The Streptomyces coelicolor small ORF trpM stimulates growth and morphological development and exerts opposite effects on actinorhodin and calcium-dependent antibiotic production

In actinomycetes, antibiotic production is often associated with a morpho-physiological differentiation program that is regulated by complex molecular and metabolic networks. Many aspects of these regulatory circuits have been already elucidated and many others still deserve further investigations. In this regard, the possible role of many small open reading frames (smORFs) in actinomycete morpho-physiological differentiation is still elusive. In Streptomyces coelicolor, inactivation of the smORF trpM (SCO2038) – whose product modulates L-tryptophan biosynthesis – impairs production of antibiotics and morphological differentiation. Indeed, it was demonstrated that TrpM is able to interact with PepA (SCO2179), a putative cytosol aminopeptidase playing a key role in antibiotic production and sporulation. In this work, a S. coelicolor trpM knock-in (Sco-trpMKI) mutant strain was generated by cloning trpM into overexpressing vector to further investigate the role of trpM in actinomycete growth and morpho-physiological differentiation. Results highlighted that trpM: (i) stimulates growth and actinorhodin (ACT) production; (ii) decreases calcium-dependent antibiotic (CDA) production; (iii) has no effect on undecylprodigiosin production. Metabolic pathways influenced by trpM knock- in were investigated by combining two-difference in gel electrophoresis/nanoliquid chromatography coupled to electrospray linear ion trap tandem mass spectrometry (2D- DIGE/nanoLC-ESI-LIT-MS/MS) and by LC-ESI-MS/MS procedures, respectively. These analyses demonstrated that over-expression of trpM causes an over-representation of factors involved in protein synthesis and nucleotide metabolism as well as a down-representation of proteins involved in central carbon and amino acid metabolism. At the metabolic level, this corresponded to a differential accumulation pattern of different amino acids – including aromatic ones but tryptophan – and central carbon intermediates. PepA was also down-represented in Sco-trpMKI. The latter was produced as recombinant His-tagged protein and was originally proven having the predicted aminopeptidase activity. Altogether, these results highlight the stimulatory effect of trpM in S. coelicolor growth and ACT biosynthesis, which are elicited through the modulation of various metabolic pathways and PepA representation, further confirming the complexity of regulatory networks that control antibiotic production in actinomycetes

A large-scale empirical exploration on refactoring activities in open source software projects

Refactoring is a well-established practice that aims at improving the internal structure of a software system without changing its external behavior. Existing literature provides evidence of how and why developers perform refactoring in practice. In this paper, we continue on this line of research by performing a large-scale empirical analysis of refactoring practices in 200 open source systems. Specifically, we analyze the change history of these systems at commit level to investigate: (i) whether developers perform refactoring operations and, if so, which are more diffused and (ii) when refactoring operations are applied, and (iii) which are the main developer-oriented factors leading to refactoring. Based on our results, future research can focus on enabling automatic support for less frequent refactorings and on recommending refactorings based on the developer's workload, project's maturity and developer's commitment to the project

Investigation of Ducted Fuel Injection Implementation in a Retrofitted Light-Duty Diesel Engine through Numerical Simulation

Ducted Fuel Injection (DFI) is a concept of growing interest to abate soot emissions in diesel combustion, based on a small duct within the combustion chamber in front of the injector nozzle. Despite the impressive potential of the DFI has been proven in literature, its application for series production and the complexity for the adaptation of existing compression-ignition (CI) engines need to be extensively investigated. In this context, the aim of this study is to numerically assess the potential of DFI implementation in a CI engine for light-duty applications, highlighting the factors which can limit or facilitate its integration in existing combustion chambers. The numerical model for combustion simulation was based on a 1D/3D-CFD coupled approach relying on a calibrated spray model, extensively validated against experimental data. Once assessed the coupling procedure by comparing the numerical results with experimental in-cylinder pressure and heat release rate data for both low and high load operating conditions, the duct impact was investigated introducing it in the computational domain. It was observed that DFI did not yield any significant advantage to engine-out soot emissions and fuel consumption with the existing combustion system. Although the soot formation was generally reduced, the soot oxidation process was partially inhibited by the duct adoption maintaining fixed the engine calibration, suggesting the need for complete optimization of the combustion system design. On the other hand, a preliminary variation of engine calibration highlighted several beneficial trends for DFI, whose operation improved with a simplified injection strategy. Present numerical results indicate that DFI retrofit solutions without specific optimization of the combustion system design do not guarantee soot reduction. Nevertheless, wide room for improvement remains in terms of DFI-targeted combustion chamber design and engine calibration towards the complete success of this technology for soot-free CI engines

An Investigation of Radiation Heat Transfer in a Light-Duty Diesel Engine

In the last two decades engine research has been mainly focused on reducing pollutant emissions. This fact together with growing awareness about the impacts of climate change are leading to an increase in the importance of thermal efficiency over other criteria in the design of internal combustion engines (ICE). In this framework, the heat transfer to the combustion chamber walls can be considered as one of the main sources of indicated efficiency diminution. In particular, in modern direct-injection diesel engines, the radiation emission from soot particles can constitute a significant component of the efficiency losses. Thus, the main of objective of the current research was to evaluate the amount of energy lost to soot radiation relative to the input fuel chemical energy during the combustion event under several representative engine loads and speeds. Moreover, the current research characterized the impact of different engine operating conditions on radiation heat transfer. For this purpose, a combination of theoretical and experimental tools were used. In particular, soot radiation was quantified with a sensor that uses two-color thermometry along with its corresponding simplified radiation model. Experiments were conducted using a 4-cylinder direct-injection light-duty diesel engine fully instrumented with thermocouples. The goal was to calculate the energy balance of the input fuel chemical energy. Results provide a characterization of radiation heat transfer for different engine loads and speeds as well as radiation trends for different engine operating conditions.Benajes Calvo, JV.; Martín Díaz, J.; García Martínez, A.; Villalta Lara, D.; Warey, A.; Doménech Llopis, V.; Vassallo, AL.... (2015). An Investigation of Radiation Heat Transfer in a Light-Duty Diesel Engine. SAE International Journal of Engines. 8(5):1-14. doi:10.4271/2015-24-2443S1148

Impact of counter-bore nozzle on the combustion process and exhaust emissions for light-duty diesel engine application

This is the author s version of a work that was accepted for publication in International Journal of Engine Research. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published as https://doi.org/10.1177/1468087418819250[EN] This article describes the main results of an investigation about counter-bore injector nozzle impact on the combustion process in a modern Euro 6 diesel engine. First, hydraulic and spray visualization tests have been performed, showing a potential advantage of such nozzle design in fuel-air mixing efficiency. Then, combustion performance has been assessed on a GM-designed 1.6-L four-cylinder engine. The engine has been installed on a dynamometric test bench and instrumented with an AVL cylinder pressure transducer for heat release rate analysis, as well as HORIBA MEXA gas analyzer for exhaust emissions and AVL 415 Smoke Meter. Engine efficiency and emissions have been analyzed on four different part-load steady-state points, representative of New European Driving Cycle and Worldwide harmonized Light duty Test Cycle certification cycles, and covering engine speeds from 1250 to 2000 r/min and brake mean effective pressure between 0.2 and 1.4 MPa. Results of indicated analysis show that counter-bore nozzles have significant differences in terms of pilot injection combustion at low load points, which in turn lead to a better ignition and shorter combustion of the main injection. In addition, an improvement of diffusive combustion is observed as load increases. Because of both, fuel consumption is reduced by approximately 1% with respect to a standard nozzle. Finally, an appreciable decrease in engine exhaust emissions has been recorded, especially in terms of particulate matter and hydrocarbon emissions. This reduction has been linked to the improvement of fuel-air mixing promoted by the counter-bore nozzle previously observed.The authors would like to thank General Motors Global Propulsion Systems-Torino S.r.l. for sponsoring the current work. Part of the equipment was purchased with the help of Generalitat Valenciana in project IDIFEDER2018 with title "Equipamiento de diagnostico optico de alta velocidad para estudiar procesos de inyeccion''.Payri, R.; De La Morena, J.; Monsalve-Serrano, J.; Pesce, FC.; Vassallo, A. (2019). Impact of counter-bore nozzle on the combustion process and exhaust emissions for light-duty diesel engine application. International Journal of Engine Research. 20(1):46-57. https://doi.org/10.1177/1468087418819250S4657201Kastengren, A. L., Tilocco, F. Z., Powell, C. F., Manin, J., Pickett, L. M., Payri, R., & Bazyn, T. (2012). ENGINE COMBUSTION NETWORK (ECN): MEASUREMENTS OF NOZZLE GEOMETRY AND HYDRAULIC BEHAVIOR. Atomization and Sprays, 22(12), 1011-1052. doi:10.1615/atomizspr.2013006309Payri, R., Viera, J. P., Gopalakrishnan, V., & Szymkowicz, P. G. (2016). The effect of nozzle geometry over internal flow and spray formation for three different fuels. Fuel, 183, 20-33. doi:10.1016/j.fuel.2016.06.041Postrioti, L., Malaguti, S., Bosi, M., Buitoni, G., Piccinini, S., & Bagli, G. (2014). Experimental and numerical characterization of a direct solenoid actuation injector for Diesel engine applications. Fuel, 118, 316-328. doi:10.1016/j.fuel.2013.11.001Payri, R., Gimeno, J., De la Morena, J., Battiston, P. A., Wadhwa, A., & Straub, R. (2016). Study of new prototype pintle injectors for diesel engine application. Energy Conversion and Management, 122, 419-427. doi:10.1016/j.enconman.2016.06.003Payri, R., Bracho, G., Marti-Aldaravi, P., & Viera, A. (2017). NEAR FIELD VISUALIZATION OF DIESEL SPRAY FOR DIFFERENT NOZZLE INCLINATION ANGLES IN NON-VAPORIZING CONDITIONS. Atomization and Sprays, 27(3), 251-267. doi:10.1615/atomizspr.2017017949Li, T., Moon, S., Sato, K., & Yokohata, H. (2017). A comprehensive study on the factors affecting near-nozzle spray dynamics of multi-hole GDI injectors. Fuel, 190, 292-302. doi:10.1016/j.fuel.2016.11.009Payri, R., Viera, J. P., Gopalakrishnan, V., & Szymkowicz, P. G. (2017). The effect of nozzle geometry over ignition delay and flame lift-off of reacting direct-injection sprays for three different fuels. Fuel, 199, 76-90. doi:10.1016/j.fuel.2017.02.075Yao, C., Geng, P., Yin, Z., Hu, J., Chen, D., & Ju, Y. (2016). Impacts of nozzle geometry on spray combustion of high pressure common rail injectors in a constant volume combustion chamber. Fuel, 179, 235-245. doi:10.1016/j.fuel.2016.03.097Hong, J. G., Ku, K. W., Kim, S. R., & Lee, C. W. (2010). EFFECT OF CAVITATION IN CIRCULAR NOZZLE AND ELLIPTICAL NOZZLES ON THE SPRAY CHARACTERISTIC. Atomization and Sprays, 20(10), 877-886. doi:10.1615/atomizspr.v20.i10.40Molina, S., Salvador, F. J., Carreres, M., & Jaramillo, D. (2014). A computational investigation on the influence of the use of elliptical orifices on the inner nozzle flow and cavitation development in diesel injector nozzles. Energy Conversion and Management, 79, 114-127. doi:10.1016/j.enconman.2013.12.015Taskiran, O. O., & Ergeneman, M. (2014). Effect of nozzle dimensions and fuel type on flame lift-off length. Fuel, 115, 833-840. doi:10.1016/j.fuel.2013.03.005He, Z., Guo, G., Tao, X., Zhong, W., Leng, X., & Wang, Q. (2016). Study of the effect of nozzle hole shape on internal flow and spray characteristics. International Communications in Heat and Mass Transfer, 71, 1-8. doi:10.1016/j.icheatmasstransfer.2015.12.002Salvador, F. J., de la Morena, J., Carreres, M., & Jaramillo, D. (2017). Numerical analysis of flow characteristics in diesel injector nozzles with convergent-divergent orifices. Proceedings of the Institution of Mechanical Engineers, Part D: Journal of Automobile Engineering, 231(14), 1935-1944. doi:10.1177/0954407017692220Baldwin, E. T., Grover, R. O., Parrish, S. E., Duke, D. J., Matusik, K. E., Powell, C. F., … Schmidt, D. P. (2016). String flash-boiling in gasoline direct injection simulations with transient needle motion. International Journal of Multiphase Flow, 87, 90-101. doi:10.1016/j.ijmultiphaseflow.2016.09.004Payri, R., Salvador, F. J., Gimeno, J., & Bracho, G. (2008). A NEW METHODOLOGY FOR CORRECTING THE SIGNAL CUMULATIVE PHENOMENON ON INJECTION RATE MEASUREMENTS. Experimental Techniques, 32(1), 46-49. doi:10.1111/j.1747-1567.2007.00188.xDesantes, J. M., Pastor, J. V., García-Oliver, J. M., & Pastor, J. M. (2009). A 1D model for the description of mixing-controlled reacting diesel sprays. Combustion and Flame, 156(1), 234-249. doi:10.1016/j.combustflame.2008.10.008PASTOR, J., JAVIERLOPEZ, J., GARCIA, J., & PASTOR, J. (2008). A 1D model for the description of mixing-controlled inert diesel sprays. Fuel, 87(13-14), 2871-2885. doi:10.1016/j.fuel.2008.04.017Payri, F., Molina, S., Martín, J., & Armas, O. (2006). Influence of measurement errors and estimated parameters on combustion diagnosis. Applied Thermal Engineering, 26(2-3), 226-236. doi:10.1016/j.applthermaleng.2005.05.00

Immunohistochemical study of basement membrane collagen IV in uterine cervix carcinoma

CONTEXT: The integrity of basement membrane (BM) is damaged during the evolution of a benign or potentially malignant lesion into a malignant one, in which it may undergo several degrees of discontinuity as a necessary condition for the invasive process. Immunostaining for collagen IV, which is exclusively found in BM, has been used to evaluate its formation in neoplastic and benign lesions of several organs.OBJECTIVE: To investigate BM continuity pattern in squamous carcinoma in situ (CIS), microinvasive (MIC) and invasive (IC) squamous cell carcinoma of the uterine cervix, and to find out if BM expression could be useful in the diagnosis of early stromal invasion (MIC).DESIGN: Archival material between 1988 and 1993 was studied at the Pathological Anatomy Department - Unicamp.PROCEDURES: The selected cases, previously formalin fixed and paraffin embedded, were reviewed retrospectively by submitting them to immunohistochemical study via the avidin-biotin-peroxidase method using a monoclonal antibody anticollagen IV.RESULTS: In all, 17 cases of CIS, 16 of MIC and 21 of IC were evaluated. All IC cases showed evident BM discontinuity, either focal or diffuse. In the CIS group, a continuous BM pattern was predominant, being focally disrupted in only 2/17 cases (11.8%). The MIC group showed an intermediate pattern, but with a clear tendency to BM discontinuity in 10/16 cases (62.5%). Inflammatory infiltrate, a variable also studied, cannot be considered responsible for BM discontinuity, since there was no statistical correlation between them.CONCLUSION: We conclude that immunostaining for collagen IV may contribute to the diagnosis of stromal invasion by BM discontinuity.CONTEXTO: A integridade da membrana basal (MB) é destruída no processo de evolução de uma lesão benigna ou potencialmente maligna para uma lesão maligna, onde ela pode sofrer vários graus de descontinuidade, como condição necessária para o processo de invasão. A imunocoloração para colágeno IV, que é exclusivamente encontrado na MB, tem sido utilizada para avaliar sua formação em processos benignos e neoplásicos de vários órgãos. OBJETIVO: Investigar o padrão de continuidade da MB no carcinoma in situ (CIS), microinvasivo (CMI) e epidermóide (CE) do colo uterino. Além disso, pretendeu-se verificar em que medida estes padrões poderiam ser úteis no diagnóstico de invasão estromal inicial (CMI). TIPO DE ESTUDO: Foi estudado o material do arquivo de tecidos dos anos de 1988 a 1993 do Departamento de Anatomia Patológica da UNICAMP. PROCEDIMENTOS: Os tecidos eram fixados em formalina e incluídos em parafina e foram revistos retrospectivamente para serem submetidos à reação imuno-histoquímica pelo método da avidina-biotina-peroxidase, com o anticorpo monoclonal anti-colágeno IV. RESULTADOS: Ao todo, foram avaliados 17 casos de CIS, 16 de CMI e 21 de CE. Todos os casos de CE mostraram evidente descontinuidade da MB, quer focal ou difusa. No grupo dos CIS foi observado um padrão contínuo de MB, sendo focalmente lesado em apenas 2/17 casos (11,8%). O grupo dos CMI mostraram um padrão intermediário, com clara tendência à descontinuidade da MB em 10/16 casos (62,5%). O infiltrado inflamatório, variável também estudada, não pode ser responsabilizado pela descontinuidade da MB, já que não houve correlação estatística entre eles. CONCLUSÃO: A imunocoloração para colágeno IV pode contribuir no diagnóstico de invasão estromal, quando houver lesão da MB.1846185

Unravelling the DNA sequences carried by Streptomyces coelicolor membrane vesicles

Membrane vesicles (MVs) are spherical particles with nanoscale dimensions and characterized by the presence of diverse cargos, such as nucleic acids, proteins, lipids, and cellular metabolites. Many examples of (micro)organisms producing MVs are reported in literature. Among them, bacterial MVs are of particular interest because they are now considered as the fourth mechanism of horizontal gene transfer. Streptomyces bacteria are well-known for their ecological roles and ability to synthesize bioactive compounds, with Streptomyces coelicolor being the model organism. It was previously demonstrated that it can produce distinct populations of MVs characterized by different protein and metabolite cargos. In this work we demonstrated for the first time that MVs of S. coelicolor carry both DNA and RNA and that their DNA content represents the entire chromosome of the bacterium. These findings suggest that MV DNA could have a role in the evolution of Streptomyces genomes and that MVs could be exploited in new strain engineering strategies

Reassessment Of Diagnostic Criteria In Cutaneous Lymphocytic Infiltrates.

Non-specific lymphocytic infiltrates of the skin pose difficulties in daily practice in pathology. There is still a lack of pathognomonic signs for the differential diagnosis between benign and malignant lymphocytic infiltrates. To evaluate the morphological and immunohistochemical profile of lymphocytic infiltrations of the skin according to clinical outcome. Retrospective; histopathological and immunohistochemical analysis. Referral center, university hospital. 28 cases of lymphocytic infiltrates of difficult differential diagnosis selected from the records. Eighteen histological variables and the immunophenotypic profile were assessed using the CD4, CD8, CD3, CD20 and CD30 lymphoid markers and compared to subsequent follow-up. The most common diagnoses were: initial mycosis fungoides (eight cases) and drug reactions (five cases). Single morphological variables did not discriminate between benign and malignant infiltrates except for the presence of Pautrier-Darier's microabscesses, which were found only in mycosis fungoides (p = 0.015). Patterns of superficial and deep infiltration (p = 0.037) and also the presence of eosinophils (p = 0.0207) were more frequently found in benign lymphocytic infiltrates. Immunohistochemical profile of T-cell subsets showed overlap between benign and malignant infiltrates with a predominance of CD4-positive (helper) lymphocytes in the majority of cases. A combination of clinical and histological features remains the most reliable approach for establishing a definite diagnosis in cases of lymphoid skin infiltrates.122161-

Antibiotic-resistant genes and bacteria as evolving contaminants of emerging concerns (e-CEC): is it time to include evolution in risk assessment?

The pressing issue of the abundance of antibiotic resistance genes and resistant bacteria in the environment (ARGs and ARB, respectively) requires procedures for assessing the risk to health. The chemo-centric environmental risk assessment models identify hazard(s) in a dose–response manner, obtaining exposure, toxicity, risk, impact and policy. However, this risk assessment approach based on ARGs/ARB evaluation from a quantitative viewpoint shows high unpredictability because ARGs/ARB cannot be considered as standard hazardous molecules: ARB duplicate and ARGs evolve within a biological host. ARGs/ARB are currently listed as Contaminants of Emerging Concern (CEC). In light of such characteristics, we propose to define ARGs/ARB within a new category of evolving CEC (or e-CEC). ARGs/ARB, like any other evolving determinants (e.g., viruses, bacteria, genes), escape environmental controls. When they do so, just one molecule left remaining at a control point can form the origin of a new dangerous and selection-responsive population. As a consequence, perhaps it is time to acknowledge this trait and to include evolutionary concepts within modern risk assessment of e-CEC. In this perspective we analyze the evolutionary responses most likely to influence risk assessment, and we speculate on the means by which current methods could measure evolution. Further work is required to implement and exploit such experimental procedures in future risk assessment protocols

The Histidine Biosynthetic Genes in the Superphylum Bacteroidota-Rhodothermota-Balneolota-Chlorobiota: Insights into the Evolution of Gene Structure and Organization

One of the most studied metabolic routes is the biosynthesis of histidine, especially in enterobacteria where a single compact operon composed of eight adjacent genes encodes the complete set of biosynthetic enzymes. It is still not clear how his genes were organized in the genome of the last universal common ancestor community. The aim of this work was to analyze the structure, organization, phylogenetic distribution, and degree of horizontal gene transfer (HGT) of his genes in the Bacteroidota-Rhodothermota-Balneolota-Chlorobiota superphylum, a group of phylogenetically close bacteria with different surviving strategies. The analysis of the large variety of his gene structures and organizations revealed different scenarios with genes organized in more or less compact—heterogeneous or homogeneous—operons, in suboperons, or in regulons. The organization of his genes in the extant members of the superphylum suggests that in the common ancestor of this group, genes were scattered throughout the chromosome and that different forces have driven the assembly of his genes in compact operons. Gene fusion events and/or paralog formation, HGT of single genes or entire operons between strains of the same or different taxonomic groups, and other molecular rearrangements shaped the his gene structure in this superphylum