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Мета: За існуючими літературними даними провести порівняльний аналіз впливу режимів перемішування на життєздатність та продуктивність міцеліальних грибів при глибинному культивуванні. А також підібрати режим культивування при якому вихід кінцевого продукту буде максимальним. Методи: У розглянутих статтях проводили дослідження режимів перемішування в лабораторних ферментерах з механічними перемішуючими пристроями. Досліди проводили для обраного типу мішалки, змінюючи частоту обертання перемішуючого пристрою в апараті. Результати: З проведеного аналізу видно, що важливу роль при глибинному культивуванні відіграє інтенсивність механічного перемішування. В середньому, кількість обертів мішалки становить 120- 180 об/хв.

AML is a stem cell disease wherein the properties of the disease-driving leukemia stem cells (LSCs) are reflected in the cellular hierarchies that they generate. We sought to understand how these hierarchies vary across patients and whether their characteristics are clinically relevant. To evaluate cellular hierarchies in AML, we re-analyzed the scRNA-seq data of 13,653 cells from 12 AML patients at diagnosis (van Galen, Cell 2019) and in particular the stem and progenitor blast populations. We identified three novel leukemia stem populations differing in their depth of quiescence, inflammatory signaling, and myeloid priming. Using the signatures of 7 malignant populations as well as 7 immune cell populations from this scRNA-seq data, we applied CIBERSORTx deconvolution (Newman, Nat Biotechnol 2019) on bulk RNA-seq data from multiple patient cohorts. This enabled determination of the relative abundance of each cell type in each patient, thereby capturing the "shape" of the leukemic hierarchies of hundreds of AML patients. AML patients within these cohorts clustered into 4 groups defined by different proportions of stem, progenitor, and mature blasts - each of which differed in their underlying genomic alterations and overall survival. Differences in chemotherapy response were mediated by specific cell types, notably a GMP-like blast population and a quiescent stem-like population (qLSPC). Dominance of the GMP-like population was associated with longer survival (HR -3.1, p=0.002), and these blasts were enriched among younger patients ( 65 years) and patients with adverse cytogenetic alterations. Critically, this qLSPC population was enriched at relapse as well as within a subset of pediatric AML patients that failed to respond to induction chemotherapy. We confirmed that qLSPCs were also enriched among functionally validated leukemia engrafting (LSC+) sorted AML fractions. Accordingly, a high LSC17 score was strongly correlated with qLSPC abundance and anti-correlated with GMP-like abundance, suggesting that the score may reflect the underlying cellular hierarchy of each AML patient. Next, we generated drug sensitivity profiles for each cell type by correlating ex vivo drug sensitivity data for each of 112 inhibitors screened in the BEAT-AML trial with the relative abundance of each cell type across individual patients. In particular, Venetoclax sensitivity correlated with the abundance of primitive cell types and anti-correlated with mature cell types, suggesting that the composition of the leukemic hierarchy in AML patients is associated with drug response. We previously demonstrated that a high LSC17 score identifies patients who do not benefit from standard chemotherapy (Ng, Nature 2016). To develop a diagnostic tool for therapy selection amongst these poor prognosis patients, we retrained the LSC17 genes against cell type abundance and derived a subscore (LSC-7) to map patients along an axis of primitive vs mature leukemic hierarchies. We show that AML samples with a high LSC-7 score (more stem-like blasts) were more sensitive to Venetoclax whereas AML patients with a low LSC-7 score (more mature blasts) benefited from treatment with Gemtuzumab-Ozogamicin (GO). Used together, the LSC17 and LSC-7 scores enable risk stratification as well as subsequent drug selection for high-risk patients, and can both be measured by a single rapid NanoString assay. To apply this approach more broadly, we identified several published studies with drug screening data from primary patient samples and accompanying RNAseq data. By correlating cell type abundance with drug response, we were able to map the critical cell types that mediate sensitivity and resistance to inhibitors of mitochondrial metabolism, histone demethylation, and the CD47-SIRPa axis, among others. Our data establish that scRNA-seq informed deconvolution of bulk expression data permits characterization of the cellular hierarchy of individual AML patients. This framework can enhance our understanding of many aspects of biological, genomic, and clinical heterogeneity in AML, and represents a powerful tool to enable personalized therapeutic decision-making in AML. Figure Disclosures Wang: Trilium Therapeutics: Patents & Royalties. Dick:Bristol-Myers Squibb/Celgene: Research Funding.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy and is the most common type of acute leukemia in adults. Although a majority of patients achieve remission following cytotoxic chemotherapy, most will relapse and ultimately die. Therapy resistance and relapse are driven by leukemia stem cells (LSC). Evidence of genetic and functional heterogeneity in the LSC compartment underscores the importance of developing therapeutic strategies that will target all subclones effectively. We previously showed that LSCs in AML depend on CD47-SIRPα interaction to evade immune surveillance (Theocharides et al, JEM 2012). CD47 acts as a "do not eat me" signal that binds to the inhibitory receptor SIRPα on macrophages and masks cancer cells from macrophage-mediated phagocytosis. TTI-621 (Trillium Therapeutics Inc., Ontario, Canada) is a human SIRPαFc protein formed by fusing the IgV doman of human SIRPα to a human IgG1-Fc moiety; it is designed to bind CD47 on leukemia cells and disrupt its interaction with SIRPα on host macrophages. Our previous studies in AML cell lines and a small number of primary AML samples demonstrated increased phagocytosis in vitro and decreased engraftment in xenotransplant models following SIRPαFc treatment (Theocharides et al, JEM 2012, Petrova et al, Clin Cancer Res 2017). Here, we tested the efficacy of TTI-621 against a broad panel of primary AML samples in xenotransplantation models to determine efficacy and response rates in this heterogeneous disease. Bulk cells obtained from the peripheral blood of 30 AML patients representing a broad range of cytogenetic and molecular subtypes were transplanted intrafemorally into sublethally-irradiated NSG mice. After a 2-week engraftment period, mice were treated with either SIRPαFc or control IgG by intraperitoneal injection 3×/week for 4 weeks, following which leukemic engraftment was determined by flow cytometry. In all but 1 sample, a significant reduction in AML engraftment was seen in SIRPαFc-treated mice compared to controls. For 23 samples defined as good responders, SIRPαFc treatment resulted in 91% (range 53-100%, p60, adverse cytogenetic risk, and secondary AML, as well as samples obtained from relapsed/resistant patients, were classified as good responders. Notably, 20 of 23 good responders had a high LSC17 score, which we have shown is associated with poor initial therapy response and short survival following standard treatments (Ng et al, Nature 2016). To determine whether SIRPαFc treatment killed LSCs, we transplanted leukemia cells harvested from primary treated mice into untreated secondary recipients at limiting dilution. For four independent samples, including three partial responders and the one non-responder, we observed a significantly lower LSC frequency (3.9-10.3 fold, p=0.002-0.024) in mice transplanted with SIRPαFc-treated cells compared to controls, indicating that SIRPαFc treatment reduced LSC numbers in primary mice, despite partial or no reduction of bulk disease. Our data demonstrate that SIRPαFc effectively targets LSCs in a human AML xenotransplantion model with high response rates across a heterogeneous cohort of primary AML samples, including samples with unfavorable risk features. SIRPαFc may be most effective in the remission setting as maintenance therapy for patients with detectable residual disease, to eradicate residual LSCs and prevent relapse. Disclosures Jin: Trillium Therapeutics: Other: licensing agreement. Wong:Trillium Therapetuics: Employment. Uger:Trillium Therapetuics: Employment. Minden:Trillium Therapetuics: Other: licensing agreement. Danska:Trillium Therapeutics: Other: licensing agreement, Research Funding. Wang:Pfizer AG Switzerland: Honoraria, Other: Travel and accommodation; Pfizer International: Honoraria, Other: Travel and accommodation; Trilium therapeutics: Other: licensing agreement, Research Funding; NanoString: Other: Travel and accommodation.

Ribosomal protein (rp)S5 belongs to the family of the highly conserved rp's that contains rpS7 from prokaryotes and rpS5 from eukaryotes. Alignment of rpS5/rpS7 from metazoans (Homo sapiens), fungi (Saccharomyces cerevisiae) and bacteria (Escherichia coli) shows that the proteins contain a conserved central/C-terminal core region and possess variable N-terminal regions. Yeast rpS5 is 69 amino acids (aa) longer than the E. coli rpS7 protein; and human rpS5 is 48 aa longer than the rpS7, respectively. To investigate the function of the yeast rpS5 and in particular the role of its N-terminal region, we obtained and characterized yeast strains in which the wild-type yeast rpS5 was replaced by its truncated variants, lacking 13, 24, 30 and 46 N-terminal amino acids, respectively. All mutant yeast strains were viable and displayed only moderately reduced growth rates, with the exception of the strain lacking 46 N-terminal amino acids, which had a doubling time of about 3 h. Biochemical analysis of the mutant yeast strains suggests that the N-terminal part of the eukaryotic and, in particular, yeast rpS5 may impact the ability of 40S subunits to function properly in translation and affect the efficiency of initiation, specifically the recruitment of initiation factors eIF3 and eIF2.

Ribosomal protein (rp) S5 belongs to a family of ribosomal proteins that includes bacterial rpS7. rpS5 forms part of the exit (E) site on the 40S ribosomal subunit and is essential for yeast viability. Human rpS5 is 67% identical and 79% similar to Saccharomyces cerevisiae rpS5 but lacks a negatively charged (pI ∼3.27) 21 amino acid long N-terminal extension that is present in fungi. Here we report that replacement of yeast rpS5 with its human homolog yielded a viable yeast strain with a 20%–25% decrease in growth rate. This replacement also resulted in a moderate increase in the heavy polyribosomal components in the mutant strain, suggesting either translation elongation or termination defects, and in a reduction in the polyribosomal association of the elongation factors eEF3 and eEF1A. In addition, the mutant strain was characterized by moderate increases in +1 and −1 programmed frameshifting and hyperaccurate recognition of the UAA stop codon. The activities of the cricket paralysis virus (CrPV) IRES and two mammalian cellular IRESs (CAT-1 and SNAT-2) were also increased in the mutant strain. Consistently, the rpS5 replacement led to enhanced direct interaction between the CrPV IRES and the mutant yeast ribosomes. Taken together, these data indicate that rpS5 plays an important role in maintaining the accuracy of translation in eukaryotes and suggest that the negatively charged N-terminal extension of yeast rpS5 might affect the ribosomal recruitment of specific mRNAs.

Expression of transcripts for the homotypic adhesion protein epithelial V-like antigen 1 (EVA1), also known as myelin protein zero like-2 (Mpzl2), is known to be present in thymic stromal cells. However, protein expression within different thymic subsets, stromal and/or lymphoid, has not been characterized due a lack of specific reagents. To address this, we generated a hybridoma (G9P3-1) secreting a monoclonal antibody (G9P3-1Mab), reactive against both human and mouse EVA1. The G9P3-1Mab was generated by immunizing Mpzl2-deficient gene-targeted mice with the extracellular domain of EVA1, followed by a conventional hybridoma fusion protocol, illustrating the feasibility of using gene-targeted mice to generate monoclonal antibodies with multiple species cross-reactivity. We confirmed expression of EVA1 on cortical and medullary epithelial cell subsets and revealed a restricted pattern of expression on CD4- CD8- double negative (DN) cell subsets, with the highest level of expression on DN3 (CD44lowCD25+) thymocytes. G9P3-1MAb is a valuable reagent to study thymic T cell development and is likely useful for the analysis of pathological conditions affecting thymopoiesis, such as thymic involution caused by stress or aging. Fil: Garabatos, Nahir. Institut d'Investigacions Biomèdiques August Pi i Sunyer; España Fil: Blanco, Jesus. Institut d'Investigacions Biomèdiques August Pi i Sunyer; España Fil: Fandos, Cesar. Institut d'Investigacions Biomèdiques August Pi i Sunyer; España Fil: Lopez, Elena. Institut d'Investigacions Biomèdiques August Pi i Sunyer; España Fil: Santamaria, Pere. Institut d'Investigacions Biomèdiques August Pi i Sunyer; España. University of Calgary; Canadá Fil: Ruiz, Andrea. Institut d'Investigacions Biomèdiques August Pi i Sunyer; España Fil: Perez Vidakovics, Maria Laura Anabella. Institut d'Investigacions Biomèdiques August Pi i Sunyer; España. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Benveniste, Patricia. University of Toronto; Canadá Fil: Galkin, Oleksandr. University of Toronto; Canadá Fil: Zuñiga Pflucker, Juan Carlos. University of Toronto; Canadá Fil: Serra, Pau. Institut d'Investigacions Biomèdiques August Pi i Sunyer; España

The 9-aminoacridine (9AA) derivative quinacrine (QC) has a long history of safe human use as an antiprotozoal and antirheumatic agent. QC intercalates into DNA and RNA and can inhibit DNA replication, RNA transcription, and protein synthesis. The extent of QC intercalation into RNA depends on the complexity of its secondary and tertiary structure. Internal ribosome entry sites (IRESs) that are required for initiation of translation of some viral and cellular mRNAs typically have complex structures. Recent work has shown that some intercalating drugs, including QC, are capable of inhibiting hepatitis C virus IRES-mediated translation in a cell-free system. Here, we show that QC suppresses translation directed by the encephalomyocarditis virus (EMCV) and poliovirus IRESs in a cell-free system and in virus-infected HeLa cells. In contrast, IRESs present in the mammalian p53 transcript that are predicted to have less-complex structures were not sensitive to QC. Inhibition of IRES-mediated translation by QC correlated with the affinity of binding between QC and the particular IRES. Expression of viral capsid proteins, replication of viral RNAs, and production of virus were all strongly inhibited by QC (and 9AA). These results suggest that QC and similar intercalating drugs could potentially be used for treatment of viral infections.

The zinc finger X-linked duplicated A (ZXDA) and ZXDC proteins are both required for robust transcription of major histocompatibility complex class II (MHC II) genes. Aside from the full length ZXDC mRNA transcript, at least one additional mRNA is produced by the ZXDC gene, in which transcription initiates within the first exon and terminates within the seventh intron. The protein product produced from this transcript, which we have named ZXDC2, is truncated on both the N- and C-terminus. We demonstrate here that ZXDC2 functions to repress MHC II transcription induced in HeLa cells treated with IFN-gamma. We further demonstrate that ZXDC2 interacts with both ZXDA and ZXDC, suggesting a mechanism by which ZXDC2 may inhibit MHC II transcription. These studies not only provide additional support for the role of ZXD proteins in regulating MHC II transcription, but also demonstrate a unique mechanism for the synthesis of a mRNA isoform.

Formation of the muscular layer of the heart, the myocardium, involves the medial movement of bilateral progenitor fields; driven primarily by shortening of the endoderm during foregut formation. Using a combination of time-lapse imaging, microsurgical perturbations and computational modeling, we show that the speed of the medial-ward movement of the myocardial progenitors is similar, but not identical to that of the adjacent endoderm. Further, the extracellular matrix microenvironment separating the two germ layers also moves with the myocardium, indicating that collective tissue motion and not cell migration drives tubular heart assembly. Importantly, as myocardial cells approach the midline, they perform distinct anterior-directed movements relative to the endoderm. Based on the analysis of microincision experiments and computational models, we propose two characteristic, autonomous morphogenetic activities within the early myocardium: 1) an active contraction of the medial portion of the heart field and 2) curling- the tendency of the unconstrained myocardial tissue to form a spherical surface with a concave ventral side. In the intact embryo, these deformations are constrained by the endoderm and the adjacent mesoderm, nevertheless the corresponding mechanical stresses contribute to the proper positioning of myocardial primordia.