Клиническое значение микробиоты легких и опыт применения ингаляционной антибактериальной терапии у детей Омского центра муковисцидоза (серия случаев)

A growth of antibiotic-resistant Pseudomonas aeruginosa from patients with cystic fibrosis decreases a lung function significantly, which requires searching new, up-to-date antibiotics. Goal. An investigation of clinical and microbiological features of respiratory lesion and an assessment of elimination therapy at chronic P. aeruginosa infection in children on the example of the Regional Cystic Fibrosis Center. Materials and methods. A retrospective analysis of case histories of children at the Omsk Cystic Fibrosis Center (n=48) was performed, with evaluation of clinical and functional parameters depending on age and microbiological status of respiratory airways. The comparative evaluation of two drugs inhaled tobramycin Bramitob and Tobramycin-Gobbi efficiency was performed. Results. An association of microorganisms was identified from the majority of bacterial cultures of cystic fibrosis patients’ sputum (66.7%). Staphylococcus aureus (18.9%) and P. aeruginosa (16.6%) with the significant prevalence of non-mucoid forms were in the lead at the group of clinically relevant pathogens. From 2011 to 2016 amount of P. aeruginosa strains, which were sensitive to the all usable antibiotics, decreased, an especially significant decline of sensitivity was registered to amikacin (32.7%) and сiprofloxacin (48.6%). Children infected with P. aeruginosa had a lot more deviations of lung function measured by the computed bronchophonography and spirometry, they more often had changes in chest computed tomography than patients free of P. aeruginosa infection. Change from inhaled tobramycin Bramitob into inhaled tobramycin Tobramycin-Gobbi in the patients’ treatment didn’t lead to the elevation of respiratory syndrome exacerbation rates, didn’t increase requirement in the intravenous antibacterial treatment and didn’t influence to the rank of microbial colonization of sputum. Conclusion. In length of time, the increase of P. aeruginosa resistance to antipseudomonal antibiotics is observed, which makes reasonable the introduction high-concentrated forms of antibiotics. The replacement of inhaled tobramycin from Bramitob to Tobramycin-Gobbi didn’t cause the impairment of patients’ clinical and functional condition and didn’t change the microbiological status.  Актуальность. Высев антибиотикорезистентной синегнойной палочки у больных муковисцидозом значительно ухудшает функцию легких, что требует поиска новых, современных антибактериальных средств. Цель. Изучение клинико-микробиологических особенностей поражения органов дыхания и оценка эффективности элиминационной терапии при хронической синегнойной инфекции у детей на примере регионального центра муковисцидоза.Материалы и методы. Проведен ретроспективный анализ историй болезни детей Омского центра муковисцидоза (n=48) с оценкой клинических и функциональных показателей в зависимости от возраста и микробиологического статуса дыхательных путей. Дана сравнительная оценка эффективности лекарственных препаратов ингаляционного тобрамицина. Результаты. В большинстве бактериальных посевов мокроты (66,7%) больных муковисцидозом микроорганизмы высевались в ассоциации. Из клинически значимых патогенов лидировали золотистый стафилококк (18,9%) и синегнойная палочка (16,6%) со значительным преобладанием немукоидных форм. За период 2011–2016 гг. произошло снижение количества чувствительных штаммов Pseudomonas aeruginosa ко всем используемым антибиотикам, из них значимое снижение отмечено к амикацину (32,7%) и ципрофлоксацину (48,6%). У детей, инфицированных синегнойной палочкой, отмечались более выраженные отклонения в показателях функции внешнего дыхания по данным компьютерной бронхофонографии, спирометрии и чаще встречались изменения при мультиспиральной компьютерной томографии органов грудной клетки, чем у больных, свободных от синегнойной инфекции. Смена ингаляционных форм тобрамицина у некоторых больных не привела к повышению частоты обострений респираторного синдрома, не увеличила потребность в назначении внутривенной антибактериальной терапии и не повлияла на степень микробной колонизации мокроты.Заключение. В динамике отмечается рост устойчивости P. aeruginosa к антисинегнойным препаратам, что делает обоснованным внедрение высококонцентрированных ингаляционных форм антибиотиков. Замена одного ингаляционного тобрамицина на другой не вызвала ухудшения клинического и функционального состояния у 4 проанализированных пациентов и не изменила их микробиологический статус.Статья издается при поддержке компании «Генфа».Авторы заявляют об отсутствии конфликта интересов, о котором необходимо сообщить

Bridging Gaps in HDR Improvement: The Role of MAD2L2, SCAI, and SCR7

This study aimed to enhance homology-directed repair (HDR) efficiency in CRISPR/Cas-mediated genome editing by targeting three key factors regulating the balance between HDR and non-homologous end joining (NHEJ): MAD2L2, SCAI, and Ligase IV. In order to achieve this, a cellular model using mutated eGFP was designed to monitor HDR events. Results showed that MAD2L2 knockdown and SCR7 treatment significantly improved HDR efficiency during Cas9-mediated HDR repair of the mutated eGFP gene in the HEK293T cell line. Fusion protein Cas9-SCAI did not improve HDR. This study is the first to demonstrate that MAD2L2 knockdown during CRISPR-mediated gene editing in HEK293T cells can increase precise correction by up to 10.2 times. The study also confirmed a moderate but consistent effect of SCR7, an inhibitor of Ligase IV, which increased HDR by 1.7 times. These findings provide valuable insights into improving HDR-based genome editing efficiency

Enzyme activity assays.

<p>Enzymatic conversion of [1-¹⁴C]-13-HPOT was analyzed by radio-HPLC. <i>Solanum tuberosum</i> AOS1 and AOC (Stumpe et al., 2006) were used as positive controls. The chromatogram shows α-ketol as well as OPDA as LjAOS1 reaction products, and OPDA as the only reaction product for LjAOC1 and LjAOC2 which were used in combination with <i>S</i>. <i>tuberosum</i> AOS1. mAU, milli absorption units. One representative of three independent experiments is shown.</p

Immunolocalization of <i>L</i>. <i>japonicus</i> AOC protein in longitudinal sections of <i>L</i>. <i>japonicus</i> nodules.

<p>The nodules come from plants from two different growth systems; (A-C, F-G) aeroponic culture, (D-E) perlite/vermiculite-grown plants (in both cases, nodules were harvest 3 weeks after inoculation). Panels (A-E) show conventional wide-field fluorescent microscopy images where the fluorescence of the secondary antibody labeling AOC is visible in green. The image in panel (F) was taken with differential interference contrast. The image in panel (G) was taken on a confocal laser scanning microscope and shows immunolabeled AOC in green, while toluidine blue-stained bacteria and nuclei are shown in red. Labels: ic, infected cells; nc, nodule cortex; uc, uninfected cells of the inner tissue. The nodule vascular bundles are labeled with asterisks. The arrows in panels (A) and (B) points at infected cells with low bacteroid density. Size bars denote (A,D) 100 μm, (B,C,E) 50 μm, and (F,G) 20 μm.</p

AOC protein levels in roots and nodules.

<p>The left panel shows a Western blot from root (LjR) and nodule (LjN) proteins from <i>L</i>. <i>japonicus</i>, developed with an antibody against tomato AOC. The right panel shows a 12% SDS-polyacrylamide gel with the same amount of protein loaded, stained with Fast Green. AOC proteins are 26 kDa in size. One representative example of three independent experiments is shown.</p

Expression analysis of <i>Ljaos1</i>, <i>Ljaos2</i>, <i>Ljaoc1</i>, <i>Ljaoc2</i> and <i>Ljaoc3</i> genes in roots (R), nodules (N), stems (S), leaves (L), flowers (F) and immature pods (P) of <i>L</i>. <i>japonicus</i> cv.

<p>Gifu using real time RT-PCR. <i>L</i>. <i>japonicus</i> ubiquitin was used as housekeeping control. Three biological replicates were used. The data are presented as box plot. The boxes show the interquartile range, and bars indicate data points below the first and above the third quartile. Lines in the boxes mark the median value.</p

Clinical Significance of Lung Microbiota and Experience of the Inhaled Antibacterial Therapy in Children of the Omsk Cystic Fibrosis Center (Сase Series)

A growth of antibiotic-resistant Pseudomonas aeruginosa from patients with cystic fibrosis decreases a lung function significantly, which requires searching new, up-to-date antibiotics. Goal. An investigation of clinical and microbiological features of respiratory lesion and an assessment of elimination therapy at chronic P. aeruginosa infection in children on the example of the Regional Cystic Fibrosis Center. Materials and methods. A retrospective analysis of case histories of children at the Omsk Cystic Fibrosis Center (n=48) was performed, with evaluation of clinical and functional parameters depending on age and microbiological status of respiratory airways. The comparative evaluation of two drugs inhaled tobramycin Bramitob and Tobramycin-Gobbi efficiency was performed. Results. An association of microorganisms was identified from the majority of bacterial cultures of cystic fibrosis patients’ sputum (66.7%). Staphylococcus aureus (18.9%) and P. aeruginosa (16.6%) with the significant prevalence of non-mucoid forms were in the lead at the group of clinically relevant pathogens. From 2011 to 2016 amount of P. aeruginosa strains, which were sensitive to the all usable antibiotics, decreased, an especially significant decline of sensitivity was registered to amikacin (32.7%) and сiprofloxacin (48.6%). Children infected with P. aeruginosa had a lot more deviations of lung function measured by the computed bronchophonography and spirometry, they more often had changes in chest computed tomography than patients free of P. aeruginosa infection. Change from inhaled tobramycin Bramitob into inhaled tobramycin Tobramycin-Gobbi in the patients’ treatment didn’t lead to the elevation of respiratory syndrome exacerbation rates, didn’t increase requirement in the intravenous antibacterial treatment and didn’t influence to the rank of microbial colonization of sputum. Conclusion. In length of time, the increase of P. aeruginosa resistance to antipseudomonal antibiotics is observed, which makes reasonable the introduction high-concentrated forms of antibiotics. The replacement of inhaled tobramycin from Bramitob to Tobramycin-Gobbi didn’t cause the impairment of patients’ clinical and functional condition and didn’t change the microbiological status

Allene oxide synthase, allene oxide cyclase and jasmonic acid levels in Lotus japonicus nodules.

Jasmonic acid (JA), its derivatives and its precursor cis-12-oxo phytodienoic acid (OPDA) form a group of phytohormones, the jasmonates, representing signal molecules involved in plant stress responses, in the defense against pathogens as well as in development. Elevated levels of JA have been shown to play a role in arbuscular mycorrhiza and in the induction of nitrogen-fixing root nodules. In this study, the gene families of two committed enzymes of the JA biosynthetic pathway, allene oxide synthase (AOS) and allene oxide cyclase (AOC), were characterized in the determinate nodule-forming model legume Lotus japonicus JA levels were to be analysed in the course of nodulation. Since in all L. japonicus organs examined, JA levels increased upon mechanical disturbance and wounding, an aeroponic culture system was established to allow for a quick harvest, followed by the analysis of JA levels in whole root and shoot systems. Nodulated plants were compared with non-nodulated plants grown on nitrate or ammonium as N source, respectively, over a five week-period. JA levels turned out to be more or less stable independently of the growth conditions. However, L. japonicus nodules formed on aeroponically grown plants often showed patches of cells with reduced bacteroid density, presumably a stress symptom. Immunolocalization using a heterologous antibody showed that the vascular systems of these nodules also seemed to contain less AOC protein than those of nodules of plants grown in perlite/vermiculite. Hence, aeroponically grown L. japonicus plants are likely to be habituated to stress which could have affected JA levels.peerReviewe