Design details for overdose education and take‐home naloxone kits: Codesign with family medicine,emergency department,addictions medicine and community

IntroductionOverdose education and naloxone distribution (OEND) programmes equip and train people who are likely to witness an opioid overdose to respond with effective first aid interventions. Despite OEND expansion across North America, overdose rates are increasing, raising questions about how to improve OEND programmes. We conducted an iterative series of codesign stakeholder workshops to develop a prototype for take‐home naloxone (THN)‐kit (i.e., two doses of intranasal naloxone and training on how to administer it).MethodsWe recruited people who use opioids, frontline healthcare providers and public health representatives to participate in codesign workshops covering questions related to THN‐kit prototypes, training on how to use it, and implementation, including refinement of design artefacts using personas and journey maps. Completed over 9 months, the workshops were audio‐recorded and transcribed with visible results of the workshops (i.e., sticky notes, sketches) archived. We used thematic analyses of these materials to identify design requirements for THN‐kits and training.ResultsWe facilitated 13 codesign workshops to identify and address gaps in existing opioid overdose education training and THN‐kits and emphasize timely response and stigma in future THN‐kit design. Using an iterative process, we created 15 prototypes, 3 candidate prototypes and a final prototype THN‐kit from the synthesis of the codesign workshops.ConclusionThe final prototype is available for a variety of implementation and evaluation processes. The THN‐kit offers an integrated solution combining ultra‐brief training animation and physical packaging of nasal naloxone to be distributed in family practice clinics, emergency departments, addiction medicine clinics and community settings.Patient or Public ContributionThe codesign process was deliberately structured to involve community members (the public), with multiple opportunities for public contribution. In addition, patient/public participation was a principle for the management and structuring of the research team.     

Manufacturing‐dependent change in biological activity of the TLR4 agonist GSK1795091 and implications for lipid A analog development

A phase I trial ({"type":"clinical-trial","attrs":{"text":"NCT03447314","term_id":"NCT03447314"}}NCT03447314; 204686) evaluated the safety and efficacy of GSK1795091, a Toll‐like receptor 4 (TLR4) agonist, in combination with immunotherapy (GSK3174998 [anti‐OX40 monoclonal antibody], GSK3359609 [anti‐ICOS monoclonal antibody], or pembrolizumab) in patients with solid tumors. The primary endpoint was safety; other endpoints included efficacy, pharmacokinetics, and pharmacodynamics (PD). Manufacturing of GSK1795091 formulation was modified during the trial to streamline production and administration, resulting in reduced PD (cytokine) activity. Fifty‐four patients received GSK1795091 with a combination partner; 32 received only the modified GSK1795091 formulation, 15 received only the original formulation, and seven switched mid‐study from the original to the modified formulation. Despite the modified formulation demonstrating higher systemic GSK1795091 exposure compared with the original formulation, the transient, dose‐dependent elevations in cytokine and chemokine concentrations were no longer observed (e.g., IP‐10, IL10, IL1‐RA). Most patients (51/54; 94%) experienced ≥1 treatment‐emergent adverse event (TEAE) during the study. Safety profiles were similar between formulations, but a higher incidence of TEAEs associated with immune responses (chills, fatigue, pyrexia, nausea, and vomiting) were observed with the original formulation. No conclusions can be made regarding GSK1795091 anti‐tumor activity due to the limited data collected. Manufacturing changes were hypothesized to have caused the change in biological activity in this study. Structural characterization revealed GSK1795091 aggregate size in the modified formulation to be twice that in the original formulation, suggesting a negative correlation between GSK1795091 aggregate size and PD activity. This may have important clinical implications for future development of structurally similar compounds.

Study Highlights

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

Toll‐like receptor 4 (TLR4) agonists, such as lipopolysaccharide (LPS) and lipid A analogs, have demonstrated anti‐cancer effects in patients. Previous studies offer conflicting evidence on the active form (monomeric vs. aggregates) of LPS/lipid A analogs and suggest that different forms can stimulate different immune pathways.

• WHAT QUESTION DID THIS STUDY ADDRESS?

This phase I study investigated the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of GSK1795091 with one of three immunotherapies in patients with solid tumors. In addition, the study addressed the effects of a manufacturing change on the biological activity of GSK1795091.

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

This study enables a better understanding of the impact of the manufacturing process on drug aggregate morphology and activity. The formulation change led to the formation of particle aggregates with globular morphology, suggesting the initial dissolution of GSK1795091 in ethanol, instead of initial dissolution using sonication, was the likely contributor to the lowered biological activity of GSK1795091. This could have implications for the development of other lipid A analogs and TLR agonists.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

Following manufacturing changes of LPS/other lipid A analogs and chemically manufactured active pharmaceutical ingredients that are prone to structural organization in solution, it is recommended to perform in vitro PD assessments to understand the impact on its biological activity prior to clinical assessment. PK and PD evaluations should be prioritized to ensure no clinically meaningful changes related to the manufacturing occur.     

Expectation effects in working memory training

There is a growing body of research focused on developing and evaluating behavioral training paradigms meant to induce enhancements in cognitive function. It has recently been proposed that one mechanism through which such performance gains could be induced involves participants’ expectations of improvement. However, no work to date has evaluated whether it is possible to cause changes in cognitive function in a long-term behavioral training study by manipulating expectations. In this study, positive or negative expectations about cognitive training were both explicitly and associatively induced before either a working memory training intervention or a control intervention. Consistent with previous work, a main effect of the training condition was found, with individuals trained on the working memory task showing larger gains in cognitive function than those trained on the control task. Interestingly, a main effect of expectation was also found, with individuals given positive expectations showing larger cognitive gains than those who were given negative expectations (regardless of training condition). No interaction effect between training and expectations was found. Exploratory analyses suggest that certain individual characteristics (e.g., personality, motivation) moderate the size of the expectation effect. These results highlight aspects of methodology that can inform future behavioral interventions and suggest that participant expectations could be capitalized on to maximize training outcomes.

There is a great deal of current scientific interest as to whether and/or how basic cognitive skills can be improved via dedicated behavioral training (1–3). This potential, if realized, could lead to substantial real-world impact. Indeed, effective training paradigms would have significant value not only for populations that show deficits in cognitive skills (e.g., individuals diagnosed with Attention Deficit Hyperactivity Disorder [ADHD] or Alzheimer’s disease and related dementias) but also, for the general public, where core cognitive capacities underpin success in both academic and professional contexts (4–6). These possible translational applications, paired with an emerging understanding of how to best unlock neuroplastic change across the life span (7, 8), have spurred hundreds of behavioral intervention studies over the past few decades. While the results have not been uniformly positive (perhaps not surprising given the massive heterogeneity in theoretical approach, methods, etc.), multiple meta-analyses suggest that it is possible for cognitive functions to be improved via some forms of dedicated behavioral training (9–11). However, while these basic science results provide optimism that real-world gains could be realized [and in fact, real-world gain is already being realized in some spheres, such as a Food and Drug Administration (FDA)–cleared video game–based treatment supplement for ADHD (12, 13)], concerns have been raised as to whether those interventions that have produced positive outcomes are truly working via the proposed mechanisms or through other nonspecific third-variable mechanisms. Several factors have been proposed to explain improvements in behavioral interventions, including selective attrition, contextual factors, regression to the mean, and practice effects to name a few (14). Here, we focus on whether expectation-based (i.e., placebo) mechanisms can explain improvements in cognitive training (15–17).In other domains, such as in clinical trials in the pharmaceutical domain for instance, expectation-based mechanisms are typically controlled for by making the experimental treatment and the control treatment perceptually indistinguishable (e.g., both might be clear fluids in an intravenous bag or a white unmarked pill). Because perceptual characteristics cannot be used to infer condition, this methodology is meant to ensure that expectations are matched between the experimental and control groups (both in terms of the expectations that the participants have and in terms of the expectations that the research team members who interact with the participants have). Under ideal circumstances, the use of such a “double-unaware” design ensures that expectations cannot be an explanatory mechanism underlying any differences between the groups’ outcomes [note that we use the double-unaware terminology in lieu of the more common “double-blind” terminology, which can be seen as ableist (18)].It is unclear whether most pharmaceutical trials do, in fact, truly meet the double-unaware standard (e.g., despite being perceptually identical, active and control treatments nonetheless often produce different patterns of side effects that could be used to infer condition) (19, 20). Yet, meeting the double-unaware standard is particularly difficult in the case of cognitive training interventions (16). Here, there is simply no way to make the experimental and control interventions perceptually indistinguishable while at the same time, ensuring that the experimental condition contains an “active ingredient” that the control condition lacks. In behavioral interventions, no matter what the active ingredient may be, it will necessarily produce a difference in look and feel as compared with a training condition that lacks the ingredient.Researchers designing cognitive training trials, therefore, typically attempt to utilize experimental and control conditions that, while differing in the proposed active ingredient, will nonetheless produce similar expectations about the likely outcomes (16, 21–24). This type of matching process, however, is inherently difficult as it is not always clear what expectations will be induced by a given type of experience. Consistent with this, there is reason to believe that expectations have not always been successfully matched. In multiple cases, despite attempts to match expectations across conditions, participants in behavioral intervention studies have nonetheless indicated the belief that the true active training task will produce more cognitive gains than the control task (25–27). Critically, the data as to whether differential expectations in these cases actually, in turn, influence the observed outcomes are decidedly mixed. In some cases, participant expectations differed between training and control conditions, and these expectations were at least partially related to differences in behavior (25). In other cases, participants expected to improve but did not show any actual improvements in cognitive skill (28), or the degree to which they improved was unrelated to their stated expectations (29).Regardless of the mixed nature of the data thus far, there is increasing consensus that training studies should 1) attempt to match the expectations generated by their experimental and control treatment conditions, 2) measure the extent to which this matching is successful and if the matching was not successful, and 3) evaluate the extent to which differential expectations explain differences in outcome (16, 30). Yet, such methods are not ideal with respect to getting to the core question of whether expectation-based mechanisms can, in fact, alter performance on cognitive tasks in the context of cognitive intervention studies in the first place. Indeed, there is a growing body of work suggesting that self-reported expectations do not necessarily fully reflect the types of predictions being generated by the brain (e.g., it is possible to produce placebo analgesia effects even in the absence of self-reported expectation of pain relief) (31, 32). Instead, addressing this question would entail purposefully maximizing the differences in expectations between groups (i.e., rather than attempting to minimize differential expectations and then, measuring the possible impact if the differences were not eliminated, as is done in most cognitive training studies).One key question then is how to maximize such expectations. In general, in those domains that have closely examined placebo effects, expectations are typically induced through two broad routes: an explicit route and an associative route. In the explicit route, as given by the name, participants are explicitly told what behavioral changes they should expect (e.g., “this pill will improve your symptoms” or “this cognitive training will improve your cognition”) (33). In the associative learning route, participants are made to experience a behavioral change associated with expected outcomes (e.g., feeling improvements of symptoms or gains in cognition) through some form of deception (34). For example, in an explicit expectation induction study, participants may first have a hot temperature probe applied to their skin, after which they are asked to rate their pain level. An inert cream is then applied that is explicitly described as an analgesic before the hot temperature probe is reapplied. If participants indicate less pain after the cream is applied, this is taken as evidence of an explicit expectation effect. In the associative expectation version, the study progresses identically as above except that when the hot temperature probe is applied the second time, it is at a physically lower temperature than it was initially (participants are not made aware of this fact). This is meant to create an associative pairing between the cream and a reduction in experienced pain (i.e., not only are they told that the cream will reduce their pain, they are provided “evidence” that the cream works as described). If then, after reapplying the cream and applying the hot temperature probe a third time (this time at the same temperature setting as the first application), if participants indicate even less pain than in the explicit condition, this is taken as evidence of an associative expectation effect. It remains to be clarified how associative learning approaches may be best applied to cognitive training; however, we suggest here that a reasonable approach to this would be to provide test sessions where test items are manipulated to provide participants with an experience where they perceive that they are performing better, or worse in the case of a nocebo, than they did at the initial test session. Notably, while there are cases where strong placebo effects have been induced via only explicit (35) or only associative methods (36), in general, the most consistent and robust effects have been induced when a combination of these methods has been utilized (37–39).Within the cognitive training field, the corresponding literature is quite sparse. Few studies have deliberately attempted to create differences in participant expectations, and of those, all have used the explicit expectation route alone, have implemented the manipulation in the context of rather short interventions (e.g., utilizing 20 min of “training” within a single session rather than the multiple hours that are typically implemented in actual training studies), or both. Of these, the results are again at best mixed, with one study suggesting that expectations alone can result in a positive impact on cognitive measures (40), while others have found no such effects (33, 41, 42). Given this critical gap in knowledge, here we examined the impact of manipulations deliberately designed to maximize the presence of differential expectations in the context of a long-term cognitive training study.     

Single-particle studies of the effects of RNA–protein interactions on the self-assembly of RNA virus particles

Understanding the pathways by which simple RNA viruses self-assemble from their coat proteins and RNA is of practical and fundamental interest. Although RNA–protein interactions are thought to play a critical role in the assembly, our understanding of their effects is limited because the assembly process is difficult to observe directly. We address this problem by using interferometric scattering microscopy, a sensitive optical technique with high dynamic range, to follow the in vitro assembly kinetics of more than 500 individual particles of brome mosaic virus (BMV)—for which RNA–protein interactions can be controlled by varying the ionic strength of the buffer. We find that when RNA–protein interactions are weak, BMV assembles by a nucleation-and-growth pathway in which a small cluster of RNA-bound proteins must exceed a critical size before additional proteins can bind. As the strength of RNA–protein interactions increases, the nucleation time becomes shorter and more narrowly distributed, but the time to grow a capsid after nucleation is largely unaffected. These results suggest that the nucleation rate is controlled by RNA–protein interactions, while the growth process is driven less by RNA–protein interactions and more by protein–protein interactions and intraprotein forces. The nucleated pathway observed with the plant virus BMV is strikingly similar to that previously observed with bacteriophage MS2, a phylogenetically distinct virus with a different host kingdom. These results raise the possibility that nucleated assembly pathways might be common to other RNA viruses.

Since the 1950s, the question of how RNA viruses self-assemble has inspired theoretical and experimental work in many fields of basic and applied science (1–5). Simple RNA viruses, which consist of a single-stranded RNA genome inside an ordered capsid made up of multiple copies of a single protein (Fig. 1A), have served as model systems for studying the physical principles of structural virology involving virus particles of all shapes and sizes (1, 2, 6, 7). However, the mechanisms and pathways by which these viruses assemble into the correct structure, while avoiding the many possible malformed structures, are not yet understood.Open in a separate windowFig. 1.Overview of the system and the measurement. (A) A 3-dimensional model of BMV reconstructed from cryoelectron microscopy data (51) shows the protein capsid (gray) surrounding the RNA (gold). The model reveals most of the icosahedral capsid but only a small portion of the RNA, the rest of which adopts a disordered arrangement within the capsid. (B) A cartoon of the experiment shows viral coat proteins assembling around RNA strands that are tethered by DNA linkages to the surface of a functionalized glass coverslip. (C) The assembling proteins are imaged at 1,000 Hz for 600 s using iSCAT microscopy. Each dark spot that appears in the images corresponds to proteins bound to an individual RNA strand. The darkness, or intensity, of each spot is proportional to the number of proteins bound to that RNA. The displayed images are the average of 1,000 consecutive frames. (D) Traces of the intensity as a function of time (1,000-frame moving average) reveal the assembly kinetics for each particle. Experimental conditions are 0.135 μmol/L protein and 250 mmol/L NaCl. The initial spike in intensity present in many of the traces is associated with vibrations introduced into the system as the coat protein is injected. The thick, black trace corresponds to the boxed particle in (C). We compare the final intensities of the traces to the estimated intensity range of full capsids, which is shown as a vertical bar to the right of the traces.Although many different RNA viruses self-assemble (8–10), our interest is in comparing the assembly of virus-like particles from two well-studied virus families: Bromoviridae, a family of plant-infecting viruses that includes brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV), and Fiersviridae (previously Leviviridae), a family of bacteria-infecting viruses that includes MS2 and Qβ. These families are as distinct phylogenetically as any two RNA virus families can be, having a last common ancestor that is thought to predate the emergence of eukaryotic cells (11). Accordingly, there are many well-established physical and biological differences among viruses in these families and virus-like particles derived from them. Yet the four most studied members—BMV, CCMV, MS2, and Qβ—do have some structural commonalities: They have icosahedral capsids with a triangulation number (T) of 3 (2), they have no lipid envelope, and each capsid surrounds approximately 3,000 to 4,000 nucleotides of single-stranded RNA.The assembly of such structures is a nontrivial process. Identical coat proteins must adopt nonequivalent positions to make a T = 3 capsid, with some arranging in pentagonal configurations and others in hexagonal configurations (2, 7, 12). Furthermore, these configurations must form in the correct proportions and positions for the capsid to close. Despite these challenges, assembly of virus-like particles of CCMV (13–15), BMV (14, 15), and MS2 (16) occurs in high yield even in vitro and in the absence of host-cell factors. The ability of viruses to avoid the many possible metastable states en route to complete assembly has been likened to the Levinthal paradox of protein folding (17, 18).But unlike proteins, RNA viruses have a template for assembly: their own RNA. Current theoretical models of RNA virus self-assembly posit markedly different roles for the RNA, depending on the relative strengths of RNA–protein and protein–protein interactions, sequence-dependent RNA–protein interactions, RNA-mediated protein–protein interactions, and several other factors (19). Although specific interactions between RNA substructures and coat proteins have been hypothesized to help the virus avoid malformed configurations (18), viruses from different families differ greatly in their RNA structures and RNA–protein interactions. It is therefore unclear whether there are common features of the assembly process for different T = 3 viruses or if there are distinct assembly pathways that depend on RNA–protein interactions.Recent measurements of assembly kinetics suggest the latter: that the assembly of viruses from different families follows different pathways. Fluorescence correlation spectroscopy experiments (20, 21) of the kinetics of binding of MS2 coat protein and RNA indicate that assembly starts with a small cluster of RNA-bound proteins that trigger a change in the hydrodynamic radius of the RNA. In contrast, cryoelectron microscopy (22) and small-angle X-ray scattering (23) experiments of the assembly of the CCMV coat protein and RNA show that disordered RNA–protein complexes formed at neutral pH anneal over several thousand seconds into well-formed capsids when the pH drops below 6.But because these experiments involve different assembly conditions and different measurement techniques, their outcomes might not reflect fundamental differences in the assembly pathways of these viruses but rather technical differences in the methods and protocols used to study them. Furthermore, most of the techniques that have been used do not measure the assembly process directly at the scale of individual particles because—one way or the other—they involve averaging over many particles. Such averaging can obscure the mechanisms and pathways that underpin stochastic assembly processes like viral assembly, in which each individual particle can follow its own unique sequence of intermediate states. Thus, it remains an open question whether a common assembly pathway might exist between these viruses.We recently demonstrated that interferometric scattering (iSCAT) microscopy (24) can resolve the assembly kinetics of individual virus-like particles (25), providing a method to directly measure and compare the assembly pathways of different viruses. To perform the iSCAT experiment, we first tether viral RNA molecules to the surface of a functionalized glass coverslip under the desired buffer conditions (26) (Fig. 1B). Next, we begin collecting iSCAT images of the RNA-decorated coverslip as we inject viral coat proteins at the desired concentration and in the appropriate buffer. As the proteins bind to the surface-tethered RNA, dark spots appear in the iSCAT images (Fig. 1C). Subtracting the intensity associated with the RNA then yields images in which the intensity of each dark spot is proportional to the number of proteins that have accrued onto each individual RNA. Previous measurements by Young and coworkers (27) show that the iSCAT intensities of protein assemblies are, in general, linearly proportional to the total mass of the assemblies. Accordingly, in our experiments, plotting the trace of the intensity of a spot as a function of time reveals the assembly kinetics for that particle, and plotting the collection of traces reveals the assembly kinetics for the ensemble of particles (Fig. 1D).In our previous work (25) we examined the assembly of bacteriophage MS2. We found that well-formed capsids could assemble around surface-tethered RNA strands and that the assembly kinetics were consistent with a nucleation-and-growth pathway in which a small cluster of RNA-bound proteins must exceed a critical size before the binding of additional proteins becomes favorable. Despite an apparently small critical nucleus size of only a few coat–protein dimers, we found that MS2 capsids grow monotonically to full or nearly full size with high yield.Although this previous study highlighted the importance of the RNA in the assembly process, the strong and specific RNA–protein interactions in MS2 (28–30), which are thought to occur at a dozen or so positions on the RNA molecule (31, 32), make it difficult to systematically address the central question of how the RNA affects the pathway. By contrast, the RNA in BMV interacts with the coat proteins primarily through nonspecific electrostatic interactions (33), with the possible exception of a single, specific RNA–protein interaction occurring at the 3′-end of the RNA (34). As a result, the strength of RNA–protein interactions in BMV can be largely tuned by changing the ionic strength of the buffer solution (22, 35, 36). BMV therefore offers not only an interesting comparison to MS2—it is phylogenetically distinct but structurally similar—but also the means to understand the role of RNA–protein interactions.In the current study, we infer the assembly pathways of BMV from iSCAT measurements under different RNA–protein interaction strengths, allowing us to critically assess of competing models of the assembly process. We follow the assembly trajectories of more than 500 individual virus particles under different assembly conditions, and we correlate the results with the absence and presence of ordered capsids as detected with negative-stain transmission electron microscopy (TEM). We find that BMV can assemble by a nucleation-and-growth process that is qualitatively similar to that of MS2. We show that the strength of RNA–protein interactions strongly affects the nucleation time but only weakly affects the growth time, suggesting that RNA plays a central role in nucleating the viral capsid but a relatively minor role in its growth kinetics. We discuss these observations in the context of recent models and hypotheses of RNA virus self-assembly.     

Factors impacting medication adherence in a birth cohort at higher risk for Hepatitis C infection

Due to the high prevalence of Hepatitis C virus (HCV) infection among individuals born between 1945 and 1965, in 2012 the Centers for Disease Control and Prevention began recommending HCV screening for this birth cohort. As adherence to HCV treatment is essential for sustained virologic response, identifying factors influencing medication adherence is important. The validated Adherence to Refills and Medications Scale (ARMS) is used to study recent medication adherence in those with chronic disease. This cross-sectional pilot study assesses factors associated with reduced adherence, indicated by higher ARMS scores, among individuals in this birth cohort. To elucidate factors associated with medication adherence, measured by the ARMS score, among a birth cohort at higher risk for HCV to guide future treatment and improve adherence. Patients born between 1945 and 1965, accessing care at an academic family medicine clinic, were recruited between April and June 2019. Demographics, prior HCV diagnosis, HCV risk factors (prior imprisonment, tattoos, and intravenous drug use), depression assessment (Patient Health Questionnaire-9), adverse childhood experiences (ACEs), and ARMS scores were collected. Mean ARMS scores were compared using t tests and analysis of variance (α = 0.05), while multiple variable models were performed using linear regression. Women comprised 58% of participants (n = 76), 52% reported depression and 37% 4 or more ACEs. The mean ARMS score was 16.3 (SD = 3.43) and 10% reported prior diagnosis of HCV. In the final multiple variable model, ARMS scores were 2.3 points higher in those with mild depression (95% CI: 0.63, 4.04), 2.0 in those with at least 4 ACEs (95% CI: 0.55, 3.49), and 1.8 in those with tattoos (95% CI: 0.30, 3.28). ACEs and food insecurity were identified as confounding variables in those with moderate to severe depression. This study found medication adherence was related to depression, ACEs, tattoos, and food insecurity among patients in this birth cohort at higher risk for HCV.     

Enhanced stability and clinical absorption of a form of encapsulated vitamin A for food fortification

Food fortification is an effective strategy to address vitamin A (VitA) deficiency, which is the leading cause of childhood blindness and drastically increases mortality from severe infections. However, VitA food fortification remains challenging due to significant degradation during storage and cooking. We utilized an FDA-approved, thermostable, and pH-responsive basic methacrylate copolymer (BMC) to encapsulate and stabilize VitA in microparticles (MPs). Encapsulation of VitA in VitA-BMC MPs greatly improved stability during simulated cooking conditions and long-term storage. VitA absorption was nine times greater from cooked MPs than from cooked free VitA in rats. In a randomized controlled cross-over study in healthy premenopausal women, VitA was readily released from MPs after consumption and had a similar absorption profile to free VitA. This VitA encapsulation technology will enable global food fortification strategies toward eliminating VitA deficiency.

Vitamin A (VitA) plays an essential role in visual health, immune function, and fetal growth and development (1). VitA deficiency (VAD) arises from diets with insufficient fat-soluble micronutrients (MNs) and is currently estimated as the second most common cause of malnutrition, after iron, globally (2). VAD can lead to xerophthalmia (preventable childhood blindness) and weakened host resistance to infection, which can increase the risk of mortality from infectious diseases, such as measles and COVID-19 (3, 4). The WHO estimated that VAD affected 190 million preschool-age children (33.3% of the preschool-age population) and >19 million pregnant women (15.3% of the pregnant population) globally in the period spanning 1995–2005 (5). The most severely affected regions still reached VAD prevalence levels of 48% in sub-Saharan Africa and 44% in South Asia in children in 2013 (6). To reduce the high burden of VAD, a VitA supplementation program was implemented worldwide in 1990 that distributed high-dose VitA capsules every 4–6 mo to over 80% of the total child population in low-income countries (7). This project effectively reduced all-cause mortality caused by severe VAD by 12% (8). However, progress toward VAD elimination was limited to a rate of improvement of only ~0.3% per year from 1990 to 2007, showing that more impactful strategies are required (9, 10).To raise and maintain serum retinol levels, frequent intake of VitA at physiological doses is proven to be more effective than one or two high doses administered over 6 mo (11). However, VitA food fortification is challenging due to its poor stability, which can lead to poor bioavailability after degradation, and fat solubility, which limits the inclusion of VitA in water-based and dry food matrices (12). To prevent VitA degradation and improve miscibility, VitA has previously been encapsulated within matrices composed of polysaccharides (13), proteins (14), and/or lipids (15); however, these materials provide limited protection during storage and cooking (16 –18) and can take up to 3 h to release in the stomach (19). Poor protection and slow release of VitA prevent effective absorption. Therefore, the ideal microparticle (MP) platform for VitA fortification should meet these criteria: i) protect VitA against degradation during storage and cooking; ii) rapidly release VitA in the gastrointestinal tract with high absorption; and iii) readily mix with various food matrices at a tunable concentration to meet the dynamic needs of the target population.We hypothesized that by encapsulating VitA with a pH-responsive hydrophobic polymer, we could enhance stability during storage and cooking and ensure its rapid release in the gastrointestinal tract for subsequent absorption. A commercially available, FDA-approved basic methacrylate copolymer (BMC), also known as either Eudragit® E PO or GRAS-status Eudraguard®, was identified from our previous work (20). BMC is generally regarded as safe with an acceptable daily intake of 20 mg/kg body weight (21). VitA-encapsulated BMC MPs were prepared by emulsion at the laboratory scale and by a commercial process at the kilogram scale. Our VitA-BMC-S MPs readily mix with flour and bouillon cubes and demonstrate enhanced stability under cooking and long-term storage conditions (over 12 mo) in comparison to a leading commercial encapsulated VitA product. The bioavailability of VitA from VitA-BMC MPs was first demonstrated in a rodent model, resulting in a ninefold increase in the accumulation of VitA in the liver from cooked VitA-BMC MPs, as compared to cooked unencapsulated free VitA. In a human clinical study, the absorption of VitA from bread fortified with VitA-BMC-S MPs was investigated, with or without the codelivery of encapsulated iron sulfate (FeSO₄) and folic acid (FA), MNs that children and pregnant women globally are also often deficient in (22, 23). The results indicate that VitA is readily released and absorbed from VitA-BMC-S MPs, and the codelivery of encapsulated iron and free FA does not influence the absorption of VitA. In total, we demonstrated scalable production of MP-encapsulated VitA with enhanced stability and good bioavailability in humans, which could potentially mitigate the high burden of VAD and be codelivered with other MNs.     

Analysis of the Effect of Machining of the Surfaces of WAAM 18Ni 250 Maraging Steel Specimens on Their Durability

It is now well-known that the interaction between surface roughness and surface-breaking defects can significantly degrade the fatigue life of additively manufactured (AM) parts. This is also aptly illustrated in the author’s recent study on the durability of wire and arc additively manufactured (WAAM) 18Ni 250 Maraging steel specimens, where it was reported that failure occurred due to fatigue crack growth that arose due to the interaction between the surface roughness and surface-breaking material defects. To improve the durability of an AM part, several papers have suggested the machining of rough surfaces. However, for complex geometries the fully machining of the entire rough surface is not always possible and the effect of the partial machining on durability is unknown. Therefore, this paper investigates if partial machining of WAAM 18Ni 250 Maraging steel surfaces will help to improve the durability of these specimens. Unfortunately, the result of this investigation has shown that partial machining may not significantly improve durability of WAAM 18Ni 250 Maraging steel specimens. Due to the order of surface roughness seen in WAAM 250 Maraging steel, the improvement to durability is only realized by full machining to completely remove the remnants of any print artefacts.     

In Vitro Diagnostic Assay to Detect SARS-CoV-2-Neutralizing Antibody in Patient Sera Using Engineered ACE-2 Mini-Protein

The recent development and mass administration of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccines allowed for disease control, reducing hospitalizations and mortality. Most of these vaccines target the SARS-CoV-2 Spike (S) protein antigens, culminating with the production of neutralizing antibodies (NAbs) that disrupt the attachment of the virus to ACE2 receptors on the host cells. However, several studies demonstrated that the NAbs typically rise within a few weeks after vaccination but quickly reduce months later. Thus, multiple booster administration is recommended, leading to vaccination hesitancy in many populations. Detecting serum anti-SARS-CoV-2 NAbs can instruct patients and healthcare providers on correct booster strategies. Several in vitro diagnostics kits are available; however, their high cost impairs the mass NAbs diagnostic testing. Recently, we engineered an ACE2 mimetic that interacts with the Receptor Binding Domain (RBD) of the SARS-2 S protein. Here we present the use of this engineered mini-protein (p-deface2 mut) to develop a detection assay to measure NAbs in patient sera using a competitive ELISA assay. Serum samples from twenty-one patients were tested. Nine samples (42.8%) tested positive, and twelve (57.1%) tested negative for neutralizing sera. The data correlated with the result from the standard commercial assay that uses human ACE2 protein. This confirmed that p-deface2 mut could replace human ACE2 in ELISA assays. Using bacterially expressed p-deface2 mut protein is cost-effective and may allow mass SARS-CoV-2 NAbs detection, especially in low-income countries where economical diagnostic testing is crucial. Such information will help providers decide when a booster is required, reducing risks of reinfection and preventing the administration before it is medically necessary.     

Attention to Stimuli of Learned versus Innate Biological Value Relies on Separate Neural Systems

The neural bases of attention, a set of neural processes that promote behavioral selection, is a subject of intense investigation. In humans, rewarded cues influence attention, even when those cues are irrelevant to the current task. Because the amygdala plays a role in reward processing, and the activity of amygdala neurons has been linked to spatial attention, we reasoned that the amygdala may be essential for attending to rewarded images. To test this possibility, we used an attentional capture task, which provides a quantitative measure of attentional bias. Specifically, we compared reaction times (RTs) of adult male rhesus monkeys with bilateral amygdala lesions and unoperated controls as they made a saccade away from a high- or low-value rewarded image to a peripheral target. We predicted that: (1) RTs will be longer for high- compared with low-value images, revealing attentional capture by rewarded stimuli; and (2) relative to controls, monkeys with amygdala lesions would exhibit shorter RT for high-value images. For comparison, we assessed the same groups of monkeys for attentional capture by images of predators and conspecifics, categories thought to have innate biological value. In performing the attentional capture task, all monkeys were slowed more by high-value relative to low-value rewarded images. Contrary to our prediction, amygdala lesions failed to disrupt this effect. When presented with images of predators and conspecifics, however, monkeys with amygdala lesions showed significantly diminished attentional capture relative to controls. Thus, separate neural pathways are responsible for allocating attention to stimuli with learned versus innate value.SIGNIFICANCE STATEMENT Valuable objects attract attention. The amygdala is known to contribute to reward processing and the encoding of object reward value. We therefore examined whether the amygdala is necessary for allocating attention to rewarded objects. For comparison, we assessed the amygdala''s contribution to attending to objects with innate biological value: predators and conspecifics. We found that the macaque amygdala is necessary for directing attention to images with innate biological value, but not for directing attention to recently learned reward-predictive images. These findings indicate that the amygdala makes selective contributions to attending to valuable objects. The data are relevant to mental health disorders, such as social anxiety disorders and small animal phobias, that arise from biased attention to select categories of objects.