Optimized dispersion of nanoparticles for biological in vitro and in vivo studies

Background: The aim of this study was to establish and validate a practical method to disperse nanoparticles in physiological solutions for biological in vitro and in vivo studies. Results: TiO(2) (rutile) dispersions were prepared in distilled water, PBS, or RPMI 1640 cell culture medium. Different ultrasound energies, various dispersion stabilizers (human, bovine, and mouse serum albumin, Tween 80, and mouse serum), various concentrations of stabilizers, and different sequences of preparation steps were applied. The size distribution of dispersed nanoparticles was analyzed by dynamic light scattering and zeta potential was measured using phase analysis light scattering. Nanoparticle size was also verified by transmission electron microscopy. A specific ultrasound energy of 4.2 x 10(5) kJ/m(3) was sufficient to disaggregate TiO(2) (rutile) nanoparticles, whereas higher energy input did not further improve size reduction. The optimal sequence was first to sonicate the nanoparticles in water, then to add dispersion stabilizers, and finally to add buffered salt solution to the dispersion. The formation of coarse TiO(2) (rutile) agglomerates in PBS or RPMI was prevented by addition of 1.5 mg/ml of human, bovine or mouse serum albumin, or mouse serum. The required concentration of albumin to stabilize the nanoparticle dispersion depended on the concentration of the nanoparticles in the dispersion. TiO(2) (rutile) particle dispersions at a concentration lower than 0.2 mg/ml could be stabilized by the addition of 1.5 mg/ml albumin. TiO(2) (rutile) particle dispersions prepared by this method were stable for up to at least 1 week. This method was suitable for preparing dispersions without coarse agglomerates (average diameter < 290 nm) from nanosized TiO(2) (rutile), ZnO, Ag, SiO(x), SWNT, MWNT, and diesel SRM2975 particulate matter. Conclusion: The optimized dispersion method presented here appears to be effective and practicable for preparing dispersions of nanoparticles in physiological solutions without creating coarse agglomerates

Proteomic Analysis of the Luteal Endometrial Secretome

Endometrium attains a secretory architecture in preparation for embryo implantation, but the identity of most endometrial secretory products remains unknown. Our objective was to characterize the endometrial secretome and compare protein expression between prereceptive (luteinizing hormone [LH]+4) receptive (LH+9) and phase endometrium. Endometrial lavage was performed in 11 participants and analyzed by difference gel electrophoresis (DIGE). LH+4 and LH+9 specimens were labeled with cyanine fluorescent dyes Cy3 and Cy5 tags, respectively, and combined. Proteins were separated using 2-dimensional gel electrophoresis, isolated, trypsin-digested, and subjected to mass spectrometry. In all, 152 proteins were identified; 82 were differentially expressed. Most proteins with increased expression on LH+9 functioned in host defense, while proteins with decreased expression had many functions. A total of 14 proteins had changes suggesting altered posttranslational modification. This article describes the first application of proteomic analysis to endometrial secretions, allowing identification of novel endometrial proteins as well as those differentially secreted in prereceptive and receptive phases

Structure and functional relevance of the Slit2 homodimerization domain

Slit proteins are secreted ligands that interact with the Roundabout (Robo) receptors to provide important guidance cues in neuronal and vascular development. Slit–Robo signalling is mediated by an interaction between the second Slit domain and the first Robo domain, as well as being dependent on heparan sulphate. In an effort to understand the role of the other Slit domains in signalling, we determined the crystal structure of the fourth Slit2 domain (D4) and examined the effects of various Slit2 constructs on chick retinal ganglion cell axons. Slit2 D4 forms a homodimer using the conserved residues on its concave face, and can also bind to heparan sulphate. We observed that Slit2 D4 frequently results in growth cones with collapsed lamellipodia and that this effect can be inhibited by exogenously added heparan sulphate. Our results show that Slit2 D4–heparan sulphate binding contributes to a Slit–Robo signalling mechanism more intricate than previously thought

A work group report on ultrafine particles (American Academy of Allergy, Asthma & Immunology): Why ambient ultrafine and engineered nanoparticles should receive special attention for possible adverse health outcomes in human subjects

Ultrafine particles (UFPs) are airborne particulates of less than 100 nm in aerodynamic diameter. Examples of UFPs are diesel exhaust particles, products of cooking, heating, and wood burning in indoor environments, and, more recently, products generated through the use of nanotechnology. Studies have shown that ambient UFPs have detrimental effects on both the cardiovascular and respiratory systems, including a higher incidence of atherosclerosis and exacerbation rate of asthma. UFPs have been found to alter and responses of the immune system to allergens and can also play a role in allergen sensitization. The inflammatory properties of UFPs can be mediated by a number of different mechanisms, including the ability to produce reactive oxygen species, leading to the generation of proinflammatory cytokines and airway inflammation. In addition, because of their small size, UFPs also have unique distribution characteristics in the respiratory tree and circulation and might be able to alter cellular function in ways that circumvent normal signaling pathways. Additionally, UFPs can penetrate intracellularly and potentially cause DNA damage. The recent advances in nanotechnology, although opening up new opportunities for the advancement of technology and medicine, could also lead to unforeseen adverse health effects in exposed human subjects. Further research is needed to clarify the safety of nanoscale particles, as well as the elucidation of the possible beneficial use of these particulates to treat disease

Endometrial CXCL13 Expression Is Cycle Regulated in Humans and Aberrantly Expressed in Humans and Rhesus Macaques With Endometriosis

C-X-C ligand 13 (CXCL13), a regulator of mucosal immunity, is secreted by human endometrial epithelium and may be involved in embryo implantation. However, cyclic expression of human endometrial CXCL13 in health and disease is not well studied. This study examines cycle stage-specific endometrial CXCL13 expression in normal humans when compared to those with biopsy-confirmed, stage 1 to 4 endometriosis using real-time reverse transcriptase, real-time polymerase chain reaction and immunohistochemistry. Eutopic endometrial CXCL13 expression was also compared between normal, control Rhesus macaques, and macaques with advanced endometriosis. In healthy women, CXLC13 messenger RNA expression was minimal in the proliferative phase and maximal in the secretory phase. However, in the presence of endometriosis, proliferative-phase endometrial expression markedly increased in both humans and rhesus subjects (P < .05). The cross-species and cross-stage concordance suggests a pathophysiologic role for CXCL13 in endometriosis and its use as a biomarker for disease

Plasminogen Activator Inhibitor-1 Promotes Neutrophil Infiltration and Tissue Injury on Ischemia–Reperfusion

Objective Ischemia-reperfusion (I/R) injury significantly contributes to organ dysfunction and failure after myocardial infarction, stroke, and transplantation. In addition to its established role in the fibrinolytic system, plasminogen activator inhibitor-1 has recently been implicated in the pathogenesis of I/R injury. The underlying mechanisms remain largely obscure. Approach and Results Using different in vivo microscopy techniques as well as ex vivo analyses and in vitro assays, we identified that plasminogen activator inhibitor-1 rapidly accumulates on microvascular endothelial cells on I/R enabling this protease inhibitor to exhibit previously unrecognized functional properties by inducing an increase in the affinity of 2 integrins in intravascularly rolling neutrophils. These events are mediated through low-density lipoprotein receptor-related protein-1 and mitogen-activated protein kinase-dependent signaling pathways that initiate intravascular adherence of these immune cells to the microvascular endothelium. Subsequent to this process, extravasating neutrophils disrupt endothelial junctions and promote the postischemic microvascular leakage. Conversely, deficiency of plasminogen activator inhibitor-1 effectively reversed leukocyte infiltration, microvascular dysfunction, and tissue injury on experimental I/R without exhibiting side effects on microvascular hemostasis. Conclusions Our experimental data provide novel insights into the nonfibrinolytic properties of the fibrinolytic system and emphasize plasminogen activator inhibitor-1 as a promising target for the prevention and treatment of I/R injury

G Protein-Coupled Estrogen Receptor (GPER) Expression in Normal and Abnormal Endometrium

Rapid estrogen effects are mediated by membrane receptors, and evidence suggests a role for both a membrane-associated form of estrogen receptor alpha (ESR1; ERα) and G-protein coupled receptor 30 (GPER; GPR30). Considering estrogen’s importance in endometrial physiology and endometriosis pathophysiology, we hypothesized that GPER could be involved in both cyclic changes in endometrial estrogen action and that aberrant expression might be seen in the eutopic endometrium of women with endometriosis. Using real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemical analysis of normal endometrium, endometrial samples demonstrated cycle-regulated expression of GPER, with maximal expression in the proliferative phase. Eutopic and ectopic endometrium from women with endometriosis overexpressed GPER as compared to eutopic endometrium of normal participants. Ishikawa cells, an adenocarcinoma cell line, expressed GPER, with increased expression upon treatment with estrogen or an ESR1 agonist, but not with a GPER-specific agonist. Decreased expression was seen in Ishikawa cells stably transfected with progesterone receptor A. Together, these data suggest that normal endometrial GPER expression is cyclic and regulated by nuclear estrogen and progesterone receptors, while expression is dysregulated in endometriosis

Antibacterial Solution Using Cinnamomum verum (Cinammon) and Cympobogon citratus (Lemongrass) Essential Oils with Hydrogen Peroxide Against Staphylococcus aureus and Escherichia coli

Amidst the pandemic, millions of impoverished Filipinos lack sufficient access to sanitation services that protect them from bacterial infections. As such, this study endeavored to formulate a disinfectant spray of hydrogen peroxide and varying concentrations of lemongrass (Cymbopogon citratus) and cinnamon (Cinnamomum verum) essential oils to produce a maximally effective solution. Due to their natural origin, these essential oils were chosen for their extensive antibacterial properties, affordable price, and low toxicity levels. Six disinfectant spray solutions containing different concentrations of either essential oil were tested against Staphylococcus aureus and Escherichia coli bacteria through the agar disk diffusion method. After analyzing the data using mean, standard deviation, and MANOVA, it was found that including cinnamon and lemongrass essential oil had no statistically significant effect on the antibacterial activity of the hydrogen peroxide solution, regardless of the concentration of essential oil used, p \u3e 0.05. However, the lemongrass samples were slightly more effective than the cinnamon solutions. Thus, future researchers are encouraged to investigate other components that can potentially increase the antibacterial activity of a disinfectant spray