Region-specific transcriptomic and functional signatures of mononuclear phagocytes in the epididymis

In the epididymis, prevention of autoimmune responses against spermatozoa and simultaneous protection against pathogens is important for male fertility. We have previously shown that mononuclear phagocytes (MPs) are located either in the epididymal interstitium or in close proximity to the epithelium. In the initial segments (IS), these ‘intraepithelial’ MPs extend slender luminal-reaching projections between epithelial cells. In this study, we performed an in-depth characterisation of MPs isolated from IS, caput–corpus and cauda epididymis of CX3CR1EGFP+/− mice that express EGFP in these cells. Flow cytometry analysis revealed region-specific subsets of MPs that express combinations of markers traditionally described in ‘dendritic cells’ or ‘macrophages’. RNA sequencing identified distinct transcriptomic signatures in MPs from each region and revealed specific genes involved in inflammatory and anti-inflammatory responses, phagosomal activity and antigen processing and presentation. Functional fluorescent in vivo labelling assays showed that higher percentages of CX3CR1+ MPs that captured and processed antigens were detected in the IS compared to other regions. Confocal microscopy showed that in the IS, caput and corpus, circulatory antigens were internalised and processed by interstitial and intraepithelial MPs. However, in the cauda only interstitial MPs internalised and processed antigens, while intraepithelial MPs did not take up antigens, indicating that all antigens have been captured before they reached the epithelial lining. Cauda MPs may thus confer a stronger protection against blood-borne pathogens compared to proximal regions. By identifying immunoregulatory mechanisms in the epididymis, our study may lead to new therapies for male infertility and epididymitis and identify potential targets for immunocontraception.

Generation of in vivo labelling tumour cell lines

The characterization of the tumour microenvironment is highly desirable in order to get a better understating on how the tumour cells exploit their neighbours to support their own growth. In Ombrato et al., Nature 2019 we describe a novel strategy that allows 4T1 breast tumour cells to label their lung metastatic niche in vivo. The labelled cells surrounding the tumour cells can be freshly isolated within the all tissue and used for ex vivo assays. Here, we describe how we generated Labelling-4T1 cells. The same procedure can be used to generate labelling cells from other cell lines, but it might require optimization. This protocol is a suggested guide.

Effect of sodium ions on conformations of the cytoplasmic loop of the PomA stator protein of Vibrio alginolyticus

The sodium driven flagellar stator of Vibrio alginolyticus is a hetero-hexamer membrane complex composed of PomA and PomB, and acts as a sodium ion channel. The conformational change in the cytoplasmic region of PomA for the flagellar torque generation, which interacts directly with a rotor protein, FliG, remains a mystery. In this study, we introduced cysteine mutations into cytoplasmic charged residues of PomA, which are highly conserved and interact with FliG, to detect the conformational change by the reactivity of biotin maleimide. In vivo labelling experiments of the PomA mutants revealed that the accessibility of biotin maleimide at position of E96 was reduced with sodium ions. Such a reduction was also seen in the D24N and the plug deletion mutants of PomB, and the phenomenon was independent in the presence of FliG. This sodium ions specific reduction was also detected in Escherichia coli that produced PomA and PomB from a plasmid, but not in the purified stator complex. These results demonstrated that sodium ions cause a conformational change around the E96 residue of loop2–3 in the biological membrane.

Fluoro-substituted cyanine for reliablein vivolabelling of amyloid-β oligomers and neuroprotection against amyloid-β induced toxicity

A fluoro-substituted cyanine showing reliable in vivo labelling of Aβ oligomers and potent neuroprotective effect against Aβ-induced toxicities is reported as a novel theranostic agent for the early diagnosis and therapy of Alzheimer's disease.

In-Vivo Labelling Studies in Patients with Chronic Lymphocytic Leukemia Studies Demonstrate the Existence of Apparently Distinct Subpopulations That Differ in Phenotype and Proliferative Capacity

It is now generally believed that proliferation of the neoplastic clone in chronic lymphocytic leukemia (CLL) takes place in lymphoid tissues where interactions involving the B-cell receptor (BCR) and other microenvironmental elements take place. Previous studies using in-vivo labelling with deuterated water have shown that recently proliferated emigrants from lymphoid tissues express low levels of the chemokine receptor CXCR4 and high levels of CD5 (CXCR4loCD5hi). It has been proposed that, following entry into the peripheral blood (PB), these cells become quiescent, re-express CXCR4 and downregulate CD5 allowing re-entry into tissues and further rounds of proliferation. In the present study we used in-vivo labelling with deuterated glucose (6,6-2H2-glucose, D2G) to investigate the proliferation and release of CLL cells into PB. In contrast to deuterated water, this technique allows pulse labelling of a distinct cohort of cells, which can then be tracked over time in-vivo. Labelling studies were performed in 10 patients with previously untreated, non-progressive CLL. Patients underwent 10 hours of labelling with oral 2DG, after which peripheral blood and lymph node compartments were serially sampled. DNA Deuterium enrichment was measured in the entire CLL population and in flow-sorted subsets defined by CXCR4/CD5 and surface IgM (sIgM) expression. Maximum release of labelled cells into PB occurred after a median of 14 days (4-56 days). The disappearance rate was very slow, with labelled cells detectable after 56 days in half of the subjects. In one case we were able to track labelled cells in both lymph node (LN) and PB and demonstrated an increase in the fraction of labelled cells in the LN between days 7 and 28, consistent with re-entry into this compartment. Subpopulations of PB CLL cells, defined by CXCR4 and CD5 expression were studied over time to investigate the dynamic nature of these molecules in circulating cells. As previously reported, maximum rapid incorporation of deuterium was observed in the CXCR4loCD5hi fraction, equivalent to 1.15 ± 0.04 %/d fractional synthesis at 7 days. Conversely, CXCR4hiCD5lo cells remained largely unlabelled throughout the 8-week study, reaching a maximum of only 0.01 ± 0.003 %/d, suggesting that they represent a non-proliferating population, not derived from the CXCR4loCD5hi subset. In contrast, CXCR4/CD5 intermediate cells exhibited delayed and intermediate labelling, peaking at 0.1 ± 0.02 %/d at 28 days. This sequential labelling pattern suggests that they derive from the CXCR4loCD5hi subset. Since both CXCR4 and CD5 expression are modulated by BCR signalling, we went on to sort cells according to sIgM expression and found maximum labelling in the sIgM high subset with little or no deuterium incorporation in the sIgM low fraction at any time. Again, the sIgM intermediate subset showed delayed labelling, suggesting that they are derived from sIgM-high cells. These observations provide further evidence for clonal heterogeneity in CLL and suggest the existence of distinct but interdependent subpopulations. Recently-divided cells are CXCR4loCD5hi, but appear to transition to an intermediate phenotype by down-regulation of CD5 and sIgM and upregulation of CXCR4 over the ensuing weeks, but do not appear to transition to CXCR4hiCD5lo cells over the time-course of the experiment. Our findings have clinical relevance, since these functionally distinct subsets might also differ in their responsiveness to therapeutic agents, such as drugs that block BCR signalling. Figure 1. Deuterium enrichment in CLL subsets over eight week study Figure 1. Deuterium enrichment in CLL subsets over eight week study Disclosures No relevant conflicts of interest to declare.

Plastoquinone-9 biosynthesis in cyanobacteria differs from that in plants and involves a novel 4-hydroxybenzoate solanesyltransferase

PQ-9 (plastoquinone-9) has a central role in energy transformation processes in cyanobacteria by mediating electron transfer in both the photosynthetic as well as the respiratory electron transport chain. The present study provides evidence that the PQ-9 biosynthetic pathway in cyanobacteria differs substantially from that in plants. We identified 4-hydroxybenzoate as being the aromatic precursor for PQ-9 in Synechocystis sp. PCC6803, and in the present paper we report on the role of the membrane-bound 4-hydroxybenzoate solanesyltransferase, Slr0926, in PQ-9 biosynthesis and on the properties of the enzyme. The catalytic activity of Slr0926 was demonstrated by in vivo labelling experiments in Synechocystis sp., complementation studies in an Escherichia coli mutant with a defect in ubiquinone biosynthesis, and in vitro assays using the recombinant as well as the native enzyme. Although Slr0926 was highly specific for the prenyl acceptor substrate 4-hydroxybenzoate, it displayed a broad specificity with regard to the prenyl donor substrate and used not only solanesyl diphosphate, but also a number of shorter-chain prenyl diphosphates. In combination with in silico data, our results indicate that Slr0926 evolved from bacterial 4-hydroxybenzoate prenyltransferases catalysing prenylation in the course of ubiquinone biosynthesis.

Ypp1/YGR198w plays an essential role in phosphoinositide signalling at the plasma membrane

Phosphoinositide signalling through the eukaryotic plasma membrane makes essential contributions to many processes, including remodelling of the actin cytoskeleton, vesicle trafficking and signalling from the cell surface. A proteome-wide screen performed in Saccharomyces cerevisiae revealed that Ypp1 interacts physically with the plasma-membrane-associated phosphoinositide 4-kinase, Stt4. In the present study, we demonstrate that phenotypes of ypp1 and stt4 conditional mutants are identical, namely osmoremedial temperature sensitivity, hypersensitivity to cell wall destabilizers and defective organization of actin. We go on to show that overexpression of STT4 suppresses the temperature-sensitive growth defect of ypp1 mutants. In contrast, overexpression of genes encoding the other two phosphoinositide 4-kinases in yeast, Pik1 and Lsb6, do not suppress this phenotype. This implies a role for Ypp1 in Stt4-dependent events at the plasma membrane, as opposed to a general role in overall metabolism of phosphatidylinositol 4-phosphate. Use of a pleckstrin homology domain sensor reveals that there are substantially fewer plasma-membrane-associated 4-phosphorylated phosphoinositides in ypp1 mutants in comparison with wild-type cells. Furthermore, in vivo labelling with [3H]inositol indicates a dramatic reduction in the level of phosphatidylinositol 4-phosphate in ypp1 mutants. This is the principal cause of lethality under non-permissive conditions in ypp1 mutants, as limiting the activity of the Sac1 phosphoinositide 4-phosphate phosphatase leads to restoration of viability. Additionally, the endocytic defect associated with elevated levels of PtdIns4P in sac1Δ cells is restored in combination with a ypp1 mutant, consistent with the opposing effects that these two mutations have on levels of this phosphoinositide.