Recombinant nucleases CEL I from celery and SP I from spinach for mutation detection

<p>Abstract</p> <p>Background</p> <p>The detection of unknown mutations is important in research and medicine. For this purpose, a mismatch-specific endonuclease CEL I from celery has been established as a useful tool in high throughput projects. Previously, CEL I-like activities were described only in a variety of plants and could not be expressed in an active form in bacteria.</p> <p>Results</p> <p>We describe expression of active recombinant plant mismatch endonucleases and modification of their activities. We also report the cloning of a CEL I ortholog from <it>Spinacia oleracea </it>(spinach) which we termed SP I nuclease. Active CEL I and SP I nucleases were expressed as C-terminal hexahistidine fusions and affinity purified from the cell culture media. Both recombinant enzymes were active in mutation detection in <it>BRCA1 </it>gene of patient-derived DNA. Native SP nuclease purified from spinach is unable to incise at single-nucleotide substitutions and loops containing a guanine nucleotide, but the recombinant SP I nuclease can cut at these sites.</p> <p>Conclusion</p> <p>The insect cell-expressed CEL I orthologs may not be identical to their native counterparts purified from plant tissues. The present expression system should facilitate further development of CEL I-based mutation detection technologies.</p

Repercussion of megakaryocyte-specific Gata1 Loss on megakaryopoiesis and the hematopoietic precursor compartment

During hematopoiesis, transcriptional programs are essential for the commitment and differentiation of progenitors into the different blood lineages. GATA1 is a transcription factor expressed in several hematopoietic lineages and essential for proper erythropoiesis and megakaryopoiesis. Megakaryocyte-specific genes, such as GP1BA, are known to be directly regulated by GATA1. Mutations in GATA1 can lead to dyserythropoietic anemia and pseudo gray-platelet syndrome. Selective loss of Gata1 expression in adult mice results in macrothrombocytopenia with platelet dysfunction, characterized by an excess of immature megakaryocytes. To specifically analyze the impact of Gata1 loss in mature committed megakaryocytes, we generated Gata1-Lox|Pf4-Cre mice (Gata1cKOMK). Consistent with previous findings, Gata1cKOMK mice are macrothrombocytopenic with platelet dysfunction. Supporting this notion we demonstrate that Gata1 regulates directly the transcription of Syk, a tyrosine kinase that functions downstream of Clec2 and GPVI receptors in megakaryocytes and platelets. Furthermore, we show that Gata1cKOMK mice display an additional aberrant megakaryocyte differentiation stage. Interestingly, these mice present a misbalance of the multipotent progenitor compartment and the erythroid lineage, which translates into compensatory stress erythropoiesis and splenomegaly. Despite the severe thrombocytopenia, Gata1cKOMK mice display a mild reduction of TPO plasma levels, and Gata1cK-OMK megakaryocytes show a mild increase in Pf4 mRNA levels; such a misbalance might be behind the general hematopoietic defects observed, affecting locally normal TPO and Pf4 levels at hematopoietic stem cell niches. © 2016 Meinders et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Identification of Novel Mt-Guab2 Inhibitor Series Active against M. tuberculosis

Tuberculosis (TB) remains a leading cause of mortality worldwide. With the emergence of multidrug resistant TB, extensively drug resistant TB and HIV-associated TB it is imperative that new drug targets be identified. The potential of Mycobacterium tuberculosis inosine monophosphate dehydrogenase (IMPDH) as a novel drug target was explored in the present study. IMPDH exclusively catalyzes the conversion of inosine monophosphate (IMP) to xanthosine monophosphate (XMP) in the presence of the cofactor nicotinamide adenine dinucleotide (NAD+). Although the enzyme is a dehydrogenase, the enzyme does not catalyze the reverse reaction i.e. the conversion of XMP to IMP. Unlike other bacteria, M. tuberculosis harbors three IMPDH-like genes, designated as Mt-guaB1, Mt-guaB2 and Mt-guaB3 respectively. Of the three putative IMPDH's, we previously confirmed that Mt-GuaB2 was the only functional ortholog by characterizing the enzyme kinetically. Using an in silico approach based on designed scaffolds, a series of novel classes of inhibitors was identified. The inhibitors possess good activity against M. tuberculosis with MIC values in the range of 0.4 to 11.4 µg mL−1. Among the identified ligands, two inhibitors have nanomolar Kis against the Mt-GuaB2 enzyme

ВАЛИДАЦИЯ ДИАГНОСТИЧЕСКОЙ ТОЧНОСТИ АЛГОРИТМА «ИСКУССТВЕННОГО ИНТЕЛЛЕКТА» ДЛЯ ВЫЯВЛЕНИЯ РАССЕЯННОГО СКЛЕРОЗА В УСЛОВИЯХ ГОРОДСКОЙ ПОЛИКЛИНИКИ

The objective of the study is to evaluate the diagnostic accuracy of an original artificial intelligence (AI) algorithm for detecting MS in the radiology department of primary (outpatient) hospital.Materials and methods. Depersonalized results of brain magnetic resonance imaging (MRI) studies performed in the period from August 22, 2019 to September 26, 2019 in 93 patients (42 men (mean age 47,5±15,9 years) and 51 women (mean age 52,3±16,8 years)) were analyzed. All patients signed a voluntary informed consent form. Brain MRIwere carried out on the VANTAGE Atlas 1,5T MRI scanner (Toshiba, Japan) under a standard protocol.Results. All MRI studies were analyzed by AI-algorithm (index-test). It decisions were compared with a  reference test (groundtruth). The sensitivity of the index-test is 100%, specificity — 75,3%, accuracy —  76,3%, negative predictive value — 100%, area under ROC-curve — 0,861. The algorithm reliably sorts out the studies without signs of MS. The algorithmshows sufficient quality and excellent reproducibility of the results on independent data.Conclusion. The developed AI algorithm ensures effective triage of MRI studies in primary care settings, maintaining an optimal index of suspicion in MS.Цель: оценить диагностическую точность оригинального алгоритма выявления РС в условиях отделения лучевой диагностики медицинской организации, оказывающей первичную (амбулаторно-поликлиническую) медицинскую помощь.Материалы и методы. Проведен анализ деперсонализированных результатов МР-исследований головного мозга, выполненных 93 пациентам в период с 22.08.2019 г. по 26.09.2019 г., из которых 42 мужчины (средний возраст 47,5±15,9 лет) и 51 женщина (средний возраст 52,3±16,8 лет); лица европеоидной расы, жители г. Москвы. Все  пациенты подписали добровольное информированное согласие. Исследования  проводились на томографе VANTAGE Atlas (Toshiba, Япония) с индукцией магнитного поля 1,5 Тл по стандартному протоколу.Результаты. Все МР-исследования проанализированы с применением оригинального  алгоритма «искусственного интеллекта» (ИИ). Решения алгоритма (индекс-теста)  сопоставлены с референс-тестом, значения которого приняты за истинный статус  обследуемых лиц. Чувствительность индекс-теста — 100%, специфичность — 75,3%,  точность — 76,3%, прогностическая ценность отрицательного результата — 100%, площадь под характеристической кривой — 0,861. Результаты свидетельствуют о надежном «отсеивании» алгоритмом результатов исследований без признаков РС.  Показано достаточное качество и отличная воспроизводимость результатов работы  алгоритма на независимых данных.Заключение. Разработанный алгоритм ИИ обеспечивает эффективную сортировку МР-исследований в условиях первичного звена здравоохранения с поддержанием оптимального уровня настороженности относительно РС