An easy method for quantification of anaerobic and microaerobic gene expression with fluorescent reporter proteins

Fluorescent proteins, such as green fluorescent proteins, are invaluable tools for detecting and quantifying gene expression in high-throughput reporter gene assays. However, they introduce significant inaccuracies in studies involving microaerobiosis or anaerobiosis, as oxygen is required for the maturation of these proteins' chromophores. In this study, the authors highlight the errors incurred by using fluorescent proteins under limited oxygenation by comparing standard fluorescence-based reporter gene assays to quantitative real-time PCR data in the study of a complex oxygen-regulated gene network. Furthermore, a solution to perform quantification of anaerobic and microaerobic gene expression with fluorescent reporter proteins using a microplate reader with an oxygen control system and applying pulses of full oxygenation before fluorescence measurements is provided

Triangular Gross-Pitaevskii breathers and Damski-Chandrasekhar shock waves

The recently proposed map [arXiv:2011.01415] between the hydrodynamic equations governing the two-dimensional triangular cold-bosonic breathers [Phys. Rev. X 9, 021035 (2019)] and the high-density zero-temperature triangular free-fermionic clouds, both trapped harmonically, perfectly explains the former phenomenon but leaves uninterpreted the nature of the initial ($t=0$) singularity. This singularity is a density discontinuity that leads, in the bosonic case, to an infinite force at the cloud edge. The map itself becomes invalid at times $t<0$. A similar singularity appears at $t = T/4$, where $T$ is the period of the harmonic trap, with the Fermi-Bose map becoming invalid at $t > T/4$. Here, we first map -- using the scale invariance of the problem -- the trapped motion to an untrapped one. Then we show that in the new representation, the solution [arXiv:2011.01415] becomes, along a ray in the direction normal to one of the three edges of the initial cloud, a freely propagating one-dimensional shock wave of a class proposed by Damski in [Phys. Rev. A 69, 043610 (2004)]. There, for a broad class of initial conditions, the one-dimensional hydrodynamic equations can be mapped to the inviscid Burgers' equation, a nonlinear transport equation. More specifically, under the Damski map, the $t=0$ singularity of the original problem becomes, verbatim, the initial condition for the wave catastrophe solution found by Chandrasekhar in 1943 [Ballistic Research Laboratory Report No. 423 (1943)]. At $t=T/8$, our interpretation ceases to exist: at this instance, all three effectively one-dimensional shock waves emanating from each of the three sides of the initial triangle collide at the origin, and the 2D-1D correspondence between the solution of [arXiv:2011.01415] and the Damski-Chandrasekhar shock wave becomes invalid.Comment: 13 pages, 2 figures. Submission to SciPos

Regulation of ribonucleotide synthesis by the Pseudomonas aeruginosa two-component system AlgR in response to oxidative stress

Ribonucleotide reductases (RNR) catalyze the last step of deoxyribonucleotide synthesis, and are therefore essential to DNA-based life. Three forms of RNR exist: classes I, II, and III. While eukaryotic cells use only class Ia RNR, bacteria can harbor any combination of classes, granting them adaptability. The opportunistic pathogen Pseudomonas aeruginosa surprisingly encodes all three classes, allowing it to thrive in different environments. Here we study an aspect of the complex RNR regulation whose molecular mechanism has never been elucidated, the well-described induction through oxidative stress, and link it to the AlgZR two-component system, the primary regulator of the mucoid phenotype. Through bioinformatics, we identify AlgR binding locations in RNR promoters, which we characterize functionally through EMSA and physically through AFM imaging. Gene reporter assays in different growth models are used to study the AlgZR-mediated control on the RNR network under various environmental conditions and physiological states. Thereby, we show that the two-component system AlgZR, which is crucial for bacterial conversion to the mucoid phenotype associated with chronic disease, controls the RNR network and directs how the DNA synthesis pathway is modulated in mucoid and non-mucoid biofilms, allowing it to respond to oxidative stress

Estudio longitudinal de lesiones deportivas en practicantes de gimnasia aeróbica de competición

Introduction: Aerobic gymnastics, since its membership in the International Gymnastics Federation, has undergone changes in its regulations. Objective: To analyze the injuries found in Spanish aerobic gymnastics athletes during different editions of the Code of Points. Methods: A descriptive, longitudinal and compara-tive study was carried out on the epidemiology of injuries in aerobic gymnastics published during different editions of the Code of Points. Results: It highlights that the number of injuries decreased from 156 to 38 last year. This decline has been related to the restriction on the number of difficulties in the exercise and the number of elements to be performed on the floor. However, they have increased the number and value of the difficulties. Conclusions: Therefore, it is concluded that the changes made in the regulations are intended to safeguard the health of athletes and ensure that competition develops at its best artistic and technical aspect.Introdução: A ginástica aeróbica, desde sua adesão à Federação Internacional de Ginástica, passou por mudanças em seus regulamentos. Objetivo: Analisar as lesões encontradas nos atletas espanhóis de ginástica aeróbica durante as diferentes edições do Código de Pontos. Métodos: Realizou-se um estudo descritivo, longitudinal e comparativo sobre a epidemiologia de lesões na ginástica aeróbica publicado durante as diferentes edições do Código de Pontos. Resultados Salienta-se que o número de lesões diminuiu de 156 para 38 no ano passado. Este declínio tem sido relacionado com a limitação do número de dificuldades no exercício e o número de elementos a serem feitos no solo. No entanto, eles aumentaram o número e valor das dificuldades. Conclusões: Portanto, concluiu-se que as modificações feitas nos regulamentos destinam-se a salvaguardar a saúde dos atletas e garantir que a competição se desenvolva no seu melhor aspecto artístico e técnico.Introducción: La gimnasia aeróbica desde su pertenencia a la Federación Internacional de Gimnasia ha sufrido cam-bios en su reglamentación. Objetivo: Analizar las lesiones que los deportistas españoles de gimnasia aeróbica presentaron durante las diferentes ediciones del Código de Puntuación. Métodos: Se ha realizado un estudio descriptivo, longitudinal y comparativo sobre la epidemiología de las lesiones en la gimnasia aeróbica publicado durante las diferentes ediciones del Código de Puntuación. Resultados: El estudio destaca la disminución del número de lesiones, de 156 a 38 en el último año. Esta disminución ha tenido relación con la restricción del número de dificultades en el ejercicio y la cantidad de elementos a realizar en el suelo. Sin embargo, han aumentado el número y el valor de las dificultades. Conclusiones: Por tanto, han concluido que las modificaciones que se realizan en la reglamentación tienen como objetivo velar por la salud de los deportistas y garantizar que la competición se desarrolle en su máximo esplendor artístico y técnico

Somatic signature of brain-specific single nucleotide variations in sporadic alzheimer's disease

© 2014 IOS Press and the authors. All rights reserved. Background: Although genome-wide association studies have shown that genetic factors increase the risk of suffering late-onset, sporadic Alzheimer's disease (SAD), the molecular mechanisms responsible remain largely unknown. Objective: The aim of the study was to investigate the presence of somatic, brain-specific single nucleotide variations (SNV) in the hippocampus of SAD samples. Methods: By using bioinformatic tools, we compared whole exome sequences in paired blood and hippocampal genomic DNAs from 17 SAD patients and from 2 controls and 2 vascular dementia patients. Results: We found a remarkable number of SNVs in SAD brains (~575 per patient) that were not detected in blood. Loci with hippocampus-specific (hs)-SNVs were common to several patients, with 38 genes being present in 6 or more patients out of the 17. While some of these SNVs were in genes previously related to SAD (e.g., CSMD1, LRP2), most hs-SNVs occurred in loci previously unrelated to SAD. The most frequent genes with hs-SNVs were associated with neurotransmission, DNA metabolism, neuronal transport, and muscular function. Interestingly, 19 recurrent hs-SNVs were common to 3 SAD patients. Repetitive loci or hs-SNVs were underrepresented in the hippocampus of control or vascular dementia donors, or in the cerebellum of SAD patients. Conclusion: Our data suggest that adult blood and brain have different DNA genomic variations, and that somatic genetic mosaicism and brain-specific genome reshaping may contribute to SAD pathogenesis and cognitive differences between individuals.BBVA Foundation and MICINN-MINECO. We also like to thank the support of the Reina Sofia Foundation, the CIEN Foundation, CIBERNED (ISCIII

The catalytic subunit of the system L1 amino acid transporter (S<i>lc7a5</i>) facilitates nutrient signalling in mouse skeletal muscle

The System L1-type amino acid transporter mediates transport of large neutral amino acids (LNAA) in many mammalian cell-types. LNAA such as leucine are required for full activation of the mTOR-S6K signalling pathway promoting protein synthesis and cell growth. The SLC7A5 (LAT1) catalytic subunit of high-affinity System L1 functions as a glycoprotein-associated heterodimer with the multifunctional protein SLC3A2 (CD98). We generated a floxed Slc7a5 mouse strain which, when crossed with mice expressing Cre driven by a global promoter, produced Slc7a5 heterozygous knockout (Slc7a5+/-) animals with no overt phenotype, although homozygous global knockout of Slc7a5 was embryonically lethal. Muscle-specific (MCK Cre-mediated) Slc7a5 knockout (MS-Slc7a5-KO) mice were used to study the role of intracellular LNAA delivery by the SLC7A5 transporter for mTOR-S6K pathway activation in skeletal muscle. Activation of muscle mTOR-S6K (Thr389 phosphorylation) in vivo by intraperitoneal leucine injection was blunted in homozygous MS-Slc7a5-KO mice relative to wild-type animals. Dietary intake and growth rate were similar for MS-Slc7a5-KO mice and wild-type littermates fed for 10 weeks (to age 120 days) with diets containing 10%, 20% or 30% of protein. In MS-Slc7a5-KO mice, Leu and Ile concentrations in gastrocnemius muscle were reduced by ∼40% as dietary protein content was reduced from 30 to 10%. These changes were associated with >50% decrease in S6K Thr389 phosphorylation in muscles from MS-Slc7a5-KO mice, indicating reduced mTOR-S6K pathway activation, despite no significant differences in lean tissue mass between groups on the same diet. MS-Slc7a5-KO mice on 30% protein diet exhibited mild insulin resistance (e.g. reduced glucose clearance, larger gonadal adipose depots) relative to control animals. Thus, SLC7A5 modulates LNAA-dependent muscle mTOR-S6K signalling in mice, although it appears non-essential (or is sufficiently compensated by e.g. SLC7A8 (LAT2)) for maintenance of normal muscle mass

DNA building blocks: keeping control of manufacture

Ribonucleotide reductase (RNR) is the only source for de novo production of the four deoxyribonucleoside triphosphate (dNTP) building blocks needed for DNA synthesis and repair. It is crucial that these dNTP pools are carefully balanced, since mutation rates increase when dNTP levels are either unbalanced or elevated. RNR is the major player in this homeostasis, and with its four different substrates, four different allosteric effectors and two different effector binding sites, it has one of the most sophisticated allosteric regulations known today. In the past few years, the structures of RNRs from several bacteria, yeast and man have been determined in the presence of allosteric effectors and substrates, revealing new information about the mechanisms behind the allosteric regulation. A common theme for all studied RNRs is a flexible loop that mediates modulatory effects from the allosteric specificity site (s-site) to the catalytic site for discrimination between the four substrates. Much less is known about the allosteric activity site (a-site), which functions as an on-off switch for the enzyme's overall activity by binding ATP (activator) or dATP (inhibitor). The two nucleotides induce formation of different enzyme oligomers, and a recent structure of a dATP-inhibited α6β2 complex from yeast suggested how its subunits interacted non-productively. Interestingly, the oligomers formed and the details of their allosteric regulation differ between eukaryotes and Escherichia coli Nevertheless, these differences serve a common purpose in an essential enzyme whose allosteric regulation might date back to the era when the molecular mechanisms behind the central dogma evolved

CD98hc facilitates B cell proliferation and adaptive humoral immunity.

The proliferation of antigen-specific lymphocytes and resulting clonal expansion are essential for adaptive immunity. We report here that B cell-specific deletion of the heavy chain of CD98 (CD98hc) resulted in lower antibody responses due to total suppression of B cell proliferation and subsequent plasma cell formation. Deletion of CD98hc did not impair early B cell activation but did inhibit later activation of the mitogen-activated protein kinase Erk1/2 and downregulation of the cell cycle inhibitor p27. Reconstitution of CD98hc-deficient B cells with CD98hc mutants showed that the integrin-binding domain of CD98hc was required for B cell proliferation but that the amino acid-transport function of CD98hc was dispensable for this. Thus, CD98hc supports integrin-dependent rapid proliferation of B cells. We propose that the advantage of adaptive immunity favored the appearance of CD98hc in vertebrates

Generalisability of deep learning models in low-resource imaging settings: A fetal ultrasound study in 5 African countries

Most artificial intelligence (AI) research have concentrated in high-income countries, where imaging data, IT infrastructures and clinical expertise are plentiful. However, slower progress has been made in limited-resource environments where medical imaging is needed. For example, in Sub-Saharan Africa the rate of perinatal mortality is very high due to limited access to antenatal screening. In these countries, AI models could be implemented to help clinicians acquire fetal ultrasound planes for diagnosis of fetal abnormalities. So far, deep learning models have been proposed to identify standard fetal planes, but there is no evidence of their ability to generalise in centres with limited access to high-end ultrasound equipment and data. This work investigates different strategies to reduce the domain-shift effect for a fetal plane classification model trained on a high-resource clinical centre and transferred to a new low-resource centre. To that end, a classifier trained with 1,792 patients from Spain is first evaluated on a new centre in Denmark in optimal conditions with 1,008 patients and is later optimised to reach the same performance in five African centres (Egypt, Algeria, Uganda, Ghana and Malawi) with 25 patients each. The results show that a transfer learning approach can be a solution to integrate small-size African samples with existing large-scale databases in developed countries. In particular, the model can be re-aligned and optimised to boost the performance on African populations by increasing the recall to $0.92 \pm 0.04$ and at the same time maintaining a high precision across centres. This framework shows promise for building new AI models generalisable across clinical centres with limited data acquired in challenging and heterogeneous conditions and calls for further research to develop new solutions for usability of AI in countries with less resources