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Nanocrystallization can improve the dissolvability of insoluble medicines in water, making them easier to administer. In the present study, sirolimus (SRL) was nanocrystallized, and the transport mechanism and efficacy of both formulations were compared in vitro and in vivo. The results showed that the 120 nm sirolimus nanocrystals (SRL NCs) had better ability to pass through the Caco-2 cell monolayer. SRL NCs were uptaken by Caco-2 cells via multiple endocytosis pathways, and the endoplasmic reticulum (ER), Golgi apparatus, and microtubules were identified as vital organelles for expelling SRL NCs out of the cells. SRL NCs decreased CD4+/CD8+ in normal mice after 14 days of daily gavage administration. Furthermore, SRL NCs enhanced the immune tolerance in an ovalbumin (OVA)-sensitized mouse model. SRL NCs significantly increased the percentage of Tregs (CD25+Foxp3+) among CD4+T cells. Collectively, these findings demonstrate that nanocrystallization of SRL enhances oral bioavailability, transcytosis, and...

Edwin Sia Sien Aun1, Joanne Yeo Suan Hui1, Julie Wong Wei Ming1, Jenny Choo Cheng Yi1, Changi Wong1, Aazani Mujahid2, Moritz Muller1*

MicroRNAs (miRNAs) are small RNAs that posttranscriptionally regulate gene expression. Their aberrant expression is commonly linked with diseased states, including hepatitis C virus (HCV) infection. Herein, we demonstrate that HCV replication induces the expression of miR-27 in cell culture and in vivo HCV infectious models. Overexpression of the HCV proteins core and NS4B independently activates miR-27 expression. Furthermore, we establish that miR-27 overexpression in hepatocytes results in larger and more abundant lipid droplets, as observed by coherent anti-Stokes Raman scattering (CARS) microscopy. This hepatic lipid droplet accumulation coincides with miR-27b's repression of peroxisome proliferator-activated receptor (PPAR)-α and angiopoietin-like protein 3 (ANGPTL3), known regulators of triglyceride homeostasis. We further demonstrate that treatment with a PPAR-α agonist, bezafibrate, is able to reverse the miR-27b-induced lipid accumulation in Huh7 cells. This miR-27b-mediated repression of PPAR-α signaling represents a novel mechanism of HCV-induced hepatic steatosis. This link was further demonstrated in vivo through the correlation between miR-27b expression levels and hepatic lipid accumulation in HCV-infected SCID-beige/Alb-uPa mice. Conclusion: Collectively, our results highlight HCV's up-regulation of miR-27 expression as a novel mechanism contributing to the development of hepatic steatosis. (Hepatology 2014;58:98–108)

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins offer a breakthrough platform for cheap, programmable, and effective sequence-specific DNA targeting. The CRISPR-Cas system is naturally equipped for targeted DNA cutting through its native nuclease activity. As such, groups researching a broad spectrum of biological organisms have quickly adopted the technology with groundbreaking applications to genomic sequence editing in over 20 different species. However, the biological code of life is not only encoded in genetics but also in epigenetics as well. While genetic sequence editing is a powerful ability, we must also be able to edit and regulate transcriptional and epigenetic code. Taking inspiration from work on earlier sequence-specific targeting technologies such as zinc fingers (ZFs) and transcription activator-like effectors (TALEs), researchers quickly expanded the CRISPR-Cas toolbox to include transcriptional activation, repression, and epigenetic modification. In this review, we highlight advances that extend the CRISPR-Cas toolkit for transcriptional and epigenetic regulation, as well as best practice guidelines for these tools, and a perspective on future applications.

Purpose: To establish if impression cytology combined with histochemical and immunocytochemical staining can be used to assess epithelium of the human upper lid margin. Methods: Following an initial eye examination of 40 healthy subjects (19 soft contact lens wearers and 21 non-contact lens wearers, aged 18–57 years), lid margin staining was assessed with lissamine green using slit lamp biomicroscopy and graded (grade 0–3). Impression cytology of the upper lid margin of both eyes was collected, fixed and stained with periodic acid Schiff (PAS) and haematoxylin for cell morphology analysis (Nelson grade) or for immunocytochemistry (keratinization-related proteins: filaggrin, transglutaminase1 (TGase1) and cytokeratin 1/10). Results: In 57% of all subjects, grade 0 lissamine green staining showed a thin line (the Marx line), just posterior to the meibomian gland ducts. Grade 2 or 3 lissamine green staining was observed in 17% of all subjects. There was no difference between contact lens and non-contact lens wearers for lid margin staining or Nelson grade (p = 0.4, Fisher’s exact test). PAS/haematoxylin staining and immunocytochemistry showed transition in epithelial cell morphology, with marginal conjunctival epithelium, mucocutaneous junction and squamous epithelium, adjacent to meibomian gland ducts. This transition in epithelium was associated with differential expression of keratinization-related proteins (filaggrin, cytokeratin 1/10 and TGase1). Conclusion: Lid margin epithelium can be successfully sampled using impression cytology and further characterized using histochemistry and immunocytochemistry staining techniques. This approach can be applied to assess lid margin changes in conditions such as dry eye and meibomian gland dysfunction.

Tombusviruses express a 19 kDa protein (p19) that, as a dimeric protein, suppresses the RNAs silencing pathway during infection by binding short-interfering RNA (siRNA) and preventing their association with the RNA-induced silencing complex (RISC). The p19 protein can bind to both endogenous and synthetic siRNAs with a high degree of size selectivity but with little sequence dependence. It also binds to other endogenous small RNAs such as microRNAs (miRNAs) but with lower affinity than to canonical siRNAs. It has become apparent, however, that miRNAs play a large role in gene regulation; their influence extends to expression and processing that affects virtually all eukaryotic processes. In order to develop new tools to study endogenous small RNAs, proteins that suppress specific miRNAs are required. Herein we describe mutational analysis of the p19 binding surface with the aim of creating p19 mutants with increased affinity for miR-122. By site-directed mutagenesis of a single residue, we describe p19 mutants with a nearly 50-fold increased affinity for miR-122 without altering the affinity for siRNA. Upon further mutational analysis of this site, we postulate that the higher affinity relies on hydrogen-bonding interactions but can be sterically hindered by residues with bulky side chains. Finally, we demonstrate the effectiveness of a mutant p19, p19-T111S, at sequestering miR-122 in human hepatoma cell lines, as compared to wild-type p19. Overall, our results suggest that p19 can be engineered to enhance its affinity toward specific small RNA molecules, particularly noncanonical miRNAs that are distinguishable based on locations of base-pair mismatches. The p19-T111S mutant also represents a new tool for the study of the function of miR-122 in post-transcriptional silencing in the human liver. © 2011 American Chemical Society.

Carnation Italian Ringspot virus (CIRV) has evolved a protein called p19 that acts as a suppressor of RNA silencing in the host cell and aids in viral persistence. This protein has been shown to be sensitive to cysteine alkylation resulting in a reduction in its ability to bind to short-interfering RNA (siRNA). To determine the sites within the protein that are sensitive to alkylation, we systematically tested the functional role of each cysteine residue using site-directed mutagenesis. Variants of the p19 protein were created at locations C110, C134 and C160 where the cysteines were replaced by an inert amino acid such as serine or isoleucine. The results from activity measurements of the purified mutant p19 proteins indicate that the mutants maintain the ability to bind siRNAs with nanomolar affinity, however, their stabilities, as measured by circular dichroism (CD), vary. Functional studies in the presence of the cysteine alkylating agent N-ethylmaleimide (NEM) indicated that p19's ability to bind siRNAs and act as a suppressor of RNA silencing is sensitive to alkylation at all three cysteine residues with the maximum effects occurring when C110 and C134 are both alkylated. These results suggest that the role of the cysteine amino acid conservation is likely to preserve the overall structural integrity of p19 for optimal thermostability and subsequent siRNA-binding activity. We find that p19 function is maximally compromised at high levels of thiol alkylation or in an oxidizing environment.

Tombusviruses use a 19 kDa protein (p19) as a suppressor of the RNA silencing pathway during infection. The p19 protein binds to short-interfering RNA (siRNA) as a dimer and shows a high selectivity for short duplex RNAs over other RNA species. Since p19 can bind to synthetic and RNA silencing generated small RNAs with little sequence dependence and with size selectivity, this protein has utility as a tool for studying RNA silencing pathways in eukaryotes. However, the ability of p 19 to serve as a tool for studying RNA silencing pathways may be complicated by the presence of other endogenous small RNAs such as micro-RNAs (miRNAs). To understand the importance of endogenous small RNA components with respect to pl9's ability to bind to siRNAs, we examined the interactions of p19 with human miR-122, a 23-nucleotide duplex miRNA containing several mismatched base pairs that is highly abundant in the liver. The binding characteristics were compared with those of an siRNA optimized against the human kinase CSK. The binding studies were performed using fluorescence polarization experiments on duplex oligonucleotides containing Cy3 dye labels at the 5'-end of one of the strands of RNA as well as electrophoretic gel mobility shift assays. Both methods indicate that the synthetic siRNA with no mismatches in base pairing bound with >3-fold selectivity over that of miR-122. Our results suggest that pl9 can distinguish between siRNAs and miRNA species, although the difference in binding constants is not so large that interactions with endogenous miRNAs can be totally ignored.

Background and Aim: Non-alcoholic fatty liver disease is the result of an imbalance in hepatic lipid partitioning that favors fatty acid synthesis and storage over fatty acid oxidation and triglyceride secretion. The progressive, inflammatory disorder of steatohepatitis can be prevented or reversed by correcting this lipid imbalance by activating peroxisome proliferator-activated receptor (PPAR) α, a transcription factor which regulates fatty acid oxidation. n-3 polyunsaturated fatty acids (PUFA), such as those found in fish oil (FO), are naturally occurring PPARα ligands which also suppress lipid synthesis. Methods: We tested the role of dietary activation of PPARα by feeding mice a n-3 PUFA-enriched FO diet in the methionine and choline deficient (MCD) model of steatohepatitis. Results were compared with mice fed the corresponding diet supplemented with monounsaturated fatty acids as olive oil (OO). Results: As expected, FO feeding led to robust hepatic PPARα activation in control mice, and decreased expression of genes involved with fatty acid synthesis. Such lipolytic gene expression profile was also clearly evident in FO MCD-fed mice, and was associated with reduced hepatic lipid accumulation in comparison with mice fed OO MCD diet. FO feeding in control mice also caused marked hepatic accumulation of lipoperoxides compared with OO and chow-fed mice. This was further exacerbated in FO MCD-fed animals, which developed steatohepatitis characterized by mild steatosis and moderate inflammation in comparison with OO MCD-fed mice; such inflammatory recruitment was not related to NF-κB activation or enhanced cyclooxygenase-2 activity. Conclusions: Feeding an n-3 PUFA-enriched diet activated PPARα and suppressed hepatic de novo lipogenesis, but failed to prevent development of steatohepatitis in the presence of methionine and choline deficiency. Instead, the very high levels of hepatic lipoperoxides may have abrogated the protection that would otherwise be conferred by PPARα activation, and could also be responsible for lipotoxic hepatocellular injury and inflammatory recruitment.

Author(s): Cheng, Tik-Chee Jenny | Abstract: Heart failure, a progressive condition that is typically undetected and often misdiagnosed, is the leading cause of deaths around the world. The disorder is characterized by the inability of the heart to keep up with the body’s demand for blood circulation. It is initiated by structural and functional changes from molecular to systemic levels that result in compensatory physiological changes, commonly termed cardiac remodeling. At first, this remodeling enables the heart to increase its cardiac output to compensate for the change. However, over time, remodeling results in cardiomyopathy, a condition with enlarged left ventricles and/or dilated ventricular walls that can lead to dysfunction and inadequate pumping of blood to the rest of the body.

Symmetric division of Gram-negative bacteria depends on the combined action of three proteins that ensure correct positioning of the cell division septum, namely, MinC, MinD, and MinE. To achieve this function, MinC and MinD form a membrane-bound complex that blocks cell division at all potential sites. Opposing this inhibition is MinE, which interacts with MinD via its N-terminal anti-MinCD domain to site-specifically counter the action of the MinCD complex. The anti-MinCD domain has been proposed to bind MinD in a helical conformation; however, little is actually known about the structure of this functionally critical region. To understand how MinE can perform its anti-MinCD function, we have therefore investigated the conformation of the full-length MinE from Neisseria gonorrhoeae by solution NMR. Although solubility considerations required the use of sample conditions that limit the observation of amide resonances to regions that are protected from solvent exchange, backbone chemical shifts from both N- and C-terminal domains could be assigned. In contrast to previous models, secondary chemical shift analysis of these solvent-protected regions shows that parts of the N-terminal anti-MinCD domain are stably folded with many functionally important residues localizing to a beta-structure. In addition, this N-terminal domain may be interacting with the C-terminal topological specificity domain, since mutations made in one domain led to NMR spectral changes in both domains. The nonfunctional MinE mutant L22D showed even larger evidence of structural perturbations in both domains, with significant destabilization of the entire MinE structure. Overall, these results suggest that there is an intimate structural association between the anti-MinCD and topological specificity domains, allowing the functional properties of the two domains to be modulated through this interaction.

This paper details a preliminary modeling study of three beach fill projects in Florida, based on planform modeling using a one line numerical model and analytical modeling techniques. The study provides two different approaches to modeling (shoreline response and volumetric response per linear measure of beach length) to compare beach performance prediction with actual measurement data. The analytical modeling theory and the one line numerical model comparison includes one hypothetical case and differences between the two types of model results are noted. A discussion of performance prediction results with actual project measured performance for the three beach fill projects is provided.

MicroRNAs (miRNAs) are small RNAs that posttranscriptionally regulate gene expression. Their aberrant expression is commonly linked with diseased states, including hepatitis C virus (HCV) infection. Herein, we demonstrate that HCV replication induces the expression of miR-27 in cell culture and in vivo HCV infectious models. Overexpression of the HCV proteins core and NS4B independently activates miR-27 expression. Furthermore, we establish that miR-27 overexpression in hepatocytes results in larger and more abundant lipid droplets, as observed by coherent anti-Stokes Raman scattering (CARS) microscopy. This hepatic lipid droplet accumulation coincides with miR-27b's repression of peroxisome proliferator-activated receptor (PPAR)-α and angiopoietin-like protein 3 (ANGPTL3), known regulators of triglyceride homeostasis. We further demonstrate that treatment with a PPAR-α agonist, bezafibrate, is able to reverse the miR-27b-induced lipid accumulation in Huh7 cells. This miR-27b-mediated repression of PPAR-α signaling represents a novel mechanism of HCV-induced hepatic steatosis. This link was further demonstrated in vivo through the correlation between miR-27b expression levels and hepatic lipid accumulation in HCV-infected SCID-beige/Alb-uPa mice.Collectively, our results highlight HCV's up-regulation of miR-27 expression as a novel mechanism contributing to the development of hepatic steatosis.

MicroRNAs (miRNAs) are an emerging class of biomarkers that are frequently deregulated in cancer cells and have shown a great promise for cancer classification and prognosis. In this work, we developed a three-way electrochemical sensor for detection and quantitation of ultra-low amounts of miRNAs in a wide dynamic range of measured concentrations. The sensor facilitates three detection modes based on hybridization (H-SENS), p19 protein binding (P-SENS) and protein displacement (D-SENS). The combined HPD sensor (HPD-SENS) identifies as low as 5 aM or 90 molecules of miRNA per 30\muL of sample without PCR amplification, and can be operated within the dynamic range from 10 aM to 1 \muM. The HPD senor is made on a commercially-available gold nanoparticles-modified electrode and suitable for analysing multiple miRNAs on a single electrode. This three-way sensor exhibits high selectivity and specificity and was successfully used for sequential analysis of miR-32 and miR-122 on one electrode. Furthermore, the P-SENS was employed for profiling of three endogenous microRNAs (miR-21, miR-32 and miR-122) in human blood serum., This paper has been withdrawn by the author due to errors

Cyclic nitrones react readily with cyclooctyne (II) to afford isoxazoline adducts in high yields.

Strain-promoted cycloadditions of cyclic nitrones with cyclooctynes proceed with rate constants up to 3.38 ± 0.31 M -1 s -1 in CD 3CN, or 59 times faster than the analogous reaction of azides. This highly specific modular labeling strategy can be applied for direct labeling of proteins and for live cell imaging of cancer cells. © The Royal Society of Chemistry 2011.

MicroRNAs (miRNAs) are endogenous posttranscriptional regulators found in all metazoa and play crucial roles in virtually all cellular processes. Their aberrant expression has been linked to several diseased states; therefore, techniques capable of sensitive and specific profiling of the miRNA milieu will have significant application in prognostics, diagnostics, and therapeutics. Here we present a method for rapid quantification of miRNA levels using p19, a tombusvirus-encoded suppressor of RNA interference with sequence-independent and size-selective affinity toward 19-bp RNA duplexes. We present a surface plasmon resonance (SPR)-based miRNA sensing method where RNA probes are immobilized on gold surfaces demonstrating p19's utility in recognition of miRNA-bound probes. This allows detection of miRNAs in the low nanomolar range. To increase the sensitivity, a bead-based enzyme immunoassay was performed, and this technique displays a lower detection limit of 1 fmol and a linear dynamic range from 1 pmol to 1 fmol. © 2011 Published by Elsevier Inc. All rights reserved.

Eukaryotes have evolved complex cellular responses to double-stranded RNA. One response that is highly conserved across many species is the RNA silencing pathway. Tombusviruses have evolved a mechanism to evade the RNA silencing pathway that involves a small protein, p19, that acts as a suppressor of RNA silencing. This protein binds specifically to small-interfering RNAs (siRNAs) with nanomolar affinity in a sequence-independent manner and with size selectivity. Here we demonstrate a new approach for rapidly determining the quantities of siRNA using fluorescence resonance energy transfer (FRET) between the Carnation Italian ringspot virus (CIRV) p19-CFP fusion protein and Cy3-labeled siRNA. The CIRV p19 fusion protein binds double-stranded siRNAs with nanomolar affinity as determined by FRET. [corrected]

Here we report that the phenanthridine derivative covalently linked to a fluorescein moiety (FLEth) can act as a fluorescence based probe for duplex short interfering RNA (siRNA) and that this probe can also be used to report on protein-RNA interactions. A fluorescence resonance energy transfer (FRET) signal that is observed at 600 nm occurs when FLEth is complexed with siRNA. At least 2 molecules of FLEth can bind to 21 nt duplex siRNA, and the dissociation constants for these interactions are reported. We find that FLEth can also report on the interaction of siRNAs with the Carnation Italian ringspot viral suppressor of RNA silencing p19. FLEth does not bind to the siRNA-p19 complex nor can p19 bind to the siRNA-FLEth complex; rather FLEth can report on the fraction of siRNA that is unbound. FLEth can also bind siRNA in delivery systems such as liposomes. Once the siRNA reaches the interior of Huh 7.5 cells, FLEth dissociates from the siRNA and is found in the nucleoli suggesting that FLEth cannot bind to siRNAs that are associated with the RNA silencing machinery.

Eukaryotes have evolved complex cellular responses to double-stranded RNA including the RNA silencing pathway. Tombusviruses have adapted a mechanism to evade RNA silencing that involves a 19 kDa dimeric protein (p19) that is a suppressor of RNA silencing. In order to develop stabilized p19 proteins, linked versions of p19 from the Carnation Italian Ringspot virus (CIRV) were constructed that joined the C-terminus of one subunit to the N-terminus of the second subunit. Like the native CIRV p19, these linked p19 proteins were able to bind to double-stranded siRNAs with nanomolar affinity and discriminate siRNA according to length. In addition, the interdomain linker improved both the stability and binding properties of the p19 dimer. The observed binding properties support the idea that the semi-rigid cross-link favors the folded, binding-competent state of p19. The cross-linked recombinant CIRV-p19s represent novel stabilized suppressors of RNA silencing and may be useful in future biophysical, immunological and cell biology studies.

It has been brought to the author's attention that the above paper containsmissing text in the Abstract and they would like to correct this. The text was inadvertently deleted during the typesetting and editing of the proofs. On page 166, the Abstract should read as follows: Eukaryotes have evolved complex cellular responses to double-stranded RNA. One response that is highly conserved across many species is the RNA silencing pathway. Tombusviruses have evolved a mechanism to evade the RNA silencing pathway that involves a small protein, p19, that acts as a suppressor of RNA silencing. This protein binds specifically to small-interfering RNAs (siRNAs) with nanomolar affinity in a sequence-independent manner and with size selectivity. Here we demonstrate a new approach for rapidly determining the quantities of siRNA using fluorescence resonance energy transfer (FRET) between the Carnation Italian ringspot virus (CIRV) p19-CFP fusion protein and Cy3-labeled siRNA. The CIRV p19 fusion protein binds double-stranded siRNAs with nanomolar affinity as determined by FRET.

Employees in nail salons, largely Vietnamese immigrant women in Boston, are exposed to a range of volatile organic chemicals from the products used in salons, including solvents, glues and polishes. Some of these chemicals have the potential to cause short and long-term adverse health effects. Only limited research has been performed on assessing occupational exposures. This project aimed to characterize total volatile organic compound (TVOC) and PM2.5 concentrations in nail salons as a function of ventilation, building characteristics, customer and employee occupancy, and type of services being performed. Students conducted sampling in 21 salons in Boston, MA from September to December, 2011. Study visits included: indoor environmental quality measurements (TVOCs, PM2.5 and carbon dioxide), site observations, and an interview. CO2 levels in 15 of 21 salons exceeded 800 ppm, suggesting that these salons may have insufficient ventilation. Higher TVOC and PM2.5 levels were found in salons with less ventilation (as estimated using CO2 concentrations). Contrary to our a priori hypothesis, average levels of TVOCs, CO2 and PM2.5 were consistent throughout salons, indicating that exposures may not be restricted to areas in the salon where work is being performed (e.g., at the manicure table). Higher TVOC concentrations were observed when tasks were being performed, yet were not dependent upon the number of tasks being performed. Improving ventilation conditions in salons to meet minimum outdoor air delivery requirements can reduce exposures to TVOCs.