Whole-genome sequencing and gene sharing network analysis powered by machine learning identifies antibiotic resistance sharing between animals,humans and environment in livestock farming

Anthropogenic environments such as those created by intensive farming of livestock, have been proposed to provide ideal selection pressure for the emergence of antimicrobial-resistant Escherichia coli bacteria and antimicrobial resistance genes (ARGs) and spread to humans. Here, we performed a longitudinal study in a large-scale commercial poultry farm in China, collecting E. coli isolates from both farm and slaughterhouse; targeting animals, carcasses, workers and their households and environment. By using whole-genome phylogenetic analysis and network analysis based on single nucleotide polymorphisms (SNPs), we found highly interrelated non-pathogenic and pathogenic E. coli strains with phylogenetic intermixing, and a high prevalence of shared multidrug resistance profiles amongst livestock, human and environment. Through an original data processing pipeline which combines omics, machine learning, gene sharing network and mobile genetic elements analysis, we investigated the resistance to 26 different antimicrobials and identified 361 genes associated to antimicrobial resistance (AMR) phenotypes; 58 of these were known AMR-associated genes and 35 were associated to multidrug resistance. We uncovered an extensive network of genes, correlated to AMR phenotypes, shared among livestock, humans, farm and slaughterhouse environments. We also found several human, livestock and environmental isolates sharing closely related mobile genetic elements carrying ARGs across host species and environments. In a scenario where no consensus exists on how antibiotic use in the livestock may affect antibiotic resistance in the human population, our findings provide novel insights into the broader epidemiology of antimicrobial resistance in livestock farming. Moreover, our original data analysis method has the potential to uncover AMR transmission pathways when applied to the study of other pathogens active in other anthropogenic environments characterised by complex interconnections between host species.     

Glucose and glutamine utilization by rat lymphocytes, monocytes and neutrophils in culture: a comparative study

Glucose and glutamine utilization and production of glutamate and lactate were determined for up to 48 h in lymphocytes, monocytes and neutrophils cultured in medium rich in metabolites and vitamins. Glucose was utilized by the three cell types in culture in the following order: neutrophils > monocytes > lymphocytes, whereas lactate was produced in the order: monocytes > neutrophils > lymphocytes. The consumption of glucose followed the activity of glucose-6-phosphate dehydrogenase but it was not related to hexokinase activity. Glutamine was consumed by the three leukocyte types in culture as follows: neutrophils > lymphocytes > or = monocytes. The consumption of glutamine was not fully related to the activity of phosphate-dependent glutaminase. The production of glutamate was not remarkably different among the three cell types. For comparison, glutamine and glucose utilization and glutamate and lactate production were also evaluated using 1-h incubated leukocytes. Under this condition, only glucose or glutamine was added to the medium. Glucose was utilized as follows: neutrophils > monocytes > lymphocytes, whereas lactate was produced in the following order: monocytes > or = neutrophils > lymphocytes. Glutamine was consumed as follows: neutrophils > lymphocytes > monocytes, whereas glutamate was produced as follows: neutrophils > or = monocytes = lymphocytes. The ratio of the amount of glucose/glutamine consumed by 1-h incubated cells was 0.5 for neutrophils, 1.5 for monocytes, and 0.3 for lymphocytes. However, the three cell types cultured for 48 h utilized glucose to a much higher degree than glutamine. The ratio of the amount of glucose/glutamine utilized by the cultured cells was 8.9 for neutrophils, 16.4 for monocytes, and 6.7 for lymphocytes. These observations support the proposition that glutamine is required in much higher amounts than glucose to accomplish the total metabolic requirement of leukocytes. Under conditions closer to physiological when the availability of a variety of metabolites and vitamins is not restricted, glucose is the preferred substrate for lymphocytes, monocytes and neutrophils.     

Interaction of soluble CD163 with activated T lymphocytes involves its association with non-muscle myosin heavy chain type A

CD163 is a monocyte/macrophage-specific scavenger receptor that undergoes ectodomain shedding upon an inflammatory stimulus. Soluble CD163 (sCD163) actively inhibits lymphocyte proliferation, but to date exactly how it interacts with these cells has remained elusive. We screened T lymphocytes and endothelial cells for proteins binding to sCD163. In both cell types a high affinity binding protein was detected. Partial sequencing of the protein revealed sequence identity to a non-muscle myosin heavy chain type A. Employing labelled sCD163 we found little specific binding of sCD163 to the extracellular domains of T lymphocytes and human umbilical vein endothelial cells (HUVEC). In activated T lymphocytes we demonstrated specific binding of sCD163 to intracellular structures as well as the presence of the native protein within the cell after co-incubation with purified sCD163. Furthermore, we developed a novel ELISA for highly specific detection of sCD163-myosin complexes. These complexes were present in activated T lymphocytes after incubation with shed sCD163. Co-localization of sCD163 and cellular myosin in T lymphocytes was further confirmed by fluorescence microscopy. Our results suggest that sCD163 associates with cellular myosin, thereby possibly modulating the cells' response to an inflammatory stimulus.     

Coumarin content and physicochemical profile of Mikania laevigata extracts

The 'guaco' lianous herb Mikania laevigata, which is widespread in Southern Brazil, is traditionally used to treat bronchitis, asthma and cough. This work investigates the influence of the extraction method, solvent:drug ratio, ethanol proportion, harvest season (summer or winter) and solvent heating on the physicochemical profile of the extracts (dry weight, density, pH) and the coumarin (1,2-benzopyrone) content determined by LC. Among the results obtained, it is observed that higher ethanol content increases the amount of coumarin in the extract. Leaves harvested in summer also produce an extract with a high coumarin yield. The most efficient method of extraction is percolation, independent of the solvent used.     

Genetic diversity of Costa Rican populations of the rice planthopper Tagosodes orizicolus (Homoptera: Delphacidae)

Tagosodes orizicolus (Homoptera: Delphacidae) is one of the main constraints of the rice production in the Neotropics. This planthopper produces severe damages as a phloem feeder, causes mechanical injury during oviposition and vectors the rice hoja blanca virus (RHBV). The main objective of this study was to determine the genetic diversity of T. orizicolus populations from three rice growing regions of Costa Rica, using RAPDs. Individuals from Guanacaste, Parrita, San Carlos and Cali-Colombia, as outgroup, were analyzed using the random primers. Phenetic relationships revealed that the Costa Rican populations were clearly separated from Cali-Colombia, sharing less than 25% similarity. Costa Rican populations were divided into two main branches separated at 30% similarity. The first branch included Guanacaste and San Carlos and the second displayed Parrita. In relation to similarity indexes within groups, the Guanacaste cluster showed the highest (over 50%) and Cali-Colombia was the most diverse (28%). The correspondence analysis confirmed the clusters of the phenogram and showed close interactions between the Parrita and San Carlos populations. The genetic separation observed could be the result of the geographic isolation among populations, but it could also be explained by the infection with the rickettsia Wolbachia pipientis. This bacterium causes cytoplasmic incompatibility in its host, which results in non-viable progeny when infected males mate with non-infected females, or when insects hosting different strains of Wolbachia mate. Then, a search for Wolbachia in previously described populations of T orizicolus was initiated. The presence of the bacteria was analyzed by PCR with 16S rDNA-specific primers for Wolbachia. The PCR analyses revealed infections of 86% in the population of San Carlos, 96% in Guanacaste, 37% in Parrita and 100% in Cali-Colombia. Crosses between individuals of T. orizicolus from Parrita and Guanacaste were performed for testing cytoplasmic incompatibility. When infected males were crossed with non-infected females within the same population, a significant reduction in progeny number was obtained as well as when crosses between infected individuals belonging to different populations were performed. These experiments showed cytoplasmic incompatibility not only caused by the presence of Wolbachia within the population, but also by the presence of different strains of the bacteria between populations.     

Protoporphyrin IX-induced structural and functional changes in human red blood cells,haemoglobin and myoglobin

Protoporphyrin IX and its derivatives are used as photosensitizers in the photodynamic therapy of cancer. Protoporphyrin IX penetrates into human red blood cells and releases oxygen from them. This leads to a change in the morphology of the cells. Spectrophotometric studies reveal that protoporphyrin IX interacts with haemoglobin and myoglobin forming ground state complexes. For both proteins, the binding affinity constant decreases, while the possible number of binding sites increases, as the aggregation state of the porphyrin is increased. The interactions lead to conformational changes of both haemoglobin and myoglobin as observed in circular dichroism studies. Upon binding with the proteins, protoporphyrin IX releases the heme-bound oxygen from the oxyproteins, which is dependent on the stoichiometric ratios of the porphyrin: protein. The peroxidase activities of haemoglobin and myoglobin are potentiated by the protein-porphyrin complexation. Possible mechanisms underlying the relation between the porphyrin-induced structural modifications of the heme proteins and alterations in their functional properties have been discussed. The findings may have a role in establishing efficacy of therapeutic uses of porphyrins as well as in elucidating their mechanisms of action as therapeutic agents.     

Microscopy images as interactive tools in cell modeling and cell biology education

The advent of genomics, proteomics, and microarray technology has brought much excitement to science, both in teaching and in learning. The public is eager to know about the processes of life. In the present context of the explosive growth of scientific information, a major challenge of modern cell biology is to popularize basic concepts of structures and functions of living cells, to introduce people to the scientific method, to stimulate inquiry, and to analyze and synthesize concepts and paradigms. In this essay we present our experience in mixing science and education in Brazil. For two decades we have developed activities for the science education of teachers and undergraduate students, using microscopy images generated by our work as cell biologists. We describe open-air outreach education activities, games, cell modeling, and other practical and innovative activities presented in public squares and favelas. Especially in developing countries, science education is important, since it may lead to an improvement in quality of life while advancing understanding of traditional scientific ideas. We show that teaching and research can be mutually beneficial rather than competing pursuits in advancing these goals.