The serum ferritin concentration is a clinical parameter measured widely for the differential diagnosis of anemia. Its levels increase with elevations of tissue iron stores and with inflammation, but studies on cellular sources of serum ferritin as well as its subunit composition, degree of iron loading and glycosylation have given rise to conflicting results. To gain further understanding of serum ferritin, we have used traditional and modern methodologies to characterize mouse serum ferritin. We find that both splenic macrophages and proximal tubule cells of the kidney are possible cellular sources for serum ferritin and that serum ferritin is secreted by cells rather than being the product of a cytosolic leak from damaged cells. Mouse serum ferritin is composed mostly of L-subunits, whereas it contains few H-subunits and iron content is low. L-subunits of serum ferritin are frequently truncated at the C-terminus, giving rise to a characteristic 17-kD band that has been previously observed in lysosomal ferritin. Taken together with the fact that mouse serum ferritin is not detectably glycosylated, we propose that mouse serum ferritin is secreted through the nonclassical lysosomal secretory pathway. (Blood. 2010;116(9):1574-1584) IntroductionFerritin in mammals is a mostly intracellular, cytosolic iron storage and detoxification protein. It consists of H-and L-subunits that assemble into a 24-subunit hollow sphere in which iron is sequestered. 1 However, ferritin can also be detected extracellularly, in cerebrospinal fluid and sinovial fluid. In serum it is an extracellular ferritin that has been used extensively in diagnostic tests. In the clinical setting, serum ferritin evaluation is most commonly used to estimate body iron stores as low serum ferritin correlates with iron depletion, whereas high serum ferritin correlates with elevated body iron stores or with inflammation in patients with normal body iron stores. 2,3 Characterization of serum ferritin has produced many controversial results regarding subunit composition, iron content and other features. It has been compared in some studies to the "natural apoferritin" fraction found in many tissues, which is essentially devoid of iron 4 while other studies have claimed that serum ferritin contains considerable amounts of iron. 5 In insects, ferritin is a secreted protein composed of 2 types of subunits and the heteropolymer functions most likely as an iron transporter in the hemolymph. Each of the subunits has a classical secretion signal and ferritin is secreted from the insect fat body. 6 In contrast, mammalian H-and L-ferritin subunits lack signals mediating endoplasmic reticulum (ER) targeting, but ferritin can traffic to the lysosomal compartment through autophagy. 7 Binding of human serum ferritin to the lectin Concanavalin A (ConA) has previously suggested that ferritin is glycosylated 8,9 and actively secreted through the ER-Golgi pathway. 10 Indeed, it was shown in hepatocyte cell-models that inhibition of ER-Golgi trafficking abolished secretion o...

The online version of this article has a Supplementary Appendix. BackgroundThe lifespan of red blood cells is terminated when macrophages remove senescent red blood cells by erythrophagocytosis. This puts macrophages at the center of systemic iron recycling in addition to their functions in tissue remodeling and innate immunity. Thus far, erythrophagocytosis has been studied by evaluating phagocytosis of erythrocytes that were damaged to mimic senescence. These studies have demonstrated that acquisition of some specific individual senescence markers can trigger erythrophagocytosis by macrophages, but we hypothesized that the mechanism of erythrophagocytosis of such damaged erythrocytes might differ from erythrophagocytosis of physiologically aged erythrocytes. Design and MethodsTo test this hypothesis we generated an erythrocyte population highly enriched in senescent erythrocytes by a hypertransfusion procedure in mice. Various erythrocyte-aging signals were analyzed and erythrophagocytosis was evaluated in vivo and in vitro. ResultsThe large cohort of senescent erythrocytes from hypertransfused mice carried numerous aging signals identical to those of senescent erythrocytes from control mice. Phagocytosis of fluorescently-labeled erythrocytes from hypertransfused mice injected into untreated mice was much higher than phagocytosis of labeled erythrocytes from control mice. However, neither erythrocytes from hypertransfused mice, nor those from control mice were phagocytosed in vitro by primary macrophage cultures, even though these cultures were able to phagocytose oxidatively damaged erythrocytes. ConclusionsThe large senescent erythrocyte population found in hypertransfused mice mimics physiologically aged erythrocytes. For effective erythrophagocytosis of these senescent erythrocytes, macrophages depend on some features of the intact phagocytosing tissue for support. ABSTRACT© F e r r a t a S t o r t i F o u n d a t i o n

The universal importance of iron, its high toxicity, and complex chemistry present a challenge to biological systems in general and to protected compartments in particular. The high mitotic rate and avid mitochondriogenesis of developing male germ cells imply high iron requirements. Yet access to germ cells is tightly regulated by the blood-testis barrier that protects the meiotic and postmeiotic germ cells. To elucidate how iron is supplied to developing male germ cells, we analyzed iron deposition and iron transport proteins in testes of mice with iron overload and with genetic ablation of the iron regulators Hfe and iron regulatory protein 2. Iron accumulated mainly around seminiferous tubules, and only small amounts localized within the seminiferous tubules. The localization and regulation of proteins involved in iron import, storage, and export such as transferrin, transferrin receptor, the divalent metal transporter-1, cytosolic ferritin, and ferroportin strongly support a model of a largely autonomous iron cycle within seminiferous tubules. We show evidence that ferritin secretion from Sertoli cells may play an important role in iron acquisition of primary spermatocytes. During spermatogenic development iron is carried along from primary spermatocytes to spermatids, and from spermatids iron is recycled to the apical compartment of Sertoli cells, which traffic it back to a new generation of spermatocytes. Losses are replenished by the peripheral circulation. Such an internal iron cycle essentially detaches the iron homeostasis within the seminiferous tubule from the periphery and protects developing germ cells from iron fluctuations. This model explains how compartmentalization can optimize cellular and systemic nutrient homeostasis.

BackgroundMosquitoes host and pass on to humans a variety of disease-causing pathogens such as infectious viruses and other parasitic microorganisms. The emergence and spread of insecticide resistance is threatening the effectiveness of current control measures for common mosquito vector borne diseases, such as malaria, dengue and Zika. Therefore, the emerging resistance to the widely used pyrethroid insecticides is an alarming problem for public health. Herein we demonstrated the use of RNA interference (RNAi) to increase susceptibility of adult mosquitoes to a widely used pyrethroid insecticide.MethodsExperiments were performed on a field-collected pyrethroid resistant strain of Ae. aegypti (Rio de Janeiro; RJ). Larvae from the resistant Ae. aegypti population were soaked with double-stranded RNAs (dsRNAs) that correspond either to voltage-gate sodium channel (VGSC), P-glycoprotein, or P450 detoxification genes and reared to adulthood. Adult mortality rates in the presence of various Deltamethrin pyrethroid concentrations were used to assess mosquito insecticide susceptibility.ResultsWe characterized the RJ Ae. aegypti strain with regard to its level of resistance to a pyrethroid insecticide and found that it was approximately 6 times more resistant to Deltamethrin compared to the laboratory Rockefeller strain. The RJ strain displayed a higher frequency of Val1016Ile and Phe1534Cys substitutions of the VGSC gene. The resistant strain also displayed a higher basal expression level of VGSC compared to the Rockefeller strain. When dsRNA-treated mosquitoes were subjected to a standard pyrethroid contact bioassay, only dsRNA targeting VGSC increased the adult mortality of the pyrethroid resistant strain. The dsRNA treatment proved effective in increasing adult mosquito susceptibility over a range of pyrethroid concentrations and these results were associated with dsRNA-specific small interfering RNAs in treated adults, and the corresponding specific down regulation of VGSC gene expression level. Finally, we demonstrated that the efficiency of our approach was further improved by ‘tiling’ along the VGSC gene in order to identify the most potent dsRNA sequences.ConclusionsThese results demonstrate that dsRNA applied to mosquito larvae retains its biological activity into adulthood. Thus, the RNAi system reported here could be a useful approach to control the widespread insecticide resistance in mosquitoes and other insect vectors of human diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1634-y) contains supplementary material, which is available to authorized users.

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We report an infant with molybdenum cofactor deficiency (MCD) and a unique clinical presentation of hemiplegia, hypotonia, dystonia, and bilateral basal ganglia changes. Biochemistry revealed absent serum homocysteine, low concentrations of plasma cystine, high levels of urinary S-sulfocysteine and sulfite, and high levels of oxypurines in serum and urine. The depletion of cysteine and cystine through reaction with sulfite suggests that other thiols and thiol-dependent proteins may be similarly depleted. Ahomocysteinemia may be a clue to the mechanism of cytotoxicity in MCD.

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The exact route of iron through the kidney and its regulation during iron overload are not completely elucidated. Under physiologic conditions, non-transferrin and transferrin bound iron passes the glomerular filter and is reabsorbed through kidney epithelial cells, so that hardly any iron is found in the urine. To study the route of iron reabsorption through the kidney, we analyzed the location and regulation of iron metabolism related proteins in kidneys of mice with iron overload, elicited by iron dextran injections. Transferrin Receptor 1 was decreased as expected, following iron overload. In contrast, the multi-ligand hetero-dimeric receptor-complex megalin/cubilin, which also mediates the internalization of transferrin, was highly up-regulated. Moreover, with increasing iron, intracellular ferritin distribution shifted in renal epithelium from an apical location to a punctate distribution throughout the epithelial cells. In addition, in contrast to many other tissues, the iron exporter ferroportin was not reduced by iron overload in the kidney. Iron accumulated mainly in interstitial macrophages, and more prominently in the medulla than in the cortex. This suggests that despite the reduction of Transferrin Receptor 1, alternative pathways may effectively mediate re-absorption of iron that cycles through the kidney during parenterally induced iron-overload. The most iron consuming process of the body, erythropoiesis, is regulated by the renal erythropoietin producing cells in kidney interstitium. We propose, that the efficient re-absorption of iron by the kidney, also during iron overload enables these cells to sense systemic iron and regulate its usage based on the systemic iron state.