Locking the GFP Fluorophore to Enhance Its Emission Intensity

Funding Information: Thanks are due to the University of Aveiro, FCT/MEC, Centro 2020 and Portugal2020, the COMPETE program, and the European Union (FEDER program) via the financial support to the LAQV-REQUIMTE (UIDB/50006/2020 and UIDP/50006/2020), to the CICECO-Aveiro Institute of Materials (UID/CTM/50011/2019, UIDB/50011/2020 and UIDP/50011/2020), financed by national funds through the FCT/MCTES, to the Portuguese NMR Network. SG is supported by national funds (OE), through FCT, I.P., in the scope of the framework contract foreseen in the numbers 4, 5, and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19. JRMF. Thanks FCT and ESF (European Social Fund) through POCH (Programa Operacional Capital Humano) for her PhD grant (UI/BD/151272/2021). Publisher Copyright: © 2022 by the authors.The Green Fluorescent Protein (GFP) and its analogues have been widely used as fluorescent biomarkers in cell biology. Yet, the chromophore responsible for the fluorescence of the GFP is not emissive when isolated in solution, outside the protein environment. The most accepted explanation is that the quenching of the fluorescence results from the rotation of the aryl–alkene bond and from the Z/E isomerization. Over the years, many efforts have been performed to block these torsional rotations, mimicking the environment inside the protein β-barrel, to restore the emission intensity. Molecule rigidification through chemical modifications or complexation, or through crystallization, is one of the strategies used. This review presents an overview of the strategies developed to achieve highly emissive GFP chromophore by hindering the torsional rotations.publishersversionpublishe

Toll like receptors gene expression of human keratinocytes cultured of severe burn injury

PURPOSE: To evaluate the expression profile of genes related to Toll Like Receptors (TLR) pathways of human Primary Epidermal keratinocytes of patients with severe burns.METHODS: After obtaining viable fragments of skin with and without burning, culture hKEP was initiated by the enzymatic method using Dispase (Sigma-Aldrich). These cells were treated with Trizol(r) (Life Technologies) for extraction of total RNA. This was quantified and analyzed for purity for obtaining cDNA for the analysis of gene expression using specific TLR pathways PCR Arrays plates (SA Biosciences).RESULTS: After the analysis of gene expression we found that 21% of these genes were differentially expressed, of which 100% were repressed or hyporegulated. Among these, the following genes (fold decrease): HSPA1A (-58), HRAS (-36), MAP2K3 (-23), TOLLIP (-23), RELA (-18), FOS (-16), and TLR1 (-6.0).CONCLUSIONS: This study contributes to the understanding of the molecular mechanisms related to TLR pathways and underlying wound infection caused by the burn. Furthermore, it may provide new strategies to restore normal expression of these genes and thereby change the healing process and improve clinical outcome.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)UNIFESP-EPM Department of SurgeryUNIFESP-EPMUNIFESPUniversidade Federal de São Paulo (UNIFESP), Escola Paulista de Medicina (EPM) Department of SurgeryUNIFESP, EPM, Department of SurgeryUNIFESP-EPMUNIFESP, EPM, Department of SurgeryFAPESP: 2011/12945-4FAPESP: 2013/10.905-0SciEL

Alternative mechanism for bacteriophage adsorption to the motile bacterium Caulobacter crescentus

2D and 3D cryo-electron microscopy, together with adsorption kinetics assays of ϕCb13 and ϕCbK phage-infected Caulobacter crescentus, provides insight into the mechanisms of infection. ϕCb13 and ϕCbK actively interact with the flagellum and subsequently attach to receptors on the cell pole. We present evidence that the first interaction of the phage with the bacterial flagellum takes place through a filament on the phage head. This contact with the flagellum facilitates concentration of phage particles around the receptor (i.e., the pilus portals) on the bacterial cell surface, thereby increasing the likelihood of infection. Phage head filaments have not been well characterized and their function is described here. Phage head filaments may systematically underlie the initial interactions of phages with their hosts in other systems and possibly represent a widespread mechanism of efficient phage propagation

How long before the end of inflation were observable perturbations produced?

We reconsider the issue of the number of e-foldings before the end of inflation at which observable perturbations were generated. We determine a plausible upper limit on that number for the standard cosmology which is around 60, with the expectation that the actual value will be up to 10 below this. We also note a special property of the $\lambda \phi^4$ model which reduces the uncertainties in that case and favours a higher value, giving a fairly definite prediction of 64 e-foldings for that model. We note an extreme (and highly implausible) situation where the number of e-foldings can be even higher, possibly up to 100, and discuss the shortcomings of quantifying inflation by e-foldings rather than by the change in $aH$. Finally, we discuss the impact of non-standard evolution between the end of inflation and the present, showing that again the expected number of e-foldings can be modified, and in some cases significantly increased.Comment: 7 pages RevTeX4 file with one figure incorporated. Minor updates to match version accepted by Physical Review

The route of infection determines Wolbachia antibacterial protection in Drosophila

International audienceBacterial symbionts are widespread among metazoans and provide a range of beneficial functions. -mediated protection against viral infection has been extensively demonstrated in In mosquitoes that are artificially transinfected with (wMel), protection from both viral and bacterial infections has been demonstrated. However, no evidence for -mediated antibacterial protection has been demonstrated in to date. Here, we show that the route of infection is key for -mediated antibacterial protection. carrying showed reduced mortality during enteric-but not systemic-infection with the opportunist pathogen -mediated protection was more pronounced in male flies and is associated with increased early expression of the antimicrobial peptide , and also increased expression of a reactive oxygen species detoxification gene (). These results highlight that the route of infection is important for symbiont-mediated protection from infection, that can protect hosts by eliciting a combination of resistance and disease tolerance mechanisms, and that these effects are sexually dimorphic. We discuss the importance of using ecologically relevant routes of infection to gain a better understanding of symbiont-mediated protection

Mental health problems and smoking among adolescents from Southern Brazil

OBJECTIVE: To analyze the association between mental health problems and smoking in adolescents. METHODS: A total of 4,325 adolescents aged 15 from the 1993 birth cohort of the city of Pelotas, Southern Brazil, was studied. Smoking was defined as having smoked one or more cigarettes in the previous 30 days. Mental health was assessed according to the total score of the Strengths and Difficulties Questionnaire. Score >; 20 points was considered positive. Data were analyzed using Poisson regression with adjustment for robust variance. RESULTS: Smoking prevalence was 6.0% and about 30% of the adolescents presented some mental health problem. In the crude analysis, the prevalence ratio for smoking was 3.3 (95%CI 2.5; 4.2). After the adjusted analysis (for sex, age, skin color, family income, mother's level of schooling, group of friends who smoke, employment in the previous year, school failure, physical activity during leisure time and experimental use of alcohol), it decreased to 1.7 (95%CI 1.2; 2.3) among those with mental health problem. CONCLUSIONS: Mental health problems in adolescence may be related to tobacco consumption.OBJETIVO: Analisar a associação entre problemas de saúde mental e uso de tabaco em adolescentes. MÉTODOS: Foram analisados 4.325 adolescentes de 15 anos da coorte de nascimentos de 1993 da cidade de Pelotas, RS. Tabagismo foi definido como fumar um ou mais cigarros nos últimos 30 dias. Saúde mental foi avaliada de acordo com o escore total do questionário Strengths and Difficulties Questionnaire e escore maior ou igual a 20 pontos foi considerado como positivo. Os dados foram analisados por regressão de Poisson, com ajuste robusto para variância. RESULTADOS: A prevalência de tabagismo foi 6,0% e cerca de 30% dos adolescentes apresentaram algum tipo de problema de saúde mental. Na análise bruta, a razão de prevalências para tabagismo foi de 3,3 (IC95% 2,5; 4,2). Após ajuste (para sexo, idade, cor da pele, renda familiar, escolaridade da mãe, grupo de amigos fumantes, trabalho no último ano, repetência escolar, atividade física de lazer e uso experimental de bebida alcoólica), diminuiu para 1,7 (IC95% 1,2; 2,3) entre aqueles com problemas de saúde mental. CONCLUSÕES: Problemas de saúde mental na adolescência podem ter relação com o consumo de tabaco.OBJETIVO: Analizar la asociación entre problemas de salud mental y uso de cigarro en adolescentes. MÉTODOS: Se analizaron 4.325 adolescentes de 15 años de la cohorte de nacimientos de 1993 de la ciudad de Pelotas, Sur de Brasil. Tabaquismo fue definido como fumar uno o más cigarros en los últimos 30 días. Salud mental fue evaluada de acuerdo con el escore total del cuestionario Strengths and Difficulties Questionnaire y escore mayor o igual a 20 puntos fue considerado como positivo. Los datos fueron analizados por regresión de Poisson, con ajuste robusto para varianza. RESULTADOS: La prevalencia de tabaquismo fue 6,0% y cerca de 30% de los adolescentes presentaron algún tipo de problema de salud mental. En el análisis bruto, la tasa de prevalencias de problema de tabaquismo de 3,3 (IC95% 2,5;4,2). Posterior al ajuste para sexo, edad, color de la piel, renta familiar, escolaridad de la madre, grupo de amigos fumadores, trabajo en el último año, repitencia escolar, actividad física de ocio y uso experimental de bebida alcohólica, disminuyó a 1,7 (IC95% 1,2;2,3) entre aquellos con problemas de salud mental. CONCLUSIONES: Problemas de salud mental en la adolescencia pueden tener relación con el consumo de tabaco

Effective and Asymptotic Critical Exponents of Weakly Diluted Quenched Ising Model: 3d Approach Versus ϵ1/2\epsilon^{1/2}-Expansion

We present a field-theoretical treatment of the critical behavior of three-dimensional weakly diluted quenched Ising model. To this end we analyse in a replica limit n=0 5-loop renormalization group functions of the $\phi^4$-theory with O(n)-symmetric and cubic interactions (H.Kleinert and V.Schulte-Frohlinde, Phys.Lett. B342, 284 (1995)). The minimal subtraction scheme allows to develop either the $\epsilon^{1/2}$-expansion series or to proceed in the 3d approach, performing expansions in terms of renormalized couplings. Doing so, we compare both perturbation approaches and discuss their convergence and possible Borel summability. To study the crossover effect we calculate the effective critical exponents providing a local measure for the degree of singularity of different physical quantities in the critical region. We report resummed numerical values for the effective and asymptotic critical exponents. Obtained within the 3d approach results agree pretty well with recent Monte Carlo simulations. $\epsilon^{1/2}$-expansion does not allow reliable estimates for d=3.Comment: 35 pages, Latex, 9 eps-figures included. The reference list is refreshed and typos are corrected in the 2nd versio

Multi-Grid Monte Carlo via XYXY Embedding. II. Two-Dimensional SU(3)SU(3) Principal Chiral Model

We carry out a high-precision simulation of the two-dimensional $SU(3)$ principal chiral model at correlation lengths $\xi$ up to $\sim 4 \times 10^5$, using a multi-grid Monte Carlo (MGMC) algorithm and approximately one year of Cray C-90 CPU time. We extrapolate the finite-volume Monte Carlo data to infinite volume using finite-size-scaling theory, and we discuss carefully the systematic and statistical errors in this extrapolation. We then compare the extrapolated data to the renormalization-group predictions. The deviation from asymptotic scaling, which is $\approx 12$ at $\xi \sim 25$, decreases to $\approx 2$ at $\xi \sim 4 \times 10^5$. We also analyze the dynamic critical behavior of the MGMC algorithm using lattices up to $256 \times 256$, finding the dynamic critical exponent $z_{int,{\cal M}^2} \approx 0.45 \pm 0.02$ (subjective 68% confidence interval). Thus, for this asymptotically free model, critical slowing-down is greatly reduced compared to local algorithms, but not completely eliminated.Comment: self-unpacking archive including .tex, .sty and .ps files; 126 pages including all figure