A high-throughput method for isolation of salicylic acid metabolic mutants

<p>Abstract</p> <p>Background</p> <p>Salicylic acid (SA) is a key defense signal molecule against biotrophic pathogens in plants. Quantification of SA levels in plants is critical for dissecting the SA-mediated immune response. Although HPLC and GC/MS are routinely used to determine SA concentrations, they are expensive and time-consuming. We recently described a rapid method for a bacterial biosensor <it>Acinetobacter </it>sp. ADPWH_<it>lux</it>-based SA quantification, which enables high-throughput analysis. In this study we describe an improved method for fast sample preparation, and present a high-throughput strategy for isolation of SA metabolic mutants.</p> <p>Results</p> <p>On the basis of the previously described biosensor-based method, we simplified the tissue collection and the SA extraction procedure. Leaf discs were collected and boiled in Luria-Bertani (LB), and then the released SA was measured with the biosensor. The time-consuming steps of weighing samples, grinding tissues and centrifugation were avoided. The direct boiling protocol detected similar differences in SA levels among pathogen-infected wild-type, <it>npr1 </it>(nonexpressor of pathogenesis-related genes), and <it>sid2 </it>(SA induction-deficient) plants as did the previously described biosensor-based method and an HPLC-based approach, demonstrating the efficacy of the protocol presented here. We adapted this protocol to a high-throughput format and identified six <it>npr1 </it>suppressors that accumulated lower levels of SA than <it>npr1 </it>upon pathogen infection. Two of the suppressors were found to be allelic to the previously identified <it>eds5 </it>mutant. The other four are more susceptible than <it>npr1 </it>to the bacterial pathogen <it>Pseudomonas syringae </it>pv. <it>maculicola </it>ES4326 and their identity merits further investigation.</p> <p>Conclusions</p> <p>The rapid SA extraction method by direct boiling of leaf discs further reduced the cost and time required for the biosensor <it>Acinetobacter </it>sp. ADPWH_<it>lux</it>-based SA estimation, and allowed the screening for <it>npr1 </it>suppressors that accumulated less SA than <it>npr1 </it>after pathogen infection in a high-throughput manner. The highly efficacious SA estimation protocol can be applied in genetic screen for SA metabolic mutants and characterization of enzymes involved in SA metabolism. The mutants isolated in this study may help identify new components in the SA-related signaling pathways.</p

A System for Assessing Cervical Readiness Using Analytics and Non-Invasive Evaluation (Crane)

Current cervical spine assessment methodologies focus solely on subjective measures, such as pain reports, and range-of-motion (ROM) testing that only measures maximum head excursion and reach (i.e., not dynamic motion). Due to report bias and the potential for negative outcomes of self-reported pain, current clinical assessment methods fail to provide valid, reliable data for medical practitioners to effectively manage long-term cervical health. Furthermore, commercial systems capable of quantitative assessment of cervical spine function are generally sparse and often immature. This paper highlights both the need and a path towards a clinical tool for objective measurement of cervical spine health and functionality. Lastly, a novel solution concept is presented to objectively assess Cervical Readiness using Analytics and Non-invasive Evaluation (CRANE). This solution concept combines cervical spine instrumentation, novel virtual reality (VR) game-based test protocols, robust analytical algorithms, and intuitive presentation of health metrics

Active muscle response contributes to increased injury risk of lower extremity in occupant–knee airbag interaction

<p><b>Objective</b>: Recent field data analysis has demonstrated that knee airbags (KABs) can reduce occupant femur and pelvis injuries but may be insufficient to decrease leg injuries in motor vehicle crashes. An enhanced understanding of the associated injury mechanisms requires accurate assessment of physiological-based occupant parameters, some of which are difficult or impossible to obtain from experiments. This study sought to explore how active muscle response can influence the injury risk of lower extremities during KAB deployment using computational biomechanical analysis.</p> <p><b>Methods</b>: A full-factorial matrix, consisting of 48 finite element simulations of a 50th percentile occupant human model in a simplified vehicle interior, was designed. The matrix included 32 new cases in combination with 16 previously reported cases. The following influencing factors were taken into account: muscle activation, KAB use, KAB design, pre-impact seating position, and crash mode. Responses of 32 lower extremity muscles during emergency braking were replicated using one-dimensional elements of a Hill-type constitutive model, with the activation level determined from inverse dynamics and validated by existing volunteer tests. Dynamics of unfolding and inflating of the KABs were represented using the state-of-the-art corpuscular particle method. Abbreviated Injury Scale (AIS) 2+ injury risks of the knee–thigh–hip (KTH) complex and the tibia were assessed using axial force and resultant bending moments. With all simulation cases being taken together, a general linear model was used to assess factor significance (<i>P</i> <.05).</p> <p><b>Results</b>: As estimated by the regression model across all simulation cases, use of KABs significantly reduced axial femur forces by 4.74 ± 0.43 kN and AIS 2+ injury risk of KTH by 47 ± 6% (<i>P</i> <.05) but did not provide substantial change to injury risk of leg fractures. Muscle activation significantly increased axial force and bending moment of the femur (3.87 ± 0.38 kN and 64.3 ± 5.9 Nm), the tibia (1.49 ± 0.12 kN and 43.0 ± 6.4 Nm), and the resultant probability of AIS 2+ tibia injuries by 36 ± 6% regardless of KAB use and crash scenario. Specifically, when counting on a relative scale, muscle activation exhibited more prominent elevation of injury risk for in-position occupants than out-of-position occupants. In a representative crash scenario—that is, using a bottom-deployed KAB in a nearside oblique impact—muscle bracing of the right leg may lead to 2.6 times higher tibia fracture risk than being relaxed for an out-of-position occupant and 5.4 times higher for an in-position occupant.</p> <p><b>Discussion and Conclusions</b>: The mechanism of higher leg injuries in the presence of KAB deployment in real-world crashes can be interpreted by the increased effective body mass, axial compression along the shafts of long bones, and altered pre-impact posture due to muscle contraction. The present analysis suggests that active muscle response can increase the risk of lower extremity injury during occupant–KAB interaction. This study demonstrated the feasibility of advanced human models to investigate the influence of physiologically based parameters on injury outcomes evidenced in field study and insight from computational examination on human variability for development of future restraint systems. Future efforts are recommended on realistic vehicle and restraint environment and advanced modeling strategies toward a full understanding of KAB efficacy.</p

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field