Glucagon stimulates exocytosis in mouse and rat pancreatic alpha-cells by binding to glucagon receptors

Glucagon, secreted by the pancreatic alpha-cells, stimulates insulin secretion from neighboring beta-cells by cAMP- and protein kinase A (PKA)-dependent mechanisms, but it is not known whether glucagon also modulates its own secretion. We have addressed this issue by combining recordings of membrane capacitance (to monitor exocytosis) in individual alpha-cells with biochemical assays of glucagon secretion and cAMP content in intact pancreatic islets, as well as analyses of glucagon receptor expression in pure alpha-cell fractions by RT-PCR. Glucagon stimulated cAMP generation and exocytosis dose dependently with an EC50 of 1.6-1.7 nm. The stimulation of both parameters plateaued at concentrations beyond 10 nm of glucagon where a more than 3-fold enhancement was observed. The actions of glucagon were unaffected by the GLP-1 receptor antagonist exendin-(9-39) but abolished by des-His1-[Glu9]-glucagon-amide, a specific blocker of the glucagon receptor. The effects of glucagon on alpha-cell exocytosis were mimicked by forskolin and the stimulatory actions of glucagon and forskolin on exocytosis were both reproduced by intracellular application of 0.1 mm cAMP. cAMP-potentiated exocytosis involved both PKA-dependent and -independent (resistant to Rp-cAMPS, an Rp-isomer of cAMP) mechanisms. The presence of the cAMP-binding protein cAMP-guanidine nucleotide exchange factor II in alpha-cells was documented by a combination of immunocytochemistry and RT-PCR and 8-(4-chloro-phenylthio)-2'-O-methyl-cAMP, a cAMP-guanidine nucleotide exchange factor II-selective agonist, mimicked the effect of cAMP and augmented rapid exocytosis in a PKA-independent manner. We conclude that glucagon released from the alpha-cells, in addition to its well-documented systemic effects and paracrine actions within the islet, also represents an autocrine regulator of alpha-cell function.     

Reconstructing the history of helminth prevalence in the UK

Intestinal helminth parasites (worms) have afflicted humans throughout history and their eggs are readily detected in archaeological deposits including at locations where intestinal parasites are no longer considered endemic (e.g. the UK). Parasites provide valuable archaeological insights into historical health, sanitation, hygiene, dietary and culinary practices, as well as other factors. Differences in the prevalence of helminths over time may help us understand factors that affected the rate of infection of these parasites in past populations. While communal deposits often contain relatively high numbers of parasite eggs, these cannot be used to calculate prevalence rates, which are a key epidemiological measure of infection. The prevalence of intestinal helminths was investigated through time in England, based on analysis of 464 human burials from 17 sites, dating from the Prehistoric to Industrial periods. Eggs from two faecal-oral transmitted nematodes (Ascaris sp. and Trichuris sp.) and the food-derived cestodes (Taenia spp. and Diphyllobothrium latum syn Dibothriocephalus latus) were identified, although only Ascaris was detected at a high frequency. The changing prevalence of nematode infections can be attributed to changes in effective sanitation or other factors that affect these faecal-oral transmitted parasites and the presence of cestode infections reflect dietary and culinary preferences. These results indicate that the impact of helminth infections on past populations varied over time, and that some locations witnessed a dramatic reduction in parasite prevalence during the industrial era (18^th-19^th century), whereas other locations continued to experience high prevalence levels. The factors underlying these reductions and the variation in prevalence provide a key historical context for modern anthelmintic programs.     

Thermostable designed ankyrin repeat proteins (DARPins) as building blocks for innovative drugs

Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development. By using in silico analysis and a rationally guided Ala scanning, we identified position 17 of the N-terminal capping repeat to play a key role in overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by 8 °C to 10 °C when Asp17 was replaced by Leu, Val, Ile, Met, Ala, or Thr. We then transferred the Asp17Leu mutation to various backgrounds, including clinically validated DARPin domains, such as the vascular endothelial growth factor-binding domain of the DARPin abicipar pegol. In all cases, these proteins showed improvements in the thermostability on the order of 8 °C to 16 °C, suggesting the replacement of Asp17 could be generically applicable to this drug class. Molecular dynamics simulations showed that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, this beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development, indicating this mutation could be partly responsible for the very high melting temperature (>90 °C) of this promising anti-COVID-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins.     

Macrophage inhibitory cytokine‐1 (MIC‐1/GDF15): a new marker of all‐cause mortality

Macrophage inhibitory cytokine‐1 (MIC‐1/GDF15) is a member of the TGF‐b superfamily, previously studied in cancer and inflammation. In addition to regulating body weight, MIC‐1/GDF15 may be used to predict mortality and/or disease course in cancer, cardiovascular disease (CVD), chronic renal and heart failure, as well as pulmonary embolism. These data suggested that MIC‐1/GDF15 may be a marker of all‐cause mortality. To determine whether serum MIC‐1/GDF15 estimation is a predictor of all‐cause mortality, we examined a cohort of 876 male subjects aged 35–80 years, selected from the Swedish Population Registry, and followed them for overall mortality. Serum MIC‐1/GDF15 levels were determined for all subjects from samples taken at study entry. A second (independent) cohort of 324 same‐sex twins (69% female) from the Swedish Twin Registry was similarly examined. All the twins had telomere length measured and 183 had serum levels of interleukin 6 (IL‐6) and C‐reactive protein (CRP) available. Patients were followed for up to 14 years and had cause‐specific and all‐cause mortality determined. Serum MIC‐1/GDF15 levels predicted mortality in the all‐male cohort with an adjusted odds ratio (OR) of death of 3.38 (95%CI 1.38–8.26). This finding was validated in the twin cohort. Serum MIC‐1/GDF15 remained an independent predictor of mortality when further adjusted for telomere length, IL‐6 and CRP. Additionally, serum MIC‐1/GDF15 levels were directly correlated with survival time independently of genetic background. Serum MIC‐1/GDF15 is a novel predictor of all‐cause mortality.     

How Many Genetic Variants Remain to Be Discovered?

A great majority of genetic markers discovered in recent genome-wide association studies have small effect sizes, and they explain only a small fraction of the genetic contribution to the diseases. How many more variants can we expect to discover and what study sizes are needed? We derive the connection between the cumulative risk of the SNP variants to the latent genetic risk model and heritability of the disease. We determine the sample size required for case-control studies in order to achieve a certain expected number of discoveries in a collection of most significant SNPs. Assuming similar allele frequencies and effect sizes of the currently validated SNPs, complex phenotypes such as type-2 diabetes would need approximately 800 variants to explain its 40% heritability. Much smaller numbers of variants are needed if we assume rare-variants but higher penetrance models. We estimate that up to 50,000 cases and an equal number of controls are needed to discover 800 common low-penetrant variants among the top 5000 SNPs. Under common and rare low-penetrance models, the very large studies required to discover the numerous variants are probably at the limit of practical feasibility. Under rare-variant with medium- to high-penetrance models (odds-ratios between 1.6 and 4.0), studies comparable in size to many existing studies are adequate provided the genotyping technology can interrogate more and rarer variants.     

Prieurianin/endosidin 1 is an actin-stabilizing small molecule identified from a chemical genetic screen for circadian clock effectors in Arabidopsis thaliana

Chemical modulators are powerful tools to investigate biological processes. To identify circadian clock effectors, we screened a natural product library in the model plant Arabidopsis thaliana. Two compounds, prieurianin (Pri) and prieurianin acetate, were identified as causing a shorter circadian period. Recently, Pri was independently identified as a vesicle trafficking inhibitor and re-named endosidin 1 (ES1). Here we show that Pri primarily affects actin filament flexibility in vivo, later resulting in reduced severing and filament depolymerization. This stabilization of the actin cytoskeleton subsequently causes changes in vesicle trafficking. Pri also affected microfilaments in mammalian cells, indicating that its target is highly conserved; however, it did not alter actin dynamics in vitro, suggesting that its activity requires the presence of actin-associated proteins. Furthermore, well-characterized actin inhibitors shortened the period length of the Arabidopsis clock in a similar way to Pri, supporting the idea that Pri affects rhythms by altering the actin network. We conclude that actin-associated processes influence the circadian system in a light-dependent manner, but their disruption does not abolish rhythmicity. In summary, we propose that the primary effect of Pri is to stabilize the actin cytoskeleton system, thereby affecting endosome trafficking. Pri appears to stabilize actin filaments by a different mechanism from previously described inhibitors, and will be a useful tool to study actin-related cellular processes.     

Influence of the Lipid Anchor Motif of N-Ras on the Interaction with Lipid Membranes: A Surface Plasmon Resonance Study

Ras GTPases play a crucial role in signal transduction cascades involved in cell differentiation and proliferation, and membrane binding is essential for their proper function. To determine the influence of the nature of the lipid anchor motif and the difference between the active (GTP) and inactive (GDP) forms of N-Ras on partitioning and localization in the lipid membrane, five different N-Ras constructs with different lipid anchors and nucleotide loading (Far/Far (GDP), HD/Far (GDP), HD/HD (GDP), Far (GDP), and HD/Far (GppNHp)) were synthesized. Using the surface plasmon resonance technique, we were able to follow the insertion and dissociation process of the lipidated proteins into and out of model membranes consisting of pure liquid-ordered (l_o) or liquid-disordered (l_d) phase and a heterogeneous two-phase mixture, i.e., a raft mixture with l_o + l_d phase coexistence. In addition, we examined the influence of negatively charged headgroups and stored curvature elastic stress on the binding properties of the lipidated N-Ras proteins. In most cases, significant differences were found for the various anchor motifs. In general, N-Ras proteins insert preferentially into a fluidlike, rather than a rigid, ordered lipid bilayer environment. Electrostatic interactions with lipid headgroups or stored curvature elastic stress of the membrane seem to have no drastic effect on the binding and dissociation processes of the lipidated proteins. The monofarnesylated N-Ras exhibits generally the highest association rate and fastest dissociation process in fluidlike membranes. Double lipidation, especially including farnesylation, of the protein leads to drastically reduced initial binding rates but strong final association. The change in the nucleotide loading of the natural N-Ras HD/Far induces a slightly different binding and dissociation kinetics, as well as stability of association, and seems to influence the tendency to segregate laterally in the membrane plane. The GDP-bound inactive form of N-Ras with an HD/Far anchor shows stronger membrane association, which might be due to a more pronounced tendency to self-assemble in the membrane matrix than is seen with the active GTP-bound form.     

Quaternary refugia of the sweet chestnut (<Emphasis Type="Italic">Castanea sativa</Emphasis> Mill.): an extended palynological approach

Knowledge about the glacial refugia of the thermophilous European Castanea sativa Mill. (sweet chestnut) is still inadequate. Its original range of distribution has been masked by strong human impact. Moreover, under natural conditions the species was probably admixed with other taxa (such as Quercus, Fraxinus, Fagus, Tilia) and thus possibly represented by low percentages in pollen records. In this paper we try to overcome the difficulties related to the scarcity and irregularity of chestnut pollen records by considering 1471 sites and extending the palynological approach to develop a Castanea refugium probability index (IRP), aimed at detecting possible chestnut refugia where chestnuts survived during the last glaciation. The results are in close agreement with the current literature on the refugia of other thermophilous European trees. The few divergences are most probably due to the large amount of new data integrated in this study, rather than to fundamental disagreements about data and data interpretation. The main chestnut refugia are located in the Transcaucasian region, north-western Anatolia, the hinterland of the Tyrrhenian coast from Liguria to Lazio along the Apennine range, the region around Lago di Monticchio (Monte Vulture) in southern Italy, and the Cantabrian coast on the Iberian peninsula. Despite the high likelihood of Castanea refugia in the Balkan Peninsula and north-eastern Italy (Colli Euganei, Monti Berici, Emilia-Romagna) as suggested by the IRP, additional palaeobotanical investigations are needed to assess whether these regions effectively sheltered chestnut during the last glaciation. Other regions, such as the Isère Département in France, the region across north-west Portugal and Galicia, and the hilly region along the Mediterranean coast of Syria and Lebanon were classified as areas of medium refugium probability. Our results reveal an unexpected spatial richness of potential Castanea refugia. It is likely that other European trees had similar distribution ranges during the last glaciation. It is thus conceivable that shelter zones with favourable microclimates were probably more numerous and more widely dispersed across Europe than so far assumed. In the future, more attention should be paid to pollen traces of sporadic taxa thought to have disappeared from a given area during the last glacial and post-glacial period.Electronic Supplementary Material Supplementary material is available in the online version of this article at . A link in the frame on the left on that page takes you directly to the supplementary material.An erratum to this article can be found at