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(Leukaemia derived) dendritic cells (DC, DC leu ) are potent stimulators of anti-leukaemic activity in acute myeloid leukaemia (AML) and can be generated with immunomodulatory kits containing granulocyte-macrophage-colony-stimulating-factor (GM-CSF), prostaglandin-E 1 (PGE 1 ), prostaglandin-E 2 (PGE 2 ) and/or picibanil (OK-321). Potential adverse effects initiated through kits, especially the proliferation of blasts, must be ruled out to ensure treatment safety. We quantified proliferating blasts with the proliferation markers CD71 and Ki-67 and the novel proliferation marker IPO-38 before and after kit treatment ex vivo. IPO-38 hereby appeared to be the most sensitive marker; a combination with CD71 may add value when assessing proliferation kinetics. Kit treatment did not or only slightly (<5%) induce blast proliferation in most cases. An induction of blast proliferation was only found in single cases and could be compensated by DC leu -induced anti-leukaemic activity in most times. Overall, we appraise kit treatment to be safe in vivo., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Modiblast Pharma GmbH (Oberhaching, Germany) holds the European Patent 15,801,987.7–1118 and US Patent 15–517,627 “Use of immunomodulatory effective compositions for the immunotherapeutic treatment of patients suffering from myeloid leukemias”, in which Helga Maria Schmetzer is involved., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Dendritic cells (DC) and leukaemia derived DC (DC leu ) are potent stimulators of anti-leukaemic activity in acute myeloid leukaemia (AML) and can be generated from mononuclear cells in vitro following standard DC/DC leu -generating protocols. With respect to future clinical applications though, DC/DC leu -generating protocols specifically designed for application in a whole-blood-(WB)-environment must be established. Therefore, we developed ten new DC/DC leu -generating protocols (kits; Kit-A/-C/-D/-E/-F/-G/-H/-I/-K/-M) for the generation of DC/DC leu from leukaemic WB, containing calcium-ionophore, granulocyte-macrophage-colony-stimulating-factor (GM-CSF), tumour-necrosis-factor-alpha, prostaglandin-E 1 (PGE 1 ), prostaglandin-E 2 (PGE 2 ) and/or picibanil (OK-432). All protocols were evaluated regarding their performance in generating DC/DC leu using refined classification and/or ranking systems; DC/DC leu were evaluated regarding their performance in stimulating anti-leukaemic activity using a cytotoxicity fluorolysis assay. Overall, we found the new kits capable to generate (mature) DC/DC leu from leukaemic WB. Through refined classification and ranking systems, we were able to select Kit-I (GM-CSF + OK-432), -K (GM-CSF + PGE 2 ) and -M (GM-CSF + PGE 1 ) as the most efficient kits in generating (mature) DC/DC leu , which are further competent to stimulate immunoreactive cells to show an improved anti-leukaemic cytotoxicity as well. This great performance of Kit-I, -K and -M in mediating DC/DC leu -based anti-leukaemic immunity in a WB-environment in vitro constitutes an important and directive step for translating DC/DC leu -based immunotherapy of AML into clinical application.

Contactins (CNTNs) are neural cell adhesion molecules that encode axon-target specificity during the patterning of the vertebrate visual and olfactory systems. Because CNTNs are tethered to the plasma membrane by a glycosylphosphatidylinositol anchor, they lack an intracellular region to communicate across the membrane. Instead, they form coreceptor complexes with distinct transmembrane proteins to transmit signals inside the cell. In particular, a complex of CNTN4 and amyloid precursor protein (APP) is known to guide the assembly of specific circuits in the visual system. Here, using in situ hybridization in zebrafish embryos, we show that CNTN4, CNTN5, and the APP homologs, amyloid beta precursor like protein 1 and amyloid beta precursor like protein 2, are expressed in olfactory pits, suggesting that these receptors may also function together in the organization of olfactory tissues. Furthermore, we use biochemical and structural approaches to characterize interactions between members of these two receptor families. In particular, APP and amyloid beta precursor like protein 1 interact with CNTN3-5, whereas amyloid beta precursor like protein 2 only binds to CNTN4 and CNTN5. Finally, structural analyses of five CNTN-amyloid pairs indicate that these proteins interact through a conserved interface involving the second fibronectin type III repeat of CNTNs and the copper-binding domain of amyloid proteins. Overall, this work sets the stage for analyzing CNTN-amyloid-mediated connectivity in vertebrate sensory circuits., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Split reporter protein-based genetic section systems are widely used to identify and characterize protein-protein interactions (PPI). The assembly of split markers that antagonize toxins, rather than required for synthesis of missing metabolites, facilitates the seeding of high density of cells and selective growth. Here we present a newly developed split chloramphenicol acetyltransferase (split-CAT) -based genetic selection system. The N terminus fragment of CAT is fused downstream of the protein of interest and the C terminus fragment is tethered upstream to its postulated partner. We demonstrate the system's advantages for the study of PPIs. Moreover, we show that co-expression of a functional ubiquitylation cascade where the target and ubiquitin are tethered to the split-CAT fragments results in ubiquitylation-dependent selective growth. Since proteins do not have to be purified from the bacteria and due to the high sensitivity of the split-CAT reporter, detection of challenging protein cascades and post-translation modifications is enabled. In addition, we demonstrate that the split-CAT system responds to small molecule inhibitors and molecular glues (GLUTACs). The absence of ubiquitylation-dependent degradation and deubiquitylation in E. coli significantly simplify the interpretation of the results. We harnessed the developed system to demonstrate that like NEDD4, UBE3B also undergoes self-ubiquitylation-dependent inactivation. We show that self-ubiquitylation of UBE3B on K665 induces oligomerization and inactivation in yeast and mammalian cells respectively. Finally, we showcase the advantages of split-CAT in the study of human diseases by demonstrating that mutations in UBE3B that cause Kaufman oculocerebrofacial syndrome exhibit clear E. coli growth phenotypes., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The Tribbles (Trib) family of pseudokinase proteins regulate cell growth, proliferation, and differentiation during normal development and in response to environmental stress. Mutations in human Trib isoforms (Trib1, 2, and 3) have been associated with metabolic disease and linked to leukemia and the formation of solid tumors, including melanomas, hepatomas, and lung cancers. Drosophila Tribbles (Trbl) was the first identified member of this sub-family of pseudokinases and shares a conserved structure and similar functions to bind and direct the degradation of key mediators of cell growth and proliferation. Common Trib targets include Akt kinase (also known as protein kinase B), C/EBP (CAAT/enhancer binding protein) transcription factors, and Cdc25 phosphatases, leading to the notion that Trib family members stand athwart multiple pathways modulating their growth-promoting activities. Recent work using the Drosophila model has provided important insights into novel facets of conserved Tribbles functions in stem cell quiescence, tissue regeneration, metabolism connected to insulin signaling, and tumor formation linked to the Hippo signaling pathway. Here we highlight some of these recent studies and discuss their implications for understanding the complex roles Tribs play in cancers and disease pathologies.

Strategies to stabilize remissions by specific elimination of residual acute myeloid leukemia (AML) blasts are needed. Leukemia-derived dendritic cell (DCleu/DC) generated from myeloid blasts improve antileukemic T-cell reactivity and install T-cell memory. Interferon (IFN)α-DC methods produce DCleu from chronic myeloid leukemia-patients (pts') blood. Various INFα-containing versus other DC methods were studied to produce DCleu (evaluated by flowcytometry) from AML-pts' blast-containing mononuclear (MNC) or whole blood (WB). After DCleu/DC stimulation in mixed lymphocyte cultures, T cells' potential to gain antileukemic cytotoxicity was studied and correlated with different DC methods and DCleu/DC counts. (1) Generation of DCleu/DC: (a) "IFN-GIT" [containing granulocyte macrophage-colony stimulating factor (GM-CSF)+IFNα+ tumor necrosis factor (TNF)-α] produced DC successfully (≥10% DC, ≥5% DCleu/cells) from AML-MNC (WB) in 54 (56%), "MCM-Mimic" in 76 (75%), "Picibanil" in 83 (64%), and "Calcium-ionophore" in 42 (67%) of cases. Proportions of DC subtypes in MNC (WB) were comparable with all DC methods, (b) IFNα combinations containing only GM-CSF+IFNα or only IFNα showed low efficiency to produce DCleu/DC from MNC (WB) compared with "IFN-GIT." (2) Antileukemic functionality: DCleu/DC-stimulated T cells showed improved leukemia cytotoxicity compared with blast cells or unstimulated T cells. The highest blast proliferation (=insufficient T cells) was seen with "IFN-GIT" DC-stimulated T cells. Probability to respond to immunotherapy or to obtain blast lysis of DC-stimulated T cells correlated with high proportions of DCleu/DC after DC culture, independent of DC-generating methods. (3) Cytokine release profiles: levels of interleukin-6, IFN-γ, and interleukin-2 were significantly lower in DC culture supernatants (from MNC/WB) with "IFN-GIT" compared with "MCM," "Pici," and "Ca" DC supernatants. Our data show that (1) WB culture simulates AML-pts' in vivo situation, (2) DC generation is possible from AML-MNC (WB) with IFNα-containing and other DC methods, (3) successful IFNα-DC generation needs GM-CSF+IFNα+TNF-α (IFN-GIT); however, "IFN-GIT" produces less DCleu/DC compared with other (non-IFNα) DC methods, (4) T cells stimulated with "IFN-GIT"-produced DCleu/DC yielded comparable antileukemic cytotoxicity; however, in cases without achieved blast lysis, an increased blast proliferation was observed.

Members of the Tribbles family of proteins are conserved pseudokinases with diverse roles in cell growth and proliferation. Both Drosophila Tribbles (Trbl) and vertebrate Trib3 proteins bind to the kinase Akt (Akt1) to block its phosphorylation activation and reduce downstream insulin-stimulated anabolism. A single nucleotide polymorphism (SNP) variant in human TRIB3, which results in a glutamine (Q) to arginine (R) missense mutation in a conserved motif at position 84, confers stronger Akt binding, resulting in reduced Akt phosphorylation, and is associated with a predisposition to Type 2 diabetes, cardiovascular disease, diabetic nephropathy, chronic kidney disease and leukemogenesis. Here, we used a Drosophila model to understand the importance of the conserved R residue in several Trbl functions. In the fly fat body, misexpression of a site-directed Q mutation at position R141 resulted in weakened binding to Drosophila Akt (dAkt), leading to increased levels of phospho-dAkt, increased cell and tissue size, and increases in the levels of stored glycogen and triglycerides. Consistent with the functional conservation of this arginine in modulating Akt activity, mouse Trib3 R84 misexpressed in the fly fat body blocked dAkt phosphorylation with a strength similar to wild-type Trbl. Limited mutational analysis shows that the R141 site dictates the strength of Akt binding but does not affect other Trbl-dependent developmental processes, suggesting a specificity that could serve as a drug target for metabolic diseases., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

New layered honeycomb tellurates, BiM(III)TeO 6 (M = Cr, Mn, Fe) were synthesized and characterized. BiM(III)TeO 6 (M = Cr, Fe) species crystallize in a trigonal space group, P3̅1c (No. 163), of edge-sharing M 3+ /Te 6+ O 6 octahedra, which form honeycomb-like double layers in the ab plane with Bi 3+ cations located between the layers. Interestingly, the structure of BiMnTeO 6 is similar to those of the Cr/Fe analogues, but with monoclinic space group, P2 1 /c (No. 14), attributed to the strong Jahn-Teller distortion of Mn 3+ cations. The crystal structure of BiM(III)TeO 6 is a superstructure of PbSb 2 O 6 -related materials (ABB'O 6 ). The Cr 3+ and Fe 3+ cations are ordered 80% and 90%, respectively, while the Mn 3+ ions are completely ordered on the B-site of the ABB'O 6 structure. BiCrTeO 6 shows a broad antiferromagnetic transition (AFM) at ∼17 K with a Weiss temperature (θ) of -59.85 K, while BiFeTeO 6 and BiMnTeO 6 show sharp AFM transitions at ∼11 K with θ of -27.56 K and at ∼9.5 K with θ of -17.57 K, respectively. These differences in the magnetic behavior are ascribed to the different concentration of magnetic nearest versus next-nearest neighbor interactions of magnetic cations due to the relative differences in the extent of M/Te ordering.