Genetic Dissection of the Regulatory Network Associated with High c-di-GMP Levels in Pseudomonas putida KT2440

Most bacteria grow in nature forming multicellular structures named biofilms. The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key player in the regulation of the transition from planktonic to sessile lifestyles and this regulation is crucial in the development of biofilms. In Pseudomonas putida KT2440, Rup4959, a multidomain response regulator with diguanylate cyclase activity, when overexpressed causes an increment in the intracellular levels of c-di-GMP that gives rise to a pleiotropic phenotype consisting of increased biofilm formation and crinkly colony morphology. In a broad genomic screen we have isolated mutant derivatives that lose the crinkly morphology, designed as cfc (crinkle free colony). A total of 19 different genes have been identified as being related with the emergence of the cfc phenotype either because the expression or functionality of Rup4959 is compromised, or due to a lack of transduction of the c-di-GMP signal to downstream elements involved in the acquisition of the phenotype. Discernment between these possibilities was investigated by using a c-di-GMP biosensor and by HPLC-MS quantification of the second messenger. Interestingly five of the identified genes encode proteins with AAA+ ATPase domain. Among the bacterial determinants found in this screen are the global transcriptional regulators GacA, AlgU and FleQ and two enzymes involved in the arginine biosynthesis pathway. We present evidences that this pathway seems to be an important element to both the availability of the free pool of the second messenger c-di-GMP and to its further transduction as a signal for biosynthesis of biopolimers. In addition we have identified an uncharacterized hybrid sensor histidine kinase whose phosphoaceptor conserved histidine residue has been shown in this work to be required for in vivo activation of the orphan response regulator Rup4959, which suggests these two elements constitute a two-component phosphorelay system.This work was supported by grants BFU2010-17946 and BFU2013-43469-P from Plan Nacional de I+D+I (Spanish Government) and by EDFR funds. OH-R was supported by a FPI fellowship and LB-M by predoctoral fund from Junta de Andalucía. MAM was supported by the Postdoctoral Juan de la Cierva Spanish Research Program (JCI-2012-11815).Peer reviewedPeer Reviewe

On-Surface Thermal Stability of a Graphenic Structure Incorporating a Tropone Moiety

On-surface synthesis, complementary to wet chemistry, has been demonstrated to be a valid approach for the synthesis of tailored graphenic nanostructures with atomic precision. Among the different existing strategies used to tune the optoelectronic and magnetic properties of these nanostructures, the introduction of non-hexagonal rings inducing out-of-plane distortions is a promising pathway that has been scarcely explored on surfaces. Here, we demonstrate that non-hexagonal rings, in the form of tropone (cycloheptatrienone) moieties, are thermally transformed into phenyl or cyclopentadienone moieties upon an unprecedented surface-mediated retro–Buchner-type reaction involving a decarbonylation or an intramolecular rearrangement of the CO unit, respectivel

Comprehensive Constitutional Genetic and Epigenetic Characterization of Lynch-Like Individuals

The causal mechanism for cancer predisposition in Lynch-like syndrome (LLS) remains unknown. Our aim was to elucidate the constitutional basis of mismatch repair (MMR) deficiency in LLS patients throughout a comprehensive (epi)genetic analysis. One hundred and fifteen LLS patients harboring MMR-deficient tumors and no germline MMR mutations were included. Mutational analysis of 26 colorectal cancer (CRC)-associated genes was performed. Pathogenicity of MMR variants was assessed by splicing and multifactorial likelihood analyses. Genome-wide methylome analysis was performed by the Infinium Human Methylation 450K Bead Chip. The multigene panel analysis revealed the presence of two MMR gene truncating mutations not previously found. Of a total of 15 additional MMR variants identified, five -present in 6 unrelated individuals- were reclassified as pathogenic. In addition, 13 predicted deleterious variants in other CRC-predisposing genes were found in 12 probands. Methylome analysis detected one constitutionalMLH1epimutation, but no additional differentially methylated regions were identified in LLS compared to LS patients or cancer-free individuals. In conclusion, the use of an ad-hoc designed gene panel combined with pathogenicity assessment of variants allowed the identification of deleterious MMR mutations as well as new LLS candidate causal genes. Constitutional epimutations in non-LS-associated genes are not responsible for LLS

Molecular Binding Mechanism of TtgR Repressor to Antibiotics and Antimicrobials

A disturbing phenomenon in contemporary medicine is the prevalence of multidrug-resistant pathogenic bacteria. Efflux pumps contribute strongly to this antimicrobial drug resistance, which leads to the subsequent failure of clinical treatments. The TtgR protein of Pseudomonas putida is a HTH-type transcriptional repressor that controls expression of the TtgABC efflux pump, which is the main contributor to resistance against several antimicrobials and toxic compounds in this microbe. One of the main strategies to modulate the bacterial resistance is the rational modification of the ligand binding target site. We report the design and characterization of four mutants-TtgRS77A, TtgRE78A, TtgRN110A and TtgRH114A - at the active ligand binding site. The biophysical characterization of the mutants, in the presence and in the absence of different antimicrobials, revealed that TtgRN110A is the variant with highest thermal stability, under any of the experimental conditions tested. EMSA experiments also showed a different dissociation pattern from the operator for TtgRN110A, in the presence of several antimicrobials, making it a key residue in the TtgR protein repression mechanism of the TtgABC efflux pump. We found that TtgRE78A stability is the most affected upon effector binding. We also probe that one mutation at the C-terminal half of helix-α4, TtgRS77A, provokes a severe protein structure distortion, demonstrating the important role of this residue in the overall protein structure and on the ligand binding site. The data provide new information and deepen the understanding of the TtgR-effector binding mechanism and consequently the TtgABC efflux pump regulation mechanism in Pseudomonas putida.This work was supported by Spanish Ministry of Economy and Competitiveness, National programme for Recruitment and Incorporation of Human Resources, Subprogramme: Ramon y Cajal RYC-2009-04570 and grant P11-CVI-7391 from Junta de Andalucía and EFDR (European Regional Development Fund)

Global impacts of Covid-19 on lifestyles and health and preparation preferences: an international survey of 30 countries

Background: The health area being greatest impacted by coronavirus disease 2019 (COVID-19) and residents' perspective to better prepare for future pandemic remain unknown. We aimed to assess and make cross-country and cross-region comparisons of the global impacts of COVID-19 and preparation preferences of pandemic. Methods: We recruited adults in 30 countries covering all World Health Organization (WHO) regions from July 2020 to August 2021. 5 Likert-point scales were used to measure their perceived change in 32 aspects due to COVID-19 (-2 = substantially reduced to 2 = substantially increased) and perceived importance of 13 preparations (1 = not important to 5 = extremely important). Samples were stratified by age and gender in the corresponding countries. Multidimensional preference analysis displays disparities between 30 countries, WHO regions, economic development levels, and COVID-19 severity levels. Results: 16 512 adults participated, with 10 351 females. Among 32 aspects of impact, the most affected were having a meal at home (mean (m) = 0.84, standard error (SE) = 0.01), cooking at home (m = 0.78, SE = 0.01), social activities (m = -0.68, SE = 0.01), duration of screen time (m = 0.67, SE = 0.01), and duration of sitting (m = 0.59, SE = 0.01). Alcohol (m = -0.36, SE = 0.01) and tobacco (m = -0.38, SE = 0.01) consumption declined moderately. Among 13 preparations, respondents rated medicine delivery (m = 3.50, SE = 0.01), getting prescribed medicine in a hospital visit / follow-up in a community pharmacy (m = 3.37, SE = 0.01), and online shopping (m = 3.33, SE = 0.02) as the most important. The multidimensional preference analysis showed the European Region, Region of the Americas, Western Pacific Region and countries with a high-income level or medium to high COVID-19 severity were more adversely impacted on sitting and screen time duration and social activities, whereas other regions and countries experienced more cooking and eating at home. Countries with a high-income level or medium to high COVID-19 severity reported higher perceived mental burden and emotional distress. Except for low- and lower-middle-income countries, medicine delivery was always prioritised. Conclusions: Global increasing sitting and screen time and limiting social activities deserve as much attention as mental health. Besides, the pandemic has ushered in a notable enhancement in lifestyle of home cooking and eating, while simultaneously reducing the consumption of tobacco and alcohol. A health care system and technological infrastructure that facilitate medicine delivery, medicine prescription, and online shopping are priorities for coping with future pandemics

Improving marketing collateral processing in a retail company

In retail, visual merchandising plays an important role in attracting and engaging customers towards making a purchase. It involves the display of merchandise along with printed forms of communication. (Gilbert, 2003, p.204) City Retailers is a local retailing company that owns the local franchise of City Sneaks, an American shoe brand that operates in stores nationwide. Visual merchandising is very important for City Sneaks. In fact, they are known for their unique way of displaying merchandise. Marketing collaterals play a large role in bringing in customers to the store because they callout the latest styles and sales promotions. This action research was conducted to address the issue of incomplete and delayed deliveries of marketing collaterals to the stores. City Retailers collateral delivery system has not been able to keep up with the fast growth of the company. Through the action research process, the City Retailers marketing team was able to determine the root cause of the problem. There appeared to be a lack of congruence between the people and the system. As a result of this research, the team was able to reduce the incidents of incomplete deliveries and shorten the processing period

On-Surface Synthesis of Non-Benzenoid Nanographenes by Oxidative Ring-Closure and Ring-Rearrangement Reactions

Nanographenes (NGs) have gained increasing attention due to their immense potential as tailor-made organic materials for nanoelectronics and spintronics. They exhibit a rich spectrum of physicochemical properties that can be tuned by controlling the size or the edge structure or by introducing structural defects in the honeycomb lattice. Here, we report the design and on-surface synthesis of NGs containing several odd-membered polycycles induced by a thermal procedure on Au(111). Our scanning tunneling microscopy, noncontact atomic force microscopy, and scanning tunneling spectroscopy measurements, complemented by computational investigations, describe the formation of two nonbenzenoid NGs (2A,B) containing four embedded azulene units in the polycyclic framework, via on-surface oxidative ring-closure reactions. Interestingly, we observe surface-catalyzed skeletal ring rearrangement reactions in the NGs, which lead to the formation of additional heptagonal rings as well as pentalene and as-indacene units in 2A,B, respectively. 2A,B on Au(111) both exhibit narrow experimental frontier electronic gaps of 0.96 and 0.85 eV, respectively, and Fermi level pinning of their HOMOs together with considerable electron transfer to the substrate. Ab initio calculations estimate moderate open-shell biradical characters for the NGs in the gas phase