TRANÇADO: RECURSOS COMPUTACIONAIS APLICADOS NO PROCESSO DE PROJETO DE UM MOBILIÁRIO URBANO

Este trabalho apresenta a pesquisa, projeto e construção do mobiliário urbano Trançado, localizado no Largo da Batata, um espaço público na cidade de São Paulo. O artigo discute o desenvolvimento do algoritmo, os parâmetros, a performance do projeto e o uso das tecnologias de prototipagem rápida e da fabricação digital para a análise do modelo e das técnicas construtivas, assim como descreve detalhes da execução e instalação final. Oferece também dados da pesquisa de pós-ocupação, assim como discute a relação entre o projeto do mobiliário e seu uso real, visto que participaram do processo agentes de diferentes áreas, como profissionais, organizações e sociedade civil. A pesquisa contribui ao relatar e discutir a qualidade do processo aplicado, não se concentrando apenas em analisar o resultado do objeto arquitetônico, mas produzir insumos para a reflexão e avanço da aplicação dos recursos computacionais no processo de projeto em arquitetura e design.This study presents the research, project and construction of the urban furniture project of Trançado, located in the Largo da Batata, a public space of the city of São Paulo. The article discusses the development of the algorithm, parameters and performance of the project and the use of rapid prototyping and digital fabrication technologies for model analysis and construction techniques, as well as details of the final assembling. It also provides post-occupation data and discusses the relationship between design process and actual use of the furniture. Stakeholders from different areas were involved: professionals, organizations and citizens. The research contributes reporting and discussing the quality of the applied process, not only focusing on analyzing the results of the final object, but producing inputs for reflection and progress of the application of computational design thinking in the architectural design process

Comparisons of Two Proteomic Analyses of Non-Mucoid and Mucoid Pseudomonas aeruginosa Clinical Isolates from a Cystic Fibrosis Patient

Pseudomonas aeruginosa chronically infects the lungs of cystic fibrosis (CF) patients. The conditions in the CF lung appear to select for P. aeruginosa with advantageous phenotypes for chronic infection. However, the mechanisms that allow the establishment of this chronic infection have not been fully characterized. We have previously reported the transcriptional analysis of two CF isolates strains 383 and 2192. Strain 2192 is a mucoid, alginate overproducing strain whereas strain 383 is non-mucoid. Mucoid strains are associated with chronic infection of the CF lung and non-mucoid strains are the typical initially infecting isolates. To elucidate novel differences between these two strains, we employed two methods of shotgun proteomics: isobaric tags for relative and absolute quantitation (iTRAQ) and two-dimensional gel electrophoresis (2-DE). iTRAQ compares the amount of protein between samples and relies on protein abundance, while 2-DE gel electrophoresis depends on selection of separated protein spots. For both these methods, mass spectrometry was then used to identify proteins differentially expressed between the two strains. The compilation of these two proteomic methods along with Western blot analysis revealed proteins of the HSI-I operon of the type 6 secretion system, showed increased expression in 383 compared to 2192, confirming the our previous transcriptional analysis. Proteomic analysis of other proteins did not fully correlate with the transcriptome but other differentially expressed proteins are discussed. Also, differences were noted between the results obtained for the two proteomic techniques. These shotgun proteomic analyses identified proteins that had been predicted only through gene identification; we now refer to these as “proteins of unknown functions” since their existence has now been established however their functional characterization remains to be elucidated

Differential in vivo labeling with barcoded antibodies allows for simultaneous transcriptomic profiling of airway, lung tissue and intravascular immune cells

Single-cell RNA sequencing (scRNA-seq) is the state-of-the-art approach to study transcriptomic signatures in individual cells in respiratory health and disease. However, classical scRNA-seq approaches provide no spatial information and are performed using either bronchoalveolar lavage fluid (BAL) or lung single cell suspensions to assess transcript levels in airway and tissue immune cells, respectively. Herein we describe a simple method to simultaneously characterize transcriptomic features of airway, lung parenchymal and intravascular immune cells based on differential in vivo labeling with barcoded antibodies. In addition to gaining basic spatial information, this approach allows for direct comparison of cells within different anatomical compartments. Furthermore, this method provides a time- and cost-effective alternative to classical scRNA-seq where lung and BAL samples are processed individually, reducing animal and reagent use. We demonstrate the feasibility of this approach in a preclinical mouse model of bacterial lung infection comparing airway, parenchymal and vasculature neutrophils early after infection

Frontline Science: Employing enzymatic treatment options for management of ocular biofilm-based infections

Pseudomonas aeruginosa-induced corneal keratitis is a sight-threatening disease. The rise of antibiotic resistance among P. aeruginosa keratitis isolates makes treatment of this disease challenging, emphasizing the need for alternative therapeutic modalities. By comparing the responses to P. aeruginosa infection between an outbred mouse strain (Swiss Webster, SW) and a susceptible mouse strain (C57BL6/N), we found that the inherent neutrophil-killing abilities of these strains correlated with their susceptibility to infection. Namely, SW-derived neutrophils were significantly more efficient at killing P. aeruginosa in vitro than C57BL6/N-derived neutrophils. To interrogate whether the distinct neutrophil killing capacities were dependent on endogenous or exogenous factors, neutrophil progenitor cell lines were generated. The in vitro differentiated neutrophils from either SW or C57BL6/N progenitors retained the differential killing abilities, illustrating that endogenous factors conferred resistance. Consistently, quantitative LC-MS/MS analysis revealed strain-specific and infection-induced alterations of neutrophil proteomes. Among the distinctly elevated proteins in the SW-derived proteomes were alpha-mannosidases, potentially associated with protection. Inhibition of alpha-mannosidases reduced neutrophil bactericidal functions in vitro. Conversely, topical application of alpha-mannosidases reduced bacterial biofilms and burden of infected corneas. Cumulatively, these data suggest novel therapeutic approaches to control bacterial biofilm assembly and improve bacterial clearance via enzymatic treatments

Neisseria gonorrhoeae O-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure

Neisseria gonorrhoeae expresses an O-linked protein glycosylation pathway that targets PilE, the major pilin subunit protein of the Type IV pilus colonization factor. Efforts to define glycan structure and thus the functions of pilin glycosylation (Pgl) components at the molecular level have been hindered by the lack of sensitive methodologies. Here, we utilized a ‘top-down’ mass spectrometric approach to characterize glycan status using intact pilin protein from isogenic mutants. These structural data enabled us to directly infer the function of six components required for pilin glycosylation and to define the glycan repertoire of strain N400. Additionally, we found that the N. gonorrhoeae pilin glycan is O-acetylated, and identified an enzyme essential for this unique modification. We also identified the N. gonorrhoeae pilin oligosaccharyltransferase using bioinformatics and confirmed its role in pilin glycosylation by directed mutagenesis. Finally, we examined the effects of expressing the PglA glycosyltransferase from the Campylobacter jejuni N-linked glycosylation system that adds N-acetylgalactosamine onto undecaprenylpyrophosphate-linked bacillosamine. The results indicate that the C. jejuni and N. gonorrhoeae pathways can interact in the synthesis of O-linked di- and trisaccharides, and therefore provide the first experimental evidence that biosynthesis of the N. gonorrhoeae pilin glycan involves a lipid-linked oligosaccharide precursor. Together, these findings underpin more detailed studies of pilin glycosylation biology in both N. gonorrhoeae and N. meningitidis, and demonstrate how components of bacterial O- and N-linked pathways can be combined in novel glycoengineering strategies

Immune stealth-driven O2 serotype prevalence and potential for therapeutic antibodies against multidrug resistant Klebsiella pneumoniae

Emerging multidrug-resistant bacteria are a challenge for modern medicine, but how these pathogens are so successful is not fully understood. Robust antibacterial vaccines have prevented and reduced resistance suggesting a pivotal role for immunity in deterring antibiotic resistance. Here, we show the increased prevalence of Klebsiella pneumoniae lipopolysaccharide O2 serotype strains in all major drug resistance groups correlating with a paucity of anti-O2 antibodies in human B cell repertoires. We identify human monoclonal antibodies to O-antigens that are highly protective in mouse models of infection, even against heavily encapsulated strains. These antibodies, including a rare anti-O2 specific antibody, synergistically protect against drug-resistant strains in adjunctive therapy with meropenem, a standard-of-care antibiotic, confirming the importance of immune assistance in antibiotic therapy. These findings support an antibody-based immunotherapeutic strategy even for highly resistant K. pneumoniae infections, and underscore the effect humoral immunity has on evolving drug resistance

Indole and 7‐hydroxyindole diminish Pseudomonas aeruginosa virulence

Indole is an extracellular biofilm signal for Escherichia coli, and many bacterial oxygenases readily convert indole to various oxidized compounds including 7‐hydroxyindole (7HI). Here we investigate the impact of indole and 7HI on Pseudomonas aeruginosa PAO1 virulence and quorum sensing (QS)‐regulated phenotypes; this strain does not synthesize these compounds but degrades them rapidly. Indole and 7HI both altered extensively gene expression in a manner opposite that of acylhomoserine lactones; the most repressed genes encode the mexGHI‐opmD multidrug efflux pump and genes involved in the synthesis of QS‐regulated virulence factors including pyocyanin (phz operon), 2‐heptyl‐3‐hydroxy‐4(1H)‐quinolone (PQS) signal (pqs operon), pyochelin (pch operon) and pyoverdine (pvd operon). Corroborating these microarray results, indole and 7HI decreased production of pyocyanin, rhamnolipid, PQS and pyoverdine and enhanced antibiotic resistance. In addition, indole affected the utilization of carbon, nitrogen and phosphorus, and 7HI abolished swarming motility. Furthermore, 7HI reduced pulmonary colonization of P. aeruginosa in guinea pigs and increased clearance in lungs. Hence, indole‐related compounds have potential as a novel antivirulence approach for the recalcitrant pathogen P. aeruginosa

Development and validation of a rabbit model of Pseudomonas aeruginosa non-ventilated pneumonia for preclinical drug development

BackgroundNew drugs targeting antimicrobial resistant pathogens, including Pseudomonas aeruginosa, have been challenging to evaluate in clinical trials, particularly for the non-ventilated hospital-acquired pneumonia and ventilator-associated pneumonia indications. Development of new antibacterial drugs is facilitated by preclinical animal models that could predict clinical efficacy in patients with these infections.MethodsWe report here an FDA-funded study to develop a rabbit model of non-ventilated pneumonia with Pseudomonas aeruginosa by determining the extent to which the natural history of animal disease reproduced human pathophysiology and conducting validation studies to evaluate whether humanized dosing regimens of two antibiotics, meropenem and tobramycin, can halt or reverse disease progression.ResultsIn a rabbit model of non-ventilated pneumonia, endobronchial challenge with live P. aeruginosa strain 6206, but not with UV-killed Pa6206, caused acute respiratory distress syndrome, as evidenced by acute lung inflammation, pulmonary edema, hemorrhage, severe hypoxemia, hyperlactatemia, neutropenia, thrombocytopenia, and hypoglycemia, which preceded respiratory failure and death. Pa6206 increased >100-fold in the lungs and then disseminated from there to infect distal organs, including spleen and kidneys. At 5 h post-infection, 67% of Pa6206-challenged rabbits had PaO2 <60 mmHg, corresponding to a clinical cut-off when oxygen therapy would be required. When administered at 5 h post-infection, humanized dosing regimens of tobramycin and meropenem reduced mortality to 17-33%, compared to 100% for saline-treated rabbits (P<0.001 by log-rank tests). For meropenem which exhibits time-dependent bactericidal activity, rabbits treated with a humanized meropenem dosing regimen of 80 mg/kg q2h for 24 h achieved 100% T>MIC, resulting in 75% microbiological clearance rate of Pa6206 from the lungs. For tobramycin which exhibits concentration-dependent killing, rabbits treated with a humanized tobramycin dosing regimen of 8 mg/kg q8h for 24 h achieved Cmax/MIC of 9.8 ± 1.4 at 60 min post-dose, resulting in 50% lung microbiological clearance rate. In contrast, rabbits treated with a single tobramycin dose of 2.5 mg/kg had Cmax/MIC of 7.8 ± 0.8 and 8% (1/12) microbiological clearance rate, indicating that this rabbit model can detect dose-response effects.ConclusionThe rabbit model may be used to help predict clinical efficacy of new antibacterial drugs for the treatment of non-ventilated P. aeruginosa pneumonia