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Leukemia patients undergo chemotherapy to combat the leukemic cells (LCs) in the bone marrow. During therapy not only the LCs, but also the blood-producing hematopoietic stem and progenitor cells (HSPCs) may be destroyed. Chemotherapeutics targeting only the LCs are urgently needed to overcome this problem and minimize life-threatening side-effects. Predictive in vitro drug testing systems allowing simultaneous comparison of various experimental settings would enhance the efficiency of drug development. Here, we present a three-dimensional (3D) human leukemic bone marrow model perfused using a magnetic, parallelized culture system to ensure media exchange. Chemotherapeutic treatment of the acute myeloid leukemia cell line KG-1a in 3D magnetic hydrogels seeded with mesenchymal stem/stromal cells (MSCs) revealed a greater resistance of KG-1a compared to 2D culture. In 3D tricultures with HSPCs, MSCs and KG-1a, imitating leukemic bone marrow, HSPC proliferation decreased while KG-1a cells remained unaffected post treatment. Non-invasive metabolic profiling enabled continuous monitoring of the system. Our results highlight the importance of using biomimetic 3D platforms with proper media exchange and co-cultures for creating in vivo-like conditions to enable in vitro drug testing. This system is a step towards drug testing in biomimetic, parallelized in vitro approaches, facilitating the discovery of new anti-leukemic drugs.

Leukemia patients undergo chemotherapy to combat the leukemic cells (LCs) in the bone marrow. During therapy not only the LCs, but also the blood-producing hematopoietic stem and progenitor cells (HSPCs) may be destroyed. Chemotherapeutics targeting only the LCs are urgently needed to overcome this problem and minimize life-threatening side-effects. Predictive

As a response to pro-inflammatory signals mesenchymal stem cells (MSCs) secrete agents and factors leading to lymphocyte recruitment, counteracting inflammation, and stimulating immunosuppression. On a molecular level, the signalling mediator TGF-β-activated kinase 1 (TAK1) is activated by many pro-inflammatory signals, plays a critical role in inflammation and regulates innate and adaptive immune responses as well. While the role of TAK1 as a signalling factor promoting inflammation is well documented, we also considered a role for TAK1 in anti-inflammatory actions exerted by activated MSCs. We, therefore, investigated the capacity of lipopolysaccharide (LPS)-treated murine MSCs with lentivirally modulated TAK1 expression levels to recruit lymphocytes. TAK1 downregulated by lentiviral vectors expressing TAK1 shRNA in murine MSCs interfered with the capacity of murine MSCs to chemoattract lymphocytes, indeed. Analysing a pool of 84 secreted factors we found that among 26 secreted cytokines/factors TAK1 regulated expression of one cytokine in LPS-activated murine MSCs in particular: interleukin-6 (IL-6). IL-6 in LPS-treated MSCs was responsible for lymphocyte recruitment as substantiated by neutralizing antibodies. Our studies, therefore, suggest that in LPS-treated murine MSCs the inflammatory signalling mediator TAK1 may exert anti-inflammatory properties via IL-6. Supplementary Information The online version contains supplementary material available at 10.1007/s11010-021-04180-8.

The Warburg effect describes the ability of cancer cells to produce energy via aerobic glycolysis instead of oxidative phosphorylation of pyruvate. This deviation in mitochondrial metabolism inhibits apoptosis, allowing increased proliferation under conditions of reduced oxygen levels. Dichloroacetate (DCA) was successfully used in several human cancer cell lines to reactivate oxidative phosphorylation in mitochondria. The aim of this study was the characterization and response of canine cancer cell lines after DCA exposure. The effect of 10 mM DCA was characterized in vitro on a set of six canine prostate adenocarcinoma and transitional cell carcinoma (TCC) derived cell lines. Cell counts, lactate levels, apoptosis, expression of apoptotic proteins, survival factors and different miRNAs were analyzed. Additionally, metabolic activity, mitochondrial activity and proliferation were investigated. DCA significantly decreased cell number of all but one utilized cell lines and leads to a significant reduction of lactate release. Decreased survivin levels were found in all cell lines, two of which presented a significant reduction in metabolic activity. Increased miR-375 levels were measured in all TCC cell lines. Reactivation of pyruvate dehydrogenase and an elevated mitochondrial activity appear to induce the transition from aerobic glycolysis back to oxidative phosphorylation. Further, these results display that DCA treatment has a suppressant effect on proliferation of canine cancer cells.

The mutation N188T in human connexin46 (hCx46) correlates with a congenital nuclear pulverulent cataract. This mutation is in the second extracellular loop, a domain involved in docking of gap junction hemichannels. To analyze the functional consequences of this mutation, we expressed hCx46N188T and the wild type (hCx46wt) in Xenopus oocytes and HeLa cells. In Xenopus oocytes, hemichannels formed by hCx46wt and hCx46N188T had similar electrical properties. Additionally, a Ca(2+) and La(3+) sensitive current was observed in HeLa cells expressing eGFP-labeled hCx46wt or eGFP-labeled hCx46N188T. These results suggest that the N188T mutation did not alter apparent expression and the membrane targeting of the protein. Cells expressing hCx46wt-eGFP formed gap junction plaques, but plaques formed by hCx46N188T were extremely rare. A reduced plaque formation was also found in cells cotransfected with hCx46N188T-eGFP and mCherry-labeled hCx46wt as well as in cocultured cells expressing hCx46N188T-eGFP and hCx46wt-mCherry. Dye transfer experiments in cells expressing hCx46N188T revealed a lower transfer rate than cells expressing hCx46wt. We postulate that the N188T mutation affects intercellular connexon docking. This hypothesis is supported by molecular modeling of hCx46 using the crystal structure of hCx26 as a template. The model indicated that N188 is important for hemichannel docking through formation of hydrogen bonds with the residues R180, T189 and D191 of the opposing hCx46. The results suggest that the N188T mutation hinders the docking of the connexons to form gap junction channels. Moreover, the finding that a glutamine substitution (hCx46N188Q) could not rescue the docking emphasizes the specific role of N188.

Musculoskeletal dysfunctions are highly prevalent due to increasing life expectancy. Consequently, novel solutions to optimize treatment of patients are required. The current major research focus is to develop innovative concepts for single tissues. However, interest is also emerging to generate applications for tissue transitions where highly divergent properties need to work together, as in bone-cartilage or bone-tendon transitions. Finding medical solutions for dysfunctions of such tissue transitions presents an added challenge, both in research and in clinics. This review aims to provide an overview of the anatomical structure of healthy adult entheses and their development during embryogenesis. Subsequently, important scientific progress in restoration of damaged entheses is presented. With respect to enthesis dysfunction, the review further focuses on inflammation. Although molecular, cellular and tissue mechanisms during inflammation are well understood, tissue regeneration in context of inflammation still presents an unmet clinical need and goes along with unresolved biological questions. Furthermore, this review gives particular attention to the potential role of a signaling mediator protein, transforming growth factor beta-activated kinase-1 (TAK1), which is at the node of regenerative and inflammatory signaling and is one example for a less regarded aspect and potential important link between tissue regeneration and inflammation.

We combine confocal Raman microscopy (CRM) of wet samples with subsequent Fluorescent in situ hybridization (FISH) without significant limitations to either technique for analyzing the same sample of a microbial community on a cell-to-cell basis. This combination of techniques allows a much deeper, more complete understanding of complex environmental samples than provided by either technique alone. The minimalistic approach is based on laboratory glassware with micro-engravings for reproducible localization of the sample at cell scale combined with a fixation and de- and rehydration protocol for the respective techniques. As proof of concept, we analyzed a floc of nitrifying activated sludge, demonstrating that the sample can be tracked with cell-scale precision over different measurements and instruments. The collected information includes the microbial content, spatial shape, variant chemical compositions of the floc matrix and the mineral microparticles embedded within. In addition, the direct comparison of CRM and FISH revealed a difference in reported cell size due to the different cell components targeted by the respective technique. To the best of our knowledge, this is the first report of a direct cell-to-cell comparison of confocal Raman microscopy and Fluorescent in situ hybridization analysis performed on the same sample. An adaptation of the method to include native samples as a starting point is planned for the near future. The micro-engraving approach itself also opens up the possibility of combining other, functionally incompatible techniques as required for further in-depth investigations of low-volume samples.

Connexin46 (Cx46), together with Cx50, forms gap junction channels between lens fibers and participates in the lens pump-leak system, which is essential for the homeostasis of this avascular organ. Mutations in Cx50 and Cx46 correlate with cataracts, but the functional relationship between the mutations and cataract formation is not always clear. Recently, it was found that a mutation at the third position of hCx46 that substituted an aspartic acid residue with a tyrosine residue (hCx46D3Y) caused an autosomal dominant zonular pulverulent cataract. We expressed EGFP-labeled hCx46wt and hCx46D3Y in HeLa cells and found that the mutation did not affect the formation of gap junction plaques. Dye transfer experiments using Lucifer Yellow (LY) and ethidium bromide (EthBr) showed an increased degree of dye coupling between the cell pairs expressing hCx46D3Y in comparison to the cell pairs expressing hCx46wt. In Xenopus oocytes, two-electrode voltage-clamp experiments revealed that hCx46wt formed voltage-sensitive hemichannels. This was not observed in the oocytes expressing hCx46D3Y. The replacement of the aspartic acid residue at the third position by another negatively charged residue, glutamic acid, to generate the mutant hCx46D3E, restored the voltage sensitivity of the resultant hemichannels. Moreover, HeLa cell pairs expressing hCx46D3E and hCx46wt showed a similar degree of dye coupling. These results indicate that the negatively charged aspartic acid residue at the third position of the N-terminus of hCx46 could be involved in the determination of the degree of metabolite cell-to-cell coupling and is essential for the voltage sensitivity of the hCx46 hemichannels.

The C-terminus (CT) of rCx46 consists of 186 residues (H230-I416). Recent studies showed that rCx46(28.2), truncated after H243, altered the formation of functional hemichannels when expressed in Xenopus oocytes, while rCx46(37.7), truncated after A333 formed gap junction hemichannels similarly to rCx46(wt). To analyze the role of the CT up to A333 in functional expression with cell imaging and dye-transfer techniques, different mutants were generated by C-terminal truncation between H243-A333, labeled with EGFP and expressed in HeLa cells. These rCx46 variants were characterized according to their compartmentalization in organelles, their presence in microscopic detectable vesicles and their ability to form gap junction plaques. rCx46 truncated after A311 (rCx46(35.3)) was compartmentalized, was found in vesicles and formed functional gap junction plaques similarly to rCx46(wt). With a truncation after P284 (rCx46(32.6)), the protein was not compartmentalized and the amount of vesicles containing the protein were reduced; however, functional gap junction plaque formation was not affected as compared to rCx46(35.3). rCx46(28.2) did not form functional gap junction plaques; it was not found in vesicles or in cellular compartments. Live-cell imaging and detection of annular junctions for rCx46(32.6) and rCx46(35.3) revealed that the truncation after P284 reduced the frequency of vesicle budding from gap junction plaques and the formation of annular junctions. These results suggest that the C-terminal region of rCx46 up to A311 (rCx46(35.3)) is necessary for its correct compartmentalization and internalization in the form of annular junctions, while the H230-P284 C-terminal region (rCx46(32.6)) is sufficient for the formation of dye coupled gap junction channels.