A JAGN1-associated severe congenital neutropenia zebrafish model revealed an altered G-CSFR signaling and UPR activation.

A variety of autosomal recessive mutations in the JAGN1 gene cause severe congenital neutropenia (CN). However, the underlying pathomechanism remains poorly understood, mainly due to the limited availability of primary hematopoietic stem cells from JAGN1-CN patients and the absence of animal models. In this study, we aimed to address these limitations by establishing a zebrafish model of JAGN1-CN. We found two paralogs of the human JAGN1 gene, jagn1a and jagn1b, which play distinct roles during zebrafish hematopoiesis. Using various approaches such as morpholino-based knockdown, CRISPR/Cas9-based gene-editing, and misexpression of a jagn1b harboring a specific human mutation, we successfully developed neutropenia while leaving other hematopoietic lineages unaffected. Further analysis of our model revealed significant upregulation of apoptosis and genes involved in unfolded protein response (UPR). However, neither UPR nor apoptosis is the primary mechanism leading to neutropenia in zebrafish. Instead, Jagn1b has a critical role in G-CSFR signaling and steady-state granulopoiesis, shedding light on the pathogenesis of neutropenia associated with JAGN1 mutations. The establishment of a zebrafish model for JAGN1-CN represents a significant advancement in understanding the specific pathological pathways underlying the disease. This model provides a valuable in vivo tool for further investigation and exploration of potential therapeutic strategies.

TH1 cytokines induce senescence in AML.

Cancer testis antigen PRAME is over-expressed in a variety of malignant cells but is not or minimally expressed in normal non-germ line cells. Adoptive transfer of PRAME-specific T cells is thus under investigation in clinical trials as an innovative therapeutic option for acute myeloid leukemia (AML). However, their senescence-inducing activity has not been studied. This study therefore examines senescence induction in AML cells by PRAME-specific TH1 cells. Analysis of cell cycle and marker expression demonstrate that the supernatants of antigen-stimulated PRAME-specific TH1 cells induce senescence in AML cell lines Kasumi and Nomo-1 through combinative IFN-γ and TNF-α. Additionally IFN-γ and TNF-α secreted by TCR-activated Vδ2+ or CMV-specific T cells can also drive these AML cell lines into terminal growth arrest. G1/0 arrest is also suggested in patient-derived AML by TH1 cytokines or supernatants from Zoledronate-stimulated or aCD3/aCD28-stimulated PBMCs. Thus, we show for the first time that senescence is induced in AML cells by combined IFN-γ and TNF-α, and that these cytokines can be derived either from TCR-engineered CD4+ T cells, or intriguingly from Virus-specific as well as innate Vδ2+ T cells responding to their cognate antigens, namely T-cell responses targeting an antigen that is NOT expressed by the leukemic cells.

Fluorescence Lifetime Imaging Microscopy (FLIM) of Intracellular Transport by Means of Doubly Labelled siRNA Architectures.

For monitoring the intracellular pathway of small interfering RNA (siRNA), both strands were labelled at internal positions by two ATTO dyes as an interstrand Förster resonance energy transfer pair. siRNA double strands show red emission and a short donor lifetime as readout, whereas siRNA antisense single strands show green emission and a long donor lifetime. This readout signals if GFP silencing can be expected (green) or not (red). We attached both dyes to three structurally different alkyne anchors by postsynthetic modifications. There is only a slight preference for the ribofuranoside anchors with the dyes at their 2'-positions. For the first time, the delivery and fate of siRNA in live HeLa cells was tracked by fluorescence lifetime imaging microscopy (FLIM), which revealed a clear relationship between intracellular transport using different transfection methods and knockdown of GFP expression, which demonstrates the potential of our siRNA architectures as a tool for future development of effective siRNA.

A zebrafish model for HAX1-associated congenital neutropenia.

Severe congenital neutropenia (CN) is a rare heterogeneous group of diseases, characterized by a granulocytic maturation arrest. Autosomal recessive mutations in the HAX1 gene are frequently detected in affected individuals. However, the precise role of HAX1 during neutrophil differentiation is poorly understood. To date, no reliable animal model has been established to study HAX1-associated CN. Here we show that knockdown of zebrafish hax1 impairs neutrophil development without affecting other myeloid cells and erythrocytes. Furthermore, we have found that interference with the Hax1 function decreases the expression level of key target genes of the granulocyte-colony stimulating factor (G-CSF) signaling pathway. The reduced neutrophil numbers in the morphants could be reversed by G-CSF, which is also the main therapeutic intervention for patients who have CN. Our results demonstrate that zebrafish is a suitable model for HAX1-associated neutropenia. We anticipate that this model will serve as an in vivo platform to identify new avenues for developing tailored therapeutic strategies for CN patients, particularly for those individuals that do not respond to the G-CSF treatment.

Postsynthetic Modifications of DNA and RNA by Means of Copper-Free Cycloadditions as Bioorthogonal Reactions.

Bioorthogonal chemistry has mainly been developed for proteins and carbohydrates. The chemistry of nucleic acids is different, and bioorthogonal labeling strategies that were successfully applied for proteins and carbohydrates cannot be simply transferred to DNA and RNA. Cycloadditions play a central role for bioorthogonal chemistry with nucleic acids. In vivo postsynthetic labeling of DNA and RNA requires copper-free variants of cycloaddition chemistry to achieve "bio"orthogonality that can be applied even in living cells. Currently, there are three major types of copper-free cycloadditions available for nucleic acids: (i) the ring-strain-promoted azide-alkyne cycloadditions, (ii) the "photoclick" 1,3-dipolar cycloadditions, and (iii) the Diels-Alder reactions with inverse electron demand. In principle, bioorthogonally reactive building blocks for postsynthetic modifications of nucleic acids by cycloaddition can be prepared by three different ways: (i) The organic synthesis of DNA and RNA applies phosphoramidites as building blocks for solid-phase automated chemistry. (ii) The biochemical preparation of DNA and RNA by primer extension (PEX) and PCR applies triphosphates as building blocks together with DNA/RNA polymerases, and works in aqueous buffer. (iii) DNA and RNA is labeled by the intrinsic metabolism in cells using bioorthogonally reactive nucleosides. In contrast to proteins and carbohydrates, for which metabolic labeling strategies are well developed, there are only a few examples in the literature for metabolic labeling of nucleic acids. In this review, we summarize the currently available DNA and RNA building blocks, both phosphoramidites and nucleotide triphosphates, for copper-free and bioorthogonal postsynthetic modification strategies.

Zebrafish and Medaka: Two Teleost Models of T-Cell and Thymic Development.

Over the past two decades, studies have demonstrated that several features of T-cell and thymic development are conserved from teleosts to mammals. In particular, works using zebrafish (Danio rerio) and medaka (Oryzias latipes) have shed light on the cellular and molecular mechanisms underlying these biological processes. In particular, the ease of noninvasive in vivo imaging of these species enables direct visualization of all events associated with these processes, which are, in mice, technically very demanding. In this review, we focus on defining the similarities and differences between zebrafish and medaka in T-cell development and thymus organogenesis; and highlight their advantages as two complementary model systems for T-cell immunobiology and modeling of human diseases.

Synthesis of Dye-Modified Oligonucleotides via Copper(I)-Catalyzed Alkyne Azide Cycloaddition Using On- and Off-Bead Approaches.

Fluorescence molecular imaging is widely used to visualize and observe different biomolecules, in particular DNA and RNA, in vivo and in real time. Typically, DNA strands are tagged with only one fluorophore, and, in the case of molecular beacons, an additional quencher is conjugated, which bears the risk of false-positive or false-negative results because only fluorescence intensities at one fluorescence wavelength (color) are compared. To address this drawback, the concept of "DNA/RNA traffic lights," which is characterized by a fluorescence color change due to energy transfer between two dyes, was developed by our working group. For these DNA and RNA systems, the oligonucleotides are post-synthetically labeled, specifically after solid-phase synthesis by chemical means, with a fluorescent dye using copper(I)-catalyzed cycloaddition at the 2' position of single uridines. In order to functionalize oligonucleotides with several different labels, an on-resin method is required to ensure the necessary selectivity. This unit describes two different CuAAC ("click") approaches-in solution (post-synthetic) and on solid phase (during synthesis)-for the attachment of fluorophores to the 2' position of DNA. © 2018 by John Wiley & Sons, Inc.