A P EDIATRIC S URVEY FOR THE N ATIONAL H IGHWAY T RAFFIC S AFETY A DMINISTRATION : E MERGENCY M EDICAL S ERVICES S YSTEM R E-ASSESSMENTS

Emergency medical services for children, or EMSC, is still a relatively underdeveloped component of most state and local EMS systems. Advocacy and funding for EMSC from the federal EMSC Program, availability of many useful EMSC products, and the rapidly enlarging literature in EMSC have created heightened awareness and interest in improving systems for pediatric emergency, trauma, and critical care. The new National Highway Traffic Safety Administration (NHTSA) EMS Technical Assistance (TA) re-assessment program, the second version of the successful original TA Program from 1988 to 1996, provides an ideal opportunity for state EMS professionals to evaluate EMSC capabilities and to integrate new EMSC products and services. The history of the TA Program reflects the evolution of EMS itself and indicates a historical inattention to children's issues, but re-assessment TA teams now have much useful intervening EMSC history to draw upon and a clear philosophical mandate to integrate children more fully in EMS system planning and management. In order to facilitate state-of-the-art reviews of EMSC within state EMS systems, a pediatric survey for the NHTSA re-assessments is presented. The survey, developed with the input of EMS administrators and physicians and approved by the National Association of State EMS Directors, follows the original ten-component model for EMS system review. It is intended for optional use within the overall EMS review process.     

Making Better Doctors: A Survey of Premedical Interns Working as Health Coaches

We operate a decision support program in a medical center in San Francisco. In this program, postbaccalaureate, premedical interns deliver decision and communication, aids to patients. We asked whether working in this program helped these premedical interns develop key physician competencies. To measure physician competencies, we adopted the standards of the Accreditation Committee on Graduate Medical Education (ACGME), which accredits residency programs in the USA. The ACGME competencies are patient care, medical knowledge, practice-based learning, interpersonal and communication skills, professionalism, and systems-based practice. We developed a survey for our program alumni to rate themselves on a scale from 0 (none) to 100 (perfect) on each competency, before and after their time in our program. The survey also solicited free-text comments regarding each competency. In June 2012, we e-mailed all 47 alumni a link to our online survey and then analyzed responses received by July 15, 2012. We visually explored the distributions of ratings and compared medians. We selected the most specific and concrete comments from the qualitative responses. Respondents (21/47 or 45 %) reported that their participation in Decision Services increased their competencies across the board. Qualitative comments suggest that this is because students accompanied patients on their clinic journeys (seeing multiple facets of the systems of care) while also actively facilitating patient physician communication. Providing decision support can improve self-ratings of crucial physician competencies. Educators should consider deploying premedical and medical students as decision support coaches to increase competencies through experiential learning.     

MicroRNAs in nasopharyngeal carcinoma

MicroRNAs（miRNAs） provide insight into both the biology and clinical behavior of many human cancers, including nasopharyngeal carcinoma（NPC）. The dysregulation of miRNAs in NPC results in a variety of tumor-promoting effects. Furthermore, several miRNAs are prognostic markers for NPC. In addition to cellular miRNAs, NPC samples also often contain miRNAs encoded by Epstein-Barr virus, and these miRNAs may impact NPC biology by targeting both cellular and viral genes. Given their numerous putative roles in NPC development and progression, a thorough understanding of the impact of miRNA dysregulation in NPC is expected to shed light on useful biomarkers and therapeutic targets for the clinical management of this disease. In this review, we describe the efforts to date to identify and characterize such miRNAs in the context of NPC.     

Single-dose vaccine against tick-borne encephalitis

Tick-borne encephalitis (TBE) virus is the most important human pathogen transmitted by ticks in Eurasia. Inactivated vaccines are available but require multiple doses and frequent boosters to induce and maintain immunity. Thus far, the goal of developing a safe, live attenuated vaccine effective after a single dose has remained elusive. Here we used a replication-defective (single-cycle) flavivirus platform, RepliVax, to generate a safe, single-dose TBE vaccine. Several RepliVax-TBE candidates attenuated by a deletion in the capsid gene were constructed using different flavivirus backbones containing the envelope genes of TBE virus. RepliVax-TBE based on a West Nile virus backbone (RV-WN/TBE) grew more efficiently in helper cells than candidates based on Langat E5, TBE, and yellow fever 17D backbones, and was found to be highly immunogenic and efficacious in mice. Live chimeric yellow fever 17D/TBE, Dengue 2/TBE, and Langat E5/TBE candidates were also constructed but were found to be underattenuated. RV-WN/TBE was demonstrated to be highly immunogenic in Rhesus macaques after a single dose, inducing a significantly more durable humoral immune response compared with three doses of a licensed, adjuvanted human inactivated vaccine. Its immunogenicity was not significantly affected by preexisting immunity against WN. Immunized monkeys were protected from a stringent surrogate challenge. These results support the identification of a single-cycle TBE vaccine with a superior product profile to existing inactivated vaccines, which could lead to improved vaccine coverage and control of the disease.     

Nonexponential T2* decay in white matter

Visualizing myelin in human brain may help the study of diseases such as multiple sclerosis. Previous studies based on T₁ and T₂ relaxation contrast have suggested the presence of a distinct water pool that may report directly on local myelin content. Recent work indicates that T₂* contrast may offer particular advantages over T₁ and T₂ contrast, especially at high field. However, the complex mechanism underlying T₂* relaxation may render interpretation difficult. To address this issue, T₂* relaxation behavior in human brain was studied at 3 and 7 T. Multiple gradient echoes covering most of the decay curve were analyzed for deviations from mono‐exponential behavior. The data confirm the previous finding of a distinct rapidly relaxing signal component (T₂* ～ 6 ms), tentatively attributed to myelin water. However, in extension to previous findings, this rapidly relaxing component displayed a substantial resonance frequency shift, reaching 36 Hz in the corpus callosum at 7 T. The component's fractional amplitude and frequency shift appeared to depend on both field strength and fiber orientation, consistent with a mechanism originating from magnetic susceptibility effects. The findings suggest that T₂* contrast at high field may be uniquely sensitive to tissue myelin content and that proper interpretation will require modeling of susceptibility‐induced resonance frequency shifts. Magn Reson Med, 2011. © 2011 Wiley‐Liss, Inc.     

Beta toxin catalyzes formation of nucleoprotein matrix in staphylococcal biofilms

Biofilms are surface-associated communities of microbes encompassed by an extracellular matrix. It is estimated that 80% of all bacterial infections involve biofilm formation, but the structure and regulation of biofilms are incompletely understood. Extracellular DNA (eDNA) is a major structural component in many biofilms of the pathogenic bacterium Staphylococcus aureus, but its role is enigmatic. Here, we demonstrate that beta toxin, a neutral sphingomyelinase and a virulence factor of S. aureus, forms covalent cross-links to itself in the presence of DNA (we refer to this as biofilm ligase activity, independent of sphingomyelinase activity) producing an insoluble nucleoprotein matrix in vitro. Furthermore, we show that beta toxin strongly stimulates biofilm formation in vivo as demonstrated by a role in causation of infectious endocarditis in a rabbit model. Together, these results suggest that beta toxin cross-linking in the presence of eDNA assists in forming the skeletal framework upon which staphylococcal biofilms are established.     

A minimally disruptive method for measuring water potential in planta using hydrogel nanoreporters

Leaf water potential is a critical indicator of plant water status, integrating soil moisture status, plant physiology, and environmental conditions. There are few tools for measuring plant water status (water potential) in situ, presenting a critical barrier for developing appropriate phenotyping (measurement) methods for crop development and modeling efforts aimed at understanding water transport in plants. Here, we present the development of an in situ, minimally disruptive hydrogel nanoreporter (AquaDust) for measuring leaf water potential. The gel matrix responds to changes in water potential in its local environment by swelling; the distance between covalently linked dyes changes with the reconfiguration of the polymer, leading to changes in the emission spectrum via Förster Resonance Energy Transfer (FRET). Upon infiltration into leaves, the nanoparticles localize within the apoplastic space in the mesophyll; they do not enter the cytoplasm or the xylem. We characterize the physical basis for AquaDust’s response and demonstrate its function in intact maize (Zea mays L.) leaves as a reporter of leaf water potential. We use AquaDust to measure gradients of water potential along intact, actively transpiring leaves as a function of water status; the localized nature of the reporters allows us to define a hydraulic model that distinguishes resistances inside and outside the xylem. We also present field measurements with AquaDust through a full diurnal cycle to confirm the robustness of the technique and of our model. We conclude that AquaDust offers potential opportunities for high-throughput field measurements and spatially resolved studies of water relations within plant tissues.

Plant life depends on water availability. In managing this demand, irrigated agriculture accounts for 70% of all human water use (1). Physiologically, the process of transpiration (E) dominates this demand for water (Fig. 1A): Solar thermal radiation and the unsaturated relative humidity in the atmosphere drive evaporation from the wet internal surfaces of leaves; this water loss pulls water up through the plant’s vascular tissue (xylem) and out of the soil. This flow occurs along a gradient in the chemical potential of water, or water potential, ψ [MPa] (2). Studies of water relations and stress physiology over the past decades have found that values of ψ along the path of E (the soil–plant–atmosphere continuum [SPAC]) correlate with plant growth, crop yield and quality, susceptibility to disease, and the balance between water loss due to E and the uptake and assimilation of carbon dioxide (water-use efficiency) (3–5).Open in a separate windowFig. 1.AquaDust as an in situ reporter of water potential (ψ). (A) Schematic representation of a maize plant undergoing transpiration (E) in a dynamic environment driven by solar thermal radiation (Qrad) and photosynthetically active radiation (PAR), wind speed (u), temperature (T), vapor pressure deficit (VPD), and soil water potential (ψsoil). Water flows through the plant (blue arrows) along a gradient in water potential (∇→ψ). Zones on the leaves infiltrated with AquaDust serve as reporters of the local leaf water potential, ψleaf, via a short (∼30 s), minimally invasive measurement of FRET efficiency (ζ) with a leaf clamp. (B) Schematic representations of infiltration of a suspension of AquaDust and of the distribution of AquaDust within the cross-section of a leaf. AquaDust passes through the stomata and localizes in the apoplastic spaces within the mesophyll; the particles are excluded from symplastic spaces and the vascular bundle. (C) Schematic diagrams showing mechanism of AquaDust response: The swollen, “wet” state when water potential in its local environment, ψenv=0 (i.e., no stress condition), results in low FRET between donor (green circles) and acceptor (yellow circles) dye (Upper); and the shrunken, “dry” state when ψenv<0 (i.e., stressed condition) results in high FRET between fluorophores, thereby altering the emission spectra (Lower). (D) Fluorescent dyes were chosen to minimize reabsorption of AquaDust emission from chlorophyll; comparison of representative fluorescent emission from AquaDust (donor peak at 520 nm and acceptor peak at 580 nm) with the absorption spectra of chlorophyll and autofluorescence of maize leaf.Due to the recognized importance of water potential in controlling plant function, plant scientists have spent considerable effort devising accurate and reliable methods to measure water potential of the soil, stem, and leaf (6). Of these, plant water potentials, and particularly leaf water potential (ψleaf), represent valuable indicators of plant water status because they integrate both environmental conditions (e.g., soil water availability and evaporative demand) and plant physiological processes (e.g., root water uptake, xylem transport, and stomatal regulation) (7, 8). To date, techniques to measure ψleaf remain either slow, destructive, or indirect. The current tools (e.g., Scholander pressure chamber, psychrometer, and pressure probe) involve disruption of the tissue, the microenvironment, or both (9–11). For example, the widely used pressure chamber requires excision of leaves or stems for the measurement of ψleaf. Other techniques, such as stem and leaf psychrometry, require intimate contact with the tissue, and accurate and repeatable measurements are difficult to obtain (9, 12). These limitations have hindered the study of spatiotemporal water-potential gradients along the SPAC and the development of high-throughput strategies to phenotype based on tissue water potential (13). Additionally, current methods for measuring ψleaf provide averages over tissues in the leaf. This characteristic makes the dissection of water relations on subleaf scales challenging, such that important questions remain, for example, about the partitioning of hydraulic resistances within leaves between the xylem and mesophyll (14–16).These outstanding challenges in the measurement of water status in planta motivated us to develop the measurement strategy presented here, AquaDust, with the following characteristics: 1) Minimally disruptive: Compatible with simple, rapid measurements on intact leaves. Fig. 1A presents our approach, in which AquaDust reporters infiltrated into the mesophyll of the leaf provide an externally accessible optical signal that correlates with the local water potential. 2) Localized: allowing for access to the values of water potential at a well-defined location along the path of transpiration in the leaf tissue. Fig. 1B shows a schematic representation of AquaDust particles localized in the apoplastic volume within the mesophyll, at the end of the hydraulic path for liquid water within the plant. 3) Sensitive and specific: capable of resolving water potentials across the physiologically relevant range (∼−3<ψ<0 MPa) and with minimal sensitivity to other physical (e.g., temperature) and chemical (e.g., pH) variables. Fig. 1C presents a schematic representation of an AquaDust particle formed of hydrogel, a highly tunable material that undergoes a structural response to changes in local water potential (swollen when wet; collapsed when dry). We couple the swelling behavior of the particle to an optical signal via the incorporation of fluorescence dyes (green and yellow circles in Fig. 1C) that undergo variable Förster Resonance Energy Transfer (FRET) as a function of spatial separation. Fig. 1D presents typical AquaDust spectra at high (wet; green curve) and low (dry; yellow curve) water potentials. A change in water potential leads to a change in the relative intensity of the two peaks in the AquaDust spectrum, such that the relative FRET efficiency, ζ=f(ID,IA), can serve as a measure of water potential. 4) Inert: nondisruptive of the physiological properties of the leaf (e.g., photosynthetic capacity, transpiration rate, etc.).In this paper, we present the development, characterization, and application of AquaDust. We show that AquaDust provides a robust, reproducible response of its fluorescence spectra to changes in leaf water potential in situ and across the usual physiological range. We apply our approach to quantify the spatial gradients of water potential along individual leaves undergoing active transpiration and across a range of soil water potentials. With these measurements, we show that the localization of AquaDust in the mesophyll allows us to quantify the importance of hydraulic resistances outside the xylem. We further use AquaDust to measure the diurnal dynamics of ψleaf under field conditions, with repeated measurements on individual, intact leaves. These measurements demonstrate the field-readiness of our techniques and validate the leaf hydraulic model we have developed. We conclude that AquaDust offers a powerful basis for tracking, spatially and temporally, water potential in planta to study the mechanisms by which it couples to both biological and physical processes to define plant function.