Magnetic nanoparticles as MRI contrast agents for the diagnosis of Alzheimer’s disease

Background Nanoparticle‐based magnetic contrast agents have opened the potential for Magnetic Resonance Imaging (MRI) to be used for early non‐invasive diagnosis of Alzheimer’s disease (AD). Current methods for clinical diagnosis in the early stages of the disease, such as Positron Emission Tomography imaging of amyloid build‐up, are limited by their availability and cost. The aim of this research is to develop a novel non‐toxic amyloid targeted nanoparticle which can successfully permeate the blood brain barrier and bind amyloid plaques resulting in enhanced contrast in the MR image and improved diagnostic sensitivity. Methods Targeted iron nanoparticles were assessed using a U‐251 cell line to determine their in vitro toxicity. Transmission electron microscopy was used to determine the movement of the nanoparticles within the cell and in vitro binding to amyloid fibrils. APPSwe/PSEN1 mice were treated with increasing doses of targeted and non‐targeted nanoparticles to evaluate acute in vivo toxicity, in addition to nanoparticle biodistribution and MRI contrast enhancement. Results The novel targeted nanoparticles have demonstrated no significant in vitro toxicity and electron microscopy results show their movement through the endocytic cycle within the cell, demonstrating an effective degradation and clearance pathway (Figure). No acute toxicity was observed in the animal model. In addition, immunohistochemistry demonstrated nanoparticles to co‐localise with plaques on ex vivo brain sections. Conclusion The present work shows promising preliminary results in the development of a targeted non‐invasive method of early AD diagnosis using contrast enhanced MRI

ER-Targeted Beclin 1 Supports Autophagosome Biogenesis in the Absence of ULK1 and ULK2 Kinases

Autophagy transports cytoplasmic material and organelles to lysosomes for degradation and recycling. Beclin 1 forms a complex with several other autophagy proteins and functions in the initiation phase of autophagy, but the exact role of Beclin 1 subcellular localization in autophagy initiation is still unclear. In order to elucidate the role of Beclin 1 localization in autophagosome biogenesis, we generated constructs that target Beclin 1 to the endoplasmic reticulum (ER) or mitochondria. Our results confirmed the proper organelle-specific targeting of the engineered Beclin 1 constructs, and the proper formation of autophagy-regulatory Beclin 1 complexes. The ULK kinases are required for autophagy initiation upstream of Beclin 1, and autophagosome biogenesis is severely impaired in ULK1/ULK2 double knockout cells. We tested whether Beclin 1 targeting facilitated its ability to rescue autophagosome formation in ULK1/ULK2 double knockout cells. ER-targeted Beclin 1 was most effective in the rescue experiments, while mitochondria-targeted and non-targeted Beclin 1 also showed an ability to rescue, but with lower activity. However, none of the constructs was able to increase autophagic flux in the knockout cells. We also showed that wild type Beclin 1 was enriched on the ER during autophagy induction, and that ULK1/ULK2 facilitated the ER-enrichment of Beclin 1 under basal conditions. The results suggest that one of the functions of ULK kinases may be to enhance Beclin 1 recruitment to the ER to drive autophagosome formation

COMPARISON OF CONSUMABLE EGGS QUALITY OF DIFFERENT PRODUCERS

Cilj ovog rada bio je usporediti kvalitetu konzumnih jaja klase L tri proizvođača, koja se nalaze u trgovačkim centrima na području grada Osijeka. Od pokazatelja kvalitete jaja u radu su uspoređene vrijednosti mase jaja i njegovih osnovnih dijelova, te su uspoređeni pokazatelji vanjske i unutarnje kvalitete jaja. Iako su jaja pripadala klasi L pronađene su razlike između proizvođača. Najveća masa jaja zabilježena je kod proizvođača B (66,14 g) dok je masa jaja proizvođača A i B bila ujednačena (65,05 g i 65,03 g; P=0,158). U masi bjelanjka nije bilo značajnih razlika između jaja različitih proizvođača (P=0,152). U vrijednostima mase žumanjka statistički značajno teže žumanjke imala su jaja podrijetlom od proizvođača B u usporedbi s proizvođačima A i C (18,19 g : 17,12g i 16,90 g ; P0,05). Analiza usporedbe kvalitete jaja različitih proizvođača pokazuje da konzumna jaja na tržištu grada Osijeka zadovoljavaju kvalitetom i odgovaraju propisima Pravilnika o kakvoći jaja u Hrvatskoj.The aim of this study was to compare consumable eggs quality class L of three producers, located in shopping malls in the city of Osijek. Indicators of egg quality compared in this work are values of the mass of eggs and its basic components and external and internal quality of eggs of three producers. Although the eggs were class L there were differences among producers. The highest weight of eggs was recorded for producer B (66.14g), while the egg mass for producers A and B were equal (65.05g and 65.03g, respectively, P=0.158). In the egg white weight there was no significant difference between eggs of different producers (P=0.152). By comparing yolk mass values, significantly heavier egg yolks originated from producer B in comparison with producer A and C (18.19g: 17.12g and 16.90g, respectively, P0.05). Analysis of the egg quality of different producers shows that consumable eggs in our market are satisfactory in with quality and correspond to the Regulations on the quality of eggs in Croatia

The versatile electron microscope : an ultrastructural overview of autophagy

Both light microscopy (LM) and electron microscopy (EM) are able to reveal important information about the formation and function of various autophagic compartments. In this article we will outline the various techniques that are emerging in EM, focusing on analyzing three-dimensional morphology, collectively known as volume electron microscopy (volume EM), as well as on methods that can be used to localize proteins and antigenic epitopes. Large cell volumes can now be visualized at the EM level by using one of the two complementary imaging techniques, namely Serial Block-face Scanning Electron Microscopy (SB-SEM) or Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). These two blockface imaging methods reveal ultrastructural information from all membrane-bound organelles such as autophagic compartments to be visualized in a three-dimensional space, in association with their surrounding organelles. Another method which falls into the volume EM category is dual-axis electron tomography (ET). This method is more suited to reconstructing smaller volumes from areas of interest that require nano-structural detail to be confirmed such as membrane contact sites (MCSs) between autophagic compartments and various organelles. Further to this, to complement the morphological identification of autophagic compartments, immunolabeling can be carried out at the EM level to confirm the nature of various autophagic compartments depending on the localization of various antigens at a sub-cellular level. To determine this, various immunolabeling techniques can be carried out, namely the pre-embedding or the post-embedding immunolabeling methods. Examples of both of these methods will be described in this chapter. Correlative light-electron microscopy (CLEM) can be used to visualize the same autophagic organelles under the LM, followed by high-resolution imaging under the EM. Finally, cryofixation has revolutionized the EM field by allowing rapid immobilization of cells and tissue in the near native state, so samples are no longer prone to artefacts induced by chemical fixation. Collectively, this chapter will discuss the aforementioned capabilities of the EM in more detail, with a particular focus on autophagy, namely the impact of EM in the study of the morphology and biogenesis of the phagophore/isolation membrane (referred to as the phagophore hereafter).Peer reviewe

Mechanistic Observation of Interactions between Macrophages and Inorganic Particles with Different Densities

Many different types of inorganic materials are processed into nano/microparticles for medical utilization. The impact of selected key characteristics of these particles, including size, shape, and surface chemistries, on biological systems, is frequently studied in clinical contexts. However, one of the most important basic characteristics of these particles, their density, is yet to be investigated. When the particles are designed for drug delivery, highly mobile macrophages are the major participants in cellular levels that process them in vivo. As such, it is essential to understand the impact of particles’ densities on the mobility of macrophages. Here, inorganic particles with different densities are applied, and their interactions with macrophages studied. A set of these particles are incubated with the macrophages and the outcomes are explored by optical microscopy. This microscopic view provides the understanding of the mechanistic interactions between particles of different densities and macrophages to conclude that the particles’ density can affect the migratory behaviors of macrophages: the higher the density of particles engulfed inside the macrophages, the less mobile the macrophages become. This work is a strong reminder that the density of particles cannot be neglected when they are designed to be utilized in biological applications

Uterine Gene Expression in the Live-Bearing Lizard, Chalcides ocellatus, Reveals Convergence of Squamate Reptile and Mammalian Pregnancy Mechanisms

Although the morphological and physiological changes involved in pregnancy in live-bearing reptiles are well studied, the genetic mechanisms that underlie these changes are not known. We used the viviparous African Ocellated Skink, Chalcides ocellatus, as a model to identify a near complete gene expression profile associated with pregnancy using RNA-Seq analyses of uterine transcriptomes. Pregnancy in C. ocellatus is associated with upregulation of uterine genes involved with metabolism, cell proliferation and death, and cellular transport. Moreover, there are clear parallels between the genetic processes associated with pregnancy in mammals and Chalcides in expression of genes related to tissue remodeling, angiogenesis, immune system regulation, and nutrient provisioning to the embryo. In particular, the pregnant uterine transcriptome is dominated by expression of proteolytic enzymes that we speculate are involved both with remodeling the chorioallantoic placenta and histotrophy in the omphaloplacenta. Elements of the maternal innate immune system are downregulated in the pregnant uterus, indicating a potential mechanism to avoid rejection of the embryo. We found a downregulation of major histocompatability complex loci and estrogen and progesterone receptors in the pregnant uterus. This pattern is similar to mammals but cannot be explained by the mammalian model. The latter finding provides evidence that pregnancy is controlled by different endocrinological mechanisms in mammals and reptiles. Finally, 88% of the identified genes are expressed in both the pregnant and the nonpregnant uterus, and thus, morphological and physiological changes associated with C. ocellatus pregnancy are likely a result of regulation of genes continually expressed in the uterus rather than the initiation of expression of unique genes

ER-Targeted Beclin 1 Supports Autophagosome Biogenesis in the Absence of ULK1 and ULK2 Kinases

Autophagy transports cytoplasmic material and organelles to lysosomes for degradation and recycling. Beclin 1 forms a complex with several other autophagy proteins and functions in the initiation phase of autophagy, but the exact role of Beclin 1 subcellular localization in autophagy initiation is still unclear. In order to elucidate the role of Beclin 1 localization in autophagosome biogenesis, we generated constructs that target Beclin 1 to the endoplasmic reticulum (ER) or mitochondria. Our results confirmed the proper organelle-specific targeting of the engineered Beclin 1 constructs, and the proper formation of autophagy-regulatory Beclin 1 complexes. The ULK kinases are required for autophagy initiation upstream of Beclin 1, and autophagosome biogenesis is severely impaired in ULK1/ULK2 double knockout cells. We tested whether Beclin 1 targeting facilitated its ability to rescue autophagosome formation in ULK1/ULK2 double knockout cells. ER-targeted Beclin 1 was most effective in the rescue experiments, while mitochondria-targeted and non-targeted Beclin 1 also showed an ability to rescue, but with lower activity. However, none of the constructs was able to increase autophagic flux in the knockout cells. We also showed that wild type Beclin 1 was enriched on the ER during autophagy induction, and that ULK1/ULK2 facilitated the ER-enrichment of Beclin 1 under basal conditions. The results suggest that one of the functions of ULK kinases may be to enhance Beclin 1 recruitment to the ER to drive autophagosome formation.Peer reviewe

ER-Targeted Beclin 1 Supports Autophagosome Biogenesis in the Absence of ULK1 and ULK2 Kinases

Autophagy transports cytoplasmic material and organelles to lysosomes for degradation and recycling. Beclin 1 forms a complex with several other autophagy proteins and functions in the initiation phase of autophagy, but the exact role of Beclin 1 subcellular localization in autophagy initiation is still unclear. In order to elucidate the role of Beclin 1 localization in autophagosome biogenesis, we generated constructs that target Beclin 1 to the endoplasmic reticulum (ER) or mitochondria. Our results confirmed the proper organelle-specific targeting of the engineered Beclin 1 constructs, and the proper formation of autophagy-regulatory Beclin 1 complexes. The ULK kinases are required for autophagy initiation upstream of Beclin 1, and autophagosome biogenesis is severely impaired in ULK1/ULK2 double knockout cells. We tested whether Beclin 1 targeting facilitated its ability to rescue autophagosome formation in ULK1/ULK2 double knockout cells. ER-targeted Beclin 1 was most effective in the rescue experiments, while mitochondria-targeted and non-targeted Beclin 1 also showed an ability to rescue, but with lower activity. However, none of the constructs was able to increase autophagic flux in the knockout cells. We also showed that wild type Beclin 1 was enriched on the ER during autophagy induction, and that ULK1/ULK2 facilitated the ER-enrichment of Beclin 1 under basal conditions. The results suggest that one of the functions of ULK kinases may be to enhance Beclin 1 recruitment to the ER to drive autophagosome formation.</p