Impact of immobilisation and image guidance protocol on planning target volume margins for supine craniospinal irradiation.

Background: The setup errors during supine-CSI (sCSI) using single or dual immobilisation (SM, DM) subsets from two institutions were reviewed to determine if DM consistently decreased the required planning target volumes (PTV) margins and to identify the optimal image guidance environments. Materials and methods: Ours and a sister institutional cohort, each with a subset of SM or DM sCSI and daily 3-dimensional online image verification sets, were reviewed for the cranial and spinal regions translational shifts. Using descriptive statistics, scatter plots and independent sample Mann-Whitney test we compared shifts in each direction for two subsets in each cohort deriving PTV margins (Van Herk: VH, Strooms: St recipes) for the cranial and spinal regions. Three image guidance (IG) protocols were simulated for two regions on the combined cohort with SM and DM subsets to identify the most optimal option with the smallest PTV margin. The IG protocols: 3F, 5F and 5FB where the systematic error correction was done using the average error from the first three, five and in the cranium alone (applied to both the cranium and spine, otherwise) for the first five set-ups, respectively. Results: 6968 image sets for 179 patients showed DM could consistently reduce the PTV margin (VH/St) for the cranium from 6/5 to 4/3.5 (31.8/30.8%) and 6/4 to 4/3.5 mm (30.5/16.8%) for primary and validation cohort, respectively. Similarly, for the spine it was 10/8.5 to 6/5.5 (38.6/38.4%) and 9/7.7 to 7/6 (21.6/21.4%), respectively. The "5F-IG" resulted in the smallest margins for both the cranial (3 mm) and spinal region (5 mm) for DM with estimated 95% CTV coverage probability. Conclusion: DM with 5F-IG would significantly reduce the required PTV margins for sCSI.

A TLC-Direct Bioautography Method for Detection of Antiurolithiatic Metabolites.

Hyperoxaluria is major urinary disorder troubling largest population throughout the world predominantly involving calcium oxalate (CaOx) crystals. Ancient Ayurvedic system of medicine in India claims better option in treatment of urolithiasis. A plant from "Pashanbheda" group is Phyllanthus niruri L., possessing antiurolithiatic activity, needed to be screened and validated. In the present study, a rapid, easy and efficient method for CaOx crystal inhibition in the agar gel system analogous to antimicrobial well diffusion assay is proposed. A novel thin-layer chromatography (TLC)-direct bioautography method was also proposed to detect the antilithiatic metabolites. It helps to localize the active metabolites in P. niruri, further the partial structure elucidation was characterized by High Resolution Liquid Chromatography by mass spectroscopy (LC-HRMS) analysis. The agar well diffusion method shows 50% inhibitory concentration (IC50) value at 228.55 and 493.529 mg/mL for tri-sodium citrate and P. niruri extract, respectively. The lowest concentration showing visible crystal inhibition (minimum inhibitory concentration, MIC) in both samples was found to be 20 mg/mL. In this study, a unique agar gel well diffusion and TLC-direct bioautography method successfully screened, detected and confirmed CaOx crystal inhibitory metabolites from P. niruri. The tuberonic acid was detected in bioactive fraction of P. niruri by LC-HRMS characterization.

Plant tissue culture independent Agrobacterium tumefaciens mediated In-planta transformation strategy for upland cotton (Gossypium hirsutum).

A new method of transgenic development called "In-planta" transformation method, where Agrobacterium is used to infect the plantlets but the steps of in vitro regeneration of plants is totally avoided. In this study, we have reported a simple In-planta method for efficient transformation of diploid cotton Gossypium hirsutum cv LRK-516 Anjali using Agrobacterium tumefaciens EHA-105 harbouring recombinant binary vector plasmid pBinAR with Arabidopsis At-NPR1 gene. Four day old plantlets were used for transformation. A vertical cut was made at the junction of cotyledonary leaves, moderately bisecting the shoot tip and exposing meristem cells at apical meristem. This site was infected with Agrobacterium inoculum. The transgenic events obtained were tested positive for the presence of At-NPR1 gene with promoter nptII gene. They are also tested negative for vector backbone integration and Agrobacterium contamination in T0 events. With this method a transformation frequency of 6.89% was reported for the cv LRK-516.

Assessment of three-dimensional set-up errors using megavoltage computed tomography (MVCT) during image-guided intensity-modulated radiation therapy (IMRT) for craniospinal irradiation (CSI) on helical tomotherapy (HT).

The purpose of this study was to assess three-dimensional (3D) set-up errors using megavoltage computed tomography (MVCT) during image-guided intensity-modulated radiation therapy (IMRT) for supine craniospinal irradiation (CSI) on helical tomotherapy (HT). Patients were immobilized in a customized 4-clamp thermoplastic head mask with or without whole-body vacuum cradle. Set-up was based primarily on a set of cranial fiducial markers. MVCT scans were acquired and co-registered with planning scan separately at three different levels (brain, upper, and lower spine) at every fraction. Only translational displacements were analysed, wherein positive sign denotes deviation in anterior, left, and superior direction; while negative sign denotes deviation in posterior, right, and inferior direction. Mean displacements, systematic, and random errors of the study population were calculated at all three levels separately. Local residual uncertainty of the upper and lower spine was also derived assuming perfect co-registration of the skull. Set-up margins for clinical target volume (CTV) to planning target volume (PTV) were derived at these three levels separately using published margin recipes. Data from 1868 co-registrations in 674 fractions on 33 patients was included. The mean displacements in the lateral, longitudinal, and vertical directions were -1.21, -1.36, and 1.38 mm; -1.25, -0.34, and 0.65 mm; and -1.47, -2.78, and 0.22 mm for the brain; upper spine; and lumbar spine respectively. The corresponding 3D vector of displacement was 2.28; 1.45; and 3.15 mm respectively. There was a distinct systematic trend towards increasing inaccuracy from the brain towards the lower spine. Using Stroom's formula, the minimum recommended CTV to PTV margins in absence of daily image-guidance were 6.5; 7.0; and 9.5 mm for the brain; upper spine; and lower spine respectively. This increased to 7.5; 8.5; and 11.5 mm using van Herk's formula. Subset and sensitivity analyses could not identify any factor predictive of increased inaccuracy. Residual uncertainty of the spinal column was lesser after daily co-registration referenced to the skull, suggesting that smaller set-up margins maybe appropriate while using daily image-guidance with an online correction protocol. Daily MVCT imaging during supine CSI on HT provides volumetric verification of the set-up process. There is substantial site-dependent variability in translational displacements that increases systematically from brain towards the lower spine with implications for differential set-up -margins for the brain, upper, and lower spine.

Efficient genomic DNA extraction protocol from medicinal rich Passiflora foetida containing high level of polysaccharide and polyphenol.

In Present work, the main objective is to develop less time consuming protocol for genomic DNA isolation from leaves of Passiflora foetida. Optimized protocol is cost effective, as it avoided use of expensive liquid nitrogen. The important parameters of CTAB buffer composition such as Polyvinylpyrrolidone PVP40000 (without PVP, 1%, 2%, 3.5%, 4.0%, 4.5%, 5.0%), CTAB (w, 1%, 2%, 3%, 4%, 5%), water bath temperature (30°C to 70°C) and duration on water bath for half hr and one and half hr has been optimized. CTAB (2%), PVP (1%), water bath temperature (70%), duration on water bath (1 hr) has efficiently yielded DNA quality of 200-1782 µg/0.5gm from leaf, stem, root, tendril and flower. However, 168 µg - 1782 µg of DNA has been obtained from 0.5 g of leaf of Passiflora foetida. Polyphenol contamination has been overcome using 5M NaCl and PVP. Acetate has been used for obtaining double-stranded DNA in stabilized form. Current DNA extraction protocol takes maximum of four hours for completion, which is many time savings. RAPD-PCR reaction parameters such as DNA concentration (100ng), Primer concentration (2 µM), Dream Taq polymerase (2 U), annealing temperature (29°C) and number of cycles for amplification of DNA has been optimized. Primer fragment Akansha 7 shows high polymorphism of 7 fragments ranges from 200bp - 2500 bp. Current optimized protocol of DNA isolation is specifically for Passiflora foetida, which can be used for downstream molecular techniques.

Abeta oligomers induce neuronal cell cycle events in Alzheimer's disease.

Neurons subject to degeneration in Alzheimer's disease (AD) exhibit evidence of re-entry into a mitotic cell cycle even before the development of substantial AD brain pathology. In efforts to identify the initiating factors underlying these cell cycle events (CCEs), we have characterized the appearance of the neuronal CCEs in the genomic-based R1.40 transgenic mouse model of AD. Notably, R1.40 mice exhibit neuronal CCEs in a reproducible temporal and spatial pattern that recapitulates the neuronal vulnerability seen in human AD. Neuronal CCEs first appear at 6 months in the frontal cortex layers II/III. This is 6-8 months before detectable amyloid beta (Abeta) deposition, suggesting that specific amyloid precursor protein (APP) processing products are responsible for the induction of neuronal CCEs. Furthermore, a reduction in the levels of Abeta (achieved by shifting the genetic background from C57BL/6 to the DBA/2 mouse strain) dramatically delays the appearance of neuronal CCEs. More significantly, elimination of beta-secretase activity blocks the appearance of CCEs, providing direct genetic evidence that the amyloidogenic processing of APP is required for the induction of CCEs. Finally, in vitro preparations of oligomeric, but not monomeric, Abeta induce DNA synthesis in dissociated cortical neurons, and this response is blocked by antioligomer specific antibodies. Together, our data suggest that low molecular weight aggregates of Abeta induce neuronal cell cycle re-entry in mouse models of Alzheimer's disease.

Using OrgAhead, a computational modeling program, to improve patient care unit safety and quality outcomes.

As part of ongoing research to investigate the impact of patient characteristics, organization characteristics and patient unit characteristics on safety and quality outcomes, we used a computational modeling program, OrgAhead, to model patient care units' achievement of patient safety (medication errors and falls) and quality outcomes. We tuned OrgAhead using data we collected from 32 units in 12 hospitals in Arizona. Validation studies demonstrated acceptable levels of correspondence between actual and virtual patient units. In this paper, we report how we used OrgAhead to develop testable hypotheses about the kinds of innovations that nurse managers might realistically implement on their patient care units to improve quality and safety outcomes. Our focus was on unit-level innovations that are likely to be easier for managers to implement. For all but the highest performing unit (for which we encountered a ceiling effect), we were able to generate practical strategies that improved performance of the virtual units that could be implemented by actual units to improve safety and quality outcomes. Nurse managers have responded enthusiastically to the additional decision support for quality improvement.

Using computational modeling to improve patient care unit safety and quality outcomes.

As part of ongoing research to investigate the impact of patient characteristics, organization characteristics and patient unit characteristics on safety and quality outcomes, we are using a computational modeling program, OrgAhead, to model patient care units' achievement of patient safety (medication errors and falls) and quality outcomes. We tuned OrgAhead using data we collected from 16 units in 5 hospitals. Subsequent validation studies demonstrated acceptable levels of correspondence between actual and virtual patient units. In this paper, we report on our initial efforts to use OrgAhead to develop testable hypotheses about the kinds of innovations that nurse managers might realistically implement on their patient care units to improve quality and safety outcomes. Our focus is on unit-level innovations that are likely to be easier for managers to implement. For all but the highest performing unit (for which we encountered a ceiling effect), we were able to generate practical strategies that improved performance of the virtual units by 6-8 percentage points. Nurse Managers have responded enthusiastically to the additional decision support for quality improvement

Using computational modeling to transform nursing data into actionable information.

Transforming organizational research data into actionable information nurses can use to improve patient outcomes remains a challenge. Available data are numerous, at multiple levels of analysis, and snapshots in time, which makes application difficult in a dynamically changing healthcare system. One potential solution is computational modeling. We describe our use of OrgAhead, a theoretically based computational modeling program developed at Carnegie Mellon University, to transform data into actionable nursing information. We calibrated the model by using data from 16 actual patient care units to adjust model parameters until performance of simulated units ordered in the same way as observed performance of the actual units 80% of the time. In future research, we will use OrgAhead to generate hypotheses about changes nurses might make to improve patient outcomes, help nurses use these hypotheses to identify and implement changes on their units, and then measure the impact of those changes on patient outcomes.

Using computational modeling to study the impact of workplace characteristics on patient safety outcomes.

How do patient characteristics, organization characteristics and patient care unit characteristics interact to affect quality, safety, and cost outcomes? What changes can nurse managers make on their units that will optimize outcomes for their patients? To answer these questions, we are collecting data from 35 nursing units in 12 hospitals in Arizona, and using the results as a basis for computational modeling. Although it has been used in clinical research, until now computational modeling has had little application to healthcare or nursing organizations. In this poster session, we describe our application of Orgahead, a computational modeling program.

Teaching evidence-based medicine skills through a residency-developed guideline.

Though many residencies have recognized the need for instruction in evidence-based medicine, the best way to teach these skills to resident physicians remains uncertain. We designed a curriculum intended to develop a resident-produced, evidence-based guideline for the care of patients with diabetes. Each resident was supervised going through the steps of evidence-based medicine: asking a clinical question, searching for the evidence to answer that question, appraising that evidence, and producing an evidence-based answer. These answers were then compiled into a guideline distributed in the residency practice. An evaluation of this curriculum using focus group and survey data showed that learners appreciated the skills and knowledge gained in devising guidelines in an evidence-based manner but were uncertain that their searches were complete. The clinical evaluation of the guideline implementation showed improvement in several clinical markers of diabetes care.

Measurement of in situ halophilic glyceraldehyde-3-phosphate dehydrogenase activity from the permeabilized cells of archaebacterium Haloarcula vallismortis.

The cells of Haloarcula vallismortis, an extreme halophilic archaebacterium, were permeabilized by various chemical, physical, and biological treatments. Biological permeabilization by lysozyme and papain showed effective results as observed by studying the in situ activity of halophilic glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) as the model enzyme. Detergents N-cetyl-N, N, N-trimethyl ammonium bromide (CTAB) and digitonin also showed significant results. Other strains of halobacteria could also be permeabilized by lysozyme. The cell morphology did not show any significant change after permeabilization as observed by phase contrast microscopy. The enzyme characteristics of hGAPDH were studied in situ using permeabilized H. vallismortis cells. The properties, like optimum pH, Km for GAP and NAD(+), inhibition by heavy metals, sulphydryl reagents, and other compounds, showed remarkable similarity with those studied in vitro.