Au(I) Catalyzed HF Transfer: Tandem Alkyne Hydrofluorination and Perfluoroarene Functionalisation

HF transfer reactions between organic substrates are an incredibly rare class of transformation. Such reactions require the development of new catalytic systems that can promote both defluorination and fluorination steps in a single reaction sequence. Herein, we report a novel catalytic protocol in which an equivalent of HF is generated from a perfluoroarene | nucleophile pair and transferred directly to an alkyne. The reaction is catalysed by [Au(IPr)NiPr2] (IPr = N,N’-1,3-Bis(2,6-diisopropylphenyl)imidazol-2-ylidene) and is 100 % atom efficient. HF transfer generates two useful products in the form of functionalised fluoroarenes and fluoroalkenes. Mechanistic studies (rate laws, KIEs, DFT calculations, competition experiments) are consistent with the Au(I) catalyst facilitating a catalytic network involving both concerted SNAr and hydrofluorination steps. The nature of the nucleophile impacts the turnover-limiting step. The cSNAr step is turnover-limiting for phenol-based nucleophiles while proteodeauration likely becomes turnover-limiting for aniline-based nucleophiles. The new approach removes the need for direct handling of HF reagents in hydrofluorination and offers new possibilities to manipulate the fluorine content of organic molecules through catalysis.

Immune Repertoire Analysis of Multiple Myeloma Research Samples Using NGS Characterization of Multiple B Cell Receptors in a Single Reaction

Introduction B cell repertoire analysis by next-generation sequencing (NGS) is at the forefront of leukemia and lymphoma research. Some advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to other methods. Importantly, in research of post-germinal B cell disorders, such as multiple myeloma (MM), NGS methods allow for the study of clonal lineage based on somatic hypermuation patterns. Current targeted NGS assays require multiple libraries to survey each B cell receptor chain (IGH, IgK, IgL), and this fact is highlighted when initial clonality detection fails due to mutations under primer binding sites. This issue can be especially true in MM which has a high rate of SHM. To address these issues, we have developed an assay for B cell analysis, based on Ion AmpliSeq™ technology, which enables efficient detection of IGH, IgK, and IgL chain rearrangements in a single reaction. Methods The B cell pan-clonality panel (Oncomine™ BCR Pan-Clonality Assay) targets the framework 3 (FR3) portion of the variable gene and the joining gene region of heavy- and light-chain loci (IGH, IgK, IgL) for all alleles found within the IMGT database, enabling readout of the complementary-determining region 3 (CDR3) sequence of each immunoglobulin chain. To maximize sensitivity, we included primers to amplify IgK loci rearrangements involving Kappa deletion element and the constant region intron. To evaluate assay performance, we conducted reproducibility studies and clonality assessment using gDNA from a total of 45 MM research samples. All MM cases examined in this work were confirmed clonal previously by light chain restriction via flow cytometry or IHC/ISH in tissue sections - 16 of the 45 MM samples were identified as lambda light chain restricted. For comparison, a small cohort of 12 B-ALL samples were also included in the study. Sequencing and repertoire analyses were performed using the Ion GeneStudio S5 System and Ion Reporter 5.16 analysis software. Results Clonality assessment of MM clinical research samples show an 93% overall positive detection rate by an assay which combines the IGH, IgK, and IgL chains in a single reaction using published guidelines for clonality assignment. Thirty-four of 45 samples show positive detection of an IGH rearrangement, while 41 of 45 showed positive detection of at least one light chain receptor. In total, 42 of 45 samples were deemed clonal by the single tube assay based on detection for one or more receptor. Clonality results for this sample set are well correlated with orthogonal data from flow, IHC/ISH, or alternate NGS assays. A clonal lambda light chain was identified in 14 of 16 samples determined to be lambda restricted by flow cytometry. In two of the lambda restricted samples only a clonal lambda rearrangement was identified, showing the benefit of including primers targeting both the kappa and lambda light chains in a pan-clonality NGS assay. Both the MM and B-ALL cohorts were evaluated for biased IGHV gene usage. IGHV3-11 was observed in 5 of 45 MM and 5 of 12 B-ALL samples. IGHV4-34, typically linked to autoreactive antibodies and underrepresented in germinal center and memory B-cells, was nonetheless found in 5 of 45 MM samples surveyed. Estimates of somatic hypermutation rates were calculated using the BCR pan-clonality assay. Most MM samples, as expected, contained some somatic hypermutation with 6 of 45 samples showing greater than 10% mutation rates. Automated lineage analysis, based on somatic hypermuation signatures within each sample, identified 8 of 45 MM samples which contained 5 or more clones in the primary clonal lineage, with one case containing a lineage with 23 clones. Two MM samples showed no somatic hypermutation as measured using the FR3 primers contained in the BCR pan-clonality assay. These samples were also evaluated using an FR1-J targeted NGS assay, which confirmed relatively low mutation rates for these MM samples at 0.44% and 1.3%, respectively. Conclusions These results demonstrate the utility of a novel assay for combined repertoire analysis of B cell receptor heavy and light chains in a single library preparation reaction. We expect this assay to simplify laboratory workflows and including analysis tools such as automated somatic hypermutation rate calculation and clonal lineage identification may open new paths for research in lymphoid cell disorders. For research use only. Disclosures Lowman: Thermo Fisher Scientific: Current Employment. Toro: Thermo Fisher Scientific: Current Employment. Pickle: Thermo Fisher Scientific: Current Employment. Ostresh: Thermo Fisher Scientific: Current Employment. Sarda: Thermo Fisher Scientific: Current Employment. Yang: Thermo Fisher Scientific: Current Employment.

DIAGNOSTICS OF THE DYNAMICS OF THE BASIC CONCEPTIN THE GERMAN LINGUISTIC CULTURE (BASED ON THE BODY OF ORAL TEXTS AND DATA OF THE ASSOCIATIVE EXPERIMENT)

Постановка задачи. Основной задачей исследования является установление динамики содержания базисного концепта Sicherheit (безопасность) в немецкой лингвокультуре. В качестве методов исследования авторы обращаются к анализу дефиниций толковых словарей, анализу сочетаемости исследуемого слова, контекстному анализу, свободному ассоциативному эксперименту. На основе разработанной модели значения авторы сопоставляют лексикографические данные и ассоциативное поле слова-стимула Sicherheit (безопасность) . Результаты. На основе сопоставления полученных данных авторы отмечают, что интегративные признаки, выделенные в сочетаемости лексем и признаки, выделенные в ассоциативном эксперименте, в значительной мере совпадают (‘источники безопасности’, ‘безопасная ситуация в мире / национальная безопасность’, ‘комфорт, стабильность’, ‘личные эмоциональные концепты’, ‘объект / предмет защиты’), хотя и различны в количественном значении. При этом сочетаемость лексемы позволяет говорить о недоверии к возможности стабильной и безопасной ситуации в целом. Однако данные свободного ассоциативного эксперимента, напротив, свидетельствуют об исключительно положительном отношении к исследуемому концепту. Согласно данным корпуса наиболее распространенной категорией является категория уверенность , которая представлена в ассоциативном эксперименте единичной реакцией. Наибольшее количество реакций ассоциативного эксперимента представляют Sicherheit как чувство защищенности от разного вида опасности. Выводы. Для носителей немецкой лингвокультуры концепт Sicherheit (безопасность) понимается как чувство защищенности от опасности, которое могут гарантировать индивиду семья или государственные органы. Гарантом безопасности выступают деньги. Для достижения безопасности необходимо соблюдение различных правил и мер безопасности. Statement of the problem. The aim of the article is to establish the dynamics of the content of the basic concept Sicherheit ( safety ) in German linguistic culture. As the main research methods, the authors turn to the analysis of definitions of explanatory dictionaries, the analysis of the compatibility of the studied word, context analysis, free associative experiment. On the basis of the developed model of meaning, the authors compare the lexicographic data and the associative field of the stimulus word Sicherheit ( safety ). Results. Based on the comparison of the obtained data, the authors note that the integrative features identified in the compatibility of lexemes and the features identified in the associative experiment largely coincide (‘sources of security’, ‘safe situation in the world / national security’, ‘comfort, stability’, ‘personal emotional concepts’, ‘object of protection’, although they are different in quantitative meaning. At the same time, we note that the compatibility of the lexeme allows us to speak of mistrust of the possibility of a stable and safe situation as a whole. However, the data of the free associative experiment, on the contrary, allow us to speak about an extremely positive attitude to the concept under study. According to the corpus, the most common category is the category of confidence, which is represented in the associative experiment by a single reaction. The largest number of reactions in the associative experiment represent Sicherheit as a feeling of protection from various types of danger. Conclusion. For representatives of the German linguistic culture, the concept of Sicherheit ( safety ) is understood as a sense of security from danger, which can be guaranteed to an individual by the family or government authorities. Money is the guarantor of security. To achieve safety, it is necessary to comply with various safety rules and measures.

Development of a novel SNP assay to detect lactase persistence associated genetic variants

Background In adulthood the activity of the lactase enzyme is inherited as autosomal dominant form associated to Single nucleotide polymorphisms (SNPs). The present research was aimed to develop a novel genetic method to test lactase non persistence more powerfully. Methods and results In our study, we selected eight different SNPs that are associated with lactase persistence from Caucasian, Arabian Bedouins, sub-Saharian Africans and Asian populations to set up an approach to detect all the eight different SNPs at the same time in the same sample. This technique is centred on the identification of SNPs with a single nucleotide primer extension method using Sanger sequencing and capillary electrophoresis. Conclusions Our method allowed us to check the genotype asset of eight SNPs related to lactase persistence simultaneously and in a very efficient manner. It could be applied to a higher number of SNPs in a single reaction.

Direct Detection of Streptococcus suis from Cerebrospinal Fluid, Positive Hemoculture, and Simultaneous Differentiation of Serotypes 1, 1/2, 2, and 14 within Single Reaction

Streptococcus suis is an emerging zoonotic bacterium causing septicemia and meningitis in humans. Due to rapid disease progression, high mortality rate, and many underdiagnosed cases by time-consuming routine identification methods, alternative diagnostic testing is essential. Among 29 broadly accepted S. suis serotypes, serotypes 2 and 14 are high prevalent; however, many PCR assays showed an inability to differentiate serotype 2 from 1/2, and 1 from 14. In this study, we developed and validated a new multiplex PCR assay that facilitates the identification of only the 29 true serotypes of S. suis and simultaneously differentiates serotypes 1, 1/2, 2, and 14 within a single reaction. Importantly, the multiplex PCR could detect S. suis directly from positive hemocultures and CSF. The results revealed high sensitivity, specificity, and 100% accuracy with almost perfect agreement (κ = 1.0) compared to culture and serotyping methods. Direct detection enables a decrease in overall diagnosis time, rapid and efficient treatment, reduced fatality rates, and proficient disease control. This multiplex PCR offers a rapid, easy, and cost-effective method that can be applied in a routine laboratory. Furthermore, it is promising for developing point-of-care testing (POCT) for S. suis detection in the future.

A New Method for the Synthesis of 2-Substituted Benzoxazoles from 2-Nitrophenol Derivatives and Aldehydes

Benzoxazole derivatives are one of the compounds with many interesting biological activities. Conventional methods are often performed under complex conditions using strong acids, expensive metal catalysts, requiring high pressure, high temperature, and under microwave irradiation. In this study, we reported a new method of benzoxazole synthesis with redox catalyst using FeCl3.6H2O and sulfur. This is a suitable, efficient, readily available and environmentally friendly catalyst system for redox and condensation reactions in one step at 100 oC. Applying this new method, we have synthesized eight 2-arylbenzoxazole derivatives with high yields (calculated according to 2-nitrophenol). This research is an important step forward in the synthesis of biologically active compounds containing the benzoxazole framework from readily available starting materials in a single reaction.

Expression of Foxp3 by T follicular helper cells in end-stage germinal centers

Germinal centers (GCs) are the site of immunoglobulin somatic hypermutation and affinity maturation, processes essential to an effective antibody response. The formation of GCs has been studied in detail, but less is known about what leads to their regression and eventual termination, factors that ultimately limit the extent to which antibodies mature within a single reaction. We show that contraction of immunization-induced GCs is immediately preceded by an acute surge in GC-resident Foxp3+ T cells, attributed at least partly to up-regulation of the transcription factor Foxp3 by T follicular helper (TFH) cells. Ectopic expression of Foxp3 in TFH cells is sufficient to decrease GC size, implicating the natural up-regulation of Foxp3 by TFH cells as a potential regulator of GC lifetimes.

Estimating Real-Time qPCR Amplification Efficiency from Single-Reaction Data

Methods for estimating the qPCR amplification efficiency E from data for single reactions are tested on six multireplicate datasets, with emphasis on their performance as a function of the range of cycles n1–n2 included in the analysis. The two-parameter exponential growth (EG) model that has been relied upon almost exclusively does not allow for the decline of E(n) with increasing cycle number n through the growth region and accordingly gives low-biased estimates. Further, the standard procedure of “baselining”—separately estimating and subtracting a baseline before analysis—leads to reduced precision. The three-parameter logistic model (LRE) does allow for such decline and includes a parameter E0 that represents E through the baseline region. Several four-parameter extensions of this model that accommodate some asymmetry in the growth profiles but still retain the significance of E0 are tested against the LRE and EG models. The recursion method of Carr and Moore also describes a declining E(n) but tacitly assumes E0 = 2 in the baseline region. Two modifications that permit varying E0 are tested, as well as a recursion method that directly fits E(n) to a sigmoidal function. All but the last of these can give E0 estimates that agree fairly well with calibration-based estimates but perform best when the calculations are extended to only about one cycle below the first-derivative maximum (FDM). The LRE model performs as well as any of the four-parameter forms and is easier to use. Its proper implementation requires fitting to it plus a suitable baseline function, which typically requires four–six adjustable parameters in a nonlinear least-squares fit.