The uncertain consequences of transferring bacterial strains between laboratories - rpoS instability as an example

Abstract\ud \ud \ud \ud Background\ud \ud Microbiological studies frequently involve exchanges of strains between laboratories and/or stock centers. The integrity of exchanged strains is vital for archival reasons and to ensure reproducible experimental results. For at least 50 years, one of the most common means of shipping bacteria was by inoculating bacterial samples in agar stabs. Long-term cultures in stabs exhibit genetic instabilities and one common instability is in rpoS. The sigma factor RpoS accumulates in response to several stresses and in the stationary phase. One consequence of RpoS accumulation is the competition with the vegetative sigma factor σ70. Under nutrient limiting conditions mutations in rpoS or in genes that regulate its expression tend to accumulate. Here, we investigate whether short-term storage and mailing of cultures in stabs results in genetic heterogeneity.\ud \ud \ud \ud Results\ud \ud We found that samples of the E. coli K-12 strain MC4100TF exchanged on three separate occasions by mail between our laboratories became heterogeneous. Reconstruction studies indicated that LB-stabs exhibited mutations previously found in GASP studies in stationary phase LB broth. At least 40% of reconstructed stocks and an equivalent proportion of actually mailed stock contained these mutations. Mutants with low RpoS levels emerged within 7 days of incubation in the stabs. Sequence analysis of ten of these segregants revealed that they harboured each of three different rpoS mutations. These mutants displayed the classical phenotypes of bacteria lacking rpoS. The genetic stability of MC4100TF was also tested in filter disks embedded in glycerol. Under these conditions, GASP mutants emerge only after a 3-week period. We also confirm that the intrinsic high RpoS level in MC4100TF is mainly due to the presence of an IS1 insertion in rssB.\ud \ud \ud \ud Conclusions\ud \ud Given that many E. coli strains contain high RpoS levels similar to MC4100TF, the integrity of such strains during transfers and storage is questionable. Variations in important collections may be due to storage-transfer related issues. These results raise important questions on the integrity of bacterial archives and transferred strains, explain variation like in the ECOR collection between laboratories and indicate a need for the development of better methods of strain transfer.We are grateful to Fundação de Amparo á Pesquisa do Estado de São Paulo (FAPESP-Brazil), who supported this study and provided a travel allowance for TF. TF was also supported by the the Australian Research Council and the US Army Research Office. We also thank K. C. Murphy and S. Kushner for respectively providing strain KM32 and plasmid pWKS130

Comparação da prova de função pulmonar e da força muscular respiratória entre idosas osteoporóticas com e sem fratura vertebral torácica = Comparation of pulmonary function and respiratory muscle strength among older osteoporotic women with and without thoracic vertebral fracture

Introduction: Osteoporosis is a disease characterized by decreased bone mass, resulting in fractures, which may occur in the thoracic vertebrae, causing changes in respiratory mechanics, impairing lung function. Objectives: To compare the spirometric, respiratory muscle strength and angle of thoracic kyphosis among elderly osteoporotic vertebral fractures with and without chest. Methods: The sample was divided into group A, elderly women with thoracic vertebral fracture (n = 5) and group B, older chest without vertebral fracture (n = 7). All were assessed by spirometry, manometer, and anthropometric measurements of the thoracic curvature. Results: The results showed an increase in thoracic curvature and a decrease in lung function in Group A compared with Group B and respiratory muscle strength deficit in Group A. Conclusion: The thoracic kyphosis deformity, caused by osteoporotic fractures, affects both the respiratory and musculoskeletal system

Desempenho do consórcio entre repolho e rabanete com pré-cultivo de crotalária, sob manejo orgânico.

Foram conduzidos dois experimentos no Sistema Integrado de Produção Agroecológica, em Seropédica, (RJ), durante dois anos consecutivos. O objetivo foi avaliar o desempenho do consórcio entre as culturas de repolho e rabanete com pré-cultivo de crotalária, sob manejo orgânico. Usou-se delineamento experimental de blocos ao acaso com sete repetições, em parcelas subdivididas, representando um fatorial 2 x 3, sendo o primeiro fator o pré-cultivo, com Crotalaria juncea e pousio (vegetação espontânea); e o segundo fator o sistema de cultivo (consórcio entre repolho e rabanete e os respectivos monocultivos). Não houve diferença significativa no tocante à produtividade do repolho ou rabanete, entre C. juncea e pousio, independentemente do tipo de manejo (monocultivos ou consórcio). O desenvolvimento do repolho não foi influenciado pela presença do rabanete, no consórcio entre essas espécies sob cultivo orgânico. O rabanete sob consórcio apresentou redução no diâmetro médio, massa média e produtividade de raízes, sem, contudo,desqualificar o padrão comercial das raízes colhidas. Considerando a média dos dois anos experimentais, o IEA atingiu 1,59, o que indicou a viabilidade do consórcio, otimizando práticas culturais, incluindo adubação,capina e irrigação

Production of poly (3-hydroxybutyrate-co-4-hydroxybutyrate) by Burkholderia sacchari using wheat straw hydrolysates and gamma-butyrolactone

“NOTICE: this is the author’s version of a work that was accepted for publication in International Journal of Biological Macromolecules. 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 in International Journal of Biological Macromolecules, Vol. 71, Special issue, (November 2014). DOI 10.1016/j.ijbiomac.2014.04.054 ""Burkholderia sacchari DSM 17165 is able to grow and produce poly(3-hydroxybutyrate) both on hexoses and pentoses. In a previous study, wheat straw lignocellulosic hydrolysates (WSH) containing high C6 and C5 sugar concentrations were shown to be excellent carbon sources for P(3HB) production. Using a similar feeding strategy developed for P(3HB) production based on WSH, fedbatch cultures were developed aiming at the production of the copolymer P(3HB-co- 4HB) (poly(3-hydroxybutyrate-co-4-hydroxybutyrate)) by B. sacchari. The ability of this strain to synthesize P(3HB-co-4HB) was first shown in shake flasks using gammabutyrolactone (GBL) as precursor of the 4HB units. Fed-batch cultures using glucose as carbon source (control) and GBL were developed to achieve high copolymer productivities and 4HB incorporations. The attained P(3HBco- 4HB) productivity and 4HB molar % were 0.7 g/(L·h) and 4.7 molar %, respectively. The 4HB incorporation was improved to 6.3 and 11.8 molar % by addition of 2 g/L propionic and acetic acid, respectively. When WSH were used as carbon source under the same feeding conditions, the values achieved were 0.5 g/(L·h) and 5.0 molar %, respectively. Burkholderia sacchari, a strain able to produce biopolymers based on xylose-rich lignocellulosic hydrolysates, is for the first time reported to produce P(3HB-co-4HB) using gamma butyrolactone as precursor."This work has received funding from the European Union 7th Framework Programme (FP7/2007-2013) under Grant Agreement number 246449 / Project: Bugworkers - New tailor-made PHB-based nanocomposites for high performance applications produced from environmentally friendly production routes.Financial support: Fundação para a Ciência e Tecnologia, Portugal (fellowships SFRH/BPD/68587/2010 and SFRH/BPD/26678/2006)

Unveiling the mechanism of action of 7α-acetoxy-6β-hydroxyroyleanone on an mrsa/visa strain: Membrane and cell wall interactions

UIDB/00100/2020 PTDC/MED-QUI/29036/2017 CEECIND/03414/2018 UIDB/04378/2020 PTDC/BIA-MIC/31645/2017 UID/DTP/04138/2019 UID/DTP/04567/2019 CBIOS/PRUID/BI1/2017 UIDB/04567/2020 UID/AMB/50017 UIDP/50017/2020 UIDB/50017/2020The number of cases of failure in the treatment of infections associated with resistant bacteria is on the rise, due to the decreasing efficacy of current antibiotics. Notably, 7α-Acetoxy-6β-hydroxyroyleanone (AHR), a diterpene isolated from different Plectranthus species, showed antibacterial activity, namely against Methicillin-resistant Staphylococcus aureus (MRSA) strains. The high antibacterial activity and low cytotoxicity render this natural compound an interesting alternative against resistant bacteria. The aim of this study is to understand the mechanism of action of AHR on MRSA, using the MRSA/Vancomycin-intermediate S. aureus (VISA) strain CIP 106760, and to study the AHR effect on lipid bilayers and on the cell wall. Although AHR interacted with lipid bilayers, it did not have a significant effect on membrane passive permeability. Alternatively, bacteria treated with this royleanone displayed cell wall disruption, without revealing cell lysis. In conclusion, the results gathered so far point to a yet undescribed mode of action that needs further investigation.publishersversionpublishe

Reusable and highly sensitive SERS immunoassay utilizing gold nanostars and a cellulose hydrogel-based platform

This work was funded by FEDER funds through the COMPETE 2020 Programme and National Funds through the FCT---Fun-dação para a Ciência e a Tecnologia, I. P., under the scope of the project. This work also received funding from the European Community’s H2020 program under grant agreement No. 716510 (ERC-2016-STG TREND), 640598 (ERC-StG-2014, NEWFUN), and 685758 (1D-Neon). This work was supported by the Applied Molecular Biosciences Unit – UCIBIO and Associate Laboratory for Green Chemistry – LAQV which are financed by Portugal national funds from /2016 and SFRH/BD/132057/2017 from the FCT/MCTES and MIT Portugal PhD Program (to I. C. and M. J. O., respectively). Professor César Laia (LAQV-REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Portugal) is acknowledged for permitting the use of DLS equipment. Professor Ludwig Krippahl (NOVA LINCS, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Portugal) is acknowledged for developing the gel analysis application eReuss. M. J. O. acknowledges Rocío Palomares Jurado PhD for visualisation/data presentation support and continued interest in the project. M. J. O. acknowledges David Peitinho MSc for help and advice in Python script. Diego Wiechers de Carvalho BSc is acknowledged for purifying the recombinant Plasmodium falciparum histidine-rich protein 2 sample and Daniela Gomes PhD for providing SEM images. Publisher Copyright: © The Royal Society of Chemistry 2021.The development of robust and sensitive point-of-care testing platforms is necessary to improve patient care and outcomes. Surface-enhanced Raman scattering (SERS)-based immunosensors are especially suited for this purpose. Here, we present a highly sensitive and selective SERS immunoassay, demonstrating for example the detection of horseradish peroxidase (HRP), in a sandwich format. The strength of our biosensor lies in merging: (i) SERS-immunotags based on gold nanostars, allowing exceptional intense SERS from attached Raman probes, covalent attachment of anti-HRP antibodies by a simple chemical method providing exceptional antigen binding activity; (ii) the ease of preparation of the capture platform from a regenerated cellulose-based hydrogel, a transparent material, ideal for microfluidics applications, with low background fluorescence and Raman signal, particularly suited for preserving high activity of the covalently bound anti-HRP antibodies. The sandwich complexes formed were characterised by atomic force microscopy, and by scanning electron microscopy coupled with electron diffraction spectroscopy; and (iii) the robustness of the simple Classical Least Squares method for SERS data analysis, resulting in superior discrimination of SERS signals from the background and much better data fitting, compared to the commonly used peak integral method. Our SERS immunoassay greatly improves the detection limits of traditional enzyme-linked immunosorbent assay approaches, and its performance is better or comparable to those of existing SERS-based immunosensors. Our approach successfully overcomes the main challenges of application at point-of-care, including increasing reproducibility, sensitivity, and specificity, associated with an environmentally friendly and robust design. Also, the proposed design withstands several cycles of regeneration, a feature absent in paper-SERS immunoassays and this opens the way for sensitive multiplexing applications on a microfluidic platform.preprintpublishe

Superpulsed low-level laser therapy protects skeletal muscle of mdx mice against damage, inflammation and morphological changes delaying dystrophy progression.

Aim: To evaluate the effects of preventive treatment with low-level laser therapy (LLLT) on progression of dystrophy in mdx mice. Methods: Ten animals were randomly divided into 2 experimental groups treated with superpulsed LLLT (904 nm, 15 mW, 700 Hz, 1 J) or placebo-LLLT at one point overlying the tibialis anterior muscle (bilaterally) 5 times per week for 14 weeks (from 6th to 20th week of age). Morphological changes, creatine kinase (CK) activity and mRNA gene expression were assessed in animals at 20th week of age. Results: Animals treated with LLLT showed very few morphological changes in skeletal muscle, with less atrophy and fibrosis than animals treated with placebo-LLLT. CK was significantly lower (p = 0.0203) in animals treated with LLLT (864.70 U.l−1, SEM 226.10) than placebo (1708.00 U.l−1, SEM 184.60). mRNA gene expression of inflammatory markers was significantly decreased by treatment with LLLT (p<0.05): TNF-α (placebo-control = 0.51 µg/µl [SEM 0.12], - LLLT = 0.048 µg/µl [SEM 0.01]), IL-1β (placebo-control = 2.292 µg/µl [SEM 0.74], - LLLT = 0.12 µg/µl [SEM 0.03]), IL-6 (placebo-control = 3.946 µg/µl [SEM 0.98], - LLLT = 0.854 µg/µl [SEM 0.33]), IL-10 (placebo-control = 1.116 µg/µl [SEM 0.22], - LLLT = 0.352 µg/µl [SEM 0.15]), and COX-2 (placebo-control = 4.984 µg/µl [SEM 1.18], LLLT = 1.470 µg/µl [SEM 0.73]). Conclusion: Irradiation of superpulsed LLLT on successive days five times per week for 14 weeks decreased morphological changes, skeletal muscle damage and inflammation in mdx mice. This indicates that LLLT has potential to decrease progression of Duchenne muscular dystrophy

Studies on the mechanism of action of anti-tumor bis(aminophenolate) ruthenium(III) complexes

Two recently published Ru(III) comp lexes bearing ( N 2 O 2 ) tetradentate bis(aminophenolate) ligands, 20 formulated as [Ru(III)( s alan )(PPh 3 )Cl] ( where s alan is the tetradentate ligand 6,6' - (1S,2S) - 21 cyclohexane - 1,2 - diylbis(azanediyl)bis(met hylene)bis(3 - methoxyphenol) in c omplex 1 , or 2,2' - (1S,2S) - 22 cyclohexane - 1,2 - diylbis(azanediyl)bis(meth ylene)bis(4 - methoxyphenol) in c omplex 2 , and PPh 3 is 23 triphenylphosphane ) were studied herein to outline their antitumor mode of action. Previously, it was 24 found that they were very active against human ovar ian and bre a st adenocarcinoma cells (on a 72 h 25 challenge ) . In this work, the human cisplatin - sensitive o varian adeno carcinoma line A2780 was used 26 as the cell model for further studies a t a 24 h challenge . B oth complexes are active , their cytotoxicity 27 being c omparable to that of c isplatin in the same conditions. 28 A s a possible target in the cell for their action, the int eraction of 1 and 2 with DNA was assessed 29 through displacement of well - established DNA fluorescent probes ( ethidium bromide , EB, and 4',6 - 30 d iamidino - 2 - p henylindole, DAPI ) through s teady - state and time - resolved fluorescence spectroscopy . 31 The complete emission spectra were analysed globally for the binary DNA ‒ p robe and ternary DNA ‒ 32 p robe ‒ Ru(III) complex systems . B oth Ru(III) complexes can displace EB and bind to DNA with 33 similar and moderate strong affinity with conditional stability constants of log K’ = ( 5.05 ± 0.01 ) for 1 34 and log K’= ( 4.79 ± 0.01 ) for 2 . The analysis of time - domain fluorescence intensity decays confirmed 35 both qualitatively and quantitatively the model used to describe the binding and competition processes. 36 Cell studies indicated that apoptosis is t he major mechanism of cell death for both complexes , with 2 37 (the mo re active complex ) promoting that process more efficiently than 1 . Transmission electron 38 micro graphs revealed clear a lteration s on intracellular organization consistent with the induction of 39 programmed cell death processes

Glucosylpolyphenols as Inhibitors of Aβ-Induced Fyn Kinase Activation and Tau Phosphorylation: Synthesis, Membrane Permeability, and Exploratory Target Assessment within the Scope of Type 2 Diabetes and Alzheimer's Disease

Despite the rapidly increasing number of patients suffering from type 2 diabetes, Alzheimer's disease, and diabetes-induced dementia, there are no disease-modifying therapies that are able to prevent or block disease progress. In this work, we investigate the potential of nature-inspired glucosylpolyphenols against relevant targets, including islet amyloid polypeptide, glucosidases, and cholinesterases. Moreover, with the premise of Fyn kinase as a paradigm-shifting target in Alzheimer's drug discovery, we explore glucosylpolyphenols as blockers of Aβ-induced Fyn kinase activation while looking into downstream effects leading to Tau hyperphosphorylation. Several compounds inhibit Aβ-induced Fyn kinase activation and decrease pTau levels at 10 μM concentration, particularly the per-O-methylated glucosylacetophloroglucinol and the 4-glucosylcatechol dibenzoate, the latter inhibiting also butyrylcholinesterase and β-glucosidase. Both compounds are nontoxic with ideal pharmacokinetic properties for further development. This work ultimately highlights the multitarget nature, fine structural tuning capacity, and valuable therapeutic significance of glucosylpolyphenols in the context of these metabolic and neurodegenerative disorders.European Commission GA 612347Fundação para a Ciência e a Tecnologia SFRH/BD/93170/2013, SFRH/BD/116614/2016, PD/BD/142847/2018, SFRH/BD/145600/2019, CEECIND/03414/2018, CEECIND/02300/2017, UIDB/00100/2020, UIDB/04046/2020, UIDB/04378/2020, IF/00780/2015Gobierno de España CTQ2016-78703-PJunta de Andalucía FQM13

Liquid-Ordered Phase Formation by Mammalian and Yeast Sterols: A Common Feature With Organizational Differences

Here, biophysical properties of membranes enriched in three metabolically related sterols are analyzed both in vitro and in vivo. Unlike cholesterol and ergosterol, the common metabolic precursor zymosterol is unable to induce the formation of a liquid ordered (lo) phase in model lipid membranes and can easily accommodate in a gel phase. As a result, Zym has a marginal ability to modulate the passive membrane permeability of lipid vesicles with different compositions, contrary to cholesterol and ergosterol. Using fluorescence-lifetime imaging microscopy of an aminostyryl dye in living mammalian and yeast cells we established a close parallel between sterol-dependent membrane biophysical properties in vivo and in vitro. This approach unraveled fundamental differences in yeast and mammalian plasma membrane organization. It is often suggested that, in eukaryotes, areas that are sterol-enriched are also rich in sphingolipids, constituting highly ordered membrane regions. Our results support that while cholesterol is able to interact with saturated lipids, ergosterol seems to interact preferentially with monounsaturated phosphatidylcholines. Taken together, we show that different eukaryotic kingdoms developed unique solutions for the formation of a sterol-rich plasma membrane, a common evolutionary trait that accounts for sterol structural diversity.Peer Reviewe