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Abstract Background Platelets are small anucleate cells that circulate in the blood in a resting state but can be activated by external cues. In case of need, platelets from blood donors can be transfused. As an alternative source, platelets can be produced from induced pluripotent stem cells (iPSCs); however, recovered numbers are low. Objectives To optimize megakaryocyte (MK) and platelet output from murine iPSCs, we investigated overexpression of the transcription factors GATA‐binding factor 1 (GATA1); nuclear factor, erythroid 2; and pre–B‐cell leukemia transcription factor 1 (Pbx1) and a hyperactive variant of the small guanosine triphosphatase RhoA (RhoAhc). Methods To avoid off‐target effects, we generated iPSCs carrying the reverse tetracycline‐responsive transactivator M2 (rtTA‐M2) in the Rosa26 locus and expressed the factors from Tet‐inducible gammaretroviral vectors. Differentiation of iPSCs was initiated by embryoid body (EB) formation. After EB dissociation, early hematopoietic progenitors were enriched and cocultivated on OP9 feeder cells with thrombopoietin and stem cell factor to induce megakaryocyte (MK) differentiation. Results Overexpression of GATA1 and Pbx1 increased MK output 2‐ to 2.5‐fold and allowed prolonged collection of MK. Cytologic and ultrastructural analyses identified typical MK with enlarged cells, multilobulated nuclei, granule structures, and an internal membrane system. However, GATA1 and Pbx1 expression did not improve MK maturation or platelet release, although in vitro–generated platelets were functional in spreading on fibrinogen or collagen‐related peptide. Conclusion We demonstrate that the use of rtTA‐M2 transgenic iPSCs transduced with Tet‐inducible retroviral vectors allowed for gene expression at later time points during differentiation. With this strategy we could identify factors that increased in vitro MK production.

The most highly regulated and abundant fatty acid in animal tissue is oleic acid (18:1n9). Oleic acid is synthesized by the Δ9 desaturase, stearoyl‐CoA desaturase‐1 (SCD1), which is responsible for the synthesis of the putative cytokine palmitoleic acid (16:1n7) and 18:2 cis‐9, trans‐11 conjugated linoleic acid. Owing to the importance of SCD1 in lipid metabolism, we generated porcine swine kidney (SK6) transgenic cell lines for sustained overexpression or knockdown of porcine stearoyl‐CoA desaturase‐1 (pSCD1) in an inducible manner by utilizing a lentiviral expression system. We successfully validated these cell culture models for expression and functionality of pSCD1 by documenting that the pSCD‐transduced cells overexpressed pSCD1 protein and mRNA. Additionally, the pSCD1‐transduced cells increased the conversion of palmitate (16:0) to palmitoleic acid nearly fourfold. The lentiviral vectors utilized in this study can be further used to generate transgenic animals to document the effects of the overexpression of SCD1 on obesity and steatosis. [ABSTRACT FROM AUTHOR]

Aurora kinases are eukaryotic serine/threonine protein kinases that regulate key events associated with chromatin condensation, centrosome and spindle function and cytokinesis. Elucidating the roles of Aurora kinases in apicomplexan parasites is crucial to understand the cell cycle control during Plasmodium schizogony or Toxoplasma endodyogeny. Here, we report on the localization of two previously uncharacterized Toxoplasma Aurora-related kinases (Ark2 and Ark3) in tachyzoites and of the uncharacterized Ark3 orthologue in Plasmodium falciparum erythrocytic stages. In Toxoplasma gondii, we show that TgArk2 and TgArk3 concentrate at specific sub-cellular structures linked to parasite division: the mitotic spindle and intranuclear mitotic structures (TgArk2), and the outer core of the centrosome and the budding daughter cells cytoskeleton (TgArk3). By tagging the endogenous PfArk3 gene with the green fluorescent protein in live parasites, we show that PfArk3 protein expression peaks late in schizogony and localizes at the periphery of budding schizonts. Disruption of the TgArk2 gene reveals no essential function for tachyzoite propagation in vitro, which is surprising giving that the P. falciparum and P. berghei orthologues are essential for erythrocyte schizogony. In contrast, knock-down of TgArk3 protein results in pronounced defects in parasite division and a major growth deficiency. TgArk3-depleted parasites display several defects, such as reduced parasite growth rate, delayed egress and parasite duplication, defect in rosette formation, reduced parasite size and invasion efficiency and lack of virulence in mice. Our study provides new insights into cell cycle control in Toxoplasma and malaria parasites and highlights Aurora kinase 3 as potential drug target. [ABSTRACT FROM AUTHOR]

The AP1 site within the tyrosine hydroxylase gene proximal promoter is essential for the response of the gene to numerous stimuli. Stimulation of this gene is often associated with induction of the AP1 transcription factor, c-Fos. However, many stimuli activate or induce multiple transcription factors that interact with this AP1 site or other sites within the gene's proximal promoter. Hence, it remains unclear whether c-Fos induction by itself is sufficient to stimulate the tyrosine hydroxylase gene. In this study we produce rat pheochromocytoma PC18 cells that overexpress c-Fos under control of the tet-inducible system. We demonstrate that induction of c-Fos leads to dramatic stimulation of tyrosine hydroxylase gene transcription rate measured using nuclear run-on assays. This stimulation is closely associated quantitatively with the induction of c-Fos and does not apparently require phosphorylation of c-Fos. The response is partially dependent on the AP1 site within the tyrosine hydroxylase proximal promoter. However, the response of the proximal promoter to c-Fos induction is relatively small compared with that of the endogenous gene. Consequently, our results suggest that c-Fos exerts its influence on the tyrosine hydroxylase gene via multiple mechanisms that are dependent and independent of the proximal promoter AP1 site. [ABSTRACT FROM AUTHOR]

Here we present a T7-driven, tetracycline-inducible system for protein expression in human pathogen Leishmania mexicana . The gene expression in this strain is tightly regulated and dose- and time-dependent. This system can be widely used by the parasitology community to analyze effects of genes of interest on biology, physiology and virulence of parasites causing cutaneous leishmaniases. [ABSTRACT FROM AUTHOR]

Background: Platelets are small anucleate cells that circulate in the blood in a resting state but can be activated by external cues. In case of need, platelets from blood donors can be transfused. As an alternative source, platelets can be produced from induced pluripotent stem cells (iPSCs); however, recovered numbers are low., Objectives: To optimize megakaryocyte (MK) and platelet output from murine iPSCs, we investigated overexpression of the transcription factors GATA-binding factor 1 (GATA1); nuclear factor, erythroid 2; and pre-B-cell leukemia transcription factor 1 (Pbx1) and a hyperactive variant of the small guanosine triphosphatase RhoA (RhoAhc)., Methods: To avoid off-target effects, we generated iPSCs carrying the reverse tetracycline-responsive transactivator M2 (rtTA-M2) in the Rosa26 locus and expressed the factors from Tet-inducible gammaretroviral vectors. Differentiation of iPSCs was initiated by embryoid body (EB) formation. After EB dissociation, early hematopoietic progenitors were enriched and cocultivated on OP9 feeder cells with thrombopoietin and stem cell factor to induce megakaryocyte (MK) differentiation., Results: Overexpression of GATA1 and Pbx1 increased MK output 2- to 2.5-fold and allowed prolonged collection of MK. Cytologic and ultrastructural analyses identified typical MK with enlarged cells, multilobulated nuclei, granule structures, and an internal membrane system. However, GATA1 and Pbx1 expression did not improve MK maturation or platelet release, although in vitro-generated platelets were functional in spreading on fibrinogen or collagen-related peptide., Conclusion: We demonstrate that the use of rtTA-M2 transgenic iPSCs transduced with Tet-inducible retroviral vectors allowed for gene expression at later time points during differentiation. With this strategy we could identify factors that increased in vitro MK production., (© 2020 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

Apicomplexans use the endolysosomal system for the biogenesis of their secretory organelles, namely, micronemes, rhoptries, and dense granules. In Toxoplasma gondii, our previous in silico search identified the HOPS tethering but not the CORVET complex and demonstrated a role of Vps11 (a common component for both complexes) in its secretory organelle biogenesis. Herein, we performed Vps11‐GFP‐Trap pull‐down assays and identified by proteomic analysis, not only the CORVET‐specific subunit Vps8 but also a BEACH domain‐containing protein (BDCP) conserved in eukaryotes. We show that knocking‐down Vps8 affects targeting of dense granule proteins, transport of rhoptry proteins, and the localization of the cathepsin L protease vacuolar compartment marker. Only a subset of micronemal proteins are affected by the absence of Vps8, shedding light on at least two trafficking pathways involved in microneme maturation. Knocking‐down BDCP revealed a restricted and particular role of this protein in rhoptry and vacuolar compartment biogenesis. Moreover, depletion of BDCP or Vps8 abolishes parasite virulence in vivo. This study identified BDCP as a novel CORVET/HOPS‐associated protein, playing specific roles and acting in concert during secretory organelle biogenesis, an essential process for host cell infection. Our results open the hypothesis for a role of BDCP in the vesicular trafficking towards lysosome‐related organelles in mammals and yeast. [ABSTRACT FROM AUTHOR]

Inducible gene expression is an important tool in molecular biology research to study protein function. Most frequently, the antibiotic doxycycline is used for regulation of so-called tetracycline (Tet)-inducible systems. In contrast to stable gene overexpression, these systems allow investigation of acute and reversible effects of cellular protein induction. Recent reports have already called for caution when using Tet-inducible systems as the employed antibiotics can disturb mitochondrial function and alter cellular metabolism by interfering with mitochondrial translation. Reprogramming of energy metabolism has lately been recognized as an important emerging hallmark of cancer and is a central focus of cancer research. Therefore, the scope of this study was to systematically analyze dose-dependent metabolic effects of doxycycline on a panel of glioma cell lines with concomitant monitoring of gene expression from Tet-inducible systems. We report that doxycycline doses commonly used with inducible expression systems (0.01–1 µg/mL) substantially alter cellular metabolism: Mitochondrial protein synthesis was inhibited accompanied by reduced oxygen and increased glucose consumption. Furthermore, doxycycline protected human glioma cells from hypoxia-induced cell death. An impairment of cell growth was only detectable with higher doxycycline doses (10 µg/mL). Our findings describe settings where doxycycline exerts effects on eukaryotic cellular metabolism, limiting the employment of Tet-inducible systems. [ABSTRACT FROM AUTHOR]

In our previous work we established a T7 polymerase-driven Tetracycline-inducible protein expression system in Leishmania mexicana (Biagi, 1953). We used this system to analyse gene expression profiles during development of L. mexicana in procyclic and metacyclic promastigotes and amastigotes. The transcription of the gene of interest and the T7 polymerase genes was significantly reduced upon cell differentiation. This regulation is not locus-specific. It depends on untranslated regions flanking open reading frames of the genes analysed. In this paper, we report that the previously established conventional inducible protein expression system may not be suitable for studies on differentiation of species of Leishmania Ross, 1903 and protein expression systems might have certain limitations.