Engineering pH-sensitive dissolution of lipid-polymer nanoparticles by Eudragit integration impacts plasmid DNA (pDNA) transfection.

Lipid-polymer nanoparticles offer a promising strategy for improving gene nanomedicines by combining the benefits of biocompatibility and stability associated with the individual systems. However, research to date has focused on poly-lactic-co-glycolic acid (PLGA) and resulted in inefficient transfection. In this study, biocompatible Eudragit constructs E100 and RS100 were formulated as lipid-polymer nanoparticles loaded with pDNA expressing red fluorescent protein (RFP) as a model therapeutic. Using a facile nanoprecipitation technique, a core-shell structure stabilised by lipid-polyethylene glycol (PEG) surfactant was produced and displayed resistance to ultracentrifugation. Both cationic polymers E100 (pH-sensitive dissolution at 5) and RS100 (pH-insensitive dissolution) produced 150-200 nm sized particles with a small positive surface charge (+3-5 mV) and high pDNA encapsulation efficiencies (EE) of 75-90 %. The dissolution properties of the Eudragit polymers significantly impacted the biological performance in human embryonic kidney cells (HEK293T). Nanoparticles composed of polymer RS100 resulted in consistently high cell viability (80-100 %), whereas polymer E100 demonstrated dose-dependent behaviour (20-90 % cell viability). The low dissolution of polymer RS100 over the full pH range and the resulting nanoparticles failed to induce RFP expression in HEK293T cells. In contrast, polymer E100-constructed nanoparticles resulted in reproducible and gradually increasing RFP expression of 26-42 % at 48-72 h. Intraperitoneal (IP) injection of the polymer E100-based nanoparticles in C57BL/6 mice resulted in targeted RFP expression in mouse testes with favourable biocompatibility one-week post-administration. These findings predicate Eudragit based lipid-polymer nanoparticles as a novel and effective carrier for nucleic acids, which could facilitate pre-clinical evaluation and translation of gene nanomedicines.

Development of peptides for targeting cell ablation agents concurrently to the Sertoli and Leydig cell populations of the testes: An approach to non-surgical sterilization.

The surgical sterilization of cats and dogs has been used to prevent their unwanted breeding for decades. However, this is an expensive and invasive procedure, and often impractical in wider contexts, for example the control of feral populations. A sterilization agent that could be administered in a single injection, would not only eliminate the risks imposed by surgery but also be a much more cost-effective solution to this worldwide problem. In this study, we sought to develop a targeting peptide that would selectively bind to Leydig cells of the testes. Subsequently, after covalently attaching a cell ablation agent, Auristatin, to this peptide we aimed to apply this conjugated product (LH2Auristatin) to adult male mice in vivo, both alone and together with a previously developed Sertoli cell targeting peptide (FSH2Menadione). The application of LH2Auristatin alone resulted in an increase in sperm DNA damage, reduced mean testes weights and mean seminiferous tubule size, along with extensive germ cell apoptosis and a reduction in litter sizes. Together with FSH2Menadione there was also an increase in embryo resorptions. These promising results were observed in around a third of all treated animals. Given this variability, we discuss how these reagents might be modified in order to increase target cell ablation and improve their efficacy as sterilization agents.

Precise control of microfluidic flow conditions is critical for harnessing the in vitro transfection capability of pDNA-loaded lipid-Eudragit nanoparticles.

Microfluidics is widely regarded as a leading technology for industrial-scale manufacture of multicomponent, gene-based nanomedicines in a reproducible manner. Yet, very few investigations detail the impact of flow conditions on the biological performance of the product, particularly biocompatibility and therapeutic efficiency. Herein, this study investigated the engineering of a novel lipid-Eudragit hybrid nanoparticle in a bifurcating microfluidics micromixer for plasmid DNA (pDNA) delivery. Nanoparticles of ~150 nm in size, with uniform polydispersity index (PDI = 0.2) and ξ-potential of 5-11 mV were formed across flow rate ratios (FRR, aqueous to organic phase) of 3:1 and 5:1, respectively. The hybrid nanoparticles maintained colloidal stability and structural integrity of loaded pDNA following recovery by ultracentrifugation. Importantly, in vitro testing in human embryonic kidney cell line (HEK293T) revealed significant differences in biocompatibility and transfection efficiency (TE). Lipid-Eudragit nanoparticles produced at FRR 3:1 displayed high cellular toxicity (0-30% viability), compared with nanoparticles prepared at FRR 5:1 (50-100% viability). Red fluorescent protein (RFP) expression was sustained for 24-72 h following exposure of cells to nanoparticles, indicating controlled release of pDNA and trafficking to the nucleus. Nanoparticles produced at FRR 5:1 resulted in markedly higher TE (12%) compared with those prepared at FRR 3:1 (2%). Notably, nanoparticles produced using the bench-scale nanoprecipitation method resulted in lower biocompatibility (30-90%) but higher RFP expression (25-38%). These findings emphasize the need for in-depth analysis of the effect of formulation and flow conditions on the physicochemical and biological performance of gene nanomedicines when transitioning from bench to clinic.

Defining a Role for Webinars in Surgical Training Beyond the COVID-19 Pandemic in the United Kingdom: Trainee Consensus Qualitative Study.

BACKGROUND: The COVID-19 pandemic posed several challenges for surgical training, including the suspension of many in-person teaching sessions in lieu of webinars. As restrictions have eased, both prepandemic and postpandemic training methods should be used. OBJECTIVE: This study investigates trainees' experiences of webinars during the COVID-19 pandemic to develop recommendations for their effective integration into surgical training going forward. METHODS: This project was led by the Association of Surgeons in Training and used an iterative process with mixed qualitative methods to consolidate arguments for and against webinars, and the drivers and barriers to their effective delivery, into recommendations. This involved 3 phases: (1) a web-based survey, (2) focus group interviews, and (3) a consensus session using a nominal group technique. RESULTS: Trainees (N=281) from across specialties and grades confirmed that the COVID-19 pandemic led to an increase in webinars for surgical training. While there were concerns, particularly around the utility for practical training (80.9%), the majority agreed that webinars had a role in training following the COVID-19 pandemic (90.2%). The cited benefits included improved access or flexibility and potential standardization of training. The majority of limitations were technical. These perspectives were refined through focus group interviews (n=18) into 25 recommendations, 23 of which were ratified at a consensus meeting, which was held at the Association of Surgeons in Training 2021 conference. CONCLUSIONS: Webinars have a role in surgical training following the COVID-19 pandemic. The 23 recommendations encompass indications and technical considerations but also discuss important knowledge gaps. They should serve as an initial framework for ensuring that webinars add value and continue to evolve as a tool for training. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR2200055325; http://www.chictr.org.cn/showprojen.aspx?proj=142802.

Feeling the music: The feel and sound of songs attenuate pain.

Background: Extensive research has demonstrated that music and touch can separately attenuate perceived pain intensity. However, little research has investigated how auditory and tactile stimulation can synergistically enhance pain attenuation by music. In the current study, we investigated whether tactile stimulation can enhance music-induced analgesia for noxious force stimulation on the fingertip. Methods: We systematically applied force to 34 listeners' fingertips to induce pain. We then compared the force measurement (in Newton) that gave rise to the same perceived moderate pain intensity when listeners were presented their self-selected liked or disliked song with auditory-only, tactile-only and auditory-tactile stimulation. Higher force indicated less perceived pain. The tactile stimulation were low-frequency modulations extracted from the songs and presented as vibrations on the wrist. Results: The results showed a significant interaction between song preference and stimulation condition. Listeners had higher force measurements at the same moderate pain for their liked compared to disliked song only in the auditory-tactile condition. They also had higher force measurements for their liked song with auditory-tactile stimulation compared to the other remaining conditions except for the liked song with auditory-only stimulation. Conclusions: The addition of tactile stimulation enhanced music-induced analgesia which reduced subjective pain intensity. The findings suggest that combined auditory and tactile stimulation may increase the affective content of self-selected preferred music, which may stimulate affective and motivation mechanisms which inhibit pain transmission.

Microfluidic formulation of lipid/polymer hybrid nanoparticles for plasmid DNA (pDNA) delivery.

Lipid/polymer hybrid nanoparticles loaded with red fluorescent protein (RFP) encoded plasmid DNA (pDNA) was formulated using poly-lactic-co-glycolic acid (PLGA), cationic lipid DC-cholesterol and surfactant mPEG2000- DSPE. A lipid/ polymer ratio of 1: 10 at 1 mg/mL surfactant concentration was found to be optimal for producing nanoparticles with diameters of 100-120 nm that remained stable upon ultracentrifugation. The production of lipid/ polymer hybrid nanoparticles was investigated using microfluidics with a toroidal mixer design. Our results showed that the flow parameters significantly influenced the physicochemical characteristics of nanoparticles and loading of pDNA was only achieved at flow rate ratio (FRR) of 3: 1. The pDNA associated with nanoparticles was demonstrated to be structurally intact using gel electrophoresis, and the encapsulation efficiency (EE) was measured to be ∼65%. The prepared hybrid nanoparticles resulted in 20% of transfection efficacy in human embryonic kidney cells (HEK293T). This study demonstrated the potential of microfluidics in the development of hybrid nanoparticles for pDNA delivery, thus facilitating the clinical translation of DNA therapeutics.

The St Thomas' Hospital Emergency Department Homeless Health Initiative: improving the quality, safety and equity of healthcare provided for homeless patients attending the ED.

We carried out a quality improvement (QI) project (QIP), aiming to improve the quality, safety and equity of healthcare provided for homeless patients attending the emergency department (ED). We used QI methodology to identify areas for improvement, and introduced and modified interventions over four Plan, Do, Study, Act cycles. We launched a departmental 'Homeless Health Initiative' (HHI), the chief intervention being the provision of 'Homeless Health Boxes' in the ED, which contained a 'Safe Discharge Checklist for Homeless Patients', maps to specialist homeless general practitioner surgeries and homeless day centres, information on other inclusion health services, copies of a local rough sleepers' magazine and oral hygiene supplies. Voluntary Homeless Link Nurses and a number of informal 'Homeless Health Champions' were appointed. The HHI was embedded in departmental awareness through regular presentations to staff and incorporation into the induction programme for new doctors. Staff satisfaction, in terms of how satisfied staff members were with the care they were able to provide for homeless patients in the ED on a 0-10 scale, improved modestly over the course of the QIP from median 6/10 to median 7/10. The number of staff who were severely dissatisfied with the care they were able to provide for homeless patients improved more markedly: first quartile staff satisfaction improved from 3.875/10 to 6.125/10. Staff compliance with the checklist was poor, with full compliance observed in only 15% of cases by the end of the QIP. An HHI is a cheap and worthwhile QI project, with the potential to significantly improve the quality, safety and equity of healthcare provided for homeless patients, while improving staff satisfaction concurrently. Similar initiatives should be considered in any ED which sees a significant number of homeless patients.

Using Phylogenomic Data to Explore the Effects of Relaxed Clocks and Calibration Strategies on Divergence Time Estimation: Primates as a Test Case.

Primates have long been a test case for the development of phylogenetic methods for divergence time estimation. Despite a large number of studies, however, the timing of origination of crown Primates relative to the Cretaceous-Paleogene (K-Pg) boundary and the timing of diversification of the main crown groups remain controversial. Here, we analysed a data set of 372 taxa (367 Primates and 5 outgroups, 3.4 million aligned base pairs) that includes nine primate genomes. We systematically explore the effect of different interpretations of fossil calibrations and molecular clock models on primate divergence time estimates. We find that even small differences in the construction of fossil calibrations can have a noticeable impact on estimated divergence times, especially for the oldest nodes in the tree. Notably, choice of molecular rate model (autocorrelated or independently distributed rates) has an especially strong effect on estimated times, with the independent rates model producing considerably more ancient age estimates for the deeper nodes in the phylogeny. We implement thermodynamic integration, combined with Gaussian quadrature, in the program MCMCTree, and use it to calculate Bayes factors for clock models. Bayesian model selection indicates that the autocorrelated rates model fits the primate data substantially better, and we conclude that time estimates under this model should be preferred. We show that for eight core nodes in the phylogeny, uncertainty in time estimates is close to the theoretical limit imposed by fossil uncertainties. Thus, these estimates are unlikely to be improved by collecting additional molecular sequence data. All analyses place the origin of Primates close to the K-Pg boundary, either in the Cretaceous or straddling the boundary into the Palaeogene.

Preventing the importation of illicit nuclear materials in shipping containers.

We develop a mathematical model to find the optimal inspection strategy for detecting a nuclear weapon (or nuclear material to make a weapon) from being smuggled into the United States in a shipping container, subject to constraints of port congestion and an overall budget. We consider an 11-layer security system consisting of shipper certification, container seals, and a targeting software system, followed by passive (neutron and gamma), active (gamma radiography), and manual testing at overseas and domestic ports. Currently implemented policies achieve a low detection probability, and improved security requires passive and active testing of trusted containers and manually opening containers that cannot be penetrated by radiography. The annual cost of achieving a high detection probability of a plutonium weapon using existing equipment in traditional ways is roughly several billion dollars if testing is done domestically, and is approximately five times higher if testing is performed overseas. Our results suggest that employing high-energy x-ray radiography and elongating the passive neutron tests at overseas ports may provide significant cost savings, and several developing technologies, radiation sensors inside containers and tamper-resistant electronic seals, should be pursued aggressively. Further effort is critically needed to develop a practical neutron interrogation scheme that reliably detects moderately shielded, highly enriched uranium.