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

Prevalence and Molecular Epidemiology of CTX-M Extended-Spectrum β-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae in Russian Hospitals

A total of 904 consecutive nosocomial isolates of Escherichia coli and Klebsiella pneumoniae collected from 28 Russian hospitals were screened for production of extended-spectrum β-lactamases (ESBLs). The ESBL phenotype was detected in 78 (15.8%) E. coli and 248 (60.8%) K. pneumoniae isolates. One hundred fifteen isolates carried the genes for CTX-M-type β-lactamases, which, as shown by PCR-restriction fragment length polymorphism analysis, were distributed into the two genetic groups of CTX-M-1 (93%)- and CTX-M-2 (7%)-related enzymes. Isolates producing the enzymes of the first group were found in 20 hospitals from geographically distant regions of the country and were characterized by considerable diversity of genetic types, as was demonstrated by enterobacterial repetitive consensus PCR typing. Within this group the CTX-M-3 and the CTX-M-15 β-lactamases were identified. In contrast, the enzymes of the CTX-M-2 group (namely, CTX-M-5) were detected only in eight clonally related E. coli isolates from a single hospital. Notably, the levels of resistance to ceftazidime were remarkably variable among the CTX-M producers. This study provides further evidence of the global dissemination of CTX-M type ESBLs and emphasizes the need for their epidemiological monitoring

FISH-Flow to quantify nascent and mature ribosomal RNA in mouse and human cells

Summary: FISH-Flow (fluorescence in situ hybridization-flow cytometry) involves hybridizing fluorescent oligos to RNA and quantifying fluorescence at a single-cell level using flow cytometry. Here, we present a FISH-Flow protocol to quantify nascent 47S and mature 18S and 28S rRNAs in mouse and human cells, including rRNA quantification across cell cycle stages using DNA staining. We describe steps for cell preparation, hybridization of fluorescent probes against rRNA, and DNA staining. We then detail procedures for flow cytometry and data analysis.For complete details on the use and execution of this protocol, please refer to Antony et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Multiple Outbreaks of Nosocomial Salmonellosis in Russia and Belarus Caused by a Single Clone of Salmonella enterica Serovar Typhimurium Producing an Extended-Spectrum β-Lactamase

Thirty-four cefotaxime-resistant Salmonella enterica serovar Typhimurium isolates representative of the isolates that caused outbreaks of gastroenteritis in 10 hospitals in seven regions of Russia and Belarus from 1994 to 2003 were analyzed. All isolates produced the CTX-M-5-like extended-spectrum β-lactamase, which confers high-level resistance to cefotaxime and ceftriaxone and decreased susceptibility to ceftazidime. The bla(CTX-M) genes were located on small (7.4- to 12-kb) non-self-transferable plasmids approximately 20 bp downstream of the ISEcp1 insertion sequences. Some isolates carried additional conjugative plasmids mediating resistance to penicillin-inhibitor combinations and various non-β-lactam agents, including tetracycline, chloramphenicol, gentamicin, tobramycin, and co-trimoxazole. Despite the minor differences in susceptibility patterns, all isolates were considered clonally related on the basis of arbitrarily primed PCR and pulsed-field gel electrophoresis analysis. The similarities of the restriction profiles of the CTX-M-coding plasmids further supported the clonal origin of these isolates

Two classes of bacterial IMPDHs according to their quaternary structures and catalytic properties.

Inosine-5'-monophosphate dehydrogenase (IMPDH) occupies a key position in purine nucleotide metabolism. In this study, we have performed the biochemical and physico-chemical characterization of eight bacterial IMPDHs, among which six were totally unexplored. This study led to a classification of bacterial IMPDHs according to the regulation of their catalytic properties and their quaternary structures. Class I IMPDHs are cooperative enzymes for IMP, which are activated by MgATP and are octameric in all tested conditions. On the other hand, class II IMPDHs behave as Michaelis-Menten enzymes for both substrates and are tetramers in their apo state or in the presence of IMP, which are shifted to octamers in the presence of NAD or MgATP. Our work provides new insights into the IMPDH functional regulation and a model for the quaternary structure modulation is proposed