A 'through-DNA' mechanism for metal uptake-vs.-efflux regulation.

Transition metals like Zn are essential for all organisms including bacteria, but fluctuations of their concentrations in the cell can be lethal. Organisms have thus evolved complex mechanisms for cellular metal homeostasis. One mechanistic paradigm involves pairs of transcription regulators sensing intracellular metal concentrations to regulate metal uptake and efflux. Here we report that Zur and ZntR, a prototypical pair of regulators for Zn uptake and efflux in E. coli , respectively, can coordinate their regulation through DNA, besides sensing cellular Zn 2+ concentrations. Using a combination of live-cell single-molecule tracking and in vitro single-molecule FRET measurements, we show that unmetallated ZntR can enhance the unbinding kinetics of Zur from DNA by directly acting on Zur-DNA complexes, possibly through forming heteromeric ternary and quaternary complexes that involve both protein-DNA and protein-protein interactions. This 'through-DNA' mechanism may functionally facilitate the switching in Zn uptake regulation when bacteria encounter changing Zn environments; it could also be relevant for regulating the uptake-vs.-efflux of various metals across different bacterial species and yeast.

Improving quality of intrapartum and immediate postpartum care in public facilities: experiences and lessons learned from Rajasthan state, India.

BACKGROUND: In spite of considerable improvement in maternal and neonatal outcomes over the past decade in India, the current maternal mortality ratio and neonatal mortality rate are far from the Sustainable Development Goal targets due to suboptimal quality of maternity care. A package of interventions for improving quality of intrapartum and immediate postpartum care was co-designed with the Ministry of Health as the Dakshata program and implemented in public sector health facilities in selected districts in the state of Rajasthan of India since June 2015. This article describes the key strategies, interventions, results and challenges from four years of Dakshata program implementation. METHODS: We have conducted secondary analysis of program data (government data) collected from 202 public facilities across 20 districts of Rajasthan state. The data collected between June-August 2015 (baseline) and the data collected between May-August 2019 (latest) were analyzed. The data sources included: facility assessments, service statistics, monthly progress reports. RESULTS: During the period of program implementation, there were 17,94,249 deliveries accounting for 70% of institutional deliveries in intervention districts. As a result of the intervention, there was a notable increase in competency of health care providers, availability of essential resources, achievement of labour room standards and adherence to evidence-based clinical standards. We also observed reductions in the proportion of referrals for pre-eclampsia/eclampsia, postpartum hemorrhage and neonatal asphyxia by 11, 8 and 3 percentage points respectively. Similarly, data revealed a reduction in stillbirth rates in Dakshata intervention facilities (19.3 vs 15.3) compared to non-Dakshata facilities (21.8 vs 18). CONCLUSIONS: Our experience and findings indicate that the quality of intrapartum and immediate postpartum care can be improved in low- and middle-income countries with the approach presented in this paper.

The role of digital clinical decision support tool in improving quality of intrapartum and postpartum care: experiences from two states of India.

BACKGROUND: Computerized clinical decision support (CDSS) -digital information systems designed to improve clinical decision making by providers - is a promising tool for improving quality of care. This study aims to understand the uptake of ASMAN application (defined as completeness of electronic case sheets), the role of CDSS in improving adherence to key clinical practices and delivery outcomes. METHODS: We have conducted secondary analysis of program data (government data) collected from 81 public facilities across four districts each in two sates of Madhya Pradesh and Rajasthan. The data collected between August -October 2017 (baseline) and the data collected between December 2019 - March 2020 (latest) was analysed. The data sources included: digitized labour room registers, case sheets, referral and discharge summary forms, observation checklist and complication format. Descriptive, univariate and multivariate and interrupted time series regression analyses were conducted. RESULTS: The completeness of electronic case sheets was low at postpartum period (40.5%), and in facilities with more than 300 deliveries a month (20.9%). In multivariate logistic regression analysis, the introduction of technology yielded significant improvement in adherence to key clinical practices. We have observed reduction in fresh still births rates and asphyxia, but these results were not statistically significant in interrupted time series analysis. However, our analysis showed that identification of maternal complications has increased over the period of program implementation and at the same time referral outs decreased. CONCLUSIONS: Our study indicates CDSS has a potential to improve quality of intrapartum care and delivery outcome. Future studies with rigorous study design is required to understand the impact of technology in improving quality of maternity care.

Acceptability and Barriers to Use of the ASMAN Provider-Facing Electronic Platform for Peripartum Care in Public Facilities in Madhya Pradesh and Rajasthan, India: A Qualitative Study Using the Technology Acceptance Model-3.

The evolving field of mobile health (mHealth) is revolutionizing collection, management, and quality of clinical data in health systems. Particularly in low- and middle-income countries (LMICs), mHealth approaches for clinical decision support and record-keeping offer numerous potential advantages over paper records and in-person training and supervision. We conducted a content analysis of qualitative in-depth interviews using the Technology Acceptance Model 3 (TAM-3) to explore perspectives of providers and health managers in Madhya Pradesh and Rajasthan, India who were using the ASMAN (Alliance for Saving Mothers and Newborns) platform, a package of mHealth technologies to support management during the peripartum period. Respondents uniformly found ASMAN easy to use and felt it improved quality of care, reduced referral rates, ensured timely referral when needed, and aided reporting requirements. The TAM-3 model captured many determinants of reported respondent use behavior, including shifting workflow and job performance. However, some barriers to ASMAN digital platform use were structural and reported more often in facilities where ASMAN use was less consistent; these affect long-term impact, sustainability, and scalability of ASMAN and similar mHealth interventions. The transitioning of the program to the government, ensuring availability of dedicated funds, human resource support, and training and integration with government health information systems will ensure the sustainability of ASMAN.

Electromagnetic (EM) catheter path tracking in ultrasound-guided brachytherapy of the breast.

PURPOSE: To evaluate a novel navigation system for breast brachytherapy, based on ultrasound (US)-guided catheter needle implantations followed by electromagnetic (EM) tracking of catheter paths. METHODS: Breast phantoms were produced, containing US-visible tumors. Ultrasound was used to localize the tumor pose and volume within the phantom, followed by planning an optimal catheter pattern through the tumor using navigation software. An electromagnetic (EM)-tracked catheter needle was used to insert the catheters in the desired pattern. The inserted catheters were visualized on a post-implant CT, serving as ground truth. Electromagnetic (EM) tracking and reconstruction of the inserted catheter paths were performed by pulling a flexible EM guidewire through each catheter, performed in two clinical brachytherapy suites. The accuracy of EM catheter tracking was evaluated by calculating the Hausdorff distance between the EM-tracked and CT-based catheter paths. The accuracy and clinical feasibility of EM catheter tracking were also evaluated in three breast cancer patients, performed in a separate experiment room. RESULTS: A total of 71 catheter needles were implanted into 12 phantoms using US guidance and EM navigation, in an average ± SD time of 8.1 ± 2.9 min. The accuracy of EM catheter tracking was dependent on the brachytherapy suite: 2.0 ± 1.2 mm in suite 1 and 0.6 ± 0.2 mm in suite 2. EM catheter tracking was successfully performed in three breast brachytherapy patients. Catheter tracking typically took less than 5 min and had an average accuracy of 1.7 ± 0.3 mm. CONCLUSION: Our preliminary results show a potential role for US guidance and EM needle navigation for implantation of catheters for breast brachytherapy. EM catheter tracking can accurately assess the implant geometry in breast brachytherapy patients. This methodology has the potential to evaluate catheter positions directly after the implantation and during the several fractions of the treatment.

The Chloroplast Tubulin Homologs FtsZA and FtsZB from the Red Alga Galdieria sulphuraria Co-assemble into Dynamic Filaments.

FtsZ is a homolog of eukaryotic tubulin and is present in almost all bacteria and many archaea, where it is the major cytoskeletal protein in the Z ring, required for cell division. Unlike some other cell organelles of prokaryotic origin, chloroplasts have retained FtsZ as an essential component of the division machinery. However, chloroplast FtsZs have been challenging to study because they are difficult to express and purify. To this end, we have used a FATT tag expression system to produce as soluble proteins the two chloroplast FtsZs from Galdieria sulphuraria, a thermophilic red alga. GsFtsZA and GsFtsZB assembled individually in the presence of GTP, forming large bundles of protofilaments. GsFtsZA also assembled in the presence of GDP, the first member of the FtsZ/tubulin superfamily to do so. Mixtures of GsFtsZA and GsFtsZB assembled protofilament bundles and hydrolyzed GTP at a rate approximately equal to the sum of their individual rates, suggesting a random co-assembly. GsFtsZA assembly by itself in limiting GTP gave polymers that remained stable for a prolonged time. However, when GsFtsZB was added, the co-polymers disassembled with enhanced kinetics, suggesting that the GsFtsZB regulates and enhances disassembly dynamics. GsFtsZA-mts (where mts is a membrane-targeting amphipathic helix) formed Z ring-like helices when expressed in Escherichia coli Co-expression of GsFtsZB (without an mts) gave co-assembly of both into similar helices. In summary, we provide biochemical evidence that GsFtsZA assembles as the primary scaffold of the chloroplast Z ring and that GsFtsZB co-assembly enhances polymer disassembly and dynamics.

Improved electromagnetic tracking for catheter path reconstruction with application in high-dose-rate brachytherapy.

PURPOSE: Electromagnetic (EM) catheter tracking has recently been introduced in order to enable prompt and uncomplicated reconstruction of catheter paths in various clinical interventions. However, EM tracking is prone to measurement errors which can compromise the outcome of the procedure. Minimizing catheter tracking errors is therefore paramount to improve the path reconstruction accuracy. METHODS: An extended Kalman filter (EKF) was employed to combine the nonlinear kinematic model of an EM sensor inside the catheter, with both its position and orientation measurements. The formulation of the kinematic model was based on the nonholonomic motion constraints of the EM sensor inside the catheter. Experimental verification was carried out in a clinical HDR suite. Ten catheters were inserted with mean curvatures varying from 0 to [Formula: see text] in a phantom. A miniaturized Ascension (Burlington, Vermont, USA) trakSTAR EM sensor (model 55) was threaded within each catheter at various speeds ranging from 7.4 to [Formula: see text]. The nonholonomic EKF was applied on the tracking data in order to statistically improve the EM tracking accuracy. A sample reconstruction error was defined at each point as the Euclidean distance between the estimated EM measurement and its corresponding ground truth. A path reconstruction accuracy was defined as the root mean square of the sample reconstruction errors, while the path reconstruction precision was defined as the standard deviation of these sample reconstruction errors. The impacts of sensor velocity and path curvature on the nonholonomic EKF method were determined. Finally, the nonholonomic EKF catheter path reconstructions were compared with the reconstructions provided by the manufacturer's filters under default settings, namely the AC wide notch and the DC adaptive filter. RESULTS: With a path reconstruction accuracy of 1.9 mm, the nonholonomic EKF surpassed the performance of the manufacturer's filters (2.4 mm) by 21% and the raw EM measurements (3.5 mm) by 46%. Similarly, with a path reconstruction precision of 0.8 mm, the nonholonomic EKF surpassed the performance of the manufacturer's filters (1.0 mm) by 20% and the raw EM measurements (1.7 mm) by 53%. Path reconstruction accuracies did not follow an apparent trend when varying the path curvature and sensor velocity; instead, reconstruction accuracies were predominantly impacted by the position of the EM field transmitter ([Formula: see text]). CONCLUSION: The advanced nonholonomic EKF is effective in reducing EM measurement errors when reconstructing catheter paths, is robust to path curvature and sensor speed, and runs in real time. Our approach is promising for a plurality of clinical procedures requiring catheter reconstructions, such as cardiovascular interventions, pulmonary applications (Bender et al. in medical image computing and computer-assisted intervention-MICCAI 99. Springer, Berlin, pp 981-989, 1999), and brachytherapy.

Probing for Binding Regions of the FtsZ Protein Surface through Site-Directed Insertions: Discovery of Fully Functional FtsZ-Fluorescent Proteins.

FtsZ, a bacterial tubulin homologue, is a cytoskeletal protein that assembles into protofilaments that are one subunit thick. These protofilaments assemble further to form a "Z ring" at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane and also serves as a scaffold to recruit cell wall remodeling proteins for complete cell division in vivo One model of the Z ring proposes that protofilaments associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of Escherichia coli FtsZ by inserting either small peptides or whole fluorescent proteins (FPs). Among the four lateral surfaces on FtsZ protofilaments, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174, located on the left and right surfaces, completely blocked function, and these sites were identified as possible sites for essential lateral interactions. However, the insert at R174 did not interfere with association of protofilaments into sheets and bundles in vitro Another goal was to find a location within FtsZ that supported insertion of FP reporter proteins while allowing the FtsZ-FPs to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by superresolution techniques. IMPORTANCE: One model for the Z-ring structure proposes that protofilaments are assembled into ribbons by lateral bonds between FtsZ subunits. Our study excluded the involvement of the front and back faces of the protofilament in essential interactions in vivo but pointed to two potential lateral bond sites, on the right and left sides. We also identified an FtsZ loop where various fluorescent proteins could be inserted without blocking function; these FtsZ-FPs functioned as the sole source of FtsZ. This advance provides improved tools for all fluorescence imaging of the Z ring and may be especially important for superresolution imaging.

Effectiveness of the WHO SCC on improving adherence to essential practices during childbirth, in resource constrained settings.

BACKGROUND: India accounts for 27 % of world's neonatal deaths. Although more Indian women deliver in facilities currently than a decade ago, early neonatal mortality has not declined, likely because of insufficient quality of care. The WHO Safe Childbirth Checklist (SCC) was developed to support health workers to perform essential practices known to reduce preventable maternal and new-born deaths around the time of childbirth. Despite promising early research many outstanding questions remain about effectiveness of the SCC in low-resource settings. METHODS: In collaboration with the Ministry of Health SCC was modified for Indian context and introduced in 101 intervention facilities in Rajasthan, India and 99 facilities served as comparison to study if it reduces mortality. This Quasi experimental Observational intervention-comparison was embedded in this larger program to test whether a program for introduction of SCC with simple implementation package was associated with increased adherence to 28 evidence-based practices. This study was conducted in 8 intervention and 8 comparison sites. Program interventions to promote appropriate use of the SCC included orienting providers to the checklist, modest modifications of the SCC to promote provider uptake and accountability, ensuring availability of essential supplies, and providing supportive supervision for helping providers in using the SCC. RESULTS: The SCC was used by providers in 86 % of 240 deliveries observed in the eight intervention facilities. Providers in the intervention group significantly adhered to practices included in the SCC than providers in the comparison group controlling for baseline scores and confounders. Women delivering in the intervention facilities received on an average 11.5 more of the 28 practices included compared with women in the comparison facilities. For selected practices provider performance in the intervention group increased as much as 93 % than comparison sites. CONCLUSION: Use of the SCC and provider performance of best practices increased in intervention facilities reflecting improvement in quality of facility childbirth care for women and new-born in low resource settings.

Metalloregulator CueR biases RNA polymerase's kinetic sampling of dead-end or open complex to repress or activate transcription.

Metalloregulators respond to metal ions to regulate transcription of metal homeostasis genes. MerR-family metalloregulators act on σ(70)-dependent suboptimal promoters and operate via a unique DNA distortion mechanism in which both the apo and holo forms of the regulators bind tightly to their operator sequence, distorting DNA structure and leading to transcription repression or activation, respectively. It remains unclear how these metalloregulator-DNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for transcription regulation. Using single-molecule FRET, we study how the copper efflux regulator (CueR)--a Cu(+)-responsive MerR-family metalloregulator--modulates RNAP interactions with CueR's cognate suboptimal promoter PcopA, and how RNAP affects CueR-PcopA interactions. We find that RNAP can form two noninterconverting complexes at PcopA in the absence of nucleotides: a dead-end complex and an open complex, constituting a branched interaction pathway that is distinct from the linear pathway prevalent for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a "biased sampling" instead of "dynamic equilibrium shifting" mechanism in regulating transcription initiation; it modulates RNAP's binding-unbinding kinetics, without allowing interconversions between the dead-end and open complexes. Instead, the apo-repressor form reinforces the dominance of the dead-end complex to repress transcription, and the holo-activator form shifts the interactions toward the open complex to activate transcription. RNAP, in turn, locks CueR binding at PcopA into its specific binding mode, likely helping amplify the differences between apo- and holo-CueR in imposing DNA structural changes. Therefore, RNAP and CueR work synergistically in regulating transcription.

Identification, synthesis and characterization of an unknown process related impurity in eslicarbazepine acetate active pharmaceutical ingredient by LC/ESI-IT/MS, 1H, 13C and 1H-1H COSY NMR / 药物分析学报

A new impurity was detected during high performance liquid chromatographic (HPLC) analysis of eslicarbazepine acetate active pharmaceutical ingredient. The structure of unknown impurity was postulated based on liquid chromatography mass spectrometry using electrospray ionization and ion trap analyzer (LC/ESI-IT/MS) analysis. Proposed structure of impurity was unambiguously confirmed by synthesis followed by characterization using 1H, 13C nuclear magnetic resonance spectrometry (NMR), 1H-1H correlation spectro-scopy (COSY) and infrared spectroscopy (IR). Based on the spectroscopic and spectrometric data, unknown impurity was characterized as 5-carbamoyl-10,11-dihydro-5H-dibenzo[b,f]azepin-10-yl propionate.

Single-molecule dynamics and mechanisms of metalloregulators and metallochaperones.

Understanding how cells regulate and transport metal ions is an important goal in the field of bioinorganic chemistry, a frontier research area that resides at the interface of chemistry and biology. This Current Topic reviews recent advances from the authors' group in using single-molecule fluorescence imaging techniques to identify the mechanisms of metal homeostatic proteins, including metalloregulators and metallochaperones. It emphasizes the novel mechanistic insights into how dynamic protein-DNA and protein-protein interactions offer efficient pathways via which MerR-family metalloregulators and copper chaperones can fulfill their functions. This work also summarizes other related single-molecule studies of bioinorganic systems and provides an outlook toward single-molecule imaging of metalloprotein functions in living cells.

Cobalt-60 tomotherapy: clinical treatment planning and phantom dose delivery studies.

PURPOSE: Investigations have shown that a Cobalt-60 (Co-60) radioactive source has the potential to play a role in intensity modulated radiation therapy (IMRT). In this paper, Co-60 tomotherapy's conformal dose delivery potential is evaluated by delivering conformal dose plans on a cylindrical homogeneous phantom containing clinical structures similar to those found in a typical head and neck (H&N) cancer. Also, the clinical potential of Co-60 tomotherapy is investigated by generating 2D clinical treatment plans for H&N and prostate anatomical regions. These plans are compared with the 6 MV based treatment plans for modalities such as linear accelerator-based tomotherapy and broad beam IMRT, and 15 MV based 3D conformal radiation therapy (3DCRT). METHODS: For experimental validation studies, clinical and nonclinical conformal dose patterns were delivered on circular, homogeneous phantoms containing GafChromic film. For clinical planning study, dose calculations were performed with the EGSnrc Monte Carlo program, where a Theratronics 780C Co-60 unit and a 6 MV linear accelerator were modeled with a MIMiC binary multileaf collimator. An inhouse inverse treatment planning system was used to optimize tomotherapy plans using the same optimization parameters for both Co-60 and 6 MV beams. The IMRT and 3DCRT plans for the clinical cases were generated entirely in the Eclipse treatment planning system based on inhouse IMRT and 3DCRT site specific protocols. RESULTS: The doses delivered to the homogeneous phantoms agreed with the calculations, indicating that it is possible to deliver highly conformal doses with the Co-60 unit. The dose distributions for Co-60 tomotherapy clinical plans for both clinical cases were similar to those obtained with 6 MV based tomotherapy and IMRT, and much more conformal compared to 3DCRT plans. The dose area histograms showed that the Co-60 plans achieve the dose objectives for the targets and organs at risk. CONCLUSIONS: These results confirm that Co-60 tomotherapy is capable of providing state-of-the-art conformal dose delivery and could be used for the treatment of targets in both small and larger separation anatomical regions.

Direct substitution and assisted dissociation pathways for turning off transcription by a MerR-family metalloregulator.

Metalloregulators regulate transcription in response to metal ions. Many studies have provided insights into how transcription is activated upon metal binding by MerR-family metalloregulators. In contrast, how transcription is turned off after activation is unclear. Turning off transcription promptly is important, however, as the cells would not want to continue expressing metal resistance genes and thus waste energy after metal stress is relieved. Using single-molecule FRET measurements we studied the dynamic interactions of the copper efflux regulator (CueR), a Cu(+)-responsive MerR-family metalloregulator, with DNA. Besides quantifying its DNA binding and unbinding kinetics, we discovered that CueR spontaneously flips its binding orientation at the recognition site. CueR also has two different binding modes, corresponding to interactions with specific and nonspecific DNA sequences, which would facilitate recognition localization. Most strikingly, a CueR molecule coming from solution can directly substitute for a DNA-bound CueR or assist the dissociation of the incumbent CueR, both of which are unique examples for any DNA-binding protein. The kinetics of the direct protein substitution and assisted dissociation reactions indicate that these two unique processes can provide efficient pathways to replace a DNA-bound holo-CueR with apo-CueR, thus turning off transcription promptly and facilely.

Dosimetry of interface region near closed air cavities for Co-60, 6 MV and 15 MV photon beams using Monte Carlo simulations.

Underdosing of treatment targets can occur in radiation therapy due to electronic disequilibrium around air-tissue interfaces when tumors are situated near natural air cavities. These effects have been shown to increase with the beam energy and decrease with the field size. Intensity modulated radiation therapy (IMRT) and tomotherapy techniques employ combinations of multiple small radiation beamlets of varying intensities to deliver highly conformal radiation therapy. The use of small beamlets in these techniques may therefore result in underdosing of treatment target in the air-tissue interfaces region surrounding an air cavity. This work was undertaken to investigate dose reductions near the air-water interfaces of 1x1x1 and 3x3x3 cm(3) air cavities, typically encountered in the treatment of head and neck cancer utilizing radiation therapy techniques such as IMRT and tomotherapy using small fields of Co-60, 6 MV and 15 MV photons. Additional investigations were performed for larger photon field sizes encompassing the entire air-cavity, such as encountered in conventional three dimensional conformal radiation therapy (3DCRT) techniques. The EGSnrc/DOSXYZnrc Monte Carlo code was used to calculate the dose reductions (in water) in air-water interface region for single, parallel opposed and four field irradiations with 2x2 cm(2) (beamlet), 10x2 cm(2) (fan beam), 5x5 and 7x7 cm(2) field sizes. The magnitude of dose reduction in water near air-water interface increases with photon energy; decreases with distance from the interface as well as decreases as the number of beams are increased. No dose reductions were observed for large field sizes encompassing the air cavities. The results demonstrate that Co-60 beams may provide significantly smaller interface dose reductions than 6 MV and 15 MV irradiations for small field irradiations such as used in IMRT and tomotherapy.

Strong hydrogen bond between glutamic acid 46 and chromophore leads to the intermediate spectral form and excited state proton transfer in the Y42F mutant of the photoreceptor photoactive yellow protein.

In the Y42F mutant of photoactive yellow protein (PYP) the photoreceptor is in an equilibrium between two dark states, the yellow and intermediate spectral forms, absorbing at 457 and 390 nm, respectively. The nature of this equilibrium and the light-induced protonation and structural changes in the two spectral forms were characterized by transient absorption, fluorescence, FTIR, and pH indicator dye experiments. In the yellow form, the oxygen of the deprotonated p-hydroxycinnamoyl chromophore is linked by a strong low-barrier hydrogen bond to the protonated carboxyl group of Glu46 and by a weaker one to Thr50. Using FTIR, we find that the band due to the carbonyl of the protonated side chain of Glu46 is shifted from 1736 cm(-1) in wild type to 1724 cm(-1) in the yellow form of Y42F, implying a stronger hydrogen bond with the deprotonated chromophore in Y42F. The FTIR data suggest moreover that in the intermediate spectral form the chromophore is protonated and Glu46 deprotonated. Flash spectroscopy (50 ns-10 s) shows that the photocycles of the two forms are essentially the same except for a transition around 5 mus that has opposite signs in the two forms and is due to the chemical relaxation between the two dark states. The two cycles are coupled, likely by excited state proton transfer. The Y42F cycle differs from wild type by the occurrence of a new intermediate with protonated chromophore between the usual I(1) and I(2) intermediates which we call I(1)H (370 nm). Transient fluorescence measurements indicate that in I(1)H the chromophore retains the orientation it had in I(1). Transient proton uptake occurs with a time constant of 230 mus and a stoichiometry of 1. No proton uptake was associated however with the formation of the I(1)H intermediate and the relaxation of the yellow/intermediate equilibrium. These protonation changes of the chromophore thus occur intramolecularly. The chromophore-Glu46 hydrogen bond in Y42F is shorter than in wild type, since the adjacent chromophore-Y42 hydrogen bond is replaced by a longer one with Thr50. This facilitates proton transfer from Glu46 to the chromophore in the dark by lowering the barrier, leading to the protonation equilibrium and causing the rapid light-induced proton transfer which couples the cycles.

The role of Cobalt-60 in modern radiation therapy: Dose delivery and image guidance.

The advances in modern radiation therapy with techniques such as intensity-modulated radiation therapy and image-guided radiation therapy (IMRT and IGRT) have been limited almost exclusively to linear accelerators. Investigations of modern Cobalt-60 (Co-60) radiation delivery in the context of IMRT and IGRT have been very sparse, and have been limited mainly to computer-modeling and treatment-planning exercises. In this paper, we report on the results of experiments using a tomotherapy benchtop apparatus attached to a conventional Co-60 unit. We show that conformal dose delivery is possible and also that Co-60 can be used as the radiation source in megavoltage computed tomography imaging. These results complement our modeling studies of Co-60 tomotherapy and provide a strong motivation for continuing development of modern Cobalt-60 treatment devices.

Practical and clinical considerations in Cobalt-60 tomotherapy.

Cobalt-60 (Co-60) based radiation therapy continues to play a significant role in not only developing countries, where access to radiation therapy is extremely limited, but also in industrialized countries. Howver, technology has to be developed to accommodate modern techniques, including image guided and adaptive radiation therapy (IGART). In this paper we describe some of the practical and clinical considerations for Co-60 based tomotherapy by comparing Co-60 and 6 MV linac-based tomotherapy plans for a head and neck (HandN) cancer and a prostate cancer case. The tomotherapy IMRT plans were obtained by modeling a MIMiC binary multi-leaf collimator attached to a Theratron-780c Co-60 unit and a 6 MV linear accelerator (CL2100EX). The EGSnrc/BEAMnrc Monte Carlo (MC) code was used for the modeling of the treatment units with the MIMiC collimator and EGSnrc/DOSXYZnrc code was used for beamlet dose data. An in-house inverse treatment planning program was then used to generate optimized tomotherapy dose distributions for the H and N and prostate cases. The dose distributions, cumulative dose area histograms (DAHs) and dose difference maps were used to evaluate and compare Co-60 and 6 MV based tomotherapy plans. A quantitative analysis of the dose distributions and dose-volume histograms shows that both Co-60 and 6 MV plans achieve the plan objectives for the targets (CTV and nodes) and OARs (spinal cord in HandN case, and rectum in prostate case).

Investigation of an efficient source design for Cobalt-60-based tomotherapy using EGSnrc Monte Carlo simulations.

Recent investigations demonstrate a strong potential for Cobalt-60 (Co-60)-based tomotherapy. Reported work suggests that Co-60-based tomotherapy offers a clinically and commercially viable alternative to megavoltage x-ray-based tomotherapy. Tomotherapy applications use a combination of intensity-modulated fan beams to deliver highly conformal radiotherapy. However, conventional Co-60 units are designed to give large uniform rectangular fields using an isotropic radioactive source in a cylindrical geometry. Such cylindrical source geometry likely provides a sub-optimal use of the radioactivity within the source volume for tomotherapy applications due to a significant loss of radiated energy outside the fan beam collimation system. To investigate a more efficient source geometry suitable for Co-60 tomotherapy applications, a computer code was written to model an isotropic source in a 6-faced polyhedron geometry such as cube, parallelepiped, prism and truncated pyramid. This code was integrated with the existing EGSnrc/BEAMnrc Monte Carlo (MC) code. The integrated source code was thoroughly tested, validated and used to investigate the energy spectra, radiation output and self-shielding properties of various rectangular-shaped (RS) Co-60 sources. Fan beam dose profiles were calculated for various cylindrical and RS Co-60 sources for a range of source-to-axis distances (SAD), multi-leaf collimator-to-isocentre distances (CID) and modified collimator systems. Fringe and penumbra distances were analysed for the simulated dose profiles. Our results demonstrate that clinically acceptable fringe and penumbra distances can be achieved by a careful selection of SAD, CID, source shape and dimensions and modified collimator system. Significant overall gain in radiation output of the 20 x 1 cm(2) fan beams can be achieved by an optimal selection of the source geometry for a given active volume of Co-60. The overall gain includes the effects of change in packing density (accounting for self-absorption) and change in source shape.