Production and analysis of the biological properties of recombinant human granulocyte colony stimulating factor chimeric form

The aim of this work was to design and study biological properties of the recombinant human granulocyte colony stimulating factor (G-CSF), «linked» to apolipoprotein A-I (apoA-I) by a peptide linker, for obtaining in perspective a prolong form of the drug based on this cytokine.Material and methods. The nucleotide sequences of the genes encoding G-CSF and apoA-I were designed and optimized for expression in Pichia pastoris yeast using several computer programs. The assembly of the gene coding for the G-CSF-apoA-I chimeric cytokine, its cloning in the pPICZa-A vector, and expression in P. pastoris cells were performed using standard genetic engineering methods. Purification of the chimeric cytokine was carried out by two-stage ion-exchange chromatography. The biological activity of the chimera was determined in vitro on rat and human bone marrow cells (BMC) using flow cytometry, cell cycle analysis and myelograms.Results. A recombinant P pastoris X-33 yeast strain producing a chimeric cytokine containing the amino acid sequence G-CSF from the N-terminus, and mature human apoA-I from the C-terminus was constructed. In experiments on BMC of rat, it was shown that G-CSF-apoA-I increases the number of granulocytes in 1.8-2 times less compared with G-CSF. At the same time, the chimeric cytokine maintained the viability of monocytic and lymphocytic cells. Unlike G-CSF, the chimera increased the number of blast cells and normalized neutrophil segmentation, reducing the number of anomalies 1.5 times more efficiently.Conclusion. A new chimeric cytokine G-CSF-apoA-I was constructed, exhibiting the properties of not only a colony-stimulating factor, but also a growth factor, supporting the viability of other types of BMC

ОПРЕДЕЛЕНИЕ БРОМИДА ЦЕТИЛТРИМЕТИЛАММОНИЯ ФОТОМЕТРИЧЕСКИМ МЕТОДОМ ЗА СЧЕТ АГРЕГАЦИИ С КАРБОЦИАНИНОВЫМ КРАСИТЕЛЕМ

It was found that the commercial carbocyanine dye IR-783 containing sulfo groups forms aggregates with cetyltrimethylammonium bromide (CTAB) in a slightly alkaline medium yielding a new absorption band, a change in the solution color from blue to yellow (Dl = 350 nm), and a change in the fluorescence intensity in the near-IR region. CTAB was determined by the photometric method  by photographing the reaction mixture in a 96-well plate with a smartphone camera or in a Camag visualizer. The difference between the intensities of the red and blue channels (R – B), corresponding to the yellow color, was used as an analytical signal. The linear range in an aqueous solution is (3 – 25)·10–6 M, the detection limit is 1.6·10–6 M, and the relative standard deviation is 2–5%. The determination is not affected with a number of non-ionic surfactants, inorganic salts and polymers; the anionic surfactants interfere. Other cationic surfactants also give analytical signals, but with different sensitivities. The characteristics of the literature method for the determination of CTAB based on the Coomassie brilliant blue G-250 dye and the proposed method are comparable. A sample of CTAB-containing lysing buffer solution was analyzed.Keywords: cetyltrimethylammonium, cationic surfactants, carbocyanine dye, aggregation, photometry DOI: http://dx.doi.org/10.15826/analitika.2022.26.3.004 Irina A. Stepanova, Anna V. Shik, Evgenii V. Skorobogatov, Anatasiya A.Bartoshevich, Mikhail K. BeklemishevDepartment of Chemistry, Lomonosov Moscow State University,Russian Federation, 119991, GSP-1, Moscow, Leninskie gory, 1, building 3Обнаружено, что коммерческий карбоцианиновый краситель IR-783, содержащий сульфогруппы, в слабощелочной среде образует агрегаты с бромидом цетилтриметиламмония (ЦТАБ) с появлением новой полосы поглощения, изменением цвета раствора с синего на желтый (Dl = 350 нм) и изменением интенсивности флуоресценции в ближней ИК области. ЦТАБ определяли фотометрическим методом, фотографируя реакционную смесь в 96-луночном планшете камерой смартфона или в визуализаторе Camag. В качестве аналитического сигнала использовали разность интенсивностей красного и синего каналов (R – B), соответствующую желтому цвету. Диапазон определяемых концентраций в водном растворе составляет (3 – 25)·10–6 М, предел обнаружения 1.6·10–6 М, относительное стандартное отклонение 2–5 %. Определению не мешает ряд неионных ПАВ, неорганических солей и полимеров, мешают анионные ПАВ. Другие катионные ПАВ также дают сигналы, но с разной чувствительностью. Характеристики литературной методики определения ЦТАБ на основе красителя кумасси бриллиантовый синий G-250 и предлагаемой сопоставимы. Проанализирован образец лизирующего буфера, содержащего ЦТАБ.Ключевые слова: цетилтриметиламмоний, катионные поверхностно-активные вещества, карбоцианиновый краситель, агрегация, фотометрияDOI: http://dx.doi.org/10.15826/analitika.2022.26.3.00

The use of NIR Fluorimetry with photographic data acquisition in the fingerprinting method with the addition of fluorophores to the samples: discrimination of apple juices

Предложено использовать красители, флуоресцирующие в ближней ИК (БИК) области спектра (700-800 нм), для распознавания объектов методом «отпечатков пальцев», основанным на добавке флуорофоров к объекту («флуоресцентный глаз»). Метод успешно применяется в классификации объектов различной природы. В данной работе метод опробован на примере дискриминации 17 образцов яблочного сока разных производителей, выпущенных в разное время. В качестве добавляемого флуорофора использовали гептаметиновый карбоцианиновый краситель индоленинового ряда в присутствии ПАВ, в качестве источника излучения - красные светодиоды, а сигнал регистрировали с помощью цифрового фотоаппарата с дополнительным ИК-светофильтром; для записи спектров применяли спектрофлуориметр с приставкой для 96-луночного флуориметрического планшета. Фотографические изображения обрабатывали с помощью стандартного программного обеспечения Unscrambler X и Excel. Результаты представили в координатах: интенсивность БИК-флуоресценции - интенсивность отражения видимого света (с использованием соответствующих фотографий). Обнаружили, что такое представление позволяет разделить образцы на группы, связанные с производителем. Получали также спектры собственной флуоресценции, в том числе с добавкой БИК-красителя, обрабатывая эти результаты методом главных компонент. По собственной эмиссии можно выделить 5-6 групп образцов, не считая контрольного, тогда как по спектрам с добавкой красителя удается добиться выделения наибольшего числа групп образцов (девять). При этом классификация с использованием спектров не позволяет группировать соки по производителям. Кроме того, получение фотографий с помощью визуализатора проще и экспресснее, чем регистрация спектров флуоресценции. Совместная обработка эмиссионных спектров и фотографий не позволяет повысить качество дискриминации образцов.The application of dyes, that fluoresce in the near infrared (NIR, 700-800 nm) region, for the recognition of samples using a fingerprinting method with the addition of fluorophores to the samples (“fluorescent eye”) is proposed. The technique has been successfully applied to the classification of samples of various nature. In the current work, this strategy has been tested on the example of discrimination of 17 samples of apple juice from different manufacturers, purchased at different times. An indolenine series heptamethine carbocyanine dye in the presence of surfactants was used as the added fluorophore, red LEDs were used as an excitation source, and the signal was recorded using a digital camera with an additional IR filter installed; a spectrofluorimeter with a 96-well plate accessory was used to record the spectra. Photographic images were processed using Unscrambler X and Excel software. The results were presented using the following coordinates: intensity of NIR fluorescence - intensity of visible light reflection (using the photographic images). It was found that such presentation allowed the samples to be divided into groups associated with the manufacturer. We have also obtained intrinsic fluorescence spectra, including those with the addition of NIR dye, and these results were processed by the principal component analysis. It was possible to distinguish 5-6 groups of samples by their intrinsic emission, not counting the blank, while the spectra with the addition of the dye allowed to isolate the largest number of groups of samples (9). At the same time, the classification using spectra did not allow juices to be grouped by the producer. Also, obtaining photographs using a visualizer was easier and faster than recording the fluorescence spectra. The joint processing of emission spectra and photographs did not improve the quality of discrimination.Работа выполнена при поддержке РНФ (грант № 20-13-00330). Авторы благодарят доцента Т.А.Подругину за предоставление карбоцианинового красителя и А.Добротворского за предоставление ИК-фотоаппарата.Current work was supported by the Russian Science Foundation (grant No 20-13-00330). The authors are grateful to Associate Professor T.A. Podrugina for providing the carbocyanine dye and A. Dobrotvorsky for providing the IR photo camera

Introduction to Integral Discriminants

The simplest partition function, associated with homogeneous symmetric forms S of degree r in n variables, is integral discriminant J_{n|r}(S) = \int e^{-S(x_1 ... x_n)} dx_1 ... dx_n. Actually, S-dependence remains the same if e^{-S} in the integrand is substituted by arbitrary function f(S), i.e. integral discriminant is a characteristic of the form S itself, and not of the averaging procedure. The aim of the present paper is to calculate J_{n|r} in a number of non-Gaussian cases. Using Ward identities -- linear differential equations, satisfied by integral discriminants -- we calculate J_{2|3}, J_{2|4}, J_{2|5} and J_{3|3}. In all these examples, integral discriminant appears to be a generalized hypergeometric function. It depends on several SL(n) invariants of S, with essential singularities controlled by the ordinary algebraic discriminant of S.Comment: 36 pages, 19 figure

Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album

The objects of the study were recombinant clones of Komagataella phaffii K51 carrying the heterologous proteinase K (PK-w) gene from Tritirachium album integrated into their genome as well as samples of recombinant proteinase K isolated from these clones. The aims of this work were i) to determine whether it is possible to create recombinant K. phaffii K51 clones overexpressing functionally active proteinase K from T. album and ii) to analyze the enzymatic activity of the resulting recombinant enzyme. The following methods were used: computational analysis of primary structure of the proteinase K gene, molecular biological methods (PCR, electrophoresis of DNA in an agarose gel, electrophoresis of proteins in an SDS polyacrylamide gel under denaturing conditions, spectrophotometry, and quantitative assays of protease activity), and genetic engineering techniques (cloning and selection of genes in bacterial cells Escherichia coli TOP10 and in the methylotrophic yeast K. phaffii K51). The gene encoding natural proteinase K (PK-w) was designed and optimized for expression in K. phaffii K51. The proteinase K gene was synthesized and cloned within the plasmid pPICZα-A vector in E. coli TOP10 cells. The proteinase K gene was inserted into pPICZα-A in such a way that – at a subsequent stage of transfection into yeast cells – it was efficiently expressed under the control of the promoter and terminator of the AOX1 gene, and the product of the exogenous gene contained the signal peptide of the Saccharomyces cerevisiae a-factor to ensure the protein’s secretion into the culture medium. The resultant recombinant plasmid (pPICZα-A/PK-w) was transfected into K. phaffii K51 cells. A recombinant K. phaffii K51 clone was obtained that carried the synthetic proteinase K gene and ensured its effective expression and secretion into the culture medium. An approximate productivity of the yeast recombinant clones for recombinant proteinase K was 25 μg/ mL after 4 days of cultivation. The resulting recombinant protease has a high specific proteolytic activity: ~5000 U/mg

Colorimetric determination of cetyltrimethylammonium bromide by using aggregation with a carbocyanine dye

Обнаружено, что коммерческий карбоцианиновый краситель IR-783, содержащий сульфогруппы, в слабощелочной среде образует агрегаты с бромидом цетилтриметиламмония (ЦТАБ) с появлением новой полосы поглощения, изменением цвета раствора с синего на желтый (Dl = 350 нм) и изменением интенсивности флуоресценции в ближней ИК области. ЦТАБ определяли фотометрическим методом, фотографируя реакционную смесь в 96-луночном планшете камерой смартфона или в визуализаторе Camag. В качестве аналитического сигнала использовали разность интенсивностей красного и синего каналов (R - B), соответствующую желтому цвету. Диапазон определяемых концентраций в водном растворе составляет (3 - 25)·10-6 М, предел обнаружения 1.6·10-6 М, относительное стандартное отклонение 2-5 %. Определению не мешает ряд неионных ПАВ, неорганических солей и полимеров, мешают анионные ПАВ. Другие катионные ПАВ также дают сигналы, но с разной чувствительностью. Характеристики литературной методики определения ЦТАБ на основе красителя кумасси бриллиантовый синий G-250 и предлагаемой сопоставимы. Проанализирован образец лизирующего буфера, содержащего ЦТАБ.It was found that the commercial carbocyanine dye IR-783 containing sulfo groups forms aggregates with cetyltrimethylammonium bromide (CTAB) in a slightly alkaline medium yielding a new absorption band, a change in the solution color from blue to yellow (Dl = 350 nm), and a change in the fluorescence intensity in the near-IR region. CTAB was determined by the photometric method by photographing the reaction mixture in a 96-well plate with a smartphone camera or in a Camag visualizer. The difference between the intensities of the red and blue channels (R - B), corresponding to the yellow color, was used as an analytical signal. The linear range in an aqueous solution is (3 - 25)·10-6 M, the detection limit is 1.6·10-6 M, and the relative standard deviation is 2-5%. The determination is not affected with a number of non-ionic surfactants, inorganic salts and polymers; the anionic surfactants interfere. Other cationic surfactants also give analytical signals, but with different sensitivities. The characteristics of the literature method for the determination of CTAB based on the Coomassie brilliant blue G-250 dye and the proposed method are comparable. A sample of CTAB-containing lysing buffer solution was analyzed.Работа выполнена при поддержке Российского научного фонда (проект № 20-13-00330). Авторы благодарят Т.А. Подругину и И.А. Дорошенко за предоставленный краситель IR-783 и Н.С. Мелик-Нубарова за краситель кумасси. Исследование проведено в рамках Программы развития Междисциплинарной научно-образовательной школы Московского университета «Будущее планеты и глобальные изменения окружающей среды».This work was supported by the Russian Science Foundation (grant No 20-13-00330). The authors are grateful to T.A. Podrugina and I.A. Doroshenko for providing the IR-783 dye and N.S. Melik-Nubarov for the Coomassie dye. The study was conducted within the framework of the Development Program of the Interdisciplinary Scientific and Educational School of Moscow University “The Future of the Planet and Global Environmental Changes”

The changing pattern of human brucellosis: clinical manifestations, epidemiology, and treatment outcomes over three decades in Georgia

<p>Abstract</p> <p>Background</p> <p>Brucellosis is an endemic infection in Georgia. We conducted a review of patient records with a suspected or confirmed diagnosis of brucellosis over three decades at the central referral hospital for brucellosis cases, the Institute of Parasitology and Tropical Medicine (IPTM) in Tbilisi. The purpose was to describe the demographic profile and clinical characteristics as well as diagnostic and treatment strategies in patients with brucellosis.</p> <p>Methods</p> <p>Data were abstracted from randomly selected patient records at the IPTM. In total, 300 records were reviewed from three time periods: 1970-73, 1988-89, and 2004-2008.</p> <p>Results</p> <p>The age distribution of patients shifted from a median age of 40 years in the first time period to 20 years in the third time period. Azeri ethnicity was an increasing proportion of the total number of cases. The frequency of relapsed infection was 14.7% (44 cases). A total of 50 patients received vaccine therapy, and although the vaccine produced immune responses, demonstrated by an increase in agglutination titers, it was not associated with improved outcome.</p> <p>Conclusion</p> <p>The demographics of brucellosis in Georgia fit a profile of persons that tend sheep. Osteoarticular complications were commonly detected, especially in children. The changing pattern of brucellosis in Georgia suggests clinicians should be updated about different trends in brucellosis in their country.</p