Receptor-mediated endocytosis of insect lipoprotein : insight into LDL receptor functioning

The extracellular transport of water-insoluble lipids through the aqueous circulatory system of animals is mediated by lipoproteins. The lipoprotein of insects, lipophorin (Lp), is homologous to that of mammalian low-density lipoprotein (LDL). Moreover, an endocytic receptor for Lp has been identified (insect lipophorin receptor, LpR) that is homologous to the LDL receptor (LDLR). LpR was cloned from mRNA isolated from insect fat body, an organ that combines the functions of mammalian liver and adipose tissue. We transfected LDLR-expressing mammalian cells with LpR cDNA to study the endocytic uptake and intracellular pathways of LDL and Lp simultaneously. Our studies provide evidence that LDL and Lp follow distinct intracellular routes after receptor-mediated endocytosis. Multicolor imaging and immunofluorescence showed that upon internalization, LDL and Lp share endocytic vesicles. Subsequently, however, Lp evacuates the LDL-containing endosomes. In contrast to LDL, that is completely degraded in lysosomes after dissociating from LDLR, both Lp and LpR converge in the endocytic recycling compartment (ERC), as confirmed by colocalization of Lp with transferrin (Tf), a ligand that is transported to the ERC upon endocytosis by its receptor (TfR). Eventually, Lp is resecreted from the ERC with a t½ of ~13 min. Insect fat body cells internalize fluorescently-labeled Lp only when LpR is expressed. Expression of LpR is down-regulated on the fourth day after an ecdysis. Consequently, Lp is no longer internalized. Starvation experiments revealed that expression of LpR is regulated by the demand of fat body tissue for lipids. In Drosophila S2 transfectants, LDLR and TfR specifically mediate endocytosis of LDL and Tf, respectively. Both ligands colocalize in endosomes immediately after endocytic uptake, as observed in mammalian cells. However, in S2 cells, the ligands also colocalize after a chase, indicating that Tf is not recycled. Similar results were obtained with Lp internalized by LpR-transfected S2 cells. In contrast, fat body tissue that was subjected to similar conditions showed a significant decrease of lipophorin-containing vesicles, indicative of recycling of the ligand. Class 5 familial hypercholesterolemia (FH) mutations in the LDLR gene impair ligand uncoupling and result in degradation of the receptor-ligand complex. To investigate whether the intracellular tail of LDLR induces degradation of LpR when Lp remains attached, the intracellular C-terminus of insect LpR was replaced by that of human LDLR. This LpR1-790LDLR791-839 hybrid receptor internalized and recycled Lp when expressed in mammalian cells. Apparently, the intracellular tail of LDLR does not elicit degradation of a ligand dissociation-deficient lipoprotein receptor. The hybrid approach was extended by generating a receptor harboring the ligand binding domain of LpR and the region from EGF domain to intracellular tail of LDLR. Although ligand endocytosis by this LpR1-342LDLR293-839 was unaffected, the receptor did not recycle. These findings suggest that latter hybrid follows an intracellular route that is similar to ligand dissociation-deficient LDLR. We studied the naturally occurring LDLR H562Y (LDLRH562Y) FH mutation in vitro, which showed constitutive recycling; the receptor, however, was degraded after LDL internalization. His562 in LDLR corresponds to Asn643 in LpR; yet, LDLRH562N did not recycle in complex with ligand, but behaves like LDLRH562Y. Substitution of the ligand binding domain of LpR by that of LDLR generates a hybrid receptor (LDLR1-292LpR343-850) that is able to bind LDL, and recycles constitutively, however, is degraded after LDL internalization. The data suggest that class 5 mutations can be divided in two distinct subclasses: (1) mutations that impair ligand dissociation (e.g. LDLRH562Y), and (2) those that prevent receptor recycling (e.g. LDLRDEGF)

Marine nature-based solutions: Where societal challenges and ecosystem requirements meet the potential of our oceans

Nature-Based Solutions (NbS), a concept introduced in the late 2000s, has developed rapidly during the last years and is now frequently appearing in a broad spectrum of policies developed within the European Union. Its role in marine policies and research programmes is however still limited, but is likely to increase as NbS are adopted as key terminology in both biodiversity strategies and the EU taxonomy for sustainable financing. This will enhance the need for scientific advisory institutions to provide evidence-based advice on potential impacts of various combinations of marine NbS. To facilitate a critical debate about the prospects and pitfalls related to the operationalisation of marine NbS in an EU context, this paper provides an analysis of core definitions, potential categories of marine NBS and a suite of case studies. Coastal waters, shelf and open oceans present multiple options for testing new and scaling up known NbS, which could support both environmental restoration simultaneously with addressing multiple societal challenges, paving the way for a new level of ecosystem-based management. However, as the acceptance of NbS types will depend on ecosystem state and thus history, it will be a significant task to consistently communicate why some solutions may count as a NbS in some areas, while not in others. To conclude, the paper therefore raises a set of research priorities and policy advice aimed at ensuring the successful advice and deployment of marine NBS in support of multiple societal goals

A negative test of orbital control of geomagnetic reversals and excursions

A ~41 Kyr periodic component has been reported in some sedimentary paleointensity records, allowing speculation that there may be some component of orbital control of geomagnetic field generation such as by obliquity modulation. However, no discernable tendency is found for astronomically-dated geomagnetic reversals in the Plio-Pleistocene (0 to 5.3 Ma) or excursions in the Brunhes (0 to 0.78 Ma) to occur at a consistent amplitude or phase of obliquity cyclicity, nor of orbital eccentricity. An implication is that paleointensity lows which are characteristically associated with these features are not distributed in a systematic way relative to obliquity and eccentricity, supporting the idea that orbital forcing does not power the geodynamo

Optimization and testing of dried antibody tube: The EuroFlow LST and PIDOT tubes as examples

Within EuroFlow, we recently developed screening tubes for hematological malignancies and immune deficiencies. Pipetting of antibodies for such 8-color 12-marker tubes however is time-consuming and prone to operational mistakes. We therefore evaluated dried formats of the lymphocytosis screening tube (LST) and of the primary immune deficiency orientation tube (PIDOT). Both tubes were evaluated on normal and/or on patient samples, comparing the mean fluorescence intensity of specific lymphocyte populations. Our data show that the dried tubes and liquid counterparts give highly comparable staining results, particularly when analyzed in multidimensional plots. In addition, the use of dried tubes may result in a reduced staining variability between different samples and thereby contributes to the generation of more robust data. Therefore, by using ready-to-use reagents in a dried single test tube format, the laboratory efficiency and quality will be improved

RNA degradome—its biogenesis and functions

RNA degradation is among the most fundamental processes that occur in living cells. The continuous decay of RNA molecules is associated not only with nucleotide turnover, but also with transcript maturation and quality control. The efficiency of RNA decay is ensured by a broad spectrum of both specific and non-specific ribonucleases. Some of these ribonucleases participate mainly in processing primary transcripts and in RNA quality control. Others preferentially digest mature, functional RNAs to yield a variety of molecules that together constitute the RNA degradome. Recently, it has become increasingly clear that the composition of the cellular RNA degradome can be modulated by numerous endogenous and exogenous factors (e.g. by stress). In addition, instead of being hydrolyzed to single nucleotides, some intermediates of RNA degradation can accumulate and function as signalling molecules or participate in mechanisms that control gene expression. Thus, RNA degradation appears to be not only a process that contributes to the maintenance of cellular homeostasis but also an underestimated source of regulatory molecules

INSGFP/w human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells

AIMS/HYPOTHESIS: We aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS⁺) cells derived in vitro. METHODS: Homologous recombination was used to insert sequences encoding green fluorescent protein (GFP) into the INS locus, to create reporter cell lines enabling the prospective isolation of viable INS⁺ cells. RESULTS: Differentiation of INS(GFP/w) hESCs using published protocols demonstrated that all GFP⁺ cells co-produced insulin, confirming the fidelity of the reporter gene. INS-GFP⁺ cells often co-produced glucagon and somatostatin, confirming conclusions from previous studies that early hESC-derived insulin-producing cells were polyhormonal. INS(GFP/w) hESCs were used to develop a 96-well format spin embryoid body (EB) differentiation protocol that used the recombinant protein-based, fully defined medium, APEL. Like INS-GFP⁺ cells generated with other methods, those derived using the spin EB protocol expressed a suite of pancreatic-related transcription factor genes including ISL1, PAX6 and NKX2.2. However, in contrast with previous methods, the spin EB protocol yielded INS-GFP⁺ cells that also co-expressed the beta cell transcription factor gene, NKX6.1, and comprised a substantial proportion of monohormonal INS⁺ cells. CONCLUSIONS/INTERPRETATION: INS(GFP/w) hESCs are a valuable tool for investigating the nature of early INS⁺ progenitors in beta cell ontogeny and will facilitate the development of novel protocols for generating INS⁺ cells from differentiating hESCs

Marine Nature-Based Solutions: where societal challenges and ecosystem requirements meet the potential of our oceans

Nature-Based Solutions (NbS), a concept introduced in the late 2000s, has developed rapidly during the last years and is now frequently appearing in a broad spectrum of policies developed within the European Union. Its role in marine policies and research programmes is however still limited, but is likely to increase as NbS are adopted as key terminology in both biodiversity strategies and the EU taxonomy for sustainable financing. This will enhance the need for scientific advisory institutions to provide evidence-based advice on potential impacts of various combinations of marine NbS. To facilitate a critical debate about the prospects and pitfalls related to the operationalisation of marine NbS in an EU context, this paper provides an analysis of core definitions, potential categories of marine NBS and a suite of case studies. Coastal waters, shelf and open oceans present multiple options for testing new and scaling up known NbS, which could support both environmental restoration simultaneously with addressing multiple societal challenges, paving the way for a new level of ecosystem-based management. However, as the acceptance of NbS types will depend on ecosystem state and thus history, it will be a significant task to consistently communicate why some solutions may count as a NbS in some areas, while not in others. To conclude, the paper therefore raises a set of research priorities and policy advice aimed at ensuring the successful advice and deployment of marine NBS in support of multiple societal goals