Gene silencing in adult Popillia japonica through feeding of double-stranded RNA (dsRNA) complexed with branched amphiphilic peptide capsules (BAPCs).

Gene silencing by feeding double-stranded (dsRNA) holds promise as a novel pest management strategy. Nonetheless, degradation of dsRNA in the environment and within the insect gut, as well as inefficient systemic delivery are major limitations to applying this strategy. Branched amphiphilic peptide capsules (BAPCs) complexed with dsRNA have been used to successfully target genes outside and inside the gut epithelium upon ingestion. This suggests that BAPCs can protect dsRNA from degradation in the gut environment and successfully shuttle it across gut epithelium. In this study, our objectives were to 1) Determine whether feeding on BAPC-dsRNA complexes targeting a putative peritrophin gene of P. japonica would result in the suppression of gut peritrophin synthesis, and 2) gain insight into the cellular uptake mechanisms and transport of BAPC-dsRNA complexes across the larval midgut of P. japonica. Our results suggest that BAPC-dsRNA complexes are readily taken up by the midgut epithelium, and treatment of the tissue with endocytosis inhibitors effectively suppresses intracellular transport. Further, assessment of gene expression in BAPC- peritrophin dsRNA fed beetles demonstrated significant downregulation in mRNA levels relative to control and/or dsRNA alone. Our results demonstrated that BAPCs increase the efficacy of gene knockdown relative to dsRNA alone in P. japonica adults. To our knowledge, this is the first report on nanoparticle-mediated dsRNA delivery through feeding in P. japonica.

Evaluation of transfection efficacy, biodistribution, and toxicity of branched amphiphilic peptide capsules (BAPCs) associated with mRNA.

Nanoparticles (NPs) have been shown to be a suitable mRNA delivery platform by conferring protection against ribonucleases and facilitating cellular uptake. Several NPs have succeeded in delivering mRNA intranasally, intratracheally, and intramuscularly in preclinical settings. However, intravenous mRNA delivery has been less explored. Only a few NPs have been tested for systemic delivery of mRNA, many of which are formulated with polyethylene glycol (PEG). The incorporation of PEG presents some tradeoffs that must be carefully considered when designing a systemic delivery model. For example, while the addition of PEG may prolong circulation time by preventing early clearance by the mononuclear phagocytic system (MPS), it has also been reported that treating patients with PEGylated drugs can result in hypersensitivity reactions due to anti-PEG antibodies. Thus, it is desirable to have alternative PEG-free delivery methods for mRNA to avoid these adverse effects while preserving the beneficial effects. Our research group developed BAPCs (branched amphiphilic peptide capsules), a peptide-based nanoparticle that resists disruption by chaotropes, proteases, and elevated temperature, thus displaying significant stability and shelf-life. In this study, we demonstrated that similarly to PEG, mRNA shields the BAPC cationic surface to avoid early clearance by the MPS. Multispectral optoacoustic tomography (MSOT) and fluorescence reflectance imaging were imaging techniques used to analyze biodistribution within major MPS organs. Analysis of pro-inflammatory cytokine expression showed that BAPC-mRNA complexes do not cause chronic inflammation. Additionally, BAPCs enhance intracellular delivery of mRNA with negligible cytotoxicity or oxidative stress. These results might pave the way for future therapeutic applications of BAPCs as a delivery platform for systemic mRNA delivery.

Insight into Cellular Uptake and Transcytosis of Peptide Nanoparticles in Spodoptera frugiperda Cells and Isolated Midgut.

Silencing genes in insects by introducing double-stranded RNA (dsRNA) in the diet holds promise as a new pest management method. It has been demonstrated that nanoparticles (NPs) can potentiate dsRNA silencing effects by promoting cellular internalization and protecting dsRNA against early degradation. However, many mysteries of how NPs and dsRNA are internalized by gut epithelial cells and, subsequently, transported across the midgut epithelium remain to be unraveled. The sole purpose of the current study is to investigate the role of endocytosis and transcytosis in the transport of branched amphipathic peptide nanocapsules (BAPCs) associated with dsRNA through midgut epithelium cells. Spodoptera frugiperda midguts and the epithelial cell line Sf9, derived from S. frugiperda, were used to study transcytosis and endocytosis, respectively. Results suggest that clathrin-mediated endocytosis and macropinocytosis are largely responsible for cellular uptake, and once within the midgut, transcytosis is involved in shuttling BAPCs-dsRNA from the lumen to the hemolymph. In addition, BAPCs were not found to be toxic to Sf9 cells or generate damaging reactive species once internalized.

Genetic counseling certificate program: A program evaluation of undergraduate exposure to genetic counseling.

Undergraduate genetic counseling exposure can generate interest in a growing field, help students prepare to apply to graduate-level programs, and introduce underrepresented populations to the career. One form of exposure that currently exists is the Genetic Counseling Certificate Program (GCCP), which is offered to undergraduate students at Rutgers University. To determine the effectiveness, benefits, and limitations of the GCCP, a program evaluation was conducted. Former GCCP students were surveyed to assess how they perceived the program. Overall, most students thought the program successfully met its objectives and thought their participation in the GCCP was beneficial. Because it is viewed favorably by former students, implementing something similar to the GCCP may be an option for institutions looking to offer additional opportunities to their undergraduates. Not only could creating programs like the GCCP enhance undergraduates' knowledge of the genetic counseling profession, but it could also contribute toward diversification of the field.

Laser-Assisted Delivery of Molecules in Fungal Cells.

Fungal infections are becoming a global health problem. A major limiting factor for the development of antifungals is the high impermeability of the rigid and thick fungal cell wall. Compared to mammalian cells, fungal cells are more resilient to perforation due to the presence of this carbohydrate armor. While a few methods have been reported to penetrate the fungal cell wall, such as electroporation, biolistics, glass beads, and the use of monovalent cations, such methods are generally time-consuming, compromise cell viability, and often lead to low permeation rates. In addition, their use remains limited to in vitro applications due to the collateral damage that these techniques could cause to healthy living tissues. Presented in this study is a delivery approach based on the generation of transient breaks, or pores, in the cell wall. Breaks are generated by cavitation and shock waves resulting from the irradiation of gold nanoparticles with a femtosecond infrared laser. Such an approach enabled the delivery of membrane impermeable molecules (i.e., calcein and plasmid DNA) into Saccharomyces cerevisiae, a fungal model organism. This method is expected to exhibit high biocompatibility and holds potential for clinical applications for the treatment of fungal infections given that neither the laser irradiation nor the nanoparticles have been found to damage cells. Mechanistical aspects of photoporation, such as the proximity needed between the nanoparticle and the cell membrane for these processes to take place, are also discussed. Hence, the laser-assisted drug delivery approach described here is suitable for further preclinical evaluation in oral, vaginal, and skin mycoses where current treatments are insufficient due to host-related adverse reactions, poor fungal cell penetration, or risk of developing antifungal resistance.

Prospects, challenges and current status of RNAi through insect feeding.

RNA interference is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into cells triggers the degradation of the complementary messenger RNA in a sequence-specific manner. Suppressing expression of vital genes could lead to insect death, therefore this technology has been considered as a potential strategy for insect pest control. There are three main routes of dsRNA administration into insects: (i) injections to the hemolymph, (ii) topical, and (iii) feeding. In this review, we focus on dsRNA administration through feeding. We summarize novel strategies that have been developed to improve the efficacy of this method, such as the use of nano-based formulations, engineered microorganisms, and transgenic plants. We also expose the hurdles that have to be overcome in order to use this technique as a reliable pest management method. © 2019 Society of Chemical Industry.

Occupational therapists working in hand therapy: the practice of holism in a cost containment environment.

OBJECTIVE: Occupational therapists specializing in hand therapy are challenged to provide care that reflects foundational concepts of occupation and holism. The researchers investigated cost containment influences on occupational therapists specializing in hand therapy to determine how therapists provide holistic care within cost containment constraints. METHOD: Five hand therapists were chosen based on selection criteria of hand therapy and holistic care practice. Using a grounded theory approach, the researchers gathered data via interviews and clinical observations. Data were analyzed through open, axial, and selective coding, and critiqued by an expert panel for emerging theory and concepts. RESULTS: Occupational therapists developed new skills or refined existing skill through adapting, educating, and strategizing in response to environmental demands. Tenets of the occupational adaptation model matched the strategies used by the participants. CONCLUSION: Occupational therapists specializing in hand therapy adapted to cost containment constraints by modifying professional settings and skills, educating multiple consumers, and implementing innovative interventions.