Small GTPases control macropinocytosis of amyloid precursor protein and cleavage to amyloid-ß.

The overproduction of the toxic peptide amyloid-beta (Aß) generated from the cleavage of amyloid precursor protein (APP) is proposed to be a critical event in the development of Alzheimer's disease. Evidence suggests that the cleavage of APP occurs after its internalization from the cell surface. Previously, we identified a novel pathway for APP internalization, which trafficks cell surface APP directly to lysosomes by macropinocytosis, leading to its processing into Aß. We also demonstrated that ADP-ribosylation factor 6 (Arf6) is required for the macropinocytosis of APP. Here, we characterized the roles of Arf6's downstream effectors Rac1, Cdc42 and RhoA. Both pharmacological inhibition and siRNA knockdown of these proteins reduced the amount of APP colocalized with LAMP1-labeled lysosomes without affecting APP transport to early endosomes. Decreases in the production of both Aß40 and Aß42 were also observed by ELISA in response to inhibitor treatment. These findings together demonstrate that Rac1, Cdc42 and RhoA are components of the mechanism regulating the macropinocytosis of APP and targeting these components can reduce the production of Aß.

Subchronic exposure to environmentally relevant concentrations of di-(2-ethylhexyl) phthalate differentially affects the colon and ileum in adult female mice.

Di(2-ethylhexyl) phthalate (DEHP) is a large-molecular-weight phthalate added to plastics to impart versatile properties. DEHP can be found in medical equipment and devices, food containers, building materials, and children's toys. Although DEHP exposure occurs most commonly by ingesting contaminated foods in the majority of the population, its effects on the gastrointestinal tract have not been well studied. Therefore, we analyzed the effects of subchronic exposure to DEHP on the ileum and colon morphology, gene expression, and immune microenvironment. Adult C57BL/6 female mice were orally dosed with corn oil (control, n = 7) or DEHP (0.02, 0.2, or 30 mg/kg, n = 7/treatment dose) for 30-34 days. Mice were euthanized during diestrus, and colon and ileum tissues were collected for RT-qPCR and immunohistochemistry. Subchronic DEHP exposure in the ileum altered the expression of several immune-mediating factors (Muc1, Lyz1, Cldn1) and cell viability factors (Bcl2 and Aifm1). Similarly, DEHP exposure in the colon impacted the gene expression of factors involved in mediating immune responses (Muc3a, Zo2, Ocln, Il6, and Il17a); and also altered the expression of cell viability factors (Ki67, Bcl2, Cdk4, and Aifm1) as well as a specialized epithelial cell marker (Vil1). Immunohistochemical analysis of the ileum showed DEHP increased expression of VIL1, CLDN1, and TNF and decreased number of T-cells in the villi. Histological analysis of the colon showed DEHP altered morphology and reduced cell proliferation. Moreover, in the colon, DEHP increased the expression of MUC2, MUC1, VIL1, CLDN1, and TNF. DEHP also increased the number of T-cells and Type 2 immune cells in the colon. These data suggest that subchronic DEHP exposure differentially affects the ileum and colon and alters colonic morphology and the intestinal immune microenvironment. These results have important implications for understanding the effects of DEHP on the gastrointestinal system.

Prenatal exposure to an environmentally relevant phthalate mixture alters ovarian steroidogenesis and folliculogenesis in the F1 generation of adult female mice.

Phthalates are a family of chemicals that can be found in plastic and personal care products used by consumers every day and they are known endocrine disrupting chemicals that can disrupt female reproduction. In previous studies, an environmentally relevant phthalate mixture was shown to affect female reproduction in a transgenerational manner. However, limited information was available on the effect of phthalate mixtures on ovarian steroidogenesis and folliculogenesis. Ovarian steroidogenesis is important for producing hormones needed for reproduction and ovarian regulation, and folliculogenesis is essential for the development of ovarian follicles and successful fertility. Thus, this study tested the hypothesis that prenatal exposure to an environmentally relevant phthalate mixture adversely affects ovarian steroidogenesis and folliculogenesis in the F1 generation of adult female mice. Pregnant dams (F0 generation) were orally dosed with vehicle control or a phthalate mixture (20 µg/kg/day-500 mg/kg/day) daily from gestational day 10 to birth, and the adult F1 females were the offspring of the dosed dams. The ovaries of the F1 generation were collected at postnatal day 60. One ovary was used for histological examination of the numbers and percent of different follicle types. The other ovary was used to measure expression of theca and granulosa cell enzymes. Additionally, sera were collected for measuring hormone levels. The results indicate that prenatal exposure to the phthalate mixture decreases hormone levels and gene expression, alters the transitioning of follicle types, and leads to a higher incidence of atresia in the F1 generation offspring.

The Impact of Di-Isononyl Phthalate Exposure on Specialized Epithelial Cells in the Colon.

Di-isononyl phthalate (DiNP) is a high-molecular-weight phthalate commonly used as a plasticizer for polyvinyl chloride and other end products, such as medical devices and construction materials. Most of our initial exposure to DiNP occurs by ingestion of DiNP-contaminated foods. However, little is known about the effects of DiNP on the colon. Therefore, the goal of this study was to test the hypothesis that DiNP exposure alters immune responses and impacts specialized epithelial cells in the colon. To test this hypothesis, adult female mice were orally dosed with corn-oil vehicle control or doses of DiNP ranging from 20 µg/kg/d to 200 mg/kg/d for 10-14 days. After the dosing period, mice were euthanized in diestrus, and colon tissues and sera were collected for histological, genomic, and proteomic analysis of various immune factors and specialized epithelial cells. Subacute exposure to DiNP significantly increased protein levels of Ki67 and MUC2, expression of a Paneth cell marker (Lyz1), and estradiol levels in sera compared with control. Gene expression of mucins (Muc1, Muc2, Muc3a, and Muc4), Toll-like receptors (Tlr4 and Tlr5), and specialized epithelial cells (ChgA, Lgr5, Cd24a, and Vil1) were not significantly different between treatment groups and control. Cytokine levels of IL-1RA and CXCL12 were also not significantly different between DiNP treatment groups and control. These data reveal that DiNP exposure increases circulating estradiol levels and gene expression in specialized epithelial cells with immune response capabilities (eg, goblet and Paneth cells) in the mouse colon, which may initiate immune responses to prevent further damage in the colon.

An evaluation of treatment options for medial, midshaft, and distal clavicle fractures: a systematic review and meta-analysis.

BACKGROUND: The majority of clavicle fractures are midshaft injuries, although fractures of the distal or medial fragment also occur. The aim of this study was to review the current evidence on these injuries to help inform future treatment plans. METHODS: We searched for studies comparing interventions for medial, midshaft, or distal clavicle fractures; however, we did not identify any comparative studies on medial fractures and performed a secondary search on this topic. We conducted Bayesian network meta-analyses, although this was not feasible with studies on medial fractures and we described their results qualitatively. RESULTS: For midshaft fractures, we found statistically significant improvements in function and time to radiographic union with plating, an elastic stable intramedullary nail (ESIN), and the Sonoma CRx intramedullary nail over nonoperative treatments. Both plating and an ESIN also showed significantly lower risks of nonunion and malunion relative to nonoperative methods. For distal fractures, a locking plate (LP) with or without coracoclavicular (CC) suturing yielded significantly better outcomes over K-wires with or without tension bands, CC suturing alone, an LP with a CC screw, a hook plate, and a sling. For medial fractures, plating may result in more favorable functional and union-related outcomes, although implant irritation may occur. In addition, K-wires, tension bands, and a screw with sutures demonstrated success when plating was technically not feasible in a few cases, whereas treatment with a sling may result in reduced function and a higher risk of complications relative to surgery. CONCLUSION: This study can provide guidance on the management of medial, midshaft, and distal clavicle fractures. The current evidence suggests that plating, an ESIN, and a CRx intramedullary nail are all good options for midshaft fractures; an LP with or without CC suturing should be preferred for distal fractures; and plating is also acceptable for medial fractures, provided that the patient is deemed suitable for surgery and has the adequate bone stock and sufficiently sized medial fragment necessary to implant the device. Patient preferences for certain outcomes should be considered, which may result in different treatment recommendations.

Photoinduced Copper-Catalyzed Coupling of Terminal Alkynes and Alkyl Iodides.

We have developed a photoinduced copper-catalyzed alkylation of terminal alkynes with primary, secondary, or tertiary alkyl iodides as electrophiles. The reaction has a broad substrate scope and can be successfully performed in the presence of ester, nitrile, aryl halide, ketone, sulfonamide, epoxide, alcohol, and amide functional groups. The alkylation is promoted by blue light (λ≈450 nm) and proceeds at room temperature in the absence of any additional metal catalysts. The use of a terpyridine ligand is essential for the success of the reaction and is shown to prevent photoinduced copper-catalyzed polymerization of the starting materials.

Arf6 controls beta-amyloid production by regulating macropinocytosis of the Amyloid Precursor Protein to lysosomes.

Alzheimer's disease (AD) is characterized by the deposition of Beta-Amyloid (Aß) peptides in the brain. Aß peptides are generated by cleavage of the Amyloid Precursor Protein (APP) by the ß - and γ - secretase enzymes. Although this process is tightly linked to the internalization of cell surface APP, the compartments responsible are not well defined. We have found that APP can be rapidly internalized from the cell surface to lysosomes, bypassing early and late endosomes. Here we show by confocal microscopy and electron microscopy that this pathway is mediated by macropinocytosis. APP internalization is enhanced by antibody binding/crosslinking of APP suggesting that APP may function as a receptor. Furthermore, a dominant negative mutant of Arf6 blocks direct transport of APP to lysosomes, but does not affect classical endocytosis to endosomes. Arf6 expression increases through the hippocampus with the development of Alzheimer's disease, being expressed mostly in the CA1 and CA2 regions in normal individuals but spreading through the CA3 and CA4 regions in individuals with pathologically diagnosed AD. Disruption of lysosomal transport of APP reduces both Aß40 and Aß42 production by more than 30 %. Our findings suggest that the lysosome is an important site for Aß production and that altering APP trafficking represents a viable strategy to reduce Aß production.