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ABSTRACT While genome-wide transposon mutagenesis screens have identified numerous essential genes in the significant human pathogen Streptococcus pyogenes (group A Streptococcus or GAS), many of their functions remain elusive. This knowledge gap is attributed in part to the limited molecular toolbox for controlling GAS gene expression and the bacterium’s poor genetic transformability. CRISPR interference (CRISPRi), using catalytically inactive GAS Cas9 (dCas9), is a powerful approach to specifically repress gene expression in both bacteria and eukaryotes, but ironically, it has never been harnessed for controlled gene expression in GAS. In this study, we present a highly transformable and fully virulent serotype M1T1 GAS strain and introduce a doxycycline-inducible CRISPRi system for efficient repression of bacterial gene expression. We demonstrate highly efficient, oligo-based single guide RNA cloning directly to GAS, enabling the construction of a gene knockdown strain in just 2 days, in contrast to the several weeks typically required. The system is shown to be titratable and functional both in vitro and in vivo using a murine model of GAS infection. Furthermore, we provide direct in vivo evidence that the expression of the conserved cell division gene ftsZ is essential for GAS virulence, highlighting its promise as a target for emerging FtsZ inhibitors. Finally, we introduce SpyBrowse (https://veeninglab.com/SpyBrowse), a comprehensive and user-friendly online resource for visually inspecting and exploring GAS genetic features. The tools and methodologies described in this work are poised to facilitate fundamental research in GAS, contribute to vaccine development, and aid in the discovery of antibiotic targets.IMPORTANCEWhile group A Streptococcus (GAS) remains a predominant cause of bacterial infections worldwide, there are limited genetic tools available to study its basic cell biology. Here, we bridge this gap by creating a highly transformable, fully virulent M1T1 GAS strain. In addition, we established a tight and titratable doxycycline-inducible system and developed CRISPR interference (CRISPRi) for controlled gene expression in GAS. We show that CRISPRi is functional in vivo in a mouse infection model. Additionally, we present SpyBrowse, an intuitive and accessible genome browser (https://veeninglab.com/SpyBrowse). Overall, this work overcomes significant technical challenges of working with GAS and, together with SpyBrowse, represents a valuable resource for researchers in the GAS field.

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The Agulhas rings transport warm and salty waters that feed the surface limb of the Atlantic Meridional Overturning Circulation. Some studies have focused on the conveying capacity of ocean eddies, and recently, the role of the Agulhas rings in advecting water masses and organisms has been explored. Here we show evidence that the Agulhas rings are responsible for the advection of mode waters from the Cape Basin to the western side of the Atlantic. We analyzed more than 3,200 temperature profiles and 2,400 salinity profiles from historical databases collocated with 52 long-lived Agulhas rings tracked from 1993 through 2016. An automated algorithm was used to identify thermostads in the profiles acquired within the rings. The data revealed mode water layers trapped inside 88% of the rings. The joint distribution of temperature and salinity indicated two types of mode waters in the range 16.2 ± 0.6°C, 35.6 ± 0.1 (Type I) and 12.9 ± 0.7°C, 35.2 ± 0.1 (Type II). The majority (67%) of the rings carrying mode waters had both types detected inside. Moreover, considering only those rings sampled west of the Mid-Atlantic Ridge, we found that 45% of them advected mode waters to the western basin. Therefore, our results demonstrate that, despite the long journey, interaction with the bottom topography and other vortices, ocean-atmosphere exchanges, and decay, the Agulhas rings are responsible for spreading mode waters initially available at the Cape Basin throughout the South Atlantic, contributing to a positive anomaly in temperature and salinity along the eddy corridor joining the Cape Basin to the Brazil Basin.

Background The increasingly widespread use of next generation sequencing protocols has brought the need for the development of user-friendly raw data processing tools. Here, we explore 2FAST2Q, a versatile and intuitive standalone program capable of extracting and counting feature occurrences in FASTQ files. Despite 2FAST2Q being previously described as part of a CRISPRi-seq analysis pipeline, in here we further elaborate on the program’s functionality, and its broader applicability and functions. Methods 2FAST2Q is built in Python, with published standalone executables in Windows MS, MacOS, and Linux. It has a familiar user interface, and uses an advanced custom sequence searching algorithm. Results Using published CRISPRi datasets in which Escherichia coli and Mycobacterium tuberculosis gene essentiality, as well as host-cell sensitivity towards SARS-CoV2 infectivity were tested, we demonstrate that 2FAST2Q efficiently recapitulates published output in read counts per provided feature. We further show that 2FAST2Q can be used in any experimental setup that requires feature extraction from raw reads, being able to quickly handle Hamming distance based mismatch alignments, nucleotide wise Phred score filtering, custom read trimming, and sequence searching within a single program. Moreover, we exemplify how different FASTQ read filtering parameters impact downstream analysis, and suggest a default usage protocol. 2FAST2Q is easier to use and faster than currently available tools, efficiently processing not only CRISPRi-seq / random-barcode sequencing datasets on any up-to-date laptop, but also handling the advanced extraction of de novo features from FASTQ files. We expect that 2FAST2Q will not only be useful for people working in microbiology but also for other fields in which amplicon sequencing data is generated. 2FAST2Q is available as an executable file for all current operating systems without installation and as a Python3 module on the PyPI repository (available at https://veeninglab.com/2fast2q).

This study proposes a numerical approach aimed at mitigating electromagnetic field intensities at ground level generated by overhead power lines. The methodology integrates the boundary element method for field evaluation with the ellipsoid method for field optimization, while adhering to critical design and safety constraints. The optimized power line configurations, derived from this integrated approach, achieved significant reductions in both electric and magnetic field magnitudes at ground level without compromising any specified constraints. Moreover, the research findings demonstrate the robustness, efficiency, and practical applicability of the proposed methodology in the design of optimized power lines, offering potential for increased power transfer capability, and decreased environmental and health impacts. [ABSTRACT FROM AUTHOR]

A methodology that combines a 24-year long (January 1993 to December 2016) global dataset of eddy trajectories, derived from altimetry, with vertical temperature and salinity profiles from the EN4.2.0 database, derived from XBTs/MBTs, CTDs and Argo floats, was used to reconstruct the mean vertical structure of North Brazil Current (NBC) rings, and to calculate some of their properties. The number of NBC rings formed each year varied from 2 to 8, with an annual-mean formation of 5.3 ± 1.5. During the analyzed period, 112 rings were sampled at least once, at various distances from the center of the rings, leading to a total of 1323 (604) temperature (salinity) profiles available to compute the mean NBC ring, depicting a large, surface intensified, and relatively shallow ring, with intense temperature and salinity anomalies. The meridional volume transport was estimated in 1.3 Sv (1Sv = 106 m3s-1) per ring, leading to an annualized transport of ~7 ± 2 Sv. The amount of South Atlantic Water (SAW) within the mean ring was estimated in ~40 to 60% of the ring volume. According to these estimates, NBC rings may be responsible in different years for approximately 20 to 80% of the northward volume transport associated with the upper limb of the Atlantic Meridional Overturning Circulation (AMOC), and approximately 15 to 55% of the meridional heat transport in the tropical Atlantic.

Among the aspects with major impacts on the energy and environmental performance of a building, the thermal insulation of the opaque elements of its envelope stands out. This work assesses the influence of the application of thermal insulation to the opaque elements of the building’s envelope on the thermal comfort conditions indoors; moreover, the influence of the thermal insulation on the energy, environmental, and economic costs over the building’s complete life cycle is evaluated. For this purpose, the three most commonly used thermal insulating materials (expanded polystyrene—EPS, extruded polystyrene—XPS, and mineral wool—MW), thicknesses between 0 (without insulation) and 40 cm, five climates (hot, warm, moderate, cold, and very cold), and six types of use (apartment, housing, clinic, school, bank branch, and supermarket) were considered. EPS reveals itself to be the most promising thermal insulation material, both in economic and environmental terms, so it was selected for this study. The EPS’ optimal thickness depends on the building’s type of use, the climate, and the perspective from which the assessment is carried out (energy, environmental, or economic). The results show that the economically optimal thicknesses of thermal insulation are significantly lower than the corresponding ones in environmental terms. Furthermore, the application of thermal insulation to the opaque building’s envelope is more beneficial in energy and environmental terms than from an economic perspective.

Coastal ocean observing and modeling systems (coastal observatories), connected with regional and global ocean systems, improve the quality of information and forecasts for effective management of safe and sustainable maritime activities. The public availability of systematic and long-term information of the ocean is an engine for the Blue Economy, boosting economic growth, employment, and innovation. An overview of some Brazilian initiatives is presented in this paper, involving universities, private companies, federal and state agencies, covering institutions from south to north of Brazil. Although these initiatives focus mainly on ocean physics, integrated efforts can extend the scope to include biogeochemistry and marine biodiversity, helping to address interdisciplinary problems. Existing initiatives can be connected, and new ones fostered, to fill in the gaps of temporal and spatial coverage of ocean monitoring in the vast oceanic area under Brazilian jurisdiction (nationally referred to as the Blue Amazon, in reference to the similar richness of the Amazon Forest). The alignment among national and regional initiatives, as well as with international programs, can be promoted if coordinated by a national-level organization, maximizing the return of public investment and socioeconomic benefits. In light of international examples, possible future institutional arrangements are discussed, leveraging from existing national public policies and international cooperation that Brazil is taking part. The United Nations Decade of Ocean Science for Sustainable Development is a timely opportunity to encourage an institutional arrangement to support and articulate an integrated network of coastal observatories in Brazil.

Medicines are the most used health technology in Long-Term Care. The prevalence of potentially inappropriate medicines amongst Long-Term Care patients is high. Pharmacists, assisted by prescribing-assessment tools, can play an important role in optimizing medication use at this level of care. Through a modified RAND/UCLA Appropriateness Method, 13 long-term care and hospital pharmacists assessed as ‘appropriate’, ‘uncertain’, or ‘inappropriate’ a collection of commonly used prescribing-assessment tools as to its suitability in assisting pharmacy practice in institutional long-term care settings. A qualitative analysis of written or transcribed comments of participants was pursued to identify relevant characteristics of prescribing-assessment tools and potential hinders in their use. From 24 different tools, pharmacists classified 9 as ‘appropriate’ for pharmacy practice targeted to long-term care patients, while 3 were classified as ‘inappropriate’. The tools feature most appreciated by study participants was the indication of alternatives to potentially inappropriate medication. Lack of time and/or pharmacists and limited access to clinical information seems to be the most relevant hinders for prescribing-assessment tools used in daily practice.

Symbiotic associations between tree species and mycorrhizal fungi may lead to better growth and nutrition of seedlings and so it becomes an important alternative to the establishment in the field, mainly in edaphic restriction areas, or to species of high nutritional demands. The cultivation of Tectona grandis (teca) in Brazil has been conducted in Cerrado areas with low fertility soils. The aim of the present study is to assess the effect of arbuscular mycorrhizal fungi (AMF) inoculation on the growth and mineral nutrition of teca seedlings. The experiment was performed in randomized blocks (six blocks), with four repetitions: 1) witness (inoculation-free mycorrhizal fungi); 2) inoculation with Rhizophagus clarus; 3) native AMF inoculum from twenty-year old teca cultivation in Mato Grosso state (MT inoculum); and 4) native AMF inoculum from clay extraction in Campos do Goytacazes (Campos inoculum). The plantlets were inoculated and cultivated in 6 dm³ pots for 135 days and assessed for stem hight and diameter of the stem base. Root samples were collected in order to assess the percentage of AMF colonization. The roots and the aerial part were dried, separately, in a forced air circulation oven at 65°C for 72 hours. Subsequently, the dry weight and the content of P, K AND S were determined. The mycorrhizal fungi inoculation, mainly the Rhizophagus clarus isolate and the Campos inoculum, has promoted better growth and K and S acquisition by teca seedlings. The inoculation using mycorrhizal fungi allowed greater nutrient use efficiency.

Overweight and obesity are growing health problems in children. The increase in fast-food consumption has greatly contributed to this phenomenon. Children are a frequent target for fast-food advertising, and the television is one of the most used marketing channels. We assessed the frequency of fast-food ingestion, television viewing time and body mass index (BMI) in children from 8 to 12 years of age. A quantitative approach was followed, using a self-report questionnaire. The sample was composed of 60 children with an age average of 9.88 years (SD=1.37). It was found that longer television viewing times were associated with higher frequency of fast-food ingestion for both school days (rS = 0.54, p < .001) and weekends/holidays (rS = 0.50, p < .001). A positive and moderate correlation between television viewing times and BMI (rS = 0.51, p < .001; rS = 0.55, p < .001) was also observed. The results indicate that television advertising makes children wanting to try the fast-food advertised (67%; n = 40), and ask parents to buy it (60%; n = 36). The good taste (72%; n = 43) and the gifts (38%; n = 23) are what the children in our study most appreciate in fast-food restaurants. Despite legal regulatory mechanisms, marketing continues to have a strong impact on the promotion of fast-food consumption in children.