Efficiency, Specificity and Temperature Sensitivity of Cas9 and Cas12a RNPs for DNA-free Genome Editing in Plants

Delivery of genome editing reagents using CRISPR-Cas ribonucleoproteins (RNPs) transfection offers several advantages over plasmid DNA-based delivery methods, including reduced off-target editing effects, mitigation of random integration of non-native DNA fragments, independence of vector constructions, and less regulatory restrictions. Compared to the use in animal systems, RNP-mediated genome editing is still at the early development stage in plants. In this study, we established an efficient and simplified protoplast-based genome editing platform for CRISPR-Cas RNP delivery, and then evaluated the efficiency, specificity, and temperature sensitivity of six Cas9 and Cas12a proteins. Our results demonstrated that Cas9 and Cas12a RNP delivery resulted in genome editing frequencies (8.7–41.2%) at various temperature conditions, 22°C, 26°C, and 37°C, with no significant temperature sensitivity. LbCas12a often exhibited the highest activities, while AsCas12a demonstrated higher sequence specificity. The high activities of CRISPR-Cas RNPs at 22° and 26°C, the temperature preferred by plant transformation and tissue culture, led to high mutagenesis efficiencies (34.0–85.2%) in the protoplast-regenerated calli and plants with the heritable mutants recovered in the next generation. This RNP delivery approach was further extended to pennycress (Thlaspi arvense), soybean (Glycine max) and Setaria viridis with up to 70.2% mutagenesis frequency. Together, this study sheds light on the choice of RNP reagents to achieve efficient transgene-free genome editing in plants.

Peptide Functionalised Nanocarriers for Bone Specific Delivery of PTH (1-34) in Osteoporosis

: Osteoporosis represents a major public health burden especially considering the aging population worldwide. Treatment modalities for osteoporosis are classified into two categories based on the effect on bone remodelling: anabolic drugs and antiresorptive drugs. Anabolic drugs are preferred as it stimulates new bone formation. Currently, PTH (1-34) is the only peptide-based drug approved as an anabolic agent for the treatment of osteoporosis by both USFDA as well as EMA. However, its non-specific delivery results in prolonged kidney exposure, causing hypercalcemia. Nanotechnology-based drug delivery systems functionalized by conjugating it with homing moieties, such as peptides, offer an advantage of targeted delivery with reduced off-target effects. Here, we propose an innovative and targeted nanovesicle approach to efficiently deliver PTH (1-34) to the bone surface using peptides as a homing moiety. The proposed innovative delivery approach will augment the specific interaction between the drug and bone surface without producing side effects. This will reduce the off-target effects of PTH (1-34), and at the same time, it will also improve the outcome of anabolic therapy. Therefore, we postulate that the proposed innovative drug delivery approach for PTH (1-34) will establish as a promising therapy for osteoporotic patients, specifically in postmenopausal women who are at greater risk of bone fracture.

Towards Continuous Deployment for Blockchain

Ensuring a production-ready state of the application under development is the immanent feature of the continuous delivery approach. In a blockchain network, nodes communicate, storing data in a decentralized manner. Each node executes the same business application but operates in a distinct execution environment. The literature lacks research, focusing on continuous practices for blockchain and distributed ledger technology. In particular, such works with support for both software development disciplines of design and deployment. Artifacts from considered disciplines have been placed in the 1 + 5 architectural views model. The approach aims to ensure the continuous deployment of containerized blockchain distributed applications. The solution has been divided into two independent components: Delivery and deployment. They interact through Git distributed version control. Dedicated GitHub repositories should store the business application and deployment configurations for nodes. The delivery component has to ensure the deployment package in the actual version of the business application with the node-specific up-to-date version of deployment configuration files. The deployment component is responsible for providing running distributed applications in containers for all blockchain nodes. The approach uses Jenkins and Kubernetes frameworks. For the sake of verification, preliminary tests have been conducted for the Electricity Consumption and Supply Management blockchain-based system for prosumers of renewable energy.

Moderate Heat-Assisted Gene Electrotransfer as a Potential Delivery Approach for Protein Replacement Therapy through the Skin

Gene-based approaches for protein replacement therapies have the potential to reduce the number of administrations. Our previous work demonstrated that expression could be enhanced and/or the applied voltage reduced by preheating the tissue prior to pulse administration. In the current study, we utilized our 16-pin multi-electrode array (MEA) and incorporated nine optical fibers, connected to an infrared laser, between each set of four electrodes to heat the tissue to 43 °C. For proof of principle, a guinea pig model was used to test delivery of reporter genes. We observed that when the skin was preheated, it was possible to achieve the same expression levels as gene electrotransfer without preheating, but with a 23% reduction of applied voltage or a 50% reduction of pulse number. With respect to expression distribution, preheating allowed for delivery to the deep dermis and muscle. This suggested that this cutaneous delivery approach has the potential to achieve expression in the systemic circulation, thus this protocol was repeated using a plasmid encoding Human Factor IX. Elevated Factor IX serum protein levels were detected by ELISA up to 100 days post gene delivery. Further work will involve optimizing protein levels and scalability in an effort to reduce application frequency.

Polymer-Based Nanosystems—A Versatile Delivery Approach

Polymer-based nanoparticles of tailored size, morphology, and surface properties have attracted increasing attention as carriers for drugs, biomolecules, and genes. By protecting the payload from degradation and maintaining sustained and controlled release of the drug, polymeric nanoparticles can reduce drug clearance, increase their cargo’s stability and solubility, prolong its half-life, and ensure optimal concentration at the target site. The inherent immunomodulatory properties of specific polymer nanoparticles, coupled with their drug encapsulation ability, have raised particular interest in vaccine delivery. This paper aims to review current and emerging drug delivery applications of both branched and linear, natural, and synthetic polymer nanostructures, focusing on their role in vaccine development.

Nucleic Acid-Based Treatments Against COVID-19: Potential Efficacy of Aptamers and siRNAs

Despite significant efforts, there are currently no approved treatments for COVID-19. However, biotechnological approaches appear to be promising in the treatment of the disease. Accordingly, nucleic acid-based treatments including aptamers and siRNAs are candidates that might be effective in COVID-19 treatment. Aptamers can hamper entry and replication stages of the SARS-CoV-2 infection, while siRNAs can cleave the viral genomic and subgenomic RNAs to inhibit the viral life cycle and reduce viral loads. As a conjugated molecule, aptamer–siRNA chimeras have proven to be dual-functioning antiviral therapy, acting both as virus-neutralizing and replication-interfering agents as well as being a siRNA targeted delivery approach. Previous successful applications of these compounds against various stages of the pathogenesis of diseases and viral infections, besides their advantages over other alternatives, might provide sufficient rationale for the application of these nucleic acid-based drugs against the SARS-CoV-2. However, none of them are devoid of limitations. Here, the literature was reviewed to assess the plausibility of using aptamers, siRNAs, and aptamer–siRNA chimeras against the SARS-CoV-2 based on their previously established effectiveness, and discussing challenges lie in applying these molecules.

Exploring the Experience of Nurses on Nursing Process Implementation in Northwest Ethiopia

BackgroundThe nursing process is a set of actions that help nurses to solve the nursing problems identified for a particular patient. Besides, it is possible to say it is an instrument for nurses in their daily activities, and at the same time, it requires the knowledge, skills, and attitude of its nurses. The study aimed to explore the nursing process implementation among nurses in selected referral hospitals of the Amhara Region. Ida Jean Orlando’s Theory of the nursing process guided the study. MethodsThe study employed a descriptive qualitative research design. After obtaining permission from respective referral hospitals and informed written consent from nurses, a semi-structured in-depth interview with sixteen nurses were carried out to gather data. NVivo 10 software was used for analysis and the findings were thematised under different headings. ResultsThree main themes were emerged from the data and stated as care delivery approach, challenges in the nursing process implementation and nurses’ preparation during training for implementing the nursing process.ConclusionThe finding showed different degrees of nursing process implementation with a huge gap in the quality. Inadequate knowledge, attitude towards the nursing process, and skill were considered a major areas of concern. The study recommends the need for coordinated efforts of all stakeholders for improving the implementation of the nursing process, as identified by this study.

Investigation of Inertial Cavitation of Sonosensitive and Biocompatible Nanoparticles in Flow - Through Tissue- Mimicking Phantoms Employing Focused Ultrasound

A promising approach to drug delivery applications for chemotherapeutics is the use of drug carriers to reduce the total amount of cytostatics, minimizing side effects. In addition, the carriers, loaded with the drug, can be guided to the tumorous tissue via the vascular system, which enables a local drug release (LDR). In our case, LDR is activated due to the sonosensitive behavior of the nanocapsules by inertial cavitation (IC) caused by focused ultrasound (FUS). Thereby, IC is excited by employing sound pressures within the recommended limit allowed for diagnostic ultrasound. In order to verify this drug delivery approach for its clinical suitability, a tissue-mimicking flow -through phantom, containing a small vessel, is used. Investigations have shown that the drug releasing cavitation effect associated with the sonosensitive and biocompatible nanocapsules also occurs in fine vessel structures, even in the case of moving particles and vessel diameters dc smaller than the wavelength λ.

Community-led disaster resilience in Nauiyu Aboriginal community

There is a need for emergency management systems in Australia to shift to a more collaborative model that involves working with communities rather than simply delivering to communities. This research paper argues that in order to address this, emergency services organisations in Australia need to continue to shift from a service delivery approach to a more localised, participatory and consultative model that acknowledges and harnesses local leadership, knowledge, skills and experience creating opportunities for community driven and fit-for-purpose emergency management. The paper makes the case for why localised approaches are important in places with diverse populations such as the Northern Territory (NT). The NT Aboriginal community of Nauiyu’s experience of evacuations due to flooding in 2015 and 2018 will be used as a case study to offer an example of locally led approaches to disaster resilience.