Plant Expression of Trans-Encapsidated Chimeric Viral Vaccines with Animal RNA Replicons: An Update.

In this protocol, we outline how to produce a chimeric viral vaccine in a biosafety level 1 (BSL1) environment. An animal viral vector RNA encapsidated with tobacco mosaic virus (TMV) coat protein can be fully assembled in planta. Agrobacterium cultures containing each component are inoculated together into tobacco leaves and the self-assembled hybrid chimeric viral vaccine is harvested 4 days later and purified with a simple PEG precipitation. The viral RNA delivery vector is derived from the BSL1 insect virus, Flock House virus (FHV), and replicates in human and animal cells but does not spread systemically. A polyethylene glycol purification protocol is also provided to collect and purify these vaccines for immunological tests. In this update, we also provide a protocol for in trans co-inoculation of a modified FHV protein A, which significantly increased the yield of in planta chimeric viral vaccine.

Validation of gavage sampling as tool for longitudinal sampling of microbiota of the mouse gastric lumen.

BACKGROUND: Fecal samples are commonly used for longitudinal studies of the gut lumen microbiome to track the course of response to infection or drug treatment, but no comparable method has been evaluated for longitudinal analysis of the gastric lumen microbiome in mice. Herein, a buffer flush of the stomach with a flexible gavage needle was used to collect gastric contents at one or several time points without harming the mouse. These samples were compared to samples collected by sacrifice and dissection of the mouse stomach. Microbiota from these samples were sequenced and evaluated in two ways: the composition of samples as measured by beta diversity and the richness of samples as measured by alpha diversity. Additionally, the effect of multiple sampling every two days on these metrics were studied. DNA was extracted from each of these samples and Illumina 16S rRNA gene sequencing was performed. RESULTS: First, taxonomic richness of gavage and dissection samples was compared. A greater number of taxa was detected in gavage samples than in dissection samples. Second, taxonomic richness was analyzed over time. No significant difference in taxonomic richness was observed with repeated gavage flushes. Third, a comparison was made of the taxonomic composition of samples collected by gavage versus dissection followed by a comparison of samples collected over multiple samplings. Nonmetric multidimensional scaling analysis revealed no clear differences between collection by gavage flushing or dissection. Using weighted Unifrac and Aitchison taxonomic distances between gavage and dissection samples were not significantly different from distances between gavage samples themselves, and no significant difference was found in the taxonomic composition of mice which were sampled repeatedly. Finally, relative abundances of specific identified taxa were compared, and eleven taxa were found to differ in frequency between collection methods. Using the more stringent Analysis of Composition of Microbiomes (ANCOM), seven was found to differ. Similarly, no significant differences were uncovered using these analyses over multiple samples by gastric flush. CONCLUSION: In summary, the consistency of the microbiota collected by gastric flushing recommends its use for microbiome analysis of gastric fluid similar to the use of fecal sampling to study the gut lumen microbiome.

Comparative Effectiveness of Mepolizumab, Benralizumab, and Dupilumab among Patients with Difficult-to-Control Asthma: A Multicenter Retrospective Propensity-matched Analysis.

Rationale: The comparative effectiveness of biologic agents used as add-on therapy in the management of difficult-to-control asthma is unclear. Objective: To compare the effectiveness of dupilumab, mepolizumab, and benralizumab among patients with difficult-to-control asthma. Methods: Retrospective multicenter cohort study of adult patients with difficult-to-control asthma starting treatment with dupilumab, mepolizumab, or benralizumab as documented in a multicenter electronic health record and claims-based database between October 19, 2018, and September 30, 2022. Propensity-score matching was used to minimize bias from nonrandomized treatment assignment; a prespecified α-level was set at 0.017 to account for three primary comparisons. The exposure of interest was the new initiation of dupilumab, benralizumab, or mepolizumab treatment. The primary outcome was the rate of asthma exacerbations in the 1 year after initiation of biologic therapy modeled using a negative binomial approach. Results: Among 893,668 patients with asthma who were prescribed an inhaled corticosteroid and were ⩾12 years old (65% female; mean age, 49 yr), 3,943 started dupilumab, 1,902 started benralizumab, and 2,012 started mepolizumab, all without an alternative indication for biologic therapy. After matching, there were 1,805 patients in each group for comparisons between dupilumab and benralizumab, 1,865 for comparisons between dupilumab and mepolizumab, and 1,721 for comparisons between mepolizumab and benralizumab. For all pairwise comparisons, covariates were well balanced after matching (all standardized mean differences <0.1). Patients who initiated dupilumab had a significantly lower rate of asthma exacerbations (1.07 per year) compared with benralizumab (1.47 per year), with a rate ratio (RR) of 0.73 (95% confidence interval, 0.63-0.85), and also had a significantly lower rate of asthma exacerbations compared with mepolizumab (1.04 per year vs. 1.45 per year), with an RR of 0.72 (0.62-0.84). There was no statistically significant difference in the rate of asthma exacerbations between mepolizumab (1.40 per year) and benralizumab (1.41 per year), with an RR of 1.00 (0.85-1.17). Conclusions: In patients with difficult-to-control asthma who had newly initiated biologic therapy, dupilumab was associated with a decreased rate of asthma exacerbations in the 1 year after initiation compared with mepolizumab or benralizumab.

Control of Helicobacter pylori with engineered probiotics secreting selective guided antimicrobial peptides.

Helicobacter pylori is the primary cause of 78% of gastric cancer cases, providing an opportunity to prevent cancer by controlling a single bacterial pathogen within the complex gastric microbiota. We developed highly selective antimicrobial agents against H. pylori by fusing an H. pylori-binding guide peptide (MM1) to broad-spectrum antimicrobial peptides. The common dairy probiotic Lactococcus lactis was then engineered to secrete these guided antimicrobial peptides (gAMPs). When co-cultured in vitro with H. pylori, the gAMP probiotics lost no toxicity compared to unguided AMP probiotics against the target, H. pylori, while losing >90% of their toxicity against two tested off-target bacteria. To test binding to H. pylori, the MM1 guide was fused to green fluorescent protein (GFP), resulting in enhanced binding compared to unguided GFP as measured by flow cytometry. In contrast, MM1-GFP showed no increased binding over GFP against five different off-target bacteria. These highly selective gAMP probiotics were then tested by oral gavage in mice infected with H. pylori. As a therapy, the probiotics outperformed antibiotic treatment, effectively eliminating H. pylori in just 5 days, and also protected mice from challenge infection as a prophylactic. As expected, the gAMP probiotics were as toxic against H. pylori as the unguided AMP probiotics. However, a strong rebound in gastric species diversity was found with both the selective gAMP probiotics and the non-selective AMP probiotics. Eliminating the extreme microbial dysbiosis caused by H. pylori appeared to be the major factor in diversity recovery. IMPORTANCE Alternatives to antibiotics in the control of Helicobacter pylori and the prevention of gastric cancer are needed. The high prevalence of H. pylori in the human population, the induction of microbial dysbiosis by antibiotics, and increasing antibiotic resistance call for a more sustainable approach. By selectively eliminating the pathogen and retaining the commensal community, H. pylori control may be achieved without adverse health outcomes. Antibiotics are typically used as a therapeutic post-infection, but a more targeted, less disruptive approach could be used as a long-term prophylactic against H. pylori or, by extension, against other gastrointestinal pathogens. Furthermore, the modular nature of the guided antimicrobial peptide (gAMP) technology allows for the substitution of different guides for different pathogens and the use of a cocktail of gAMPs to avoid the development of pathogen resistance.

Child and Adolescent Psychiatry and Underrepresented Youth With School Attendance Problems: Integration With Systems of Care, Advocacy, and Future Directions.

School attendance problems (SAPs) include full-day and partial absences (eg, missing classes, tardiness) as well as difficulties going to or remaining in school. SAPs also include nonparticipation in distance/hybrid learning formats or lack of access to necessary technology or equipment. SAPs are particularly prevalent among students of color, students in poverty, students with disabilities, English language learners, and migrant populations. SAPs are often part of a complicated clinical picture of mental health (eg, emotional, neurodevelopmental, conduct disorders) and somatic (eg, abdominal, cardiovascular, respiratory problems) challenges. These challenges are exacerbated by disparities in socioeconomic status, childhood adversities, family structure, and neighborhood-level factors that have an impact on mental health outcomes.1 SAPs have serious negative consequences in childhood (eg, lower academic achievement, greater risk of dropout) and in adulthood (eg, lower lifetime earning potential, greater occupational and mental health problems). Unfortunately, underrepresented youth with SAPs often have less access to proper care, especially psychiatric care.

Exploring the relationships between resilience, mindfulness, and experiential avoidance after the use of a mindfulness- and acceptance-based mobile app for posttraumatic stress disorder.

PURPOSE: This study used a mindfulness- and acceptance-based mobile app to examine the relationships between resilience, mindfulness, experiential avoidance, and posttraumatic stress disorder (PTSD) symptoms. DESIGN AND METHODS: A quasi-experimental pre-posttest, single-group study design was used. A total of 23 college student veterans used the app for 4 weeks. Outcomes of resilience, mindfulness, experiential avoidance, and PTSD were measured at three time-points (baseline, end of Week 2, and end of Week 4). FINDINGS: All outcomes significantly improved at postintervention. Improvements in resilience and PTSD significantly correlated with improvement in mindfulness. PRACTICE IMPLICATIONS: Mindfulness- and acceptance-based mobile apps can be safely used by individuals with PTSD as a complementary approach to enhance resilient coping with PTSD.

The Heterogeneity of Selective Mutism: A Primer for a More Refined Approach.

Selective mutism is a persistent and debilitating psychiatric disorder in which a child fails to speak in situations where speaking is expected. Although listed as an anxiety disorder, the multifaceted and heterogeneous nature of selective mutism indicates that a more accurate conceptualization may be as a neurodevelopmental disorder. This article serves as a primer of historical and clinical presentations, empirical clinical profiles, clinical distinctions, assessment, and treatment related to the complexity of selective mutism. The article includes a brief discussion of selective mutism within a developmental psychopathology perspective with an eye toward reformed efforts for prevention, assessment, and treatment regarding this population.

A multi-tiered systems of support blueprint for re-opening schools following COVID-19 shutdown.

The COVID-19 pandemic will create enormous disruptions for youth and families with respect to economic and health status, social relationships, and education for years to come. The process of closing and intermittently reopening schools adds to this disruption and creates confusion for parents and school officials who must balance student educational progress with health and safety concerns. One framework that may serve as a roadmap in this regard is a multi-tiered systems of support (MTSS) model. This article briefly addresses four main domains of functioning (adjustment, traumatic stress, academic status, health and safety) across three tiers of support (universal, targeted, intensive). Each section draws on existing literature bases to provide specific recommendations for school officials who must address various and changing logistical, academic, and health-based challenges. The recommendations are designed to be flexible given fluctuations in the current crisis as well as focused on maximum-value targets. An MTSS approach adapted for contemporary circumstances can also be used to help address longstanding disparities that have been laid bare by the pandemic.

The use of functionally deficient viral vectors as visualization tools to reveal complementation patterns between plant viruses and the silencing suppressor p19.

Plant virus transport complementation is classically observed as a helper virus allowing another virus to regain cell-to-cell or systemic movement through a restrictive host plant (Malyshenko et al., 1989). The complementation effect is usually studied by observing virus infection after co-infection or super-inoculation of the helper virus. We herein demonstrate the utility of functionally deficient viral vectors as tools to determine the contribution of individual viral genes to plant viral transport complementation. Two functionally deficient viral vectors were engineered that derive from foxtail mosaic potexvirus and sunn-hemp mosaic tobamovirus, namely FECT (FoMV Eliminate CP and TGB, (Liu and Kearney, 2010)) and SHEC (SHMV Eliminate CP gene, (Liu and Kearney, 2010)), respectively. FECT had all the ORFs removed except for the replicase and thus is defective for both long-distance and cell-to-cell movement. SHEC lacked only the coat protein ORF and retained the movement protein (MP) and is functional for cell-to-cell movement. When FECT and SHEC vectors were inoculated with the silencing suppressor p19 in different zones of the same leaf, FECT was enabled to express its reporter gene beyond the original inoculation zone. When FECT, SHEC, and p19 were individually inoculated in separate zones, both FECT and SHEC reporter gene expression was observed within the p19 zone, distant from the original virus inoculation points. These observations indicate that SHEC movement protein could create a trafficking network to allow viral RNAs of FECT and SHEC and p19/p19 transcript to move from cell to cell. This system provides a tool to visually monitor the movement of viruses and silencing suppressors as well as to identify the effects of individual viral components on virus movement.

The School Climate and Academic Mindset Inventory (SCAMI): Confirmatory Factor Analysis and Invariance Across Demographic Groups.

School climate is a multidimensional construct of the quality of a student's academic environment, often subsuming dimensions such as safety, instructional practices, social relationships, school facilities, and school connectedness. Positive school climate has beneficial effects on a wide range of adjustment variables in youth, including academic achievement, mental health, school attendance and graduation, and school-based behavior. Studies regarding school climate assessment have burgeoned in recent years but remain marked by limited sample sizes, narrow developmental levels, restricted items, unclear psychometric strength across multiple demographic groups, and lack of integration with key student education contextual factors such as academic mindset and social emotional learning. The present study evaluated a comprehensive measure of aspects of school climate, academic mindset, and social emotional learning in a sample of 105,379 diverse students (M age = 13.47 years; SD = 2.47). The 9-factor structure of the School Climate and Academic Mindset Inventory was supported via confirmatory factor analysis. A trimmed model displayed adequate goodness-of-fit for males and females, younger age groups, and European American, biracial/multiracial, Hispanic, Native American, and Native Hawaiian/Pacific Islander students. The trimmed model was slightly less strong for older age groups as well as for African American and Asian American students. The scale may be useful for assessing school climate interventions, longitudinal climate patterns, and school-based algorithms of future performance, though additional validation of the scale remains necessary.

The readily transformable Impatiens walleriana efficiently attracts nectar feeding with Aedes and Culex mosquitoes in simulated outdoor garden settings in Mississippi and Florida.

Mosquitoes rely upon plant nectars for their energy needs, a trait that has the potential to allow nectar to serve as a platform for producing and delivering toxins to nuisance and/or vector mosquito species. Impatiens walleriana (Order: Ericales, Family: Balsaminaceae) is a readily transformable and widely planted nectar plant that has been previously shown to attract mosquito nectar-feeding. However, those feeding studies were only conducted indoors and did not test if variable environmental conditions will affect nectar feeding. In this study, we tested incidence of nectar feeding from the extrafloral nectaries of I. walleriana with the mosquitoes Aedes aegypti, Ae. albopictus and Culex quinquefasciatus (Order: Diptera, Family: Culicidae) in simulated, outdoor garden settings in Mississippi and Florida. I. walleriana and other common garden plants (in a 1:4 ratio) were placed into a mesh-lined 4'x7' mesocosm along with 50 mosquitoes. To track nectar feeding, the nectar of I. walleriana was tagged with red dye and mosquitoes were analyzed for red dye fluorescence after feeding. Fluorescence analysis demonstrated that 81.9% of male and 86.6% of female mosquitoes fed on the nectar of I. walleriana within 24 h. This suggests that mosquitoes may readily feed on impatiens nectar in outdoor garden settings at temperate and semi-tropical sites, even when alternate common garden plants are available. This attraction capacity is essential for the further consideration of I. walleriana for development as a transgenic, mosquitocidal nectar plant.

A Multidimensional, Multi-tiered System of Supports Model to Promote School Attendance and Address School Absenteeism.

School attendance and school completion are important benchmarks of successful development. Unfortunately, school absenteeism and school dropout remain debilitating and prevalent conditions among youth. Stakeholders invested in promoting school attendance and reducing school absenteeism generally agree that multifaceted ecological frameworks are needed to account for these heterogeneous problems as well as differences across local education agencies and broader jurisdictions. A multi-tiered system of supports (MTSS) framework emphasizes many aspects that match well with school attendance and its problems, including prevention and a continuum of supports, screening, evidence-based assessment and intervention, problem-solving and data-based decision-making, implementation fidelity, and natural embedding into extant school improvement plans. This article outlines a multidimensional MTSS model for school attendance and absenteeism to account for recent developments regarding service delivery within schools. Such developments include integrated models of multi-tiered service delivery to concurrently address multiple domains of functioning, the development of more nuanced approaches for students with various challenges, and consideration of three-dimensional (pyramidal) perspectives to allow simultaneous and yet nuanced strategies for several domain clusters. Sample domain clusters common to the literature that could populate the multiple dimensions or sides of a MTSS pyramid model for school attendance and absenteeism are presented. These domain clusters include (1) school refusal/truancy/school withdrawal/school exclusion, (2) functional profiles and analysis, (3) preschool/elementary/middle/high school, (4) ecological levels of impact on school attendance and its problems, and (5) low/moderate/high absenteeism severity. Recommendations are made as well regarding broader MTSS integration and implementation science vis-à-vis school attendance and its problems.

Repurposing a drug targeting peptide for targeting antimicrobial peptides against Staphylococcus.

OBJECTIVES: Targeted therapies seek to selectively eliminate a pathogen without disrupting the resident microbial community. However, with selectivity comes the potential for developing bacterial resistance. Thus, a diverse range of targeting peptides must be made available. RESULTS: Two commonly used antimicrobial peptides (AMPs), plectasin and eurocin, were genetically fused to the targeting peptide A12C, which selectively binds to Staphylococcus species. The targeting peptide did not decrease activity against the targeted Staphylococcus aureus and Staphylococcus epidermidis, but drastically decreased activity against the nontargeted species, Enterococcus faecalis, Bacillus subtilis, Lactococcus lactis and Lactobacillus rhamnosus. This effect was equally evident across two different AMPs, two different species of Staphylococcus, four different negative control bacteria, and against both biofilm and planktonic forms of the bacteria. CONCLUSIONS: A12C, originally designed for targeted drug delivery, was repurposed to target antimicrobial peptides. This illustrates the wealth of ligands, both natural and synthetic, which can be adapted to develop a diverse array of targeting antimicrobial peptides.

Making plants into cost-effective bioreactors for highly active antimicrobial peptides.

As antibiotic-resistant bacterial pathogens become an ever-increasing concern, antimicrobial peptides (AMPs) have grown increasingly attractive as alternatives. Potentially, plants could be used as cost-effective AMP bioreactors; however, reported heterologous AMP expression is much lower in plants than in E. coli expression systems and often results in plant cytotoxicity, even for AMPs fused to carrier proteins. This suggests that there may be a physical characteristic of the previously described heterologous AMPs which impedes efficient expression in plants. Using a meta-analysis of protein databases, this study has determined that native plant AMPs were significantly less cationic than AMPs native to other taxa. To apply this finding to plant expression, the transient expression of 10 different heterologous AMPs, ranging in charge from +7 to -5, was tested in the tobacco, Nicotiana benthamiana. Elastin-like polypeptide (ELP) was used as the carrier protein for AMP expression. ELP fusion allowed for a simple, cost-effective temperature shift purification. Using this system, all five anionic AMPs expressed well, with two at unusually high levels (375 and 563 µg/gfw). Furthermore, antimicrobial activity against Staphylococcus epidermidis was an order of magnitude greater (average minimum inhibitory concentration MIC of 0.26µM) than that typically seen for AMPs expressed in E. coli systems and was associated with the uncleaved fusion peptide. In summary, this study describes a means of expressing AMP fusions in plants in high yield, purified by a simple temperature-shift protocol, resulting in a fusion peptide with high antimicrobial activity and without the need for a peptide cleavage step.

Reconciling Contemporary Approaches to School Attendance and School Absenteeism: Toward Promotion and Nimble Response, Global Policy Review and Implementation, and Future Adaptability (Part 2).

As noted in Part 1 of this two-part review, school attendance is an important foundational competency for children and adolescents, and school absenteeism has been linked to myriad short- and long-term negative consequences, even into adulthood. Categorical and dimensional approaches for this population have been developed. This article (Part 2 of a two-part review) discusses compatibilities of categorical and dimensional approaches for school attendance and school absenteeism and how these approaches can inform one another. The article also poses a multidimensional multi-tiered system of supports pyramid model as a mechanism for reconciling these approaches, promoting school attendance (and/or prevention of school absenteeism), establishing early warning systems for nimble response to school attendance problems, assisting with global policy review and dissemination and implementation, and adapting to future changes in education and technology.