Targeting dCas9‐SunTag to a Susceptibility Gene Promoter Is Sufficient for CRISPR Interference

ABSTRACT Cassava production in sub‐Saharan Africa is severely impacted by diseases. Most pathogens require interaction with host susceptibility factors to complete their life cycles and cause disease. Targeted DNA methylation is an epigenetic strategy to alter gene expression in plants, and we previously reported that a zinc‐finger fused to DMS3 could establish methylation at the promoter of MeSWEET10a , a bacterial susceptibility gene, and this resulted in decreased disease. Here, we attempt a similar strategy for cassava brown streak disease. This disease is caused by the ipomoviruses CBSV and UCBSV. These viruses belong to the family Potyviridae , which has been shown extensively to require host eIF4E‐family proteins to infect plants and cause disease. We previously found that cassava plants with simultaneous knockout mutations in two eIF4E genes, nCBP‐1 and nCBP‐2 , resulted in decreased susceptibility to CBSD. Here, we report successful simultaneous targeting of both promoters with methylation using a dCas9‐DRMcd‐SunTag system. However, in contrast to our previous work with MeSWEET10a, controls indicate that CRISPR interference is occurring in these lines and is sufficient for the reduction of gene expression. Future research will use genetic crosses to segregate away the DNA methylation reagents and, if DNA methylation proves heritable, assess whether methylation alone is sufficient to increase resistance to CBSD.

In‐reach rehabilitation is feasible and led to functional gains in selected heart and/or lung transplantation recipients

Abstract Background In‐reach rehabilitation is a relatively new model of care available in a small number of Australian public hospitals. These multidisciplinary teams deliver evidence‐based structured rehabilitation to carefully selected patients during acute care. There are no published rehabilitation outcomes in heart and/or lung transplant recipients. Objective To describe the rehabilitation outcomes of a cohort of heart and/or lung transplant recipients. Design Retrospective cohort study. Setting One metropolitan institution with the largest heart and lung transplant service in Australia. Patients Between 2014 and 2023, a total of 957 heart and/or lung transplants were performed at this institution. Intervention In‐reach rehabilitation was delivered to selected patients during the 10‐year period. However, from 2019 onwards, patients were proactively screened by the rehabilitation team for eligibility as well as referred from the acute transplant teams. Main outcome measures Functional independence measure (FIM) changes and percentage of patients going to inpatient rehabilitation after completion of acute care. Results In‐reach rehabilitation was received by 223 (24.3%) patients. With the exception of three patients (who died), the vast majority were able to complete an in‐reach rehabilitation program and were discharged to the community ( n  = 98, 43.9%), inpatient rehabilitation ( n  = 119, 53.4%), or transferred to another hospital ( n  = 3, 1.3%), demonstrating feasibility. Across the cohort, the median admission and discharge FIM scores were 77.0 (interquartile range, 60–94.8) and 100 (interquartile range, 77–118), respectively, demonstrating significant functional improvements from start to finish of the in‐reach rehabilitation program ( p  < .001). Over this period, the number of patients discharged to inpatient rehabilitation decreased as a proportion of the total number of transplants (25.7% in 2014, 47.8% in 2017, 34.7% in 2019, 26.2% in 2021, 8.0% in 2023). Proactive rehabilitation screening implemented from 2019 allowed for earlier and longer program delivery to more patients. Conclusions In‐reach rehabilitation is feasible in acute care after heart and/or lung transplantation and was associated with functional improvements. The addition of proactive rehabilitation screening appeared to improve the effectiveness of the in‐reach rehabilitation program.

Assessing knowledge about the Americans with Disabilities Act and comfort level in treating persons with disabilities among fourth‐year medical students following a clerkship in physical medicine and rehabilitation

Abstract Background All physicians will care for persons with disabilities over the course of their careers. However, most medical school curricula do not adequately prepare students to recognize and address the specific needs of persons with disabilities. Despite physician requirements under the Americans with Disabilities Act (ADA), patients with disabilities may receive substandard care due to a lack of accommodations. Objective To examine the effect of a physical medicine and rehabilitation (PM&R) clerkship on fourth‐year medical students' understanding of physician responsibilities under the ADA as well as comfort levels when caring for persons with disabilities. Design Subjective pre‐ and postintervention surveys. Setting A single institution U.S. allopathic medical school. Participants All fourth‐year medical students enrolled in the mandatory PM&R clerkship during the 2022–2023 academic year were eligible for participation in the study. Surveys were distributed to all eligible students and 99 students successfully completed both surveys for inclusion in the study. Interventions For the 2022–2023 academic year, the mandatory 2‐week PM&R clerkship included didactic lectures on the ADA and additional interactive, first‐hand accounts from persons living with a disability. Main Outcome Measures The change from medical students' baseline to postclerkship knowledge regarding the ADA responsibilities for physicians and overall comfort in working with persons with a physical disability. Results Medical students' overall knowledge of the clinical and financial responsibilities of physicians under the ADA and comfort in interacting with persons with disabilities improved following the clerkship ( p  < .001). Conclusions These findings highlight the importance of disability education as part of medical school curricula to improve medical student knowledge about physician responsibilities under the ADA and overall comfort level in caring for persons with disability. Further studies are needed to determine if these results carry over into clinical practice to combat the health care disparities experienced by persons with disabilities.

Response to Letter Re: Comment on “Assessing knowledge about the Americans with Disabilities Act and comfort level in treating persons with disabilities among fourth‐year medical students following a clerkship in physical medicine and rehabilitation”

Meta‐analysis on effects of Bt‐maize on nontarget invertebrates – Data transportability across continents

Societal impact statement Maize varieties producing insecticidal proteins from Bacillus thuringiensis (Bt) have become an important component of integrated pest management worldwide. For regulatory approval of such plants, risks to the environment need to be assessed. To make such assessments less expensive and time‐consuming, conclusions drawn from data collected in one region could be used in regulatory submissions in other regions. By comparing published data of invertebrates recorded in Bt maize field experiments worldwide, we contribute to the discussion of data transportability across continents. The results are of value to regulatory authorities throughout the world and ultimately of benefit to growers and consumers. Summary For insecticidal crops, adverse effects on non‐target invertebrates including beneficial decomposers, predators, and parasitoids are of particular concern. This work focuses on data transportability across continents by comparing non‐target invertebrate data from published Bt maize field studies. Data derived from a comprehensive global database were summarized for taxonomic composition and subjected to meta‐analyses considering taxonomy, Bt maize target insect order, and ecological functional group. Each dataset represents a replicated comparison of an invertebrate population recorded in Bt maize with the respective population in non‐Bt (control) maize. Taxonomic composition at order or higher taxonomic level was comparable across continents. Meta‐analyses revealed that most analyzed invertebrates were equally abundant in Bt and non‐Bt maize, while robust adverse effects were only observed on specialized parasitoids of target pests. The conclusions drawn from the North American data were confirmed for Europe and Asia. The similarity of species communities at order level as well as outcomes of meta‐analyses across continents indicate that conclusions from field studies are generally transportable across geographies. High‐quality, well‐designed, well‐described, and independent studies from multiple locations and years, and open‐access data availability (transparency), increase trust in the conclusions drawn and the usefulness for submissions to multiple regulatory systems.

A manifesto for plant science education

Societal Impact Statement Plants provide oxygen, food, shelter, medicines and environmental services, without which human society could not exist. Tackling pressing and global challenges requires well‐trained plant scientists and plant‐aware individuals. This manifesto provides a practical evidence‐based vision to strengthen plant science education, focused on five strategic priorities. It is relevant to all stakeholders within plant science and beyond: from frontline educators to institutional leaders; from commercial or charitable professionals to entrepreneurs and donors; from individual community members to their legislative representatives. Strengthening plant science education demands concrete actions from all stakeholders, ultimately to the benefit of us all. Summary Plant science education needs urgent attention. Skilled plant scientists are needed to address major environmental and societal challenges, and global communities require plant‐aware professionals to drive impactful policy, research and environmental stewardship. This manifesto was collaboratively generated by a community of educators who gathered to reflect on the state of plant science education. The forward‐facing document provides a clear strategy for plant science education, complementing existing research strategies. Five themes were identified as essential for meeting the evolving needs of plant science, educators and learners: (i) plants must be at the centre of an education that addresses global challenges and societal values; (ii) plant science education must prepare students for their futures using bold and effective pedagogies; (iii) equity, diversity and inclusion must be robustly embedded in educational practices; (iv) local and strategic partnerships (with industry and beyond) are required to strengthen academic education; and (v) plant science educators need resources and opportunities to develop and connect. The manifesto is intended as a framework for change. Educators, funders, publishers, industry representatives, policymakers and all other members of our communities must commit to sustained investment in plant science education. By proactively and collectively embracing the recommendations provided, the sector has an opportunity to cultivate a new generation equipped with the knowledge, skills and passion to unlock the full potential of photosynthetic organisms.

A Constitutive Model for the Non‐shock Ignition and Mechanical Response of PBX Under Low Velocity Impact

ABSTRACT A physical framework is constructed to characterize the viscoelastic‐plastic response, crack damage, and ignition characteristics of polymer‐bonded explosives (PBXs) under low‐velocity impact conditions. This approach enhances the existing viscoelastic statistical crack mechanics model (Visco‐SCRAM) through multiple key advancements. Damage progression is determined using the generalized Griffith criterion for instability, focusing on the most critical (i.e., maximally unstable) crack size instead of an orientation‐averaged crack dimension. The collective effect of numerous microcracks on the dynamic mechanical response of microcrack‐containing materials is captured in a statistical sense through a dominant microcrack represented as an internal variable. A confined Steven test simulation is conducted to validate the model and to investigate the mechanical and thermal responses of PBXs. The analysis reveals that although the radius has a minimal effect on the temperature rise of the specimen, the thickness plays a significant role in determining the velocity threshold, the location of the hot spot, and the extent of temperature increase. The simulation results show that the critical impact velocities are 45 m/s for the oval projectile, 95 m/s for the flat projectile, and 24 m/s for the pin projectile. A comprehensive analysis is provided regarding the differences in temperature rise among the three types of projectiles. Moreover, the study demonstrates that frictional work is the dominant ignition mechanism during low‐velocity impacts. This research presents a new perspective for understanding and predicting the ignition behavior of PBX under such impact conditions.

Contrasting Chemical Kinetic Parameters for Near In‐Service‐Temperature Aging and High‐Temperature Thermal Decomposition of Triaminotrinitrobenzene Formulations

ABSTRACT Triaminotrinitrobenzene‐based high explosives such as LX‐17 offer high energy density and exceptional safety, yet their long‐term aging behavior at low temperatures remains poorly understood. In this study, several legacy and new production lots of LX‐17 were subjected to accelerated aging experiments below 100°C, during which the formation rate of the initial degradation product, monofurazan (F1), was monitored. Kinetic analysis was performed using a sample‐age‐aware computational approach, yielding activation energy estimates of 82–91 kJ mol −1 for the low‐temperature initiation step—markedly lower than the ∼200 kJ mol −1 associated with high‐temperature thermal decomposition. Extrapolation from established cookoff models supports the conclusion that the dominant degradation mechanism at low temperatures differs from that at high temperatures. Our results provide a unified kinetic framework that bridges the gap between in‐service conditions and high‐temperature damage.

Design and characterization of calprotectin tetramerization variants for probing the role of oligomerization in receptor activation

Abstract Calprotectin is a heterodimer of the S100A8 and S100A9 EF‐hand calcium binding proteins, which activates cell surface receptors that signal through the NF‐κB inflammatory signaling pathway. Like all S100 proteins, calcium‐induced conformational changes in calprotectin are required for binding to partner proteins. In the case of calprotectin, the addition of calcium correlates with the formation of a dimer of heterodimers (heterotetramer). Ligand‐induced receptor oligomerization has been proposed as a mechanism of receptor activation. Conversely, it has also been suggested that calprotectin tetramerization can inhibit binding to receptors and serve as an autoinhibitory mechanism. In order to investigate the biological relevance of calprotectin tetramerization and facilitate in‐depth biophysical and structural analysis, we have prepared three tetramerization‐deficient variants: two single‐site S100A8 mutations of hydrophobic isoleucine residues mediating the tetramer interface to lysine (I60K, I73K) and the corresponding double‐site mutant (I60K/I70K). Dynamic light scattering, small‐angle x‐ray scattering, and nuclear magnetic resonance spectroscopy showed that all three tetramer‐deficient variants remain as dimers in solution even in the presence of 40‐fold excess calcium and undergo calcium‐induced conformational changes. The crystal structure of I73K was determined to atomic‐level resolution and confirms that the mutations cause only subtle, localized effects on the structure. Together, the results indicate that these tetramerization‐deficient mutants will be useful reagents for discerning the functional role of calprotectin oligomerization in the activation of inflammatory receptors.

Streptococcus pneumoniae HtrA is a dynamic and monomeric virulence factor capable of forming larger oligomeric complexes

Abstract High‐temperature requirement A (HtrA) proteases are a conserved family of serine proteases central to protein quality control and bacterial virulence. While Gram‐negative and human HtrAs are structurally well studied, Gram‐positive homologs remain essentially uncharacterized. Here, we present the first integrated structural and mechanistic analysis of a Gram‐positive HtrA, from Streptococcus pneumoniae , a virulence factor essential for adhesion and infection in vivo. Proteomic profiling of an htrA knockout and cleavage assays demonstrate that S. pneumoniae HtrA is required for protein quality control, with the PDZ domain mediating substrate recognition. Biochemically, S. pneumoniae HtrA exists exclusively as a monomer in solution, a striking divergence from canonical trimeric HtrAs that we show is shared with other Gram‐positive homologs. NMR analyses reveal that the monomer dynamically samples open and closed conformations, while cryo‐EM of a catalytic mutant identifies a hexamer stabilized by a unique LoopA–PDZ interaction. Together, these findings define S. pneumoniae HtrA as a dynamic monomer with interdomain coupling between its protease and PDZ domains, establishing Gram‐positive HtrAs as a mechanistically divergent subgroup within the HtrA family.