Optimisation of culture conditions for a producer clone coexpressing arylsulfatase B and a formylglycine-generating enzyme in order to increase the yield of arylsulfatase B

Maroteaux—Lamy syndrome (mucopolysaccharidosis type VI) is an orphan genetic disease caused by mutations in the arylsulfatase B gene (ARSB), which encodes the lysosomal enzyme arylsulfatase B (ASB). The relevance of the study lies in the need of a Russian recombinant ASB product for patients with the disease in the Russian Federation. Previously, the authors have developed producer lines coexpressing the target ASB enzyme with an auxiliary formylglycine-generating enzyme (FGE), based on Chinese hamster ovary (CHO) cells. Further development of the recombinant ASB preparation places priority on increasing the enzyme yield. The aim of this study was to increase the productivity of producer clones by optimising the culture process and adding calcium chloride and copper sulfate to the culture medium. Materials and methods: a suspension-adapted CHO cell line was used. Monoclonal cell lines were developed using Cell Metric and ClonePix FL systems. The concentration of ASB in the culture liquid was determined using the enzyme-linked immunosorbent assay (ELISA). The authors analysed batch culture and/or fed-batch culture in media supplemented with various concentrations of copper sulfate and calcium chloride. Results: the combined addition of copper sulfate and calcium chloride at concentrations of 300 μM during batch culture of producer clones coexpressing ASB and FGE increases viability and specific productivity of the cells up to 4.58±1.62 pg/ (cell×day). The cultivation of the lead producer clone coexpressing ASB and FGE under fed-batch conditions for 12 days and the addition of copper sulfate to the growth medium at the concentration of 300 μM allow for increasing the yield of the active lysosomal enzyme, arylsulfatase B, to 420 mg/L. Conclusions: the cultivation of producer clones coexpressing ASB and FGE under fed-batch conditions with copper sulfate added to the medium significantly improves cell line growth properties and the ASB yield. This approach to the selection of culture conditions for producer cell lines can be applied to other enzymes of the sulfatase family

The Product Produced by Interaction of the Cis-diammin(cyclobutane 1,1 dicarboxylato) Platinum(II) with Arabinogalactan

Получен продукт взаимодействия комплекса цис-диамин(циклобутан-1,1-дикарбоксилат- О,О`) платина(II) с арабиногалактаном, который охарактеризован методами РФА, рентгенофлуоресцентным, ИК-спектроскопии, термогравиметрии и УФ-спектрофотоментии, установлен его состав. Показано, что цис-диамин(циклобутан-1,1-дикарбоксилат-О,О`) платина(II) предположительно связан с арабиногалактаном водородными связями, образованными группами -С-О-С- и NH3-группами исходного комплексаThe obtained new product of reaction of the complex cis-diammin (cyclobutane 1,1 dicarboxylato) platinum(II) with arabinogalactan was characterized by XRD, IR-spectroscopy, UF-spectrophotometry and thermogravimetry. The composition and structure of this product was studied. It has been shown that cis-diammin(cyclobutane 1,1 dicarboxylato) platinum(II) associated with arabinogalactan through communications -C-О-C- groups by hydrogen of the original comple

Modelling social identification and helping in evacuation simulation

Social scientists have criticised computer models of pedestrian streams for their treatment of psychological crowds as mere aggregations of individuals. Indeed most models for evacuation dynamics use analogies from physics where pedestrians are considered as particles. Although this ensures that the results of the simulation match important physical phenomena, such as the deceleration of the crowd with increasing density, social phenomena such as group processes are ignored. In particular, people in a crowd have social identities and share those social identities with the others in the crowd. The process of self categorisation determines norms within the crowd and influences how people will behave in evacuation situations. We formulate the application of social identity in pedestrian simulation algorithmically. The goal is to examine whether it is possible to carry over the psychological model to computer models of pedestrian motion so that simulation results correspond to observations from crowd psychology. That is, we quantify and formalise empirical research on and verbal descriptions of the effect of group identity on behaviour. We use uncertainty quantification to analyse the model’s behaviour when we vary crucial model parameters. In this first approach we restrict ourselves to a specific scenario that was thoroughly investigated by crowd psychologists and where some quantitative data is available: the bombing and subsequent evacuation of a London underground tube carriage on July 7th 2005

Методический подход к разработке стандартных образцов фазового состава технологических продуктов золотоизвлекательных фабрик

The paper describes the results of standardless quantitative XRD phase analysis (QPA) of 5 reference samples of elemental composition of sulphidic ore and technological products from Olimpiada gold deposit. The conformance between measured mineral composition and certified elemental composition is evaluated. Possibilities of the standardless QPA for determination of phase composition of the reference samples are discussed.Описываются результаты бесстандартного количественного рентгенофазового анализа минерального состава пяти стандартных образцов элементного состава сульфидной руды и технологических продуктов золотоизвлекательных фабрик Олимпиадинского ГОК. Оценивается соответствие полученного минерального и аттестованного элементного составов. Делается вывод о возможности создания на основе стандартных образцов элементного состава стандартных образцов фазового состава для настройки автоматизированных градуировочных рентгенофазовых методов анализа минерального состава руды и технологических продуктов золотоизвлекательных фабрик. Обсуждаются методические аспекты разработки стандартных образцов фазового состава

Оптимизация условий культивирования клона-продуцента, коэкспрессирующего арилсульфатазу B и формилглицин-генерирующий фермент, с целью повышения выхода фермента арилсульфатазы B

Maroteaux—Lamy syndrome (mucopolysaccharidosis type VI) is an orphan genetic disease caused by mutations in the arylsulfatase B gene (ARSB), which encodes the lysosomal enzyme arylsulfatase B (ASB). The relevance of the study lies in the need of a Russian recombinant ASB product for patients with the disease in the Russian Federation. Previously, the authors have developed producer lines coexpressing the target ASB enzyme with an auxiliary formylglycine-generating enzyme (FGE), based on Chinese hamster ovary (CHO) cells. Further development of the recombinant ASB preparation places priority on increasing the enzyme yield. The aim of this study was to increase the productivity of producer clones by optimising the culture process and adding calcium chloride and copper sulfate to the culture medium. Materials and methods: a suspension-adapted CHO cell line was used. Monoclonal cell lines were developed using Cell Metric and ClonePix FL systems. The concentration of ASB in the culture liquid was determined using the enzyme-linked immunosorbent assay (ELISA). The authors analysed batch culture and/or fed-batch culture in media supplemented with various concentrations of copper sulfate and calcium chloride. Results: the combined addition of copper sulfate and calcium chloride at concentrations of 300 μM during batch culture of producer clones coexpressing ASB and FGE increases viability and specific productivity of the cells up to 4.58±1.62 pg/ (cell×day). The cultivation of the lead producer clone coexpressing ASB and FGE under fed-batch conditions for 12 days and the addition of copper sulfate to the growth medium at the concentration of 300 μM allow for increasing the yield of the active lysosomal enzyme, arylsulfatase B, to 420 mg/L. Conclusions: the cultivation of producer clones coexpressing ASB and FGE under fed-batch conditions with copper sulfate added to the medium significantly improves cell line growth properties and the ASB yield. This approach to the selection of culture conditions for producer cell lines can be applied to other enzymes of the sulfatase family.Синдром Марото—Лами (мукополисахаридоз VI типа) — орфанное генетическое заболевание, вызванное мутациями гена ARSB, который кодирует лизосомальный фермент арилсульфатазу В. Актуальность исследования заключается в необходимости разработки отечественного препарата рекомбинантной арилсульфатазы В для лечения пациентов с данным заболеванием в Российской Федерации. Ранее были получены клеточные линии-продуценты, коэкспрессирующие целевой фермент арилсульфатазу В и вспомогательный формилглицин-генерирующий фермент на основе клеточной линии СНО. Однако для дальнейшей разработки препарата рекомбинантной арилсульфатазы В представляется важным повышение выхода фермента. Цель работы: увеличение продуктивности клонов-продуцентов за счет оптимизации процесса культивирования и добавления в культуральную среду хлорида кальция и сульфата меди. Материалы и методы: использовали суспензионную клеточную линию СНО. Моноклональные клеточные линии получали с использованием систем Cell Metric и Clone Pix FL. Концентрацию арилсульфатазы В в культуральной жидкости определяли методом иммуноферментного анализа. Использовали периодическое культивирование (batch culture) и/или периодическое культивирование с подпиткой (fedbatch culture) в среде с добавлением различных концентраций сульфата меди и хлорида кальция. Результаты: продемонстрировано, что одновременное добавление сульфата меди и хлорида кальция в концентрации 300 мкM при периодическом культивировании клонов-продуцентов, коэкспрессирующих арилсульфатазу В и формилглицин-генерирующий фермент, увеличивает жизнеспособность культур и повышает удельную продуктивность клеток до 4,58±1,62 пг/(клетка×сут). При культивировании лидерного клона-продуцента, коэкспрессирующего арилсульфатазу В и формилглицин-генерирующий фермент, в условиях периодического культивирования с подпиткой длительностью 12 сут достигнуто увеличение выхода активного лизосомального фермента арилсульфатазы В до 420 мг/л при добавлении в ростовую среду сульфата меди в концентрации 300 мкM. Выводы: культивирование клонов-продуцентов, коэкспрессирующих арилсульфатазу В и формилглицин-генерирующий фермент, в условиях периодического культивирования с подпиткой и с добавлением в среду сульфата меди приводит к значительному улучшению ростовых свойств клеточной линии и выхода целевого фермента. Данный подход в подборе условий культивирования продуцентов можно применять к другим ферментам подкласса сульфатаз

Arabidopsis CPR5 Independently Regulates Seed Germination and Postgermination Arrest of Development through LOX Pathway and ABA Signaling

The phytohormone abscisic acid (ABA) and the lipoxygenases (LOXs) pathway play important roles in seed germination and seedling growth and development. Here, we reported on the functional characterization of Arabidopsis CPR5 in the ABA signaling and LOX pathways. The cpr5 mutant was hypersensitive to ABA in the seed germination, cotyledon greening and root growth, whereas transgenic plants overexpressing CPR5 were insensitive. Genetic analysis demonstrated that CPR5 gene may be located downstream of the ABI1 in the ABA signaling pathway. However, the cpr5 mutant showed an ABA independent drought-resistant phenotype. It was also found that the cpr5 mutant was hypersensitive to NDGA and NDGA treatment aggravated the ABA-induced delay in the seed germination and cotyledon greening. Taken together, these results suggest that the CPR5 plays a regulatory role in the regulation of seed germination and early seedling growth through ABA and LOX pathways independently

A CLASP-modulated cell edge barrier mechanism drives cell-wide cortical microtubule organization in Arabidopsis

It is well known that the parallel order of microtubules in the plant cell cortex defines the direction of cell expansion, yet it remains unclear how microtubule orientation is controlled, especially on a cell-wide basis. Here we show through 4D imaging and computational modelling that plant cell polyhedral geometry provides spatial input that determines array orientation and heterogeneity. Microtubules depolymerize when encountering sharp cell edges head-on, whereas those oriented parallel to those sharp edges remain. Edge-induced microtubule depolymerization, however, is overcome by the microtubule-associated protein CLASP, which accumulates at specific cell edges, enables microtubule growth around sharp edges and promotes formation of microtubule bundles that span adjacent cell faces. By computationally modelling dynamic 'microtubules on a cube' with edges differentially permissive to microtubule passage, we show that the CLASP-edge complex is a 'tuneable' microtubule organizer, with the inherent flexibility to generate the numerous cortical array patterns observed in nature

Transgenerational Stress Memory Is Not a General Response in Arabidopsis

Adverse conditions can trigger DNA damage as well as DNA repair responses in plants. A variety of stress factors are known to stimulate homologous recombination, the most accurate repair pathway, by increasing the concentration of necessary enzymatic components and the frequency of events. This effect has been reported to last into subsequent generations not exposed to the stress. To establish a basis for a genetic analysis of this transgenerational stress memory, a broad range of treatments was tested for quantitative effects on homologous recombination in the progeny. Several Arabidopsis lines, transgenic for well-established recombination traps, were exposed to 10 different physical and chemical stress treatments, and scored for the number of somatic homologous recombination (SHR) events in the treated generation as well as in the two subsequent generations that were not treated. These numbers were related to the expression level of genes involved in homologous recombination and repair. SHR was enhanced after the majority of treatments, confirming previous data and adding new effective stress types, especially interference with chromatin. Compounds that directly modify DNA stimulated SHR to values exceeding previously described induction rates, concomitant with an induction of genes involved in SHR. In spite of the significant stimulation in the stressed generations, the two subsequent non-treated generations only showed a low and stochastic increase in SHR that did not correlate with the degree of stimulation in the parental plants. Transcripts coding for SHR enzymes generally returned to pre-treatment levels in the progeny. Thus, transgenerational effects on SHR frequency are not a general response to abiotic stress in Arabidopsis and may require special conditions

A Link among DNA Replication, Recombination, and Gene Expression Revealed by Genetic and Genomic Analysis of TEBICHI Gene of Arabidopsis thaliana

Spatio-temporal regulation of gene expression during development depends on many factors. Mutations in Arabidopsis thaliana TEBICHI (TEB) gene encoding putative helicase and DNA polymerase domains-containing protein result in defects in meristem maintenance and correct organ formation, as well as constitutive DNA damage response and a defect in cell cycle progression; but the molecular link between these phenotypes of teb mutants is unknown. Here, we show that mutations in the DNA replication checkpoint pathway gene, ATR, but not in ATM gene, enhance developmental phenotypes of teb mutants, although atr suppresses cell cycle defect of teb mutants. Developmental phenotypes of teb mutants are also enhanced by mutations in RAD51D and XRCC2 gene, which are involved in homologous recombination. teb and teb atr double mutants exhibit defects in adaxial-abaxial polarity of leaves, which is caused in part by the upregulation of ETTIN (ETT)/AUXIN RESPONSIVE FACTOR 3 (ARF3) and ARF4 genes. The Helitron transposon in the upstream of ETT/ARF3 gene is likely to be involved in the upregulation of ETT/ARF3 in teb. Microarray analysis indicated that teb and teb atr causes preferential upregulation of genes nearby the Helitron transposons. Furthermore, interestingly, duplicated genes, especially tandemly arrayed homologous genes, are highly upregulated in teb or teb atr. We conclude that TEB is required for normal progression of DNA replication and for correct expression of genes during development. Interplay between these two functions and possible mechanism leading to altered expression of specific genes will be discussed

11β-HSD1 inhibition in men mitigates prednisolone-induced adverse effects in a proof-of-concept randomised double-blind placebo-controlled trial

Glucocorticoids prescribed to limit inflammation, have significant adverse effects. As 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) regenerates active glucocorticoid, we investigated whether 11β-HSD1 inhibition with AZD4017 could mitigate adverse glucocorticoid effects without compromising their anti-inflammatory actions. We conducted a proof-of-concept, randomized, double-blind, placebo-controlled study at Research Unit, Churchill Hospital, Oxford, UK (NCT03111810). 32 healthy male volunteers were randomized to AZD4017 or placebo, alongside prednisolone treatment. Although the primary endpoint of the study (change in glucose disposal during a two-step hyperinsulinemic, normoglycemic clamp) wasn’t met, hepatic insulin sensitivity worsened in the placebo-treated but not in the AZD4017-treated group. Protective effects of AZD4017 on markers of lipid metabolism and bone turnover were observed. Night-time blood pressure was higher in the placebo-treated but not in the AZD4017-treated group. Urinary (5aTHF+THF)/THE ratio was lower in the AZD4017-treated but remained the same in the placebo-treated group. Most anti-inflammatory actions of prednisolone persisted with AZD4017 co-treatment. Four adverse events were reported with AZD4017 and no serious adverse events. Here we show that co-administration of AZD4017 with prednisolone in men is a potential strategy to limit adverse glucocorticoid effects