Clustering and internalization of toxic amylin oligomers in pancreatic cells require plasma membrane cholesterol

Self-assembly of the human pancreatic hormone amylin into toxic oligomers and aggregates is linked to dysfunction of islet β-cells and pathogenesis of type 2 diabetes mellitus. Recent evidence suggests that cholesterol, an essential component of eukaryotic cells membranes, controls amylin aggregation on model membranes. However, the pathophysiological consequence of cholesterol-regulated amylin polymerization on membranes and biochemical mechanisms that protect β-cells from amylin toxicity are poorly understood. Here, we report that plasma membrane (PM) cholesterol plays a key role in molecular recognition, sorting, and internalization of toxic amylin oligomers but not monomers in pancreatic rat insulinoma and human islet cells. Depletion of PM cholesterol or the disruption of the cytoskeleton network inhibits internalization of amylin oligomers, which in turn enhances extracellular oligomer accumulation and potentiates amylin toxicity. Confocal microscopy reveals an increased nucleation of amylin oligomers across the plasma membrane in cholesterol-depleted cells, with a 2-fold increase in cell surface coverage and a 3-fold increase in their number on the PM. Biochemical studies confirm accumulation of amylin oligomers in the medium after depletion of PM cholesterol. Replenishment of PM cholesterol from intracellular cholesterol stores or by the addition of water-soluble cholesterol restores amylin oligomer clustering at the PM and internalization, which consequently diminishes cell surface coverage and toxicity of amylin oligomers. In contrast to oligomers, amylin monomers followed clathrin-dependent endocytosis, which is not sensitive to cholesterol depletion. Our studies identify an actin-mediated and cholesterol-dependent mechanism for selective uptake and clearance of amylin oligomers, impairment of which greatly potentiates amylin toxicity.     

Expression of Drosophila neoplastic tumor suppressor genes discslarge,scribble, and lethal giant larvae in the mammalian ovary

The similarities and differences in molecular mechanisms regulating invertebrate and mammalian folliculogenesis are starting to be deciphered. In Drosophila, the neoplastic tumor suppressor gene discslarge is crucial for suppressing proliferation and movement of follicle cells relative to the growing oocyte. Lethal giant larvae and scribble play similar roles and have been suggested to collaborate intimately with discslarge. We have identified and determined the expression pattern of murine homologs of these Drosophila genes. In situ data shows that murine discslarge-1, discslarge-3, discslarge-4, lethal giant larvae, and scribble are expressed in both overlapping and distinct patterns in oocytes and granulosa cells in maturing follicles. Disclarge-4 is expressed in the surface epithelium and is lost in mouse carcinogenic surface epithelial cells. All of these genes, as well as discslarge-2 and discslarge-5, are expressed in human ovaries. Our data suggests that as in Drosophila, these tumor suppressors may cooperate during mammalian folliculogenesis, but also have distinct functions.     

Comparison and quantitative verification of mapping algorithms for whole-genome bisulfite sequencing

Coupling bisulfite conversion with next-generation sequencing (Bisulfite-seq) enables genome-wide measurement of DNA methylation, but poses unique challenges for mapping. However, despite a proliferation of Bisulfite-seq mapping tools, no systematic comparison of their genomic coverage and quantitative accuracy has been reported. We sequenced bisulfite-converted DNA from two tissues from each of two healthy human adults and systematically compared five widely used Bisulfite-seq mapping algorithms: Bismark, BSMAP, Pash, BatMeth and BS Seeker. We evaluated their computational speed and genomic coverage and verified their percentage methylation estimates. With the exception of BatMeth, all mappers covered >70% of CpG sites genome-wide and yielded highly concordant estimates of percentage methylation (r² ≥ 0.95). Fourfold variation in mapping time was found between BSMAP (fastest) and Pash (slowest). In each library, 8–12% of genomic regions covered by Bismark and Pash were not covered by BSMAP. An experiment using simulated reads confirmed that Pash has an exceptional ability to uniquely map reads in genomic regions of structural variation. Independent verification by bisulfite pyrosequencing generally confirmed the percentage methylation estimates by the mappers. Of these algorithms, Bismark provides an attractive combination of processing speed, genomic coverage and quantitative accuracy, whereas Pash offers considerably higher genomic coverage.     

A Study on the Safety and Effects of Amorpha fruticosa Fruit Extract on Spontaneously Hypertensive Rats with Induced Type 2 Diabetes

Metabolic syndrome is characterized by a variety of diagnostic criteria: obesity, dyslipidemia, type 2 diabetes, and arterial hypertension. They contribute to the elevated risk of cardiovascular morbidity and mortality. The potential for Amorpha fruticosa L. (Fabaceae) to improve diabetes and metabolic disease is promising, based on in vitro tests. This is why a further investigation of the species is needed. Additionally, a toxicity review in relation to safety revealed that to date, there are no published data regarding the toxicity of A. fruticosa towards humans. This species could provide abundant and cheap resources because it is an aggressive invasive plant that grows almost unrestrictedly. The objective of this study was to evaluate the acute toxicity of a purified extract of A. fruticosa (EAF), and to assess its antioxidant, antihypertensive, and antihyperglycemic activity in streptozotocin-induced diabetic spontaneously hypertensive rats (SHRs). The EAF was slightly toxic (LD₅₀ = 2121 mg/kg, b.w.) when administered orally, and moderately toxic (LD₅₀ = 316 mg/kg, b.w.) at intraperitoneal administration, both in mice. The oral administration of EAF (100 mg/kg) for 35 days to SHRs caused significant decreases in the systolic pressure, blood glucose levels, and MDA quantity. It also increased the hepatic level of the endogenous antioxidant GSH, not only in diabetic SHRs, but also in the control group. An additional potential benefit to human health might be conferred through the environmental management of A. fruticosa based on its large-scale use for medicinal purposes, as this aggressive invasive species brings problems to natural habitats in many European countries.     

Anticancer Secondary Metabolites: From Ethnopharmacology and Identification in Native Complexes to Biotechnological Studies in Species of Genus Astragalus L. and Gloriosa L.

Some of the most effective anticancer compounds are still derived from plants since the chemical synthesis of chiral molecules is not economically efficient. Rapid discovery of lead compounds with pronounced biological activity is essential for the successful development of novel drug candidates. This work aims to present the chemical diversity of antitumor bioactive compounds and biotechnological approaches as alternative production and sustainable plant biodiversity conservation. Astragalus spp., (Fabaceae) and Gloriosa spp. (Liliaceae) are selected as research objects within this review because they are known for their anticancer activity, because they represent two of the largest families respectively in dicots and monocots, and also because many of the medicinally important plants are rare and endangered. We summarized the ethnobotanical data concerning their anticancer application, highlighted the diversity of their secondary metabolites possessing anticancer properties such as saponins, flavonoids, and alkaloids, and revealed the potential of the in vitro cultures as an alternative way of their production. Since the natural supply is limited, it is important to explore the possibility of employing plant cell or organ in vitro cultures for the biotechnological production of these compounds as an alternative.     

Further evidence of the Asian mud-daubing wasps in Europe (Hymenoptera: Sphecidae)

We report the further south‐eastward range extension of the invasive Asian mud‐daubing wasp, Sceliphron curvatum (Smith), initially introduced into Austria in the late 1970s. We document its presence in the central Balkan Peninsula from 1997 onwards, and we discuss the circumstances and course of its recent expansion in the area. We also report the first finding of another Asian species, Sceliphron deforme (Smith), in Europe, and we consider various aspects of this case, given the possibility that the record from southern Montenegro represents a successful introduction.