Development of monoclonal antibodies specific to ribosomal protein S6 kinase 2

Ribosomal protein S6 kinase 2 (S6K2) is a serine/threonine kinase that belongs to the family of AGC kinases, which includes PKB/Akt, PKC, PDK1, and SGK1. Mammalian cells express two isoforms of S6K, termed S6K1 and S6K2. Each of these has nuclear and cytoplasmic spicing variants, which originate from different initiation start codons. Nuclear isoforms of S6K1 and S6K2 are slightly longer, as they possess additional sequences at the N-terminus with nuclear localization signals. Biochemical and genetic studies implicated S6Ks in the regulation of cell size, growth, and energy metabolism. Deregulation of S6K signaling has been linked to various human pathologies, making them excellent targets for drug discovery. The aim of this study was to produce monoclonal antibodies directed at the N-terminal regulatory region of S6K2, which shows very low homology to S6K1 or other members of the AGC family. To achieve this goal, two S6K2 fragments covering 1-64aa and 14-64aa N-terminal sequences were expressed in bacteria as GST/6His fusion proteins. Affinity purified recombinant proteins were used as antigens for immunization, hybridoma screening, and analysis of generated clones. We produced a panel of S6K2-specific antibodies, which recognized recombinant S6K2 proteins in ELISA and Western blot analysis. Further analysis of selected clones revealed that three clones, termed B1, B2, and B4, specifically recognized not only recombinant, but also endogenous S6K2 in Western blot analysis of HEK293 cell lysates. Specificity of B2 clone has been confirmed in additional commonly used immunoassays, including immunoprecipitation and immunocytochemistry. These properties make B2 MAb particularly valuable for elucidating signal transduction pathways involving S6K2 signaling under physiological conditions and in human pathologies

Malaria pigment hemozoin impairs gm-csf receptor expression and function by 4-hydroxynonenal

Malarial pigment hemozoin (HZ) generates the lipoperoxidation product 4-hydroxynonenal (4-HNE), which is known to cause dysregulation of the immune response in malaria. The inhibition of granulocyte macrophage colony-stimulating factor (GM-CSF)-dependent differentiation of dendritic cells (DC) by HZ and 4-HNE was previously described in vitro, and the GM-CSF receptor (GM-CSF R) was hypothesised to be a primary target of 4-HNE in monocytes. In this study, we show the functional impact of HZ on GM-CSF R in monocytes and monocyte-derived DC by (i) impairing GM-CSF binding by 50 ± 9% and 65 ± 14%, respectively (n = 3 for both cell types); (ii) decreasing the expression of GM-CSF R functional subunit (CD116) on monocyte’s surface by 36 ± 11% (n = 6) and in cell lysate by 58 ± 16% (n = 3); and (iii) binding of 4-HNE to distinct amino acid residues on CD116. The data suggest that defective DC differentiation in malaria is caused by GM-CSF R dysregulation and GM-CSF R modification by lipoperoxidation product 4-HNE via direct interaction with its CD116 subunit

Convergence of the all-time supremum of a L\'evy process in the heavy-traffic regime

In this paper we derive a technique of obtaining limit theorems for suprema of L\'evy processes from their random walk counterparts. For each $a>0$, let $\{Y^{(a)}_n:n\ge 1\}$ be a sequence of independent and identically distributed random variables and $\{X^{(a)}_t:t\ge 0\}$ be a L\'evy processes such that $X_1^{(a)}\stackrel{d}{=} Y_1^{(a)}$, $\mathbb E X_1^{(a)}<0$ and $\mathbb E X_1^{(a)}\uparrow0$ as $a\downarrow0$. Let $S^{(a)}_n=\sum_{k=1}^n Y^{(a)}_k$. Then, under some mild assumptions, $\Delta(a)\max_{n\ge 0} S_n^{(a)}\stackrel{d}{\to} R\iff\Delta(a)\sup_{t\ge 0} X^{(a)}_t\stackrel{d}{\to} R$, for some random variable $R$ and some function $\Delta(\cdot)$. We utilize this result to present a number of limit theorems for suprema of L\'evy processes in the heavy-traffic regime

Влияние температуры микроволнового спекания на структуру и механические свойства гидроксиапатитной керамики

Досліджено особливості мікрохвильового спікання біогенного гідроксиапатиту, призначеного для використання в медичній практиці для заміщення кісткових дефектів різного походження. Зразки отримували у високотемпературній мікрохвильовій муфельній печі (1,5 кВт, 2,45 ГГц) за температур 800, 900, 1000 і 1100 °С. Встановлено, що підвищення температури при мікрохвильовому спіканні не призводить до стрімкого росту розміру зерна та утворення агломератів. Отримані матеріали мають рівномірну дрібнозернисту структуру із середнім розміром зерен 0,42-0,56 мкм і пор ~ 0,5 мкм. Встановлено, що пористість отриманих матеріалів, отриманих в інтервалі температур 800-1000 °С, становить ~ 40 %, а при 1100 °С – зменшується до 33 %. Міцність на стиск зростає від 31 до 59 МПа з підвищенням температури і є близькою до міцності губчатої кістки людини. Дослідження in vіtro показали, що розчинність матеріалів у фізіологічному розчині не залежить від температури спікання і становить 0,1-0,15 мас. %/добу Таким чином, методом мікрохвильового спікання при 800-1100 °С можна отримувати гідроксиапатитну кераміку медичного призначення з достатніми структурно-механічними властивостями для заміщення дефектів кісткової тканини.The aim of the paper was to investigate features of microwave sintering of biogenic hydroxyapatite obtained for filling of various bone defects in medicine. The samples were prepared at high-temperature muffle furnace (1,5 kW, 2,45 GHz) at 800, 900, 1000 and 1100 °C. It was established that increasing of microwave sintering temperature did not lead to a rapid increasing of grain size and agglomerate formation. The prepared materials have homogeneous fine-grained structure with average grain size 0,42-0,56 mm and average pore size is equal to 0,5 mm. It was established that the porosity of materials prepared at 800-1000 °C was equal to 40 % and for materials prepared at 1100 °C reduced down to 33 %. The compressive strength increased from 31 to 59 MPa with increasing of temperature and was close to that of cancellous human bone. Studies in vіtro showed that the solubility of the materials in physiological solution did not depend on sintering temperature and was equal to 0,1-0,15 wt. %/day. Thus, the possibility of preparation of ceramics based on biogenic hydroxyapatite by microwave sintering at 800-1100 °C with sufficient structural-mechanical properties for medical use for the substitution of bone defects was shown in the present work.Исследованы особенности микроволнового спекания биогенного гидроксиапатита, предназначенного для использования в медицинской практике для замещения костных дефектов различного происхождения. Образцы получали в высокотемпературной микроволновой муфельной печи (1,5 кВт, 2,45 ГГц) при температурах 800, 900, 1000 и 1100 °С. Установлено, что повышение температуры при микроволновом спекании не приводит к стремительному росту размера зерна и образованию агломератов. Полученные материалы имеют равномерную мелкозернистую структуру со средним размером зерен 0,42-0,56 мкм и пор ~ 0,5 мкм. Установлено, что пористость материалов, полученных в интервале температур 800-1000 °С, составляет ~ 40 %, а при 1100 °С – уменьшается до 33 %. Прочность на сжатие возрастает от 31 до 59 МПа с повышением температуры и является близкой к прочности губчатой кости человека. Исследования in vіtro показали, что растворимость материалов в физиологическом растворе не зависит от температуры спекания и составляет 0,1-0,15 мас. %/сутки. Таким образом, методом микроволнового спекания при 800-1100 °С можно получать гидроксиапатитную керамику медицинского назначения с достаточными структурно-механическими свойствами для замещения дефектов костной ткани

Епізоотична ситуація щодо грипу птиці в Україні впродовж 2020–2021 років

Avian influenza viruses (AIVs) are spread globally by wild migratory birds that are reservoirs of AIVs. Epidemics of highly pathogenic avian influenza (HPAI) can devastate the poultry industry and result in severe trade restrictions. Many routes of wild migratory birds pass through the territory of Ukraine, therefore studying the circulation of the viruses is important for the prevention of AIV spreading. The aim of the investigation was to summarize and analyze results of AI tests carried out by state laboratories during 2020–2021. The tests were conducted in the framework of state control of AI aimed to early detection and prevention of HPAI virus spreading on the territory of Ukraine. 27&nbsp;354 samples of biological material from wild and domestic birds (11&nbsp;115 samples – in 2020 and 16&nbsp;239 – in 2021) were tested by PCR in State Scientific and Research Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise and regional state laboratories. For PCR testing commercial kits and reagents were used: IndiSpin Pathogen Kit (INDICAL BIOSCIENCE), VetMAX™-Gold AIV Detection Kit (Thermo Fisher Scientific), AIV H5-H7 REAL TIME (ADIAVET), AgPath-ID™ One-Step RT-PCR Reagents (Ambion) with N8 RT-PCR primers and probe. In 2020, 84 positive samples on AI type A subtype H5N8 from poultry of Vinnytsia, Kherson, Kyiv and Mykolaiv oblasts was detected. In 2021, the presence of AIV RNA subtype H5N8 and H5N1 was detected in 87 samples (72 samples from poultry, 11 from wild birds and 4 from zoo birds) of Mykolaiv, Kyiv, Donetsk, Ternopil and Kherson oblasts. In 2020, 9 outbreaks of AI were registered in Vinnytsia (1), Mykolaiv(5), Kyiv (2) and Kherson (1) oblasts; in 2021, 11 outbreaks were registered in Mykolaiv (4), Kyiv (2), Donetsk&nbsp;(2), Ternopil (1) and Kherson (2) oblasts. In general, during 2020–2021, 20 outbreaks of AI subtype H5 (3 – commercial poultry farms, 13 – from backyards, 3 – in wild birds and 1 – in zoo birds) were registered in 6 oblasts of Ukraine. RNA of AIV in the samples from imported birds was not detected. Wild migratory birds are the most likely source of AIV in Ukraine since most outbreaks were registered in oblasts located at the crossing of wild bird migratory routes.Віруси грипу питці (ГП) поширюються по всьому світу дикими перелітними птахами, які є їх резервуарами. Епізоотії високопатогенного грипу птиці (ВПГП) можуть завдавати значної шкоди птахівництву і призводити до торговельних обмежень. Через територію України проходить багато міграційних шляхів дикої перелітної птиці, тому вивчення циркуляції збудника ГП є важливим для запобігання поширенню ВПГП. Метою дослідження було вивчення епізоотологічної ситуації щодо ГП на території України на основі результатів лабораторних досліджень патологічного/біологічного матеріалів від птиці методом ПЛР, отриманих впродовж 2020–2021 рр. Випробування проводилися в рамках державного контролю ГП з метою раннього виявлення та запобігання поширенню вірусу ВПГП на території України. 27 354 зразки біологічного матеріалу від диких та домашніх птахів (11 115 зразків – у 2020 році та 16 239 – у 2021 році) було досліджено методом ПЛР у Державному науково-дослідному інституті лабораторної діагностики та ветеринарно-санітарної експертизи (ДНДІЛДВСЕ) і обласних державних лабораторіях. У 2020 році було виявлено 84 позитивних щодо грипу типу А субтипу H5N8 зразки від свійської птиці Вінницької, Херсонської, Київської та Миколаївської областей. У 2021 році наявність РНК вірусу грипу типу А субтипів H5N8 та H5N1 виявлено у 87 зразках (72 зразки від свійської птиці, 11 – від дикої птиці та 4 – від зоопаркової птиці) Миколаївської, Київської, Донецької, Тернопільської та Херсонської областей. У 2020 році було зареєстровано 9 спалахів ВПГП у Вінницькій (1), Миколаївській(5), Київській (2) та Херсонській (1) областях; у 2021 році зареєстровано 11 спалахів у Миколаївській (4), Київській (2), Донецькій (2), Тернопільській (1) та Херсонській (2) областях. Загалом впродовж 2020–2021 рр. у 6 областях України зареєстровано 20 спалахів грипу типу А субтипу Н5 (3 – на птахофабриках, 13 – в приватному секторі, 3 – серед дикої птиці та 1 – у зоопаркової птиці). РНК вірусу ГП у зразках від імпортованої птиці не було виявлено. Дикі перелітні птахи є найбільш вірогідним джерелом занесення і розповсюдження вірусу ГП на території України, оскільки більшість спалахів зареєстровано в областях, розташованих на перетині найбільших міграційних шляхів дикої птиці

Inherited Glutathione Reductase Deficiency and Plasmodium falciparum Malaria—A Case Study

In Plasmodium falciparum-infected red blood cells (RBCs), the flavoenzyme glutathione reductase (GR) regenerates reduced glutathione, which is essential for antioxidant defense. GR utilizes NADPH produced in the pentose phosphate shunt by glucose-6-phosphate dehydrogenase (G6PD). Thus, conditions affecting host G6PD or GR induce increased sensitivity to oxidants. Hereditary G6PD deficiency is frequent in malaria endemic areas and provides protection against severe malaria. Furthermore, GR deficiency resulting from insufficient saturation of the enzyme with its prosthetic group FAD is common. Based on these naturally occurring phenomena, GR of malaria parasites and their host cells represent attractive antimalarial drug targets. Recently we were given the opportunity to examine invasion, growth, and drug sensitivity of three P. falciparum strains (3D7, K1, and Palo Alto) in the RBCs from three homozygous individuals with total GR deficiency resulting from mutations in the apoprotein. Invasion or growth in the GR-deficient RBCs was not impaired for any of the parasite strains tested. Drug sensitivity to chloroquine, artemisinin, and methylene blue was comparable to parasites grown in GR-sufficient RBCs and sensitivity towards paraquat and sodium nitroprusside was only slightly enhanced. In contrast, membrane deposition of hemichromes as well as the opsonizing complement C3b fragments and phagocytosis were strongly increased in ring-infected RBCs of the GR-deficient individuals compared to ring-infected normal RBCs. Also, in one of the individuals, membrane-bound autologous IgGs were significantly enhanced. Thus, based on our in vitro data, GR deficiency and drug-induced GR inhibition may protect from malaria by inducing enhanced ring stage phagocytosis rather than by impairing parasite growth directly

Patterns in random walks and Brownian motion

We ask if it is possible to find some particular continuous paths of unit length in linear Brownian motion. Beginning with a discrete version of the problem, we derive the asymptotics of the expected waiting time for several interesting patterns. These suggest corresponding results on the existence/non-existence of continuous paths embedded in Brownian motion. With further effort we are able to prove some of these existence and non-existence results by various stochastic analysis arguments. A list of open problems is presented.Comment: 31 pages, 4 figures. This paper is published at http://link.springer.com/chapter/10.1007/978-3-319-18585-9_

Overview: Recent advances in the understanding of the northern Eurasian environments and of the urban air quality in China – a Pan-Eurasian Experiment (PEEX) programme perspective

The Pan-Eurasian Experiment (PEEX) Science Plan, released in 2015, addressed a need for a holistic system understanding and outlined the most urgent research needs for the rapidly changing Arctic-boreal region. Air quality in China, together with the long-range transport of atmospheric pollutants, was also indicated as one of the most crucial topics of the research agenda. These two geographical regions, the northern Eurasian Arctic-boreal region and China, especially the megacities in China, were identified as a “PEEX region”. It is also important to recognize that the PEEX geographical region is an area where science-based policy actions would have significant impacts on the global climate. This paper summarizes results obtained during the last 5 years in the northern Eurasian region, together with recent observations of the air quality in the urban environments in China, in the context of the PEEX programme. The main regions of interest are the Russian Arctic, northern Eurasian boreal forests (Siberia) and peatlands, and the megacities in China. We frame our analysis against research themes introduced in the PEEX Science Plan in 2015. We summarize recent progress towards an enhanced holistic understanding of the land–atmosphere–ocean systems feedbacks. We conclude that although the scientific knowledge in these regions has increased, the new results are in many cases insufficient, and there are still gaps in our understanding of large-scale climate–Earth surface interactions and feedbacks. This arises from limitations in research infrastructures, especially the lack of coordinated, continuous and comprehensive in situ observations of the study region as well as integrative data analyses, hindering a comprehensive system analysis. The fast-changing environment and ecosystem changes driven by climate change, socio-economic activities like the China Silk Road Initiative, and the global trends like urbanization further complicate such analyses. We recognize new topics with an increasing importance in the near future, especially “the enhancing biological sequestration capacity of greenhouse gases into forests and soils to mitigate climate change” and the “socio-economic development to tackle air quality issues”

Pan-Eurasian Experiment (PEEX): Towards a holistic understanding of the feedbacks and interactions in the land-Atmosphere-ocean-society continuum in the northern Eurasian region

The northern Eurasian regions and Arctic Ocean will very likely undergo substantial changes during the next decades. The Arctic-boreal natural environments play a crucial role in the global climate via albedo change, carbon sources and sinks as well as atmospheric aerosol production from biogenic volatile organic compounds. Furthermore, it is expected that global trade activities, demographic movement, and use of natural resources will be increasing in the Arctic regions. There is a need for a novel research approach, which not only identifies and tackles the relevant multi-disciplinary research questions, but also is able to make a holistic system analysis of the expected feedbacks. In this paper, we introduce the research agenda of the Pan-Eurasian Experiment (PEEX), a multi-scale, multi-disciplinary and international program started in 2012 (https://www.atm.helsinki.fi/peex/). PEEX sets a research approach by which large-scale research topics are investigated from a system perspective and which aims to fill the key gaps in our understanding of the feedbacks and interactions between the land-Atmosphere-Aquatic-society continuum in the northern Eurasian region. We introduce here the state of the art for the key topics in the PEEX research agenda and present the future prospects of the research, which we see relevant in this context