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Extracellular vesicles (EVs) are naturally generated nanovesicles which potentially mediate the intercellular communication and interorgan crosstalk. EVs have recently gained significant interest as a promising material for delivery of therapeutics. Small RNAs, including small interfering RNA (siRNA) and microRNA (miRNA), provide a great therapeutic strategy for treating human diseases. However, it remains a challenge to deliver unconjugated small RNAs to the target tissue or cells. The delivery of small RNAs in an EV-encapsulating manner has a number of advantages, such as enhancing the concentration of small RNAs, improving the uptake of small RNAs by the recipient cells, and potentially achieving a cell-specific delivery. https://www.selleckchem.com/products/l-mimosine.html In this chapter, a protocol is provided for EV preparation and loading with small RNAs. Additionally, a detailed experimental protocol for tracking and validating small RNA delivery into the lungs is described. Overall, the described protocols are valuable for delivering functional small RNAs both in vitro and in vivo.Polyamidoamine dendrimers (PAMAM) form positively charged nanoparticles that function as nonviral delivery vectors for gene therapy. They protect nucleic acids from enzymatic degradation and facilitate endocytosis and endosomal escape. In this chapter, we describe the preparation and in vitro evaluation of small interfering RNA (siRNA)-PAMAM dendrimers. The physicochemical properties of the designed formulations were evaluated by size and zeta potential assessment and atomic force microscopy (AFM). The binding and release of the siRNA molecules from the PAMAM dendrimers were also assessed. Visualization and quantitative analysis of the siRNA-PAMAM dendrimers in live cells were analyzed by fluorescence microscopy and flow cytometry, respectively. Improving siRNA delivery to human cells through PAMAM dendrimers should accelerate the clinical applications of RNA interference.The silencing of an oncogene with a small interfering RNA (siRNA) is a promising way for cancer therapy. Its efficacy can be further enhanced by integrating with other therapeutics; however, transporting siRNA and other active ingredients to the same location at the same time is challenging. Here, we report a novel multifunctional nanodelivery platform by sequentially layering several functional ingredients, such as siRNAs, microRNAs, peptides, and targeting ligands, onto a core through charge-charge interaction. The prepared nanovectors effectively and programmably delivered multiple active components to maximize therapeutic combination with minimal off-targeting effects.RNA interference (RNAi) is a powerful tool for suppressing gene expression associated with various diseases that are not amenable to treatment with low molecular weight drugs. Despite significant progress in this area, the potential for therapeutic use of RNAi in humans is limited due to the lack of efficient delivery systems. Bioconjugation is one of the most promising methods for delivering siRNA to cells and tissues, since conjugation of siRNA with molecules capable of penetrating cells through natural transport mechanisms can provide specificity of delivery without toxic effects and unwanted immunostimulation. Here we describe the design, preparation, and in vivo evaluation of cholesterol-containing siRNA conjugates able to accumulate in the tumor, penetrate into cells without a carrier, and suppress the expression of the target genes.Chemically modified oligonucleotides (ONs) are routinely used in the laboratory to assess gene function, and clinical advances are rapidly progressing as continual efforts are being made to optimize ON efficacy. Over the years, RNA interference (RNAi) has become one of the main tools used to inhibit RNA expression across a wide variety of species. Efforts have been made to improve the exogenous delivery of the double-stranded RNA components to the endogenous intracellular RNAi machinery to direct efficacious degradation of a user-defined RNA target. More recently, synthetic RNA ONs are being used to mimic the bacterial-derived CRISPR/Cas system to direct specific editing of the mammalian genome. Both of these techniques rely on the use of various chemical modifications to the RNA phosphate backbone or sugar in specific positions throughout the ONs to improve the desired biological outcome. Relevant chemical modifications also include conjugated targeting ligands to assist ON delivery to specific cell types. Chemical modifications are most beneficial for therapeutically relevant ONs, as they serve to enhance target binding, increase drug longevity, facilitate cell-specific targeting, improve internalization into productive intracellular compartments, and mitigate both sequence-specific as well as immune-related off-target effects (OTEs). The knowledge gained from years of optimizing RNAi reagents and characterizing the biochemical and biophysical properties of each chemical modification will hopefully accelerate the CRISPR/Cas technology into the clinic, as well as further expand the use of RNAi to treat currently undruggable diseases. This review discusses the most commonly employed chemical modifications in RNAi reagents and CRISPR/Cas guide RNAs and provides an overview of select publications that have demonstrated success in improving ON efficacy and/or mitigating undesired OTEs.RNA interference (RNAi), a natural gene silencing process, is a widely used technique in basic research, preclinical studies, and drug development strategies. Although the technique has great potential to generate new human therapies and treat undruggable diseases, the clinical application of RNAi is still challenging primarily because of the delivery problem and potential off-target effects. Over the past two decades, great efforts have been undertaken to develop delivery agents and chemical modifications to overcome these challenges. Such advances in RNA delivery and chemical modifications have benefited researchers who are developing gene-editing therapies based on CRISPR-Cas9, an RNA-guided endonuclease, which is already having a major impact on biology and medicine. Here, I review the discovery of these two interference tools, identify the technical challenges yet to be overcome and provide some perspectives on how these two RNA-based technologies can be harnessed to treat human diseases.PURPOSE To examine longitudinal associations of intuitive eating (IE), defined as eating according to internal hunger and satiety cues, with psychological health outcomes and disordered eating behaviors. METHODS Data from a diverse sample of 1491 participants (54.1% female, 19.7% non-Hispanic white) followed from adolescence (baseline; Mage = 14.5 years) into young adulthood (follow-up; Mage = 22.2 years) came from the population-based EAT 2010-2018 (Eating and Activity over Time) study. Logistic regression models predicting psychological health outcomes and disordered eating behaviors at follow-up simultaneously included baseline IE and change in IE from baseline to follow-up as predictors, adjusting for demographic covariates, body mass index, and outcome at baseline. RESULTS Greater baseline IE and increases in IE from baseline to follow-up were both associated with lower odds of high depressive symptoms, low self-esteem, high body dissatisfaction, unhealthy weight control behaviors (e.g., fasting, skipping meals), extreme weight control behaviors (e.g., taking diet pills, vomiting), and binge eating at 8-year follow-up. Particularly strong protective associations were observed for binge eating, such that a one-point higher IE score at baseline was associated with 74% lower odds of binge eating at follow-up, and a one-point higher increase in IE score from baseline to follow-up was associated with 71% lower odds of binge eating at follow-up. CONCLUSIONS These results indicate that IE longitudinally predicts better psychological and behavioral health across a range of outcomes and suggest that IE may be a valuable intervention target for improving psychological health and reducing disordered eating behaviors, particularly binge eating. LEVEL OF EVIDENCE Level III, cohort study.BACKGROUND The number of original and supplemental ANDAs, BLAs, NDAs, and Biosimilars FDA drug/biologic approvals (Approvals) has risen dramatically in the recent years, incidentally, so has the number of issued FDA guidances (Guidances). It is hypothesized that if the structures of the two timeseries are similar and/or concomitantly co-evolving, then there is a relationship between the two variables that may be worthy of further investigation. METHODS Structural breakpoint (SBP) and cointegration (CI) analyses are used to provide insights into the relatedness of the two timeseries (Approvals, Guidances). Various descriptive statistics (e.g., nonparametric correlation testing, decomposition, unit testing, stationarity, and maximum order of integration) were also performed to better understand the nature of the timeseries understudy. RESULTS Structural breaks were identified with the following dates Approvals (1983, 1989, 1996, 2004, and 2012) and Guidances (1995 and 2012). Approvals and Guidances were (medium) correlative, nonstationary, and cointegrated with a maximum order of integration of one (I(1)). Descriptive statistical markers suggest additional similarities (e.g., seasonal variation) between the two timeseries. CONCLUSIONS To the author's knowledge, this is the first work to empirically investigate Guidances and their relationship with Approvals. The similarity in the structure of the timeseries (e.g., seasonal variation, SBPs and CI) suggests a deeper relationship between Guidances and Approvals, including the existence of a "long-run" equilibrium (wherein one or more exogenous factors restrain the divergence) between the two variables. This work offers an exciting opportunity for further research into the processes influencing the rates of Approvals and Guidances. A discussion on the limitations of the approach is also presented.BACKGROUND Double-J stents are used to treat ureteric outflow obstruction. Deployed in antegrade or retrograde fashion, they relieve ureteric obstruction in several conditions including ureteric calculi, strictures and malignancy. Traditionally exchanged in an operating theatre (OT) under general anaesthetic (GA), more recently described is the technique of using fluoroscopic guidance under sedation. AIMS To assess the efficacy and safety of retrograde double-J stent exchange in an interventional radiology (IR) setting in a tertiary oncology referral centre over a 7-year period. METHODS Clinical data on 460 double-J stent exchanges in 126 female patients was acquired from the hospital electronic patient record. Four fellowship-trained interventional radiologists performed the procedures. A standard approach was used in conjunction with conscious sedation using midazolam and fentanyl. Use of the technique with certain anatomical variations is also described. RESULTS Technical success rate was 96%. The main reasons for failure included failure to snare the stent (1.8%) and patient discomfort (1.1%). The overall complication rate was 5% 5 category 1 (minor) and 18 category 3 outcomes, with the latter group requiring further intervention. Average screening time was 9.65 min and the average radiation dose was 2018.24 mGy/m2. We also demonstrate the successful use of this method in patients with unusual anatomy and ileal conduits. CONCLUSION Fluoroscopic-guided retrograde double-J stent exchange is a safe and effective procedure that can be performed with a high degree of success using equipment and techniques used in daily IR practice. This approach precludes the need for GA, reduces OT utilisation and is well tolerated in a patient group for whom this procedure is typically palliative.