Enzymatic transformations of cellulose assessed by quantitative high-throughput fourier transform infrared spectroscopy (QHT-FTIR).

Enzymatic hydrolysis of bacterial microcrystalline cellulose was performed with the thermophile enzyme system of Thermobifida fusca Cel5A (a classical endocellulase), Cel6B (a classical exocellulase), Cel9A (a processive endoglucanase), and a synergistic mixture of endo- and exocellulases. Different concentrations of enzymes were used to vary the extent of hydrolysis. Following standardization, the concentration of cellulose was directly correlated to the absorbance of the cellulose signals. Crystallinity indexes (Lateral Order Index (LOI), Total Crystallinity Index, Hydrogen Bonding Index), allomorphic composition, conversion of specific atomic bonds (including the ß-glucosidic bonds) were extracted from the spectral data obtained by QHT-FTIR. By quantifying the disruption of the H-bonding in complement to the sugar production, a more dynamic and complex picture of the role of cellulases in the hydrolysis of cellulose was demonstrated. The disruption of the H-bonding within the cellulose matrix appears as a quantifiable activity of the enzymes which was not correlated with the production of sugars in solution. The results also demonstrate that Cel9A activities from the cellulose transformation standpoint were partially similar to the activities of the synergistic mixture. In addition, Cel9A preferentially degraded the I(α) fraction of the crystalline cellulose while the Cel5A and Cel6B synergistic mixture preferentially degraded the I(ß) fraction.

Labeling and purification of cellulose-binding proteins for high resolution fluorescence applications.

The study of enzymatic reactions through fluorescence spectroscopy requires the use of bright, functional fluorescent molecules. In the case of proteins, labeling with fluorescent dyes has been carried out through covalent reactions with specific amino acids. However, these reactions are probabilistic and can yield mixtures of unlabeled and labeled enzymes with catalytic activities that can be modified by the addition of fluorophores. To have meaningful interpretations of results from the study of labeled enzymes, it is then necessary to reduce the variability in physical, chemical, and biological characteristics of the labeled products. In this paper, a solid phase labeling protocol is described as an advantageous alternative to free solution labeling of cellulose-binding proteins and is applied to tag cellulases with three different fluorophores. The products from the labeling reactions were purified to remove the unreacted dye and separate labeled and unlabeled enzymes. Characterization of the catalytic and spectroscopic properties of the isolated labeled species confirmed that highly homogeneous populations of labeled cellulases can be achieved. The protocol for the separation of labeled products is applicable to any mixture of labeled proteins, making this an attractive methodology for the production of labeled proteins suitable for single molecule fluorescence spectroscopy.

Clinical Implementation of Magnetic Resonance Imaging Systems for Simulation and Planning of Pediatric Radiation Therapy.

AIM: To describe the clinical implementation and optimization of magnetic resonance imaging (MRI) systems installed in a radiation oncology department for dedicated use in radiotherapy (RT) simulation and treatment planning for pediatric patients. METHODS: Two wide-bore MRI systems were installed and commissioned in 2016. Patient setups, coil placements, and scan protocols were developed to image various anatomic sites in children. Patients with brain tumors were routinely imaged using a pair of flexible loop coils and a posterior receiver coil integrated into the patient couch. The integrated posterior coil and the flexible anterior torso coil supported by the coil bridge were used together when imaging the abdomen, pelvis, or spine. A three-dimensional acquisition was most often performed, given the benefit of high-resolution multiplanar reformation as well as elimination of B0-related distortions in the slice selection direction. RESULTS: We performed 542 MRI studies (265 for planning and 277 for monitoring on-treatment tumor changes) on pediatric patients in the first year after system installation. Multisequence images of pediatric RT patients with ependymoma, medulloblastoma, craniopharyngioma, rhabdomyosarcoma, or Ewing sarcoma were shown to illustrate the image quality obtainable with optimized planning sequences. CONCLUSIONS: Magnetic resonance imaging (MRI) of pediatric patients in their treatment positions with setup devices in place can be performed with coil arrangements that include flexible coils. The resulting image quality is suitable for treatment planning and on-treatment monitoring. We provide optimized site-specific sequence parameters to support the continued improvement of MRI for pediatric RT planning.

Quantifying potential reduction in contrast dose with monoenergetic images synthesized from dual-layer detector spectral CT.

OBJECTIVE: To estimate the potential dose reduction in iodinated contrast when interpreting monoenergetic images from spectral CT. METHODS: 51 paediatric patients received contrast-enhanced CT simulation for radiation therapy using a single-source, dual-layer detector spectral CT. The contrast-to-noise ratios (CNRs) of blood vessels were measured relative to surrounding soft tissue. CNRs on monoenergetic 40-70 keV images were compared with polychromatic 120 kVp images. To compare with in vivo results, a phantom with iodine inserts (2-20 mg ml-1 concentration) was scanned and CNRs were calculated relative to water background. RESULTS: Monoenergetic keV and body site had significant effects on CNR ratio (p < 0.0001). Across all body sites, the mean CNR ratio (monoenergetic/polychromatic CNR) was 3.3 (20th percentile [%20] 2.6), 2.4 (%20 2.1), 1.7 (%20 1.5), 1.2 (%20 1.0) for 40, 50, 60 and 70 keV images, respectively. Image noise was highest at 40 keV and lowest at 70 keV. Phantom measurements indicated that the same CNR as 120 kVp images can be achieved with a 4.0-fold lower iodine concentration on 40 keV images and 2.5-fold lower on 50 keV images. CONCLUSION: 50 keV monoenergetic images provided the best balance of improved CNR on all studies (mean 2.4-fold increase in vivo) for enhancing vessels vs image noise. A 50% reduction in contrast dose on a 50 keV image should maintain comparable or better CNR as compared with polychromatic CT in over 80% of CT studies. Advances in knowledge: Use of a novel, single-source, dual-layer detector spectral CT scanner to improve visualization of contrast-enhanced blood vessels will reduce the amount of iodinated contrast required for radiation oncology treatment planning.