MINIstock: Model for INsect Inclusion in sustainable agriculture: USDA-ARS's research approach to advancing insect meal development and inclusion in animal diets.

Animal agriculture is under pressure to increase efficiency, sustainability, and innovation to meet the demands of a rising global population while decreasing adverse environmental effects. Feed cost and availability are 2 of the biggest hurdles to sustainable production. Current diets depend on sources of grain and animal byproduct protein for essential amino acids which have limited sustainability. Insects have arisen as an attractive, sustainable alternative protein source for animal diets due to their favorable nutrient composition, low space and water requirements, and natural role in animal diets. Additionally, insects are capable of bioremediating waste streams including agricultural and food waste, manure, and plastics helping to increase their sustainability. The insect rearing industry has grown rapidly in recent years and shows great economic potential. However, state-of-the-art research is urgently needed to overcome barriers to adoption in commercial animal diets such as regulatory restrictions, production scale issues, and food safety concerns. To address this need, the USDA Agricultural Research Service "MINIstoc: Model for INsect Inclusion" project was created to bring together diverse scientists from across the world to synergistically advance insect meal production and inclusion in animal diets. Here, we provide a short review of insects as feed while describing the MINIstock project which serves as the inspiration for the Journal of Economic Entomology Special Collection "Insects as feed: sustainable solutions for food waste and animal production practices."

CPR Quality Officer role to improve CPR quality: A multi-centred international simulation randomised control trial.

Background: An out-of-hospital cardiac arrest requires early recognition, prompt and quality clinical interventions, and coordination between different clinicians to improve outcomes. Clinical team leaders and clinical teams have high levels of cognitive burden. We aimed to investigate the effect of a dedicated Cardio-Pulmonary Resuscitation (CPR) Quality Officer role on team performance. Methods: This multi-centre randomised control trial used simulation in universities from the UK, Poland, and Norway. Student Paramedics participated in out-of-hospital cardiac arrest scenarios before randomisation to either traditional roles or assigning one member as the CPR Quality Officer. The quality of CPR was measured using QCPR® and Advanced Life Support (ALS) elements were evaluated. Results: In total, 36 teams (108 individuals) participated. CPR quality from the first attempt (72.45%, 95% confidence interval [CI] 64.94 to 79.97) significantly increased after addition of the CPR Quality role (81.14%, 95% CI 74.20 to 88.07, p = 0.045). Improvement was not seen in the control group. The time to first defibrillation had no significant difference in the intervention group between the first attempt (53.77, 95% CI 36.57-70.98) and the second attempt (48.68, 95% CI 31.31-66.05, p = 0.84). The time to manage an obstructive airway in the intervention group showed significant difference (p = 0.006) in the first attempt (168.95, 95% CI 110.54-227.37) compared with the second attempt (136.95, 95% CI 87.03-186.88, p = 0.1). Conclusion: A dedicated CPR Quality Officer in simulated scenarios improved the quality of CPR compressions without a negative impact on time to first defibrillation, managing the airway, or adherence to local ALS protocols.

Acidification-Induced Structure Evolution of Lipid Nanoparticles Correlates with Their In Vitro Gene Transfections.

The rational design of lipid nanoparticles (LNPs) for enhanced gene delivery remains challenging because of incomplete knowledge of their formulation-structure relationship that impacts their intracellular behavior and consequent function. Small-angle neutron scattering has been used in this work to investigate the structure of LNPs encapsulating plasmid DNA upon their acidification (from pH 7.4 to 4.0), as would be encountered during endocytosis. The results revealed the acidification-induced structure evolution (AISE) of the LNPs on different dimension scales, involving protonation of the ionizable lipid, volume expansion and redistribution of aqueous and lipid components. A similarity analysis using an LNP's structural feature space showed a strong positive correlation between function (measured by intracellular luciferase expression) and the extent of AISE, which was further enhanced by the fraction of unsaturated helper lipid. Our findings reveal molecular and nanoscale changes occurring during AISE that underpin the LNPs' formulation-nanostructure-function relationship, aiding the rational design of application-directed gene delivery vehicles.

Determining current approaches to the evaluation of the quality of healthcare simulation-based education provision: a scoping review.

Background: With an increase in simulation being used in healthcare education, there is a need to ensure the quality of simulation-based education is high. This scoping review was conducted to answer the question: What are the current approaches to the evaluation of the quality of health-care simulation-based education provision? Methods: Databases PubMed, Cochrane, ERIC, CINAHL and Medline were searched in March 2023 to retrieve peer-reviewed healthcare research and review articles written in the English language within the last 20 years. All data were extracted from six studies, themed and presented in the main text and in tabular form. Results: Two scoping reviews, one systematic review and three research articles were included. Three main themes were found: adherence to existing design frameworks, lack of validation of these frameworks and lack of evaluation frameworks, and a proposed evaluation framework. Many of the excluded articles focussed on gaining participant feedback to evaluate simulation activities, rather than evaluating the quality of the design and implementation of the simulation. Conclusions: Benchmarking of current United Kingdom (UK) healthcare simulation against UK and international simulation standards is required to increase its quality, therefore, an agreed UK template framework to evaluate simulation packages is recommended.

Hepatic transcriptome analyses of juvenile white bass (Morone chrysops) when fed diets where fish meal is partially or totally replaced by alternative protein sources.

White bass (Morone chrysops) are a popular sportfish throughout the southern United States, and one parent of the commercially-successful hybrid striped bass (M. chrysops ♂ x M. saxatilis ♀). Currently, white bass are cultured using diets formulated for other carnivorous fish, such as largemouth bass (Micropterus salmoides) or hybrid striped bass and contain a significant percentage of marine fish meal. Since there are no studies regarding the utilization of alternative proteins in this species, we evaluated the global gene expression of white bass fed diets in which fish meal was partially or totally replaced by various combinations of soybean meal, poultry by-product meal, canola meal, soy protein concentrate, wheat gluten, or a commercial protein blend (Pro-Cision™). Six isonitrogenous (40% protein), isolipidic (11%), and isocaloric (17.1 kJ/g) diets were formulated to meet the known nutrient and energy requirements of largemouth bass and hybrid striped bass using nutrient availability data for most of the dietary ingredients. One of the test diets consisted exclusively of plant protein sources. Juvenile white bass (40.2 g initial weight) were stocked into a flow-through aquaculture system (three tanks/diet; 10 fish/tank) and fed the test diets twice daily to satiation for 60 days. RNA sequencing and bioinformatic analyses revealed significant differentially expressed genes between all test diets when compared to fish meal control. A total of 1,260 differentially expressed genes were identified, with major ontology relating to cell cycle and metabolic processes as well as immune gene functions. This data will be useful as a resource for future refinements to moronid diet formulation, as marine fish meal becomes limiting and plant ingredients are increasingly added as a reliable protein source.

Antibody-oligonucleotide conjugate achieves CNS delivery in animal models for spinal muscular atrophy.

Antisense oligonucleotides (ASOs) have emerged as one of the most innovative new genetic drug modalities. However, their high molecular weight limits their bioavailability for otherwise-treatable neurological disorders. We investigated conjugation of ASOs to an antibody against the murine transferrin receptor, 8D3130, and evaluated it via systemic administration in mouse models of the neurodegenerative disease spinal muscular atrophy (SMA). SMA, like several other neurological and neuromuscular diseases, is treatable with single-stranded ASOs that modulate splicing of the survival motor neuron 2 (SMN2) gene. Administration of 8D3130-ASO conjugate resulted in elevated levels of bioavailability to the brain. Additionally, 8D3130-ASO yielded therapeutic levels of SMN2 splicing in the central nervous system of adult human SMN2-transgenic (hSMN2-transgenic) mice, which resulted in extended survival of a severely affected SMA mouse model. Systemic delivery of nucleic acid therapies with brain-targeting antibodies offers powerful translational potential for future treatments of neuromuscular and neurodegenerative diseases.

FRAGLER: A Fragment Recycler Application Enabling Rapid and Scalable Modular DNA Assembly.

Rapid and flexible plasmid construct generation at scale is one of the most limiting first steps in drug discovery projects. These hurdles can partly be overcome by adopting modular DNA design principles, automated sequence fragmentation, and plasmid assembly. To this end we have designed a robust, multimodule golden gate based cloning platform for construct generation with a wide range of applications. The assembly efficiency of the system was validated by splitting sfGFP and sfCherry3C cassettes and expressing them in E. coli followed by fluorometric assessment. To minimize timelines and cost for complex constructs, we developed a software tool named FRAGLER (FRAGment recycLER) that performs codon optimization, multiple sequence alignment, and automated generation of fragments for recycling. To highlight the flexibility and robustness of the platform, we (i) generated plasmids for SarsCoV2 protein reagents, (ii) automated and parallelized assemblies, and (iii) built modular libraries of chimeric antigen receptors (CARs) variants. Applying the new assembly framework, we have greatly streamlined plasmid construction and increased our capacity for rapid generation of complex plasmids.

Bioinformatic Design of Dendritic Cell-Specific Synthetic Promoters.

Next-generation DNA vectors for cancer immunotherapies and vaccine development require promoters eliciting predefined transcriptional activities specific to target cell types, such as dendritic cells (DCs), which underpin immune response. In this study, we describe the de novo design of DC-specific synthetic promoters via in silico assembly of cis-transcription factor response elements (TFREs) that harness the DC transcriptional landscape. Using computational genome mining approaches, candidate TFREs were identified within promoter sequences of highly expressed DC-specific genes or those exhibiting an upregulated expression during DC maturation. Individual TFREs were then screened in vitro in a target DC line and off-target cell lines derived from skeletal muscle, fibroblast, epithelial, and endothelial cells using homotypic (TFRE repeats in series) reporter constructs. Based on these data, a library of heterotypic promoter assemblies varying in the TFRE composition, copy number, and sequential arrangement was constructed and tested in vitro to identify DC-specific promoters. Analysis of the transcriptional activity and specificity of these promoters unraveled underlying design rules, primarily TFRE composition, which govern the DC-specific synthetic promoter activity. Using these design rules, a second library of exclusively DC-specific promoters exhibiting varied transcriptional activities was generated. All DC-specific synthetic promoter assemblies exhibited >5-fold activity in the target DC line relative to off-target cell lines, with transcriptional activities ranging from 8 to 67% of the nonspecific human cytomegalovirus (hCMV-IE1) promoter. We show that bioinformatic analysis of a mammalian cell transcriptional landscape is an effective strategy for de novo design of cell-type-specific synthetic promoters with precisely controllable transcriptional activities.

Aß31-35 Decreases Neprilysin-Mediated Alzheimer's Amyloid-ß Peptide Degradation.

Alzheimer's disease is associated with the deposition of extracellular senile plaques, made primarily of amyloid-ß (Aß), particularly peptides Aß1-42 and Aß1-40. Neprilysin, or neutral endopeptidase (NEP), catalyzes proteolysis of the amyloid peptides (Aß) and is recognized as one of the major regulators of the levels of these peptides in the brain, preventing Aß accumulation and plaque formation. Here, we used a combination of techniques to elucidate the mechanism of Aß binding and cleavage by NEP. Our findings indicate that the Aß31-X cleavage products remain bound to the neprilysin active site, reducing proteolytic activity. Interestingly, it was already shown that this Aß31-35 sequence is also critical for recognition of Aß peptides by other targets, such as the serpin-enzyme complex receptor in neuronal cells.

Brain pharmacokinetics of anti-transferrin receptor antibody affinity variants in rats determined using microdialysis.

Receptor-mediated transcytosis (RMT) is used to enhance the delivery of monoclonal antibodies (mAb) into the central nervous system (CNS). While the binding to endogenous receptors on the brain capillary endothelial cells (BCECs) may facilitate the uptake of mAbs in the brain, a strong affinity for the receptor may hinder the efficiency of transcytosis. To quantitatively investigate the effect of binding affinity on the pharmacokinetics (PK) of anti-transferrin receptor (TfR) mAbs in different regions of the rat brain, we conducted a microdialysis study to directly measure the concentration of free mAbs at different sites of interest. Our results confirmed that bivalent anti-TfR mAb with an optimal dissociation constant (KD) value (76 nM) among four affinity variants can have up to 10-fold higher transcytosed free mAb exposure in the brain interstitial fluid (bISF) compared to lower and higher affinity mAbs (5 and 174 nM). This bell-shaped relationship between KD values and the increased brain exposure of mAbs was also visible when using whole-brain PK data. However, we found that mAb concentrations in postvascular brain supernatant (obtained after capillary depletion) were almost always higher than the concentrations measured in bISF using microdialysis. We also observed that the increase in mAb area under the concentration curve in CSF compartments was less significant, which highlights the challenge in using CSF measurement as a surrogate for estimating the efficiency of RMT delivery. Our results also suggest that the determination of mAb concentrations in the brain using microdialysis may be necessary to accurately measure the PK of CNS-targeted antibodies at the site-of-actions in the brain.

Differential susceptibility of white bass (Morone chrysops), striped bass (Morone saxatilis) and hybrid striped bass (M. chrysops × M. saxatilis) to Flavobacterium columnare and effects of mucus on bacterial growth and biofilm development.

Columnaris disease generates substantial losses of many freshwater fish species; one is the hybrid striped bass. The ubiquitous aquatic bacterium Flavobacterium columnare can be highly effective in biofilm formation on fish skin and gills. Previous research showed a difference between columnaris disease susceptibility of hybrid striped bass (Morone saxatilis × M. chrysops) and white bass (M. chrysops). To understand these differential susceptibilities and possible mucosal relationship, we assessed total bacterial growth and biofilm formation with mucus derived from each moronid parental species: white bass and striped bass (M. saxatilis). Differential susceptibility was confirmed of the other parent species, the striped bass (M. saxatilis). In addition to intraspecies investigations, individual hybrid striped bass mucosal affects were also studied for deferential responses to bacterial growth and biofilm formation. Species- and concentration-dependent differences were detected in the total growth of the bacteria to host mucus. Our data suggest that bass mucus can significantly affect biofilm formation with the F. columnare isolate tested. There appears to be a correlation between the bacteria's response of growth and biofilms and bass species susceptibility. This study provides insight into our understanding of the host-pathogen interaction between F. columnare and moronids.

Antibodies binding the head domain of P2X4 inhibit channel function and reverse neuropathic pain.

P2X4 is a ligand-gated ion channel implicated in neuropathic pain. Drug discovery efforts targeting P2X4 have been unsuccessful largely because of the difficulty in engineering specificity and selectivity. Here, we describe for the first time the generation of a panel of diverse monoclonal antibodies (mAbs) to human and mouse P2X4, capable of both positive and negative modulation of channel function. The affinity-optimised anti-P2X4 mAb IgG#151-LO showed exquisite selectivity for human P2X4 and induced potent and complete block of P2X4 currents. Site-directed mutagenesis of P2X4 revealed the head domain as a key interaction site for inhibitory mAbs. Inhibition of spinal P2X4 either by intrathecal delivery of an anti-P2X4 mAb or by systemic delivery of an anti-P2X4 bispecific mAb with enhanced blood-spinal cord barrier permeability produced long-lasting (>7 days) analgesia in a mouse model of neuropathic pain. We therefore propose that inhibitory mAbs binding the head domain of P2X4 have therapeutic potential for the treatment of neuropathic pain.

A peptide derived from melanotransferrin delivers a protein-based interleukin 1 receptor antagonist across the BBB and ameliorates neuropathic pain in a preclinical model.

Delivery of biologic drugs across the blood-brain barrier is becoming a reality. However, the solutions often involve the assembly of complex multi-specific antibody molecules. Here we utilize a simple 12 amino-acid peptide originating from the melanotransferrin (MTf) protein that has shown improved brain delivery properties. 3D confocal fluorescence microscopic analysis demonstrated brain parenchymal localisation of a fluorescently labelled antibody (NIP228) when chemically conjugated to either the MTf peptide or full-length MTf protein. Measurement of plasma kinetics demonstrated the MTf peptide fusions had very similar kinetics to an unmodified NIP228 control antibody, whereas the fusion to MTf protein had significantly reduced plasma exposure most likely due to a higher tissue distribution in the periphery. Brain exposure for the MTf peptide fusions was significantly increased for the duration of the study, exceeding that of the fusions to full length MTf protein. Using a neuropathic pain model, we have demonstrated that fusions to interleukin-1 receptor antagonist (IL-1RA) are able to induce significant and durable analgesia following peripheral administration. These data demonstrate that recombinant and chemically conjugated MTf-based brain delivery vectors can deliver therapeutic levels of drug to the central nervous system.

Intracellular sorting and transcytosis of the rat transferrin receptor antibody OX26 across the blood-brain barrier in vitro is dependent on its binding affinity.

The blood-brain barrier (BBB) is a formidable obstacle to the delivery of therapeutics to the brain. Antibodies that bind transferrin receptor (TfR), which is enriched in brain endothelial cells, have been shown to cross the BBB and are being developed as fusion proteins to deliver therapeutic cargos to brain targets. Various antibodies have been developed for this purpose and their in vivo evaluation demonstrated that either low affinity or monovalent receptor binding re-directs their transcellular trafficking away from lysosomal degradation and toward improved exocytosis on the abluminal side of the BBB. However, these studies have been performed with antibodies that recognize different TfR epitopes and have different binding characteristics, preventing inter-study comparisons. In this study, the efficiency of transcytosis in vitro and intracellular trafficking in endosomal compartments were evaluated in an in vitro BBB model for affinity variants (Kd from 5 to174 nM) of the rat TfR-binding antibody, OX26. Distribution in subcellular fractions of the rat brain endothelial cells was determined using both targeted quantitative proteomics-selected reaction monitoring and fluorescent imaging with markers of early- and late endosomes. The OX26 variants with affinities of 76 and 108 nM showed improved trancytosis (Papp values) across the in vitro BBB model compared with a 5 nM OX26. Although ~40% of the 5 nM OX26 and ~35% of TfR co-localized with late-endosome/lysosome compartment, 76 and 108 nM affinity variants showed lower amounts in lysosomes and a predominant co-localization with early endosome markers. The study links bivalent TfR antibody affinity to mechanisms of sorting and trafficking away from late endosomes and lysosomes, resulting in improvement in their transcytosis efficiency. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14193.

Enhanced Delivery of Galanin Conjugates to the Brain through Bioengineering of the Anti-Transferrin Receptor Antibody OX26.

The blood-brain barrier (BBB) is a formidable obstacle for brain delivery of therapeutic antibodies. However, antibodies against the transferrin receptor (TfR), enriched in brain endothelial cells, have been developed as delivery carriers of therapeutic cargoes into the brain via a receptor-mediated transcytosis pathway. In vitro and in vivo studies demonstrated that either a low-affinity or monovalent binding of these antibodies to the TfR improves their release on the abluminal side of the BBB and target engagement in brain parenchyma. However, these studies have been performed with mouse-selective TfR antibodies that recognize different TfR epitopes and have varied binding characteristics. In this study, we evaluated serum pharmacokinetics and brain and CSF exposure of the rat TfR-binding antibody OX26 affinity variants, having KDs of 5 nM, 76 nM, 108 nM, and 174 nM, all binding the same epitope in bivalent format. Pharmacodynamic responses were tested in the Hargreaves chronic pain model after conjugation of OX26 affinity variants with the analgesic and antiepileptic peptide, galanin. OX26 variants with affinities of 76 nM and 108 nM showed enhanced brain and cerebrospinal fluid (CSF) exposure and higher potency in the Hargreaves model, compared to a 5 nM affinity variant; lowering affinity to 174 nM resulted in prolonged serum pharmacokinetics, but reduced brain and CSF exposure. The study demonstrates that binding affinity optimization of TfR-binding antibodies could improve their brain and CSF exposure even in the absence of monovalent TfR engagement.

Correction to: Current research into brain barriers and the delivery of therapeutics for neurological diseases: a report on CNS barrier congress London, UK, 2017.

After publication of the article [1], it has been brought to our attention that there are some errors in the formatting of names in the final version of the article.

Central inhibition of granulocyte-macrophage colony-stimulating factor is analgesic in experimental neuropathic pain.

With less than 50% of patients responding to the current standard of care and poor efficacy and selectivity of current treatments, neuropathic pain continues to be an area of considerable unmet medical need. Biological therapeutics such as monoclonal antibodies (mAbs) provide better intrinsic selectivity; however, delivery to the central nervous system (CNS) remains a challenge. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is well described in inflammation-induced pain, and early-phase clinical trials evaluating its antagonism have exemplified its importance as a peripheral pain target. Here, we investigate the role of this cytokine in a murine model of traumatic nerve injury and show that deletion of the GM-CSF receptor or treatment with an antagonizing mAb alleviates pain. We also demonstrate enhanced analgesic efficacy using an engineered construct that has greater capacity to penetrate the CNS. Despite observing GM-CSF receptor expression in microglia and astrocytes, the gliosis response in the dorsal horn was not altered in nerve injured knockout mice compared with wild-type littermate controls as evaluated by ionized calcium binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein, respectively. Functional analysis of glial cells revealed that pretreatment with GM-CSF potentiated lipopolysaccharide-induced release of proinflammatory cytokines. In summary, our data indicate that GM-CSF is a proinflammatory cytokine that contributes to nociceptive signalling through driving spinal glial cell secretion of proinflammatory mediators. In addition, we report a successful approach to accessing CNS pain targets, providing promise for central compartment delivery of analgesics.

Isolation of blood-brain barrier-crossing antibodies from a phage display library by competitive elution and their ability to penetrate the central nervous system.

The blood-brain barrier (BBB) is a formidable obstacle for delivery of biologic therapeutics to central nervous system (CNS) targets. Whilst the BBB prevents passage of the vast majority of molecules, it also selectively transports a wide variety of molecules required to maintain brain homeostasis. Receptor-mediated transcytosis is one example of a macromolecule transport system that is employed by cells of the BBB to supply essential proteins to the brain and which can be utilized to deliver biologic payloads, such as antibodies, across the BBB. In this study, we performed phage display selections on the mouse brain endothelial cell line, bEND.3, to enrich for antibody single-chain variable fragments (scFvs) that could compete for binding with a known BBB-crossing antibody fragment, FC5. A number of these scFvs were converted to IgGs and characterized for their ability to bind to mouse, rat and human brain endothelial cells, and subsequent ability to transport across the BBB. We demonstrated that these newly identified BBB-targeting IgGs had increased brain exposure when delivered peripherally in mice and were also able to transport a biologically active molecule, interleukin-1 receptor antagonist (IL-1RA), into the CNS. The antagonism of the interleukin-1 system within the CNS can result in the relief of neuropathic pain. We demonstrated that the BBB-targeting IgGs were able to elicit an analgesic response in a mouse model of nerve ligation-induced hypersensitivity when fused to IL-1RA.

Current research into brain barriers and the delivery of therapeutics for neurological diseases: a report on CNS barrier congress London, UK, 2017.

This is a report on the CNS barrier congress held in London, UK, March 22-23rd 2017 and sponsored by Kisaco Research Ltd. The two 1-day sessions were chaired by John Greenwood and Margareta Hammarlund-Udenaes, respectively, and each session ended with a discussion led by the chair. Speakers consisted of invited academic researchers studying the brain barriers in relation to neurological diseases and industry researchers studying new methods to deliver therapeutics to treat neurological diseases. We include here brief reports from the speakers.

The effect of temperature on the mucosal IgM antibody response to DNP-KLH in channel catfish (Ictalurus punctatus).

Bath immersion remains a practical route for immunizing against disease in channel catfish; however research efforts in this area have revealed variable results when activating mucosal Ab responses with different antigens. This is likely due to a number of factors including the individual species, age of the fish, preparation of the immunogens, and differences in the overall dosage and the duration of exposure to vaccines. The current study sought to evaluate the effect of water temperature on the in vivo mucosal adaptive immune response in channel catfish to a protein-hapten antigen, DNP-KLH. Fish were bath immersed at different water temperatures and periodically evaluated over an eighteen week period for the development of serum and mucosal IgM antibodies to DNP-KLH using an indirect enzyme-linked immunosorbent assay. None of the temperature groups produced a serum antibody response; however there were detectable DNP-KLH specific IgM antibodies in the mucus starting at week eight. The extent of the mucosal antibody response and duration differed between the treatments. Our results show that there are intrinsic differences in the capacity to generate in vivo mucosal Ab responses in the skin at different water temperatures and the implications of these findings to channel catfish farming will be discussed.