airway remodeling, cytokines, and NF-κB. Oxidative stress disturbance was measured from ROS, 8-isoprostane, NADPH oxidase, TBARS, SOD, catalase, HO-1, and Nrf2. RESULTS: Lr attenuated cellularity, mucus, collagen, cytokines, ROS, 8-isoprostane, NADPH oxidase, and TBARS. Otherwise, SOD, catalase, HO-1, and Nrf2 were upregulated in Lr-treated COPD mice. Anti-cytokine and antioxidant effects of butyrate also occurred in CSE-exposed macrophages. GLPG-094 rendered Lr and butyrate less effective. CONCLUSIONS: Lr attenuates lung inflammation and oxidative stress in COPD mice, suggesting the presence of a GPR43 receptor-dependent mechanism also found in macrophages.

Transcriptional profiling of a fungal granuloma reveals a low metabolic activity of Paracoccidioides brasiliensis yeasts and an actively regulated host immune response.

Granulomas are important immunological structures in the host defense against the fungus Paracoccidioides brasiliensis, the main etiologic agent of Paracoccidioidomycosis (PCM), a granulomatous systemic mycosis endemic in Latin America. We have performed transcriptional and proteomic studies of yeasts present in the pulmonary granulomas of PCM aiming to identify relevant genes and proteins that act under stressing conditions. C57BL/6 mice were infected with 1x106 yeasts and after 8- and 12-weeks of infection, granulomatous lesions were obtained for extraction of fungal and murine RNAs and fungal proteins. Dual transcriptional profiling was done comparing lung cells and P. brasiliensis yeasts from granulomas with uninfected lung cells and the original yeast suspension used in the infection, respectively. Mouse transcripts indicated a lung malfunction, with low expression of genes related to muscle contraction and organization. In addition, an increased expression of transcripts related to the activity of neutrophils, eosinophils, macrophages, lymphocytes as well as an elevated expression of IL-1ß, TNF-α, IFN-γ, IL-17 transcripts were observed. The increased expression of transcripts for CTLA-4, PD-1 and arginase-1, provided evidence of immune regulatory mechanisms within the granulomatous lesions. Also, our results indicate iron as a key element for the granuloma to function, where a high number of transcripts related to fungal siderophores for iron uptake was observed, a mechanism of fungal virulence not previously described in granulomas. Furthermore, transcriptomics and proteomics analyzes indicated a low fungal activity within the granuloma, as demonstrated by the decreased expression of genes and proteins related to energy metabolism and cell cycle.

A snapshot of Bothrops jararaca snake venom gland subcellular proteome.

Snake venom protein synthesis undergoes finely regulated processes in the specialized secretory epithelium within the venom gland. Such processes occur within a defined period in the cell and at specific cellular locations. Thus, the determination of subcellular proteomes allows the characterization of protein groups for which the site may be relevant to their biological roles, thereby allowing the deconvolution of complex biological circuits into functional information. In this regard, we performed subcellular fractionation of proteins from B. jararaca venom gland, focusing on nuclear proteins since this cellular compartment comprises key effectors that shape gene expression. Our results provided a snapshot of B. jararaca's subcellular venom gland proteome and pointed to a 'conserved' proteome core among different life stages (newborn and adult) and between sexes (adult male and female). Overall, the top 15 highly abundant proteins identified in B. jararaca venom glands mirrored the panel of highly expressed genes in human salivary glands. Therefore, the expression profile observed for such a protein set could be considered a conserved core signature of salivary gland secretory epithelium. Moreover, the newborn venom gland displayed a unique expression signature of transcription factors involved in regulating transcription and biosynthetic processes and may mirror biological constraints of the ontogenetic development of B. jararaca, contributing to venom proteome diversity.

ArtinM Cytotoxicity in B Cells Derived from Non-Hodgkin's Lymphoma Depends on Syk and Src Family Kinases.

Receptors on the immune cell surface have a variety of glycans that may account for the immunomodulation induced by lectins, which have a carbohydrate recognition domain (CRD) that binds to monosaccharides or oligosaccharides in a specific manner. ArtinM, a D-mannose-binding lectin obtained from Artocarpus heterophyllus, has affinity for the N-glycans core. Immunomodulation by ArtinM toward the Th1 phenotype occurs via its interaction with TLR2/CD14 N-glycans on antigen-presenting cells, as well as recognition of CD3γ N-glycans on murine CD4+ and CD8+ T cells. ArtinM exerts a cytotoxic effect on Jurkat human leukemic T-cell line and human myeloid leukemia cell line (NB4). The current study evaluated the effects of ArtinM on murine and human B cells derived from non-Hodgkin's lymphoma. We found that murine B cells are recognized by ArtinM via the CRD, and the ArtinM stimulus did not augment the proliferation rate or production of IL-2. However, murine B cell incubation with ArtinM augmented the rate of apoptosis, and this cytotoxic effect of ArtinM was also seen in human B cell-lines sourced from non-Hodgkin's lymphoma Raji cell line. This cytotoxic effect was inhibited by the phosphatase activity of CD45 on Lck, and the protein kinases of the Src family contribute to cell death triggered by ArtinM.

A snapshot of Bothrops jararaca snake venom gland subcellular proteome

Snake venom protein synthesis undergoes finely regulated processes in the specialized secretory epithelium within the venom gland. Such processes occur within a defined period in the cell and at specific cellular locations. Thus, the determination of subcellular proteomes allows the characterization of protein groups for which the site may be relevant to their biological roles, thereby allowing the deconvolution of complex biological circuits into functional information. In this regard, we performed subcellular fractionation of proteins from B. jararaca venom gland, focusing on nuclear proteins since this cellular compartment comprises key effectors that shape gene expression. Our results provided a snapshot of B. jararaca's subcellular venom gland proteome and pointed to a ‘conserved’ proteome core among different life stages (newborn and adult) and between sexes (adult male and female). Overall, the top 15 highly abundant proteins identified in B. jararaca venom glands mirrored the panel of highly expressed genes in human salivary glands. Therefore, the expression profile observed for such a protein set could be considered a conserved core signature of salivary gland secretory epithelium. Moreover, the newborn venom gland displayed a unique expression signature of transcription factors involved in regulating transcription and biosynthetic processes and may mirror biological constraints of the ontogenetic development of B. jararaca, contributing to venom proteome diversity.

The rate and role of pseudogenes of the Mycobacterium tuberculosis complex.

Whole-genome sequence analyses have significantly contributed to the understanding of virulence and evolution of the Mycobacterium tuberculosis complex (MTBC), the causative pathogens of tuberculosis. Most MTBC evolutionary studies are focused on single nucleotide polymorphisms and deletions, but rare studies have evaluated gene content, whereas none has comprehensively evaluated pseudogenes. Accordingly, we describe an extensive study focused on quantifying and predicting possible functions of MTBC and Mycobacterium canettii pseudogenes. Using NCBI's PGAP-detected pseudogenes, we analysed 25 837 pseudogenes from 158 MTBC and M. canetii strains and combined transcriptomics and proteomics of M. tuberculosis H37Rv to gain insights about pseudogenes' expression. Our results indicate significant variability concerning rate and conservancy of in silico predicted pseudogenes among different ecotypes and lineages of tuberculous mycobacteria and pseudogenization of important virulence factors and genes of the metabolism and antimicrobial resistance/tolerance. We show that in silico predicted pseudogenes contribute considerably to MTBC genetic diversity at the population level. Moreover, the transcription machinery of M. tuberculosis can fully transcribe most pseudogenes, indicating intact promoters and recent pseudogene evolutionary emergence. Proteomics of M. tuberculosis and close evaluation of mutational lesions driving pseudogenization suggest that few in silico predicted pseudogenes are likely capable of neofunctionalization, nonsense mutation reversal, or phase variation, contradicting the classical definition of pseudogenes. Such findings indicate that genome annotation should be accompanied by proteomics and protein function assays to improve its accuracy. While indels and insertion sequences are the main drivers of the observed mutational lesions in these species, population bottlenecks and genetic drift are likely the evolutionary processes acting on pseudogenes' emergence over time. Our findings unveil a new perspective on MTBC's evolution and genetic diversity.

A proteomics-MM/PBSA dual approach for the analysis of SARS-CoV-2 main protease substrate peptide specificity.

The main protease Mpro of SARS-CoV-2 is a well-studied major drug target. Additionally, it has been linked to this virus' pathogenicity, possibly through off-target effects. It is also an interesting diagnostic target. To obtain more data on possible substrates as well as to assess the enzyme's primary specificity a two-step approach was introduced. First, Terminal Amine Isobaric Labeling of Substrates (TAILS) was employed to identify novel Mpro cleavage sites in a mouse lung proteome library. In a second step, using a structural homology model, the MM/PBSA variant MM/GBSA (Molecular Mechanics Poisson-Boltzmann/Generalized Born Surface Area) free binding energy calculations were carried out to determine relevant interacting amino acids. As a result, 58 unique cleavage sites were detected, including six that displayed glutamine at the P1 position. Furthermore, modeling results indicated that Mpro has a far higher potential promiscuity towards substrates than expected. The combination of proteomics and MM/PBSA modeling analysis can thus be useful for elucidating the specificity of Mpro, and thus open novel perspectives for the development of future peptidomimetic drugs against COVID-19, as well as diagnostic tools.

The rate and role of pseudogenes of the Mycobacterium tuberculosis complex

Whole-genome sequence analyses have significantly contributed to the understanding of virulence and evolution of the Mycobacterium tuberculosis complex (MTBC), the causative pathogens of tuberculosis. Most MTBC evolutionary studies are focused on single nucleotide polymorphisms and deletions, but rare studies have evaluated gene content, whereas none has comprehensively evaluated pseudogenes. Accordingly, we describe an extensive study focused on quantifying and predicting possible functions of MTBC and Mycobacterium canettii pseudogenes. Using NCBI’s PGAP-detected pseudogenes, we analysed 25 837 pseudogenes from 158 MTBC and M. canetii strains and combined transcriptomics and proteomics of M. tuberculosis H37Rv to gain insights about pseudogenes' expression. Our results indicate significant variability concerning rate and conservancy of in silico predicted pseudogenes among different ecotypes and lineages of tuberculous mycobacteria and pseudogenization of important virulence factors and genes of the metabolism and antimicrobial resistance/tolerance. We show that in silico predicted pseudogenes contribute considerably to MTBC genetic diversity at the population level. Moreover, the transcription machinery of M. tuberculosis can fully transcribe most pseudogenes, indicating intact promoters and recent pseudogene evolutionary emergence. Proteomics of M. tuberculosis and close evaluation of mutational lesions driving pseudogenization suggest that few in silico predicted pseudogenes are likely capable of neofunctionalization, nonsense mutation reversal, or phase variation, contradicting the classical definition of pseudogenes. Such findings indicate that genome annotation should be accompanied by proteomics and protein function assays to improve its accuracy. While indels and insertion sequences are the main drivers of the observed mutational lesions in these species, population bottlenecks and genetic drift are likely the evolutionary processes acting on pseudogenes' emergence over time. Our findings unveil a new perspective on MTBC’s evolution and genetic diversity.

A proteomics-MM/PBSA dual approach for the analysis of SARS-CoV-2 main protease substrate peptide specificity

The main protease Mpro of SARS-CoV-2 is a well-studied major drug target. Additionally, it has been linked to this virus’ pathogenicity, possibly through off-target effects. It is also an interesting diagnostic target. To obtain more data on possible substrates as well as to assess the enzyme’s primary specificity a two-step approach was introduced. First, Terminal Amine Isobaric Labeling of Substrates (TAILS) was employed to identify novel Mpro cleavage sites in a mouse lung proteome library. In a second step, using a structural homology model, the MM/PBSA variant MM/GBSA (Molecular Mechanics Poisson-Boltzmann/Generalized Born Surface Area) free binding energy calculations were carried out to determine relevant interacting amino acids. As a result, 58 unique cleavage sites were detected, including six that displayed glutamine at the P1 position. Furthermore, modeling results indicated that Mpro has a far higher potential promiscuity towards substrates than expected. The combination of proteomics and MM/PBSA modeling analysis can thus be useful for elucidating the specificity of Mpro, and thus open novel perspectives for the development of future peptidomimetic drugs against COVID-19, as well as diagnostic tools.

Community-based network analyses reveal emerging connectivity patterns of protein-protein interactions in murine melanoma secretome.

Protein-protein interaction networks (PPINs) are static representations of protein connections in which topological features such as subgraphs (communities) may contain proteins functionally related, revealing an additional layer of interactome complexity. We created two PPINs from the secretomes of a paired set of murine melanocytes (a normal melanocyte and its transformed phenotype). Community structures, identified by a graph clustering algorithm, resulted in the identification of subgraphs in both networks. Interestingly, the underlying structure of such communities revealed shared and exclusive proteins (core and exclusive nodes, respectively), in addition to proteins that changed their location within each community (rewired nodes). Functional enrichment analysis of core nodes revealed conserved biological functions in both networks whereas exclusive and rewired nodes in the tumoral phenotype network were enriched in cancer-related processes, including TGFß signaling. We found a remarkable shift in the tumoral interactome, resulting in an emerging pattern which was driven by the presence of exclusive nodes and may represent functional network motifs. Our findings suggest that the rearrangement in the tumoral interactome may be correlated with the malignant transformation of melanocytes associated with substrate adhesion impediment. The interactions found in core and new/rewired nodes might potentially be targeted for therapeutic intervention in melanoma treatment. SIGNIFICANCE: Malignant transformation is a result of synergistic action of multiple molecular factors in which genetic alterations as well as protein expression play paramount roles. During oncogenesis, cellular crosstalk through the secretion of soluble mediators modulates the phenotype of transformed cells which ultimately enables them to successfully disrupt important signaling pathways, including those related to cell growth and proliferation. Therefore, in this work we profiled the secretomes of a paired set of normal and transformed phenotypes of a murine melanocyte. After assembling the two interactomes, clusters of functionally related proteins (network communities) were observed as well as emerging patterns of network rewiring which may represent an interactome signature of transformed cells. In summary, the significance of this study relies on the understanding of the repertoire of 'normal' and 'tumoral' secretomes and, more importantly, the set of interacting proteins (the interactome) in both of these conditions, which may reveal key components that might be potentially targeted for therapeutic intervention.

Community-based network analyses reveal emerging connectivity patterns of protein-protein interactions in murine melanoma secretome

Protein-protein interaction networks (PPINs) are static representations of protein connections in which topological features such as subgraphs (communities) may contain proteins functionally related, revealing an additional layer of interactome complexity. We created two PPINs from the secretomes of a paired set of murine melanocytes (a normal melanocyte and its transformed phenotype). Community structures, identified by a graph clustering algorithm, resulted in the identification of subgraphs in both networks. Interestingly, the underlying structure of such communities revealed shared and exclusive proteins (core and exclusive nodes, respectively), in addition to proteins that changed their location within each community (rewired nodes). Functional enrichment analysis of core nodes revealed conserved biological functions in both networks whereas exclusive and rewired nodes in the tumoral phenotype network were enriched in cancer-related processes, including TGFβ signaling. We found a remarkable shift in the tumoral interactome, resulting in an emerging pattern which was driven by the presence of exclusive nodes and may represent functional network motifs. Our findings suggest that the rearrangement in the tumoral interactome may be correlated with the malignant transformation of melanocytes associated with substrate adhesion impediment. The interactions found in core and new/rewired nodes might potentially be targeted for therapeutic intervention in melanoma treatment. Significance: Malignant transformation is a result of synergistic action of multiple molecular factors in which genetic alterations as well as protein expression play paramount roles. During oncogenesis, cellular crosstalk through the secretion of soluble mediators modulates the phenotype of transformed cells which ultimately enables them to successfully disrupt important signaling pathways, including those related to cell growth and proliferation. Therefore, in this work we profiled the secretomes of a paired set of normal and transformed phenotypes of a murine melanocyte. After assembling the two interactomes, clusters of functionally related proteins (network communities) were observed as well as emerging patterns of network rewiring which may represent an interactome signature of transformed cells. In summary, the significance of this study relies on the understanding of the repertoire of ‘normal’ and ‘tumoral’ secretomes and, more importantly, the set of interacting proteins (the interactome) in both of these conditions, which may reveal key components that might be potentially targeted for therapeutic intervention.

Evaluating the X chromosome-specific diversity of Colombian populations using insertion/deletion polymorphisms.

The European and African contribution to the pre-existing Native American background has influenced the complex genetic pool of Colombia. Because colonisation was not homogeneous in this country, current populations are, therefore, expected to have different proportions of Native American, European and African ancestral contributions. The aim of this work was to examine 11 urban admixed populations and a Native American group, called Pastos, for 32 X chromosome indel markers to expand the current knowledge concerning the genetic background of Colombia. The results revealed a highly diverse genetic background comprising all admixed populations, harbouring important X chromosome contributions from all continental source populations. In addition, Colombia is genetically sub-structured, with different proportions of European and African influxes depending on the regions. The samples from the North Pacific and Caribbean coasts have a high African ancestry, showing the highest levels of diversity. The sample from the South Andean region showed the lowest diversity and significantly higher proportion of Native American ancestry than the other samples from the North Pacific and Caribbean coasts, Central-West and Central-East Andean regions, and the Orinoquian region. The results of admixture analysis using X-chromosomal markers suggest that the high proportion of African ancestry in the North Pacific coast was primarily male driven. These men have joined to females with higher Native American and European ancestry (likely resulting from a classic colonial asymmetric mating type: European male x Amerindian female). This high proportion of male-mediated African contributions is atypical of colonial settings, suggesting that the admixture occurred during a period when African people were no longer enslaved. In the remaining regions, the African contribution was primarily female-mediated, whereas the European counterpart was primarily male driven and the Native American ancestry contribution was not gender biased.

Comparison of the genetic background of different Colombian populations using the SNPforID 52plex identification panel.

Various strategies for analysing SNP markers and genotyping have been published with the goal of obtaining informative profiles from biological samples that contain only small amounts of template and/or degraded DNA. In this study, a multiplex assay of 52 autosomal single-nucleotide polymorphisms (SNPs) was used to analyse 438 individuals from urban populations from different regions of Colombia, as well as a sample of 50 Native American individuals of the Pastos ethnic group from Nariño. To determine if significant differences in these 52 SNPs exist between the distinct regions of Colombia, genetic distance and admixture analyses were performed based on the available data for 17 different Colombian population groups and for population groups from Africa, Europe and America. The results demonstrate significant differences between the populations from the Southwest Andean, Central-West Andean, Central-East Andean, Orinoquian and northern Colombian Pacific Coast regions. Most of the regions exhibited a European and Native American admixture. One exception is the population from the region of Chocó (on the northern Pacific Coast), which exhibits a high proportion of African admixture (54 %). From the observed genetic backgrounds, it is possible to conclude that a single reference database for the entire country would not be suitable for forensic purposes. The allele frequencies and the forensically relevant parameters were calculated for all of the markers in each Colombian region with significant values for the combined matching probability (power of discrimination ≥0.99999999999999990) and the combined probability of exclusion (≥0.9990) in trios that were obtained from all of the population groups.

El concepto de "padre" en la teoría psicoanalítica / The concept of "father" in psychoanalytic theory

Algo que es patente en un seguimiento de las múltiples referencias freudianas en torno al padre, es la interrogación constante por su estatuto. Desde algunos apartes del Proyecto de psicología en donde el padre ya es colocado como un rival que recoge los celos del hijo varón, hasta los elementos posibles de extraer del Moisés y la religión monoteísta.

Something that is evident in a follow-up of the many Freudian references around the father, is the constant interrogation by his statute. From some sections of the Psychology Project where the father is already placed as a rival that collects the jealousy of the male child, to the possible elements of extracting from Moses and the monotheistic religion.