Dynamic calcium signals mediate the feeding response of the carnivorous sundew plant.

Some of the most spectacular examples of botanical carnivory-in which predator plants catch and digest animals presumably to supplement the nutrient-poor soils in which they grow-occur within the Droseraceae family. For example, sundews of the genus Drosera have evolved leaf movements and enzyme secretion to facilitate prey digestion. The molecular underpinnings of this behavior remain largely unknown; however, evidence suggests that prey-induced electrical impulses are correlated with movement and production of the defense hormone jasmonic acid (JA), which may alter gene expression. In noncarnivorous plants, JA is linked to electrical activity via changes in cytoplasmic Ca2+. Here, we find that dynamic Ca2+ changes also occur in sundew (Drosera spatulata) leaves responding to prey-associated mechanical and chemical stimuli. Furthermore, inhibition of these Ca2+ changes reduced expression of JA target genes and leaf movements following chemical feeding. Our results are consistent with the presence of a conserved Ca2+-dependent JA signaling pathway in the sundew feeding response and provide further credence to the defensive origin of plant carnivory.

Rasch Calibration of the International Knee Documentation Committee Subjective Knee Form.

CONTEXT: Among numerous knee-related patient-reported outcome measures, the International Knee Documentation Committee Subjective Knee Form (IKDC-SKF) has been used across a wide variety of knee pathologies. However, traditional validation procedures (classical test theory) and existing studies (estimating item parameters) have limitations in establishing the measurement properties of the IKDC-SKF. Rasch analysis reveals a strong validation approach to improve IKDC-SKF clinical interpretation with larger samples. OBJECTIVE: To assess psychometric properties, including differential item functioning, of the IKDC-SKF as a patient-reported measure of knee function. DESIGN: Cross-sectional study. SETTING: Secondary data. Data were extracted from the cloud-based orthopedic and sports medicine global registry Surgical Outcome System (Arthrex). PATIENTS: A total of 1725 individuals who underwent an arthroscopic knee procedure and completed all items on the IKDC-SKF. MAIN OUTCOME MEASURE(S): Rasch analysis including model-data fit, rating scale's function, item-person map (distribution of item difficulty and person ability), and differential item functioning (sex and age groups) was used to evaluate the psychometric properties of the IKDC-SKF. RESULTS: Ten misfit items were found and removed. The 5-point Likert scale of the 9-item IKDC-SKF worked well. Item difficulty ranged from 0.58 to 0.81 logits, and person's knee function had ranged from -5.56 to 4.86 logits, with a wide distribution. The IKDC-SKF was found to function similarly for sex (male vs female) and age. CONCLUSIONS: Rasch analysis identified a unidimensional structure retaining 9 of the original IKDC-SKF items; however, a more comprehensive inventory is necessary to assess a wider range of knee function and improve measurement validity.

Heritable Phytohormone Profiles of Poplar Genotypes Vary in Resistance to a Galling Aphid.

Insect galls are highly specialized structures arising from atypical development of plant tissue induced by insects. Galls provide the insect enhanced nutrition and protection against natural enemies and environmental stresses. Galls are essentially plant organs formed by an intimate biochemical interaction between the gall-inducing insect and its host plant. Because galls are plant organs, their development is likely to be governed by phytohormones involved in normal organogenesis. We characterized concentrations of both growth and defensive phytohormones in ungalled control leaves and galls induced by the aphid Pemphigus betae on narrowleaf cottonwood Populus angustifolia that differ genotypically in resistance to this insect. We found that susceptible trees differed from resistant trees in constitutive concentrations of both growth and defense phytohormones. Susceptible trees were characterized by significantly higher constitutive cytokinin concentrations in leaves, significantly greater ability of aphids to elicit cytokinin increases, and significantly lower constitutive defense phytohormone concentrations than observed in resistant trees. Phytohormone concentrations in both constitutive and induced responses in galled leaves exhibited high broad-sense heritability that, respectively, ranged from 0.39 to 0.93 and from 0.28 to 0.66, suggesting that selection can act upon these traits and that they might vary across the landscape. Increased cytokinin concentrations may facilitate forming strong photosynthate sinks in the galls, a requirement for galling insect success. By characterizing for the first time the changes in 15 phytohormones belonging to five different classes, this study offers a better overview of the signaling alteration occurring in galls that has likely been important for their ecology and evolution. Copyright © 2019 The Author(s). This is an open-access article distributed under the CC BY-NC-ND 4.0 International license .

Two- and three-input TALE-based AND logic computation in embryonic stem cells.

Biological computing circuits can enhance our ability to control cellular functions and have potential applications in tissue engineering and medical treatments. Transcriptional activator-like effectors (TALEs) represent attractive components of synthetic gene regulatory circuits, as they can be designed de novo to target a given DNA sequence. We here demonstrate that TALEs can perform Boolean logic computation in mammalian cells. Using a split-intein protein-splicing strategy, we show that a functional TALE can be reconstituted from two inactive parts, thus generating two-input AND logic computation. We further demonstrate three-piece intein splicing in mammalian cells and use it to perform three-input AND computation. Using methods for random as well as targeted insertion of these relatively large genetic circuits, we show that TALE-based logic circuits are functional when integrated into the genome of mouse embryonic stem cells. Comparing construct variants in the same genomic context, we modulated the strength of the TALE-responsive promoter to improve the output of these circuits. Our work establishes split TALEs as a tool for building logic computation with the potential of controlling expression of endogenous genes or transgenes in response to a combination of cellular signals.

Evaluating Psychometric Properties of the International Knee Documentation Committee Subjective Knee Form in a Heterogeneous Sample of Post-Operative Patients.

Background: The International Knee Document Committee Subjective Knee Form (IKDC-SKF) is a patient-reported outcome measure used in orthopedics and sports medicine. Further psychometric assessment is necessary to confirm measurement properties in a large, heterogenous sample. Purpose: The purpose of the study was to assess the psychometric properties of the IKDC-SKF in a large, heterogenous sample. Study Design: Cross-Sectional Study. Methods: An exploratory factor analysis (EFA) was conducted to identify a sound latent structure and to assess internal consistency in a large sample of patients who underwent knee arthroscopy. A confirmatory factor analysis (CFA) was conducted to confirm structural validity. Multi-group invariance was conducted to assess factorial stability across sex and age groups, while longitudinal invariance procedures were performed to assess stability over time. Results: A 3-factor, 9-item IKDC-SKF short form was identified with EFA procedures. The model was confirmed with CFA (CFI = 0.983; TLI = 0.975; IFI = 0.983; RMSEA = 0.057), while a sound 2-factor, 6-item model was also identified (CFI = 1.0; TLI = 0.999; IFI = 1.0; RMSEA = 0.11). The 9-item IKDC-SKF short form was invariant across groups but not time; removal of a single item (i.e., 8-item IKDC-SKF short form) resulted in longitudinal invariance. The 6-item IKDC-SKF short form was invariant across groups and time. Conclusion: The 6-item, 8-item, and 9-item short form versions of the IKDC-SKF exceed contemporary fit recommendations and present as plausible alternatives to the IKDC-SKF with improved measurement properties, reduced scale response burden, and evidence of multi-group and longitudinal invariance. Further, the 6- and 8-item IKDC-SKF short forms may be used to assess group differences or change across time.# Level of evidence hereLevel 3©The Author(s).

Enhancing Precision and Efficiency of Cas9-Mediated Knockin Through Combinatorial Fusions of DNA Repair Proteins.

Cas9 targets genomic loci with high specificity. For knockin with double-strand break repair, however, Cas9 often leads to unintended on-target knockout rather than intended edits. This imprecision is a barrier for direct in vivo editing where clonal selection is not feasible. In this study, we demonstrate a high-throughput workflow to comparatively assess on-target efficiency and precision of editing outcomes. Using this workflow, we screened combinations of donor DNA and Cas9 variants, as well as fusions to DNA repair proteins. This yielded novel high-performance double-strand break repair editing agents and combinatorial optimizations, yielding increases in knockin efficiency and precision. Cas9-RC, a novel fusion Cas9 flanked by eRad18 and CtIP[HE], increased knockin performance in vitro and in vivo in the developing mouse brain. Continued comparative assessment of editing efficiency and precision with this framework will further the development of high-performance editing agents for in vivo knockin and future genome therapeutics.

Rapid modeling of an ultra-rare epilepsy variant in wild-type mice by in utero prime editing.

Generating animal models for individual patients within clinically-useful timeframes holds great potential toward enabling personalized medicine approaches for genetic epilepsies. The ability to rapidly incorporate patient-specific genomic variants into model animals recapitulating elements of the patient's clinical manifestations would enable applications ranging from validation and characterization of pathogenic variants to personalized models for tailoring pharmacotherapy to individual patients. Here, we demonstrate generation of an animal model of an individual epilepsy patient with an ultra-rare variant of the NMDA receptor subunit GRIN2A, without the need for germline transmission and breeding. Using in utero prime editing in the brain of wild-type mice, our approach yielded high in vivo editing precision and induced frequent, spontaneous seizures which mirrored specific elements of the patient's clinical presentation. Leveraging the speed and versatility of this approach, we introduce PegAssist, a generalizable workflow to generate bedside-to-bench animal models of individual patients within weeks. The capability to produce individualized animal models rapidly and cost-effectively will reduce barriers to access for precision medicine, and will accelerate drug development by offering versatile in vivo platforms to identify compounds with efficacy against rare neurological conditions.

Moss PIEZO homologs have a conserved structure, are ubiquitously expressed, and do not affect general vacuole function.

The PIEZO protein family was first described in animals where these mechanosensitive calcium channels perform numerous essential functions, including the perception of light touch, shear, and compressive forces. PIEZO homologs are present in most eukaryotic lineages and recently we reported that two PIEZO homologs from moss Physcomitrium patens localize to the vacuolar membrane and modulate its morphology in tip-growing caulonemal cells. Here we show that predicted structures of both PpPIEZO1 and PpPIEZO2 are very similar to that of mouse Piezo2. Furthermore, we show that both moss PIEZO genes are ubiquitously expressed in moss vegetative tissues and that they are not required for normal vacuolar pH or intracellular osmotic potential. These results suggest that moss PIEZO proteins are widely expressed mechanosensory calcium channels that serve a signaling rather than maintenance role in vacuoles.

Unbiased proteomic and forward genetic screens reveal that mechanosensitive ion channel MSL10 functions at ER-plasma membrane contact sites in Arabidopsis thaliana.

Mechanosensitive (MS) ion channels are an evolutionarily conserved way for cells to sense mechanical forces and transduce them into ionic signals. The channel properties of Arabidopsis thaliana MscS-Like (MSL)10 have been well studied, but how MSL10 signals remains largely unknown. To uncover signaling partners of MSL10, we employed a proteomic screen and a forward genetic screen; both unexpectedly implicated endoplasmic reticulum-plasma membrane contact sites (EPCSs) in MSL10 function. The proteomic screen revealed that MSL10 associates with multiple proteins associated with EPCSs. Of these, only VAMP-associated proteins (VAP)27-1 and VAP27-3 interacted directly with MSL10. The forward genetic screen, for suppressors of a gain-of-function MSL10 allele (msl10-3G, MSL10S640L), identified mutations in the synaptotagmin (SYT)5 and SYT7 genes. We also found that EPCSs were expanded in leaves of msl10-3G plants compared to the wild type. Taken together, these results indicate that MSL10 associates and functions with EPCS proteins, providing a new cell-level framework for understanding MSL10 signaling. In addition, placing a mechanosensory protein at EPCSs provides new insight into the function and regulation of this type of subcellular compartment.

Should we offer disaster preparedness and response training workshops across Idaho? A feasibility study.

INTRODUCTION: It is important for individuals and families to prepare for potential disasters to enable communities to generate a consolidated response. It is estimated that 30 percent of residents of the fourth largest city in Idaho, Idaho Falls, are not prepared to deal with disasters. A 1-day training workshop for healthcare professionals and students at Idaho State University in Pocatello was organized to build their capacity for acute disaster response and preparedness. This study assesses the impact of the workshop in improving knowledge and attitudes of the participants toward disaster management. METHODS: A mixed methods study design was employed. Pre- and post-tests were administered to the participants (n = 18) to examine change in self-perceived understanding of disasters, and disaster preparedness and management. Core competencies encompassing knowledge of disaster preparedness were assessed via 18 multiple-choice questions. Participant attitudes toward disaster training exercises in colleges, universities, and healthcare facilities were recorded, and written feedback regarding the workshop obtained. Chi-square and paired t-tests were used to examine changes in disaster preparedness and cumulative knowledge score. Quantitative variables and comments were analyzed using Stata.v.13 and Maxqda, respectively. RESULTS: Our assessment indicates an improvement in mean knowledge score [pretest: 10.7 (2.8), post-test: 12.5 (2.9); p = 0.007] and self-perceived disaster preparedness and management. The attitude of audience toward training workshops was wholly positive in both the pre- and post-tests. Participants commented that the workshop was "excellent," helped them polish their knowledge and skills, trained them to build emergency kits and communication plans, and perform triage amidst crises. CONCLUSION: Training workshops should be offered to communities to build their capacity to prepare for and respond to disasters.

Advancing scientific excellence through inclusivity in the NIH BRAIN Initiative.

Leveraging breadth and depth of the scientific workforce invites creativity, relevance, and differing views that directly tie into innovation and problem solving. The NIH BRAIN Initiative is using a multi-pronged strategy to enhance diversity and inclusion toward promoting the best science.

Plant PIEZO homologs modulate vacuole morphology during tip growth.

In animals, PIEZOs are plasma membrane-localized cation channels involved in diverse mechanosensory processes. We investigated PIEZO function in tip-growing cells in the moss Physcomitrium patens and the flowering plant Arabidopsis thaliana PpPIEZO1 and PpPIEZO2 redundantly contribute to the normal growth, size, and cytoplasmic calcium oscillations of caulonemal cells. Both PpPIEZO1 and PpPIEZO2 localized to vacuolar membranes. Loss-of-function, gain-of-function, and overexpression mutants revealed that moss PIEZO homologs promote increased complexity of vacuolar membranes through tubulation, internalization, and/or fission. Arabidopsis PIEZO1 also localized to the tonoplast and is required for vacuole tubulation in the tips of pollen tubes. We propose that in plant cells the tonoplast has more freedom of movement than the plasma membrane, making it a more effective location for mechanosensory proteins.

Using network analysis to conduct a system-wide program evaluation within a university.

OBJECTIVE: To conduct a system-wide assessment using social network analysis (SNA) to examine how 14 important issues (e.g., consent; racism) are addressed through education, training, and programming at a university. METHODS: Evaluators conducted interviews with campus departments responsible for educating/training on the 14 issues. Interviews revealed which programs (n = 52) were offered that addressed the 14 issues, and data on audience characteristics, date of delivery, and which issues were covered in each program were collected. SNA was used to calculate degree and create visualization graphs illustrating patterns of content-coverage across all 52 programs. RESULTS: The average degree was 19.38 (SD = 9.70), meaning programs overlapped in topic area with nearly 20 other programs, on average. Most programs (n = 36; 69.2 %) were attended by audiences of 500 people or less. "Diversity and inclusion" represented the topic area with the most programs (n = 23), whereas "suicide" and "bullying/hazing" had the least number of programs (n = 3). Degree was negatively correlated with attendance numbers (r=-.310, p < .001), indicating the more a program overlapped in content with other programs, the smaller the audience. CONCLUSIONS: This study supports the use of network analysis in conducting systemic evaluations of programs offered at a university, complementing the work of ongoing, local-level program evaluations.

Complete Genome Sequence of Klebsiella pneumoniae Siphophage Sanco.

Klebsiella pneumoniae is an opportunistic pathogen that is the cause of several hospital-acquired infections. Bacteriophages that target this bacterium could be used therapeutically as novel antimicrobial agents. Here, we present the complete genome sequence of the T1-like K. pneumoniae phage Sanco.

Genetic and physical interactions between the organellar mechanosensitive ion channel homologs MSL1, MSL2, and MSL3 reveal a role for inter-organellar communication in plant development.

Plant development requires communication on many levels, including between cells and between organelles within a cell. For example, mitochondria and plastids have been proposed to be sensors of environmental stress and to coordinate their responses. Here we present evidence for communication between mitochondria and chloroplasts during leaf and root development, based on genetic and physical interactions between three Mechanosensitive channel of Small conductance-Like (MSL) proteins from Arabidopsis thaliana. MSL proteins are Arabidopsis homologs of the bacterial Mechanosensitive channel of Small conductance (MscS), which relieves cellular osmotic pressure to protect against lysis during hypoosmotic shock. MSL1 localizes to the inner mitochondrial membrane, while MSL2 and MSL3 localize to the inner plastid membrane and are required to maintain plastid osmotic homeostasis during normal growth and development. In this study, we characterized the phenotypic effect of a genetic lesion in MSL1, both in wild type and in msl2 msl3 mutant backgrounds. msl1 single mutants appear wild type for all phenotypes examined. The characteristic leaf rumpling in msl2 msl3 double mutants was exacerbated in the msl1 msl2 msl3 triple mutant. However, the introduction of the msl1 lesion into the msl2 msl3 mutant background suppressed other msl2 msl3 mutant phenotypes, including ectopic callus formation, accumulation of superoxide and hydrogen peroxide in the shoot apical meristem, decreased root length, and reduced number of lateral roots. All these phenotypes could be recovered by molecular complementation with a transgene containing a wild type version of MSL1. In yeast-based interaction studies, MSL1 interacted with itself, but not with MSL2 or MSL3. These results establish that the abnormalities observed in msl2 msl3 double mutants is partially dependent on the presence of functional MSL1 and suggest a possible role for communication between plastid and mitochondria in seedling development.

Morphometric analysis of young petiole galls on the narrow-leaf cottonwood, Populus angustifolia, by the sugarbeet root aphid, Pemphigus betae.

An insect-induced gall is a highly specialized structure resulting from atypical development of plant tissue induced by a reaction to the presence and activity of an insect. The insect induces a differentiation of tissues with features and functions of an ectopic organ, providing nutrition and protection to the galling insect from natural enemies and environmental stresses. In this anatomical and cytological study, we characterized how the gall-inducing aphid Pemphigus betae reshapes the leaf morphology of the narrow-leaf cottonwood Populus angustifolia to form a leaf fold gall. Young galls displayed a bend on one side of the midvein toward the center of the leaf and back to create a fold on the abaxial side of the leaf. This fold was formed abaxially by periclinal and anticlinal divisions, effectively eliminating intercellular spaces from the spongy parenchyma. Galls at this stage exhibited both cell hypertrophy and tissue hyperplasia. Cells on the adaxial surface were more numerous and smaller than cells near the abaxial surface were, creating the large fold that surrounds the insect. Mesophyll cells exhibited some features typical of nutritive cells induced by other galling insects, including conspicuous nucleolus, reduced and fragmented vacuole, smaller and degraded chloroplasts, and dense cytoplasm compared to ungalled tissue. Even though aphids feed on the contents of phloem and do not directly consume the gall tissue, they induce changes in the plant vascular system, which lead to nutrient accumulation to support the growing aphid numbers in mature galls.