Development of a technology for the preparation of a dry nutrient medium for anthrax vaccine production

Currently, submerged cultivation of the Bacillus anthracis STI-1 strain for live anthrax vaccine production requires liquid nutrient media, which have disadvantages of a short shelf life (no more than one month) and a narrow range of storage temperatures (2–8 °С). Dry media, in contrast, have a number of indisputable advantages: such media are transportable and easy to use, have a standard capability to retain properties, and can be stored without preservatives at 2–30 °С for 2–5 years. The aim of this work was to develop a technology for the preparation of a dry nutrient medium for anthrax vaccine production. Materials and methods: The study used the Bacillus anthracis STI-1 vaccine strain and a nutrient medium for its cultivation, containing a 70:30 mixture of an enzymatic digest of casein and a pre-processed corn extract solution. Drying of the nutrient medium was carried out on a spray-drying unit. The authors evaluated physicochemical parameters of experimental medium batches. The shelf life was determined by an accelerated stability study. The dry nutrient medium was used to produce a live anthrax vaccine. Quality attributes of the vaccine were assessed for compliance with regulatory requirements. Results: The authors developed the dry media production technology. According to it, the liquid nutrient medium is fed to the drying unit at a rate of 20–25 dm3/h. The spray air pressure is 0.02 MPa. Temperatures at the drying chamber inlet and outlet are 118–122 °С and 85–90 °С, respectively. The technology was used to obtain 3 experimental batches of the dry medium. The study results demonstrate that the technology is reproducible, and the tested quality attributes of experimental medium batches are consistent with the requirements. According to the accelerated stability study, the shelf life of the dry nutrient medium at 2–30 °С is at least 3 years. Experiments demonstrated the possibility of using the dry nutrient medium for live anthrax vaccine production. Critical quality attributes of the vaccine obtained with the medium met regulatory requirements. Conclusions: The developed technology allows for the production of a standard dry nutrient medium with a prolonged shelf life, which is convenient for live anthrax vaccine production

Eukaryotic-like Ser/Thr Protein Kinases SpkC/F/K Are Involved in Phosphorylation of GroES in the Cyanobacterium Synechocystis

Serine/threonine protein kinases (STPKs) are the major participants in intracellular signal transduction in eukaryotes, such as yeasts, fungi, plants, and animals. Genome sequences indicate that these kinases are also present in prokaryotes, such as cyanobacteria. However, their roles in signal transduction in prokaryotes remain poorly understood. We have attempted to identify the roles of STPKs in response to heat stress in the prokaryotic cyanobacterium Synechocystis sp. PCC 6803, which has 12 genes for STPKs. Each gene was individually inactivated to generate a gene-knockout library of STPKs. We applied in vitro Ser/Thr protein phosphorylation and phosphoproteomics and identified the methionyl-tRNA synthetase, large subunit of RuBisCO, 6-phosphogluconate dehydrogenase, translation elongation factor Tu, heat-shock protein GrpE, and small chaperonin GroES as the putative targets for Ser/Thr phosphorylation. The expressed and purified GroES was used as an external substrate to screen the protein extracts of the individual mutants for their Ser/Thr kinase activities. The mutants that lack one of the three protein kinases, SpkC, SpkF, and SpkK, were unable to phosphorylate GroES in vitro, suggesting possible interactions between them towards their substrate. Complementation of the mutated SpkC, SpkF, and SpkK leads to the restoration of the ability of cells to phosphorylate the GroES. This suggests that these three STPKs are organized in a sequential order or a cascade and they work one after another to finally phosphorylate the GroES

Положительное давление в конце выдоха и ингаляция оксида азота при лечении острого респираторного дистресс-синдрома

Objective: to evaluate the impact of positive end-expiratory pressure (PEEP) on hemodynamic and gas-exchange effects of inhaled nitric oxide (iNO) in patients with acute respiratory distress syndrome (ARDS).Subjects and methods. The study included 27 cardiosurgical patients with ARDS who were divided into 2 groups, depending on the increment (A) in PaO2/FiO2 during iNO delivery (5 ppm): A PaO2/FiO2 > 15% [Group A (n=13)] and APaO2/FiO2 < 15% [Group B (n=14)]. The impact of different end-expiratory pressures (0, 4, 8, 12, and 16 cm H2O) on the parameters of pulmonary gas-exchange function, hemodynamics, O2 transport-uptake, respiratory biomechanics in the presence and absence of iNO delivery was examined.Results. In Groups A and B patients receiving no iNO, there were increases in PaO2/FiO2 and thoracopulmonary compliance (p<0.05), with end-expiratory pressure being elevated from 0 to 8 cm H2O. The rise of PEEP from 8 to 12 and 16 cm H2O in the absence of iNO delivery was not further accompanied by increased PaO2/FiO2 and thoracopulmonary compliance. Only in Group A, the effect of iNO on pulmonary oxygenating function was potentiated with end-expiratory pressure being increased from 0 to 8 cm: the increment of PaO2/FiO2 during iNO delivery accordingly increased from 21±3 to 38±6 mm Hg. In this group, a subsequent increase of PEEP to 12—16 cm H2O did not lead to the enhanced effect of iNO on arterial oxygenation.Conclusion. PEEP creation attended by increased thoracopulmonary compliance potentiates the effect of iNO on the pulmonary oxygenating function only in patients in whom the use of iNO is effective when end-expiratory pressure is zero. Цель исследования — оценка влияния положительного давления в конце выдоха на гемодинамические и газообменные эффекты иNO у больных ОРДС.Материалы и методы. В исследование включили 27 кардиохирургических больных с ОРДС, которые были разделены на 2 группы в зависимости от прироста (A) PaO2/FiO2 на фоне подачи иNO (5 ppm): APaO2/FiO2 > 15% (группа А, n=13) и APaO2/FiO2 < 15% (группа Б, n=14). Изучалось влияние различного уровня давления в конце выдоха (0, 4, 8, 12 и 16 см вод. ст.) на показатели газообменной функции лёгких, гемодинамики, транспорт-потребление О2, биомеханики дыхания на фоне и в отсутствие подачи иNO.Результаты. Без иNO у пациентов обеих групп выявлено увеличение (p<0,05) PaO2/FiO2 и торакопульмональной податливости при увеличении конечно-экспираторного давления с 0 до 8 см вод. ст. Повышение ПДКВ с 8 до 12 и 16 см вод. ст. в отсутствие подачи иNO не сопровождалось дальнейшим увеличением PaO2/FiO2 и торакопульмональной податливости. Только у пациентов группы А усиление влияния иNO на оксигенирующую функцию лёгких произошло при повышении конечно-экспираторного давления с 0 до 8 см вод. ст.: прирост PaO2/FiO2 на фоне подачи иNO увеличился (p<0,05), соответственно, с 21±3 до 38±6 мм рт. ст. Дальнейшее повышение ПДКВ до 12—16 см вод. ст. в этой группе не привело к усилению влияния иNO на артериальную оксигенацию.Заключение. Создание ПДКВ, сопровождаемое увеличением торакопульмональной податливости, усиливает действия иNO на оксигенирующую функцию лёгких только у больных, у которых применение иNO было эффективным и при нулевом конечно-экспираторном давлении.

Improvement of regeneration in pepper: a recalcitrant species

[EN] Organogenesis is influenced by factors like genotype, type of explant, culture medium components, and incubation conditions. The influence of ethylene, which can be produced in the culture process, can also be a limiting factor in recalcitrant species like pepper. In this work, bud induction was achieved from cotyledons and hypocotyls-from eight pepper cultivars-on Murashige and Skoog (MS) medium supplemented with 22.2 mu M 6-benzyladenine (6BA) and 5.71 mu M indole-3-acetic acid (IAA), in media with or without silver nitrate (SN) (58.86 mu M), a suppressor of ethylene action. In the SN-supplemented medium, the frequencies of explants with buds and with callus formation were lower in both kinds of explant, but higher numbers of developed shoots were isolated from explants cultured on SN. Bud elongation was better in medium with gibberellic acid (GA(3)) (2.88 mu M) than in medium free of growth regulators or supplemented with 1-aminocyclopropane-1-carboxylic acid (ACC) at 34.5 mu M. However, isolation of shoots was difficult and few plants were recovered. The effect of adding SN following bud induction (at 7 d) and that of dark incubation (the first 7 d of culture) was also assessed in order to improve the previous results. When SN was added after bud induction, similar percentages of bud induction were found for cotyledons (average frequency 89.37% without SN and 94.37% with SN) whereas they doubled in hypocotyls (50% without SN and 87.7% with SN). In addition, in both kinds of explant, the number of developed plants able to be transferred to soil (developed and rooted) was greatly increased by SN. Dark incubation does not seem to improve organogenesis in pepper, and hypocotyl explants clearly represent a better explant choice-with respect to cotyledonary explants-for the pepper cultivars assayed.We thank the COMAV germplasm bank at Universitat Politecnica de Valencia and the Arid Lands Institute for pepper seeds and the Tunisian Ministry of Higher Education and Scientific Research who fund N. Gammoudi's stay.Gammoudi, N.; San Pedro-Galan, T.; Ferchichi, A.; Gisbert Domenech, MC. (2018). Improvement of regeneration in pepper: a recalcitrant species. In Vitro Cellular & Developmental Biology - Plant. 54(2):145-153. https://doi.org/10.1007/s11627-017-9838-1S145153542Ashrafuzzaman M, Hossain MM, Razi Ismail M, Shahidul Haque M, Shahidullah SM, Uz Zaman S (2009) Regeneration potential of seedling explants of chilli (Capsicum annuum). Afr J Biotechnol 8:591–596Bortesi L, Fischer R (2015) The CRISPR/Cas9 system for plant genome editing and beyond. 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Разработка технологии приготовления сухой питательной среды для производства сибиреязвенной вакцины

Currently, submerged cultivation of the Bacillus anthracis STI-1 strain for live anthrax vaccine production requires liquid nutrient media, which have disadvantages of a short shelf life (no more than one month) and a narrow range of storage temperatures (2–8 °С). Dry media, in contrast, have a number of indisputable advantages: such media are transportable and easy to use, have a standard capability to retain properties, and can be stored without preservatives at 2–30 °С for 2–5 years. The aim of this work was to develop a technology for the preparation of a dry nutrient medium for anthrax vaccine production. Materials and methods: The study used the Bacillus anthracis STI-1 vaccine strain and a nutrient medium for its cultivation, containing a 70:30 mixture of an enzymatic digest of casein and a pre-processed corn extract solution. Drying of the nutrient medium was carried out on a spray-drying unit. The authors evaluated physicochemical parameters of experimental medium batches. The shelf life was determined by an accelerated stability study. The dry nutrient medium was used to produce a live anthrax vaccine. Quality attributes of the vaccine were assessed for compliance with regulatory requirements. Results: The authors developed the dry media production technology. According to it, the liquid nutrient medium is fed to the drying unit at a rate of 20–25 dm3/h. The spray air pressure is 0.02 MPa. Temperatures at the drying chamber inlet and outlet are 118–122 °С and 85–90 °С, respectively. The technology was used to obtain 3 experimental batches of the dry medium. The study results demonstrate that the technology is reproducible, and the tested quality attributes of experimental medium batches are consistent with the requirements. According to the accelerated stability study, the shelf life of the dry nutrient medium at 2–30 °С is at least 3 years. Experiments demonstrated the possibility of using the dry nutrient medium for live anthrax vaccine production. Critical quality attributes of the vaccine obtained with the medium met regulatory requirements. Conclusions: The developed technology allows for the production of a standard dry nutrient medium with a prolonged shelf life, which is convenient for live anthrax vaccine production.В настоящее время при производстве вакцины сибиреязвенной живой для глубинного выращивания штамма Bacillus anthracis СТИ-1 используется жидкая питательная среда, недостатками которой являются малый срок годности — не более одного месяца и узкий диапазон температуры ее хранения: от 2 до 8 °С. Сухие питательные среды (ПС) обладают рядом неоспоримых преимуществ: их можно хранить от 2 до 5 лет при температуре от 2 до 30 °С без консервантов; они транспортабельны, удобны в применении и стандартны в сохранении свойств. Цель работы: разработка технологии приготовления сухой ПС для производства сибиреязвенной вакцины. Материалы и методы: в исследованиях использовали вакцинный штамм B. anthracis СТИ-1 и ПС, состоящую из смеси ферментативного гидролизата казеина и раствора обработанного кукурузного экстракта в соотношении 70 и 30%, для культивирования сибиреязвенного микроба. Обезвоживание ПС осуществляли на установке распылительного типа. Экспериментальные серии сухой ПС оценивали по физико-химическим показателям на соответствие требованиям нормативной документации. Срок годности определяли методом «ускоренного старения». С использованием сухой ПС готовили вакцину сибиреязвенную живую и проводили оценку показателей качества препарата на соответствие требованиям нормативной документации. Результаты: разработана технология приготовления сухой ПС (скорость подачи ПС на сушку от 20 до 25 дм3/ч, давление сжатого воздуха в распылителе 0,02 МПа, температура воздуха на входе в сушильную камеру от 118 до 122 °С, температура воздуха на выходе — от 85 до 90 °С). По этой технологии получены 3 серии экспериментальной сухой ПС. Показано, что разработанная технология воспроизводима, а экспериментальные серии сухой ПС по изученным показателям отвечают предъявляемым требованиям. Срок годности сухой ПС, установленный с использованием метода «ускоренного старения», не менее 3 лет при температуре хранения от 2 до 30 °С. Экспериментально подтверждена возможность использования сухой ПС в технологии производства сибиреязвенной вакцины. Приготовленный препарат по основным показателям качества отвечает требованиям нормативной документации. Выводы: разработанная технология позволяет получить сухую ПС стандартную с увеличенным сроком хранения и удобную при использовании в производстве вакцины сибиреязвенной живой