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Inter-satellite interferometric missions are critical in the ongoing monitoring of climate change. Next-generation Earth geodesy missions are opportunities to improve on mission cost and measurement sensitivity through revised design. To be considered feasible, mission architectures must meet an optical power requirement that factors in both shot noise and laser frequency noise. Reference-transponder mission configurations, like the Gravity Recovery and Climate Experiment-Follow On (GRACE-FO) mission, are designed for measurement down to a received carrier-to-noise density ratio of 70 dB-Hz—1.9 picowatts in shot-noise-limited detection. This work shows, through modeling and simulation, that the optical power level required to perform robust measurement varies significantly between mission configurations. Alternate configurations, such as retro-reflector-based schemes, can operate robustly down to much lower carrier-to-noise density ratios, with the example parameters considered here: down to 29 dB-Hz—150 attowatts in shot-noise-limited detection. These results motivate exploration of alternate missions configurations with revised optical power requirements, increasing the feasibility of new designs.

Abstract Satellite geodesy uses the measurement of the motion of one or more satellites to infer precise information about the Earth’s gravitational field. In this work, we consider the achievable precision limits on such measurements by examining approximate models for the three main noise sources in the measurement process of the current Gravitational Recovery and Climate Experiment (GRACE) Follow-On mission: laser phase noise, accelerometer noise and quantum noise. We show that, through time-delay interferometry, it is possible to remove the laser phase noise from the measurement, allowing for almost three orders of magnitude improvement in the signal-to-noise ratio. Several differential mass satellite formations are presented which can further enhance the signal-to-noise ratio through the removal of accelerometer noise. Finally, techniques from quantum optics have been studied, and found to have great promise for reducing quantum noise in other alternative mission configurations. We model the spectral noise performance using an intuitive 1D model and verify that our proposals have the potential to greatly enhance the performance of near-future satellite geodesy missions.

The pulmonary surfactant protein A (SP-A) is a constitutively expressed immune-protective collagenous lectin (collectin) in the lung. It binds to the cell membrane of immune cells and opsonizes infectious agents such as bacteria, fungi, and viruses through glycoprotein binding. SARS-CoV-2 enters airway epithelial cells by ligating the Angiotensin Converting Enzyme 2 (ACE2) receptor on the cell surface using its Spike glycoprotein (S protein). We hypothesized that SP-A binds to the SARS-CoV-2 S protein and this binding interferes with ACE2 ligation. To study this hypothesis, we used a hybrid quantum and classical in silico modeling technique that utilized protein graph pruning. This graph pruning technique determines the best binding sites between amino acid chains by utilizing the Quantum Approximate Optimization Algorithm (QAOA)-based MaxCut (QAOA-MaxCut) program on a Near Intermediate Scale Quantum (NISQ) device. In this, the angles between every neighboring three atoms were Fourier-transformed into microwave frequencies and sent to a quantum chip that identified the chemically irrelevant atoms to eliminate based on their chemical topology. We confirmed that the remaining residues contained all the potential binding sites in the molecules by the Universal Protein Resource (UniProt) database. QAOA-MaxCut was compared with GROMACS with T-REMD using AMBER, OPLS, and CHARMM force fields to determine the differences in preparing a protein structure docking, as well as with Goemans-Williamson, the best classical algorithm for MaxCut. The relative binding affinity of potential interactions between the pruned protein chain residues of SP-A and SARS-CoV-2 S proteins was assessed by the ZDOCK program. Our data indicate that SP-A could ligate the S protein with a similar affinity to the ACE2-Spike binding. Interestingly, however, the results suggest that the most tightly-bound SP-A binding site is localized to the S2 chain, in the fusion region of the SARS-CoV-2 S protein, that is responsible for cell entry Based on these findings we speculate that SP-A may not directly compete with ACE2 for the binding site on the S protein, but interferes with viral entry to the cell by hindering necessary conformational changes or the fusion process.

Abstract Autologous bone grafts are considered the gold standard grafting material for the treatment of nonunion, but in very large bone defects, traditional autograft alone is insufficient to induce repair. Recombinant human bone morphogenetic protein 2 (rhBMP-2) can stimulate bone regeneration and enhance the healing efficacy of bone grafts. The delivery of rhBMP-2 may even enable engineered synthetic scaffolds to be used in place of autologous bone grafts for the treatment of critical size defects, eliminating risks associated with autologous tissue harvest. We here demonstrate that an osteoinductive scaffold, fabricated by combining a 3D printed rigid polymer/ceramic composite scaffold with an rhBMP-2-eluting collagen sponge can treat extremely large-scale segmental defects in a pilot feasibility study using a new sheep metatarsus fracture model stabilized with an intramedullary nail. Bone regeneration after 24 weeks was evaluated by micro-computed tomography, mechanical testing, and histological characterization. Load-bearing cortical bridging was achieved in all animals, with increased bone volume observed in sheep that received osteoinductive scaffolds compared to sheep that received an rhBMP-2-eluting collagen sponge alone.

Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Here, the authors develop a monoclonal antibody that specifically inhibits APC’s anticoagulant function without compromising its cytoprotective function, and shows efficacy in animal models.

A novel canine tibial plateau leveling osteotomy (TPLO) fixation device was recently developed with design features such as titanium alloy (TA) material, distal monocortical screw fixation, and a point contact undersurface specifically targeted to reduce surgical site infection rates by ensuring tissue perfusion under the plate. The strength of the novel TPLO construct was compared with that of a predicate stainless steel (SS) locking plate construct with bicortical screws in 16 paired cadaveric canine limbs. The mean loads to failure were 716.71 ± 109.50 N (range 455.69–839.69 N) and 629.50 ± 176.83 N (range 272.58–856.18 N) in the TA and SS groups, respectively. The average ratio of the loads to failure of the paired specimens was 1.18 (p = 0.031). No failure of the TA constructs involved the distal fixation with monocortical screws. Substantial mechanical equivalence of this novel TA monocortical/bicortical fixation construct to an established SS bicortical screw fixation construct is demonstrated. Clinical investigation of potential merits of this novel TA, monocortical/bicortical locking screw/plate system is now warranted.

Future space-based geodesy missions such as the Mass Change Mission and the Next Generation Gravity Mission are expected to rely on laser ranging as their primary instrument. Short-term laser frequency stability has previously been achieved on the GRACE Follow On mission by stabilizing the lasers to an optical cavity. The development of a technique to provide long-term laser frequency stability is expected to be required. We have previously demonstrated a technique to track long-term frequency changes by using measurements of the optical cavity’s free spectral range. In this paper, we calibrate this technique to absolute frequency by using an atomic reference. We have also validated an approach for on-ground calibration to allow the absolute frequency to be determined whilst in orbit.

We present our results regarding the cosmetic outcome achieved and the rate of infection using the MammoSite breast brachytherapy applicator to treat patients with partial breast irradiation. In addition, factors associated with cosmetic outcome and infection are analyzed. The study population consisted of 30 patients with early stage breast cancer treated using the MammoSite device from October 28, 2002, to February 13, 2004. Cosmetic outcome was analyzed for its association with the following parameters: volume of the balloon, balloon-to-skin distance, maximal skin point dose per fraction, V100 (percent of volume that received 100% of the prescription dose), V150 (percent of volume that received 150% of the prescription dose), and V200 (percent of volume that received 200% of the prescription dose). The occurrence of infection at the time of treatment and during follow-up was also recorded. At a median follow-up of 13 months (range 1-16 months), 53.3% of the patients (16/30) were reported to have an excellent cosmetic outcome and 40.0% (12/30) had a good cosmetic outcome. Excellent cosmetic outcome was associated with a greater mean balloon-to-skin distance compared to those who achieved a good cosmetic outcome (1.5 cm versus 1.2 cm) (p = 0.164). The mean V100, V150, and V200 of those in the excellent cosmetic outcome group were 92.1%, 34.5%, and 7.6% versus 93.0%, 34.7%, and 7.6% in the good cosmetic outcome group (p = 0.642, 0.926, and 0.853), The mean balloon volumes were 47.7 cm3 and 56.9 cm3, respectively (p = 0.063) in the excellent and good outcome groups. The mean maximal skin doses per fraction in the excellent and good outcome groups were 354.8 cGy and 422.3 cGy (p = 0.286), respectively. Infection occurred in 13.3% of the patients (4/30). An excellent or good cosmetic outcome was achieved in 93.3% of patients and infection occurred in 13.3% of patients treated with the MammoSite breast brachytherapy applicator. Excellent cosmetic outcome was associated with a greater balloon-to-skin distance, lower maximal skin dose per fraction, and smaller mean balloon volume; however, the results did not reach statistical significance. [ABSTRACT FROM AUTHOR]

To expand the known spectrum of genes that maintain genome stability, we screened a recently released collection of temperature sensitive (Ts) yeast mutants for a chromosome instability (CIN) phenotype. Proteasome subunit genes represented a major functional group, and subsequent analysis demonstrated an evolutionarily conserved role in CIN. Analysis of individual proteasome core and lid subunit mutations showed that the CIN phenotype at semi-permissive temperature is associated with failure of subunit localization to the nucleus. The resultant proteasome dysfunction affects chromosome stability by impairing the kinetics of double strand break (DSB) repair. We show that the DNA repair protein Mms22 is required for DSB repair, and recruited to chromatin in a ubiquitin-dependent manner as a result of DNA damage. Moreover, subsequent proteasome-mediated degradation of Mms22 is necessary and sufficient for cell cycle progression through the G(2)/M arrest induced by DNA damage. Our results demonstrate for the first time that a double strand break repair protein is a proteasome target, and thus link nuclear proteasomal activity and DSB repair.

Background: Absorbable hydrogel spacer injected between prostate and rectum is gaining popularity for rectal sparing. The spacer alters patient anatomy and thus requires new auto-contouring models., Purpose: To report the development and comprehensive evaluation of two deep-learning models for patients injected with a radio-transparent (model I) versus radiopaque (model II) spacer., Methods and Materials: Model I was trained and cross-validated by 135 cases with transparent spacer and tested on 24 cases. Using refined training methods, model II was trained and cross-validated by the same dataset, but with the Hounsfield Unit distribution in the spacer overridden by that obtained from ten cases with opaque spacer. Model II was tested on 64 cases. The models auto-contour eight regions of interest (ROIs): spacer, prostate, proximal seminal vesicles (SVs), left and right femurs, bladder, rectum, and penile bulb. Qualitatively, each auto contour (AC), as well as the composite set, was assessed against manual contour (MC), by a radiation oncologist using a 1 (accepted directly or after minor editing), 2 (accepted after moderate editing), 3 (accepted after major editing), and 4 (rejected) scoring scale. The efficiency gain was characterized by the mean score as nearly complete [1-1.75], substantial (1.75-2.5], meaningful (2.5-3.25], and no (3.25-4.00]. Quantitatively, the geometric similarity between AC and MC was evaluated by dice similarity coefficient (DSC) and mean distance to agreement (MDA), using tolerance recommended by AAPM TG-132 Report. The results by the two models were compared to examine the outcome of the refined training methods. The large number of testing cases for model II allowed further investigation of inter-observer variability in clinical dataset. The correlation between score and DSC/MDA was studied on the ROIs with 10 or more counts of each acceptable score (1, 2, 3)., Results: For model I/model II: the mean score was 3.63/1.30 for transparent/opaque spacer, 2.71/2.16 for prostate, 3.25/2.44 for proximal SVs, 1.13/1.02 for both femurs, 2.25/1.25 for bladder, 3.00/2.06 for rectum, 3.38/2.42 for penile bulb, and 2.79/2.20 for the composite set; the mean DSC was 0.52/0.84 for spacer, 0.84/0.85 for prostate, 0.60/0.62 for proximal SVs, 0.94/0.96 for left femur, 0.95/0.96 for right femur, 0.91/0.95 for bladder, 0.81/0.84 for rectum, and 0.65/0.65 for penile bulb; and the mean MDA was 2.9/0.9 mm for spacer, 1.9/1.7 mm for prostate, 2.4/2.3 mm for proximal SVs, 0.8/0.5 mm for left femur, 0.7/0.5 mm for right femur, 1.5/0.9 mm for bladder, 2.3/1.9 mm for rectum, and 2.2/2.2 mm for penile bulb. Model II showed significantly improved scores for all ROIs, and metrics for spacer, femurs, bladder, and rectum. Significant inter-observer variability was only found for prostate. Highly linear correlation between the score and DSC was found for the two qualified ROIs (prostate and rectum)., Conclusions: The overall efficiency gain was meaningful for model I and substantial for model II. The ROIs meeting the clinical deployment criteria (mean score below 3.25, DSC above 0.8, and MDA below 2.5 mm) included prostate, both femurs, bladder and rectum for both models, and spacer for model II., (© 2023 American Association of Physicists in Medicine.)

Purpose: To identify policy and system related weaknesses in treatment planning and plan check work-flows., Methods: The authors' web deployed plan check automation solution, PlanCheck, which works with all major planning and record and verify systems (demonstrated here for mosaiq only), allows them to compute violation rates for a large number of plan checks across many facilities without requiring the manual data entry involved with incident filings. Workflows and failure modes are heavily influenced by the type of record and verify system used. Rather than tackle multiple record and verify systems at once, the authors restricted the present survey to mosaiq facilities. Violations were investigated by sending inquiries to physicists running the program., Results: Frequent violations included inadequate tracking in the record and verify system of total and prescription doses. Infrequent violations included incorrect setting of patient orientation in the record and verify system. Peaks in the distribution, over facilities, of violation frequencies pointed to suboptimal policies at some of these facilities. Correspondence with physicists often revealed incomplete knowledge of settings at their facility necessary to perform thorough plan checks., Conclusions: The survey leads to the identification of specific and important policy and system deficiencies that include: suboptimal timing of initial plan checks, lack of communication or agreement on conventions surrounding prescription definitions, and lack of automation in the transfer of some parameters.

Purpose: For patients with left-sided breast cancers, radiation treatment to the intact breast results in high doses to significant volumes of the heart, increasing the risk of cardiac morbidity, particularly in women with unfavorable cardiac anatomy. We compare helical tomotherapy (TOMO) and inverse planned intensity modulated radiation therapy (IMRT) with three-dimensional conformal radiotherapy using opposed tangents (3D-CRT) for reductions in cardiac volumes receiving high doses., Methods and Materials: Fifteen patients with left-sided breast cancers and unfavorable cardiac anatomy, determined by a maximum heart depth (MHD) of >or=1.0 cm within the tangent fields, were planned for TOMO and IMRT with five to seven beam angles, in addition to 3D-CRT. The volumes of heart and left ventricle receiving >or=35 Gy (V35) were compared for the plans, as were the mean doses to the contralateral breast and the volume receiving >or=20 Gy (V20) for the ipsilateral lung., Results: The mean MHD was 1.7 cm, and a significant correlation was observed between MHD and both heart and left ventricle V35. The V35s for IMRT (0.7%) and TOMO (0.5%) were significantly lower than for 3D-CRT (3.6%). The V20 for IMRT (22%) was significantly higher than for 3D-CRT (15%) or TOMO (18%), but the contralateral breast mean dose for TOMO (2.48 Gy) was significantly higher than for 3D-CRT (0.93 Gy) or IMRT (1.38 Gy)., Conclusions: Both TOMO and IMRT can significantly reduce cardiac doses, with modest increases in dose to other tissues in left-sided breast cancer patients with unfavorable cardiac anatomy., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Introduction: Electron beam radiation is the modality most often used to deliver an operative bed boost to breast cancer patients after completing whole breast radiation. However, electrons can potentially provide inadequate coverage. The MammoSite breast brachytherapy applicator may provide dosimetric advantages in the delivery of an operative bed boost and its role in this setting is not yet defined., Materials and Methods: The study population consisted of 15 patients with early stage breast cancer treated with partial breast irradiation (PBI) using the MammoSite device. For each patient, a theoretical boost plan using electrons and a second theoretical boost plan using the MammoSite applicator were created. To assess the adequacy of each boost plan, the PTV V90, PTV V95, and PTV V100 were calculated. To assess dose to normal tissues, the ipsilateral breast V50, ipsilateral lung V30, and heart V20 were calculated., Results: The mean PTV V100 for the MammoSite boost was 95.5%, compared to 77.4% for the electron boost (p<0.001). The mean PTV V95 was 97.8%, compared to 93.3% for the electron boost (p=0.02). The mean PTV V90, mean breast V50, mean lung V30, and mean heart V20 were not statistically different for MammoSite compared to electrons., Conclusions: A tumor bed boost using the MammoSite breast brachytherapy applicator provides superior target coverage and delivers similar doses to the ipsilateral breast and lung compared to a boost delivered with electrons. More investigation into the role of balloon brachytherapy in the delivery of a breast boost is warranted., (Copyright 2009 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.)

Patients with breast cancer are often treated with radiation to the breast (or chest wall) and draining regional lymph nodes. This is typically performed with a three-field technique in which an anterior supraclavicular field is matched to opposed tangent fields. A single isocenter technique is not always possible. Several techniques have been described to create a perfect match using a conventional simulator. We describe and test a simple, fast and accurate technique to estimate the couch and collimator angles required for a perfect geometric match using 3-D treatment planning software. This method requires no mathematical formulae and is verifiable relative to patient anatomy. An external skin contour is created on the axial slice at the match line and displayed with a 3-D representation. Using a beam's eye view (BEV) of a tangent field, small couch and collimator rotation adjustments are made sequentially until the contour edges are superimposed. The virtual external contour technique was easy to use, gave verification of the match in the BEV and yielded estimates of couch and collimator rotations very close to those calculated using published formulae.

Objectives: Static field intensity modulated radiation therapy (IMRT) has demonstrated dosimetric and clinical benefits over 3-dimensional conformal radiation therapy. TomoTherapy is a unique form of IMRT that may offer further improvements., Methods: The study population consisted of 15 patients with low-risk prostate cancer treated at Rush University with TomoTherapy (n = 7) or IMRT (n = 8). For each patient, both a TomoTherapy plan and an IMRT plan were generated using identical planning objectives. The planning target volume (PTV) was defined as the prostate and proximal seminal vesicles plus a margin. The prescription dose was 7740 cGy in 43 fractions. Radiation Therapy Oncology Group (RTOG) normal tissue guidelines were used as constraints, and the PTV coverage was made equivalent for the paired plans by equalizing the PTV V100. RTOG benchmark DVH values for the rectum and bladder and mean dose to the penile bulb were recorded. The volume of PTV receiving ≥ 105% of the prescription dose was measured., Results: The mean DVH values for each of the RTOG constraints for rectum and bladder were significantly improved using TomoTherapy. The volume of the PTV that received at least 105% of the dose was higher with IMRT (11.7% vs. 0.2%, <0.001). The mean dose to the penile bulb was higher with TomoTherapy (40.4 Gy vs. 27.4 Gy, P = 0.005)., Conclusions: TomoTherapy offers a more favorable dose distribution to the bladder and rectum, as well as improved target homogeneity in comparison with IMRT.

We hypothesized that use of a true 3D display providing easy visualization of patient anatomy and dose distribution would lead to the production of better quality radiation therapy treatment plans. We report on a randomized prospective multi-institutional study to evaluate a novel 3D display for treatment planning.The Perspecta Spatial 3D System produces 360 degrees holograms by projecting crosssectional images on a diffuser screen rotating at 900 rpm. Specially-developed software allows bi-directional transfer of image and dose data between Perspecta and the Pinnacle planning system.Thirty-three patients previously treated at three institutions were included in this IRB-approved study. Patient data were de-identified, randomized, and assigned to different planners. A physician at each institution reviewed the cases and established planning objectives. Two treatment plans were then produced for each patient, one based on the Pinnacle system alone and another in conjunction with Perspecta. Plan quality was then evaluated by the same physicians who established the planning objectives. All plans were viewable on both Perspecta and Pinnacle for review. Reviewing physicians were blinded to the planning device used. Data from a 13-patient pilot study were also included in the analysis.Perspecta plans were considered better in 28 patients (61%), Pinnacle in 14 patients (30%), and both were equivalent in 4 patients. The use of non-coplanar beams was more common with Perspecta plans (82% vs. 27%). The mean target dose differed by less than 2% between rival plans. Perspecta plans were somewhat more likely to have the hot spot located inside the target (43% vs. 33%). Conversely, 30% of the Pinnacle plans had the hot spot outside the target compared with 18% for Perspecta plans. About 57% of normal organs received less dose from Perspecta plans. No statistically significant association was found between plan preference and planning institution or planner.The study found that use of the holographic display leads to radiotherapy plans preferred in a majority of cases over those developed with 2D displays. These data indicate that continued development of this technology for clinical implementation is warranted.

In this report, we describe a novel technique used to plan and administer external beam radiation therapy to a patient in the upright position. A patient required reirradiation for thymic carcinoma but was unable to tolerate the supine position due to bilateral phrenic nerve injury and paralysis of the diaphragm. Computed tomography (CT) images in the upright position were acquired at the Northern Illinois University Institute for Neutron Therapy at Fermilab. The CT data were imported into a standard 3-dimensional (3D) treatment planning system. Treatment was designed to deliver 24 Gy to the target volume while respecting normal tissue tolerances. A custom chair that locked into the treatment table indexing system was constructed for immobilization, and port films verified the reproducibility of setup. Radiation was administered using mixed photon and electron AP fields.

Purpose: A new form of partial breast irradiation (PBI), ClearPath (CP) breast brachytherapy, has been introduced. We present our results of a dosimetric comparison of MammoSite (MS) and CP PBI., Methods and Materials: The dimensions of the CP device were reconstructed onto the MS planning CT scans for 15 previously treated patients. The mean %V(100), %V(150), %V(200) (percent of the PTV that received 100%, 150%, and 200% of the prescription dose, respectively), ipsilateral breast %V(50) (percent of the ipsilateral normal breast that received 50% of the prescription dose), ipsilateral lung %V(30) (percent of the ipsilateral lung that received 30% of the prescription dose), the heart %V(5) (percent of the heart that received 5% of the prescription dose), and the maximum skin point dose per fraction were then determined for each patient using the two methods of balloon-based PBI., Results: The mean %V(100) was 96.5% vs. 96.5%, the mean %V(150) was 42.1% vs. 42.9% (p=ns), and the mean V(200) was 11.4% vs. 15.2% (p<.05) for the MS and CP methods, respectively. The mean ipsilateral breast %V(50) was 19.8% vs.18.0% (p<.05), the mean ipsilateral lung %V(30) was 3.7% vs. 2.8% (p<.05), the mean heart %V(5) was 57.0% vs. 54.3% (p<.05), and the maximum skin point dose per fraction was 312.2 and 273.6cGy (p<.05) for the MS and CP methods, respectively., Conclusions: The MS and CP methods of PBI offer comparable target volume coverage; however, the CP device achieves increased normal tissue sparing.

Purpose: This analysis was undertaken to dosimetrically compare iridium-192 high-dose-rate brachytherapy (IB) and Xoft Axxent Electronic Brachytherapy (XB; Xoft Inc., Sunnyvale, CA) in the treatment of endometrial cancer., Methods and Materials: The planning CT scans from 11 patients previously treated with IB were used to construct hypothetical treatment plans using the source characteristics of the XB device. The mean V95, V100, and V150 (percent of the planning target volume that received 95%, 100%, and 150% of the prescription dose) were calculated. For both the bladder and rectum, the V35 (percent of the organ that received 35% of the prescription dose) and V50 (percent of the organ that received 50% of the prescription dose) were calculated for each patient using both methods of vaginal brachytherapy., Results: The mean %V95 was 99.7% vs. 99.6% (p = ns) and the mean %V100 was 99.0% vs. 99.1% (p = ns) for the IB and XB methods, respectively. The mean %V150 was 35.8% vs. 58.9% (p < 0.05) for the IB and XB methods, respectively. The mean bladder %V35 was 47.7% vs. 27.4% (p < 0.05) and the mean bladder %V50 was 26.5% vs. 15.9% (p < 0.05) for the IB and XB methods, respectively. The mean rectal %V35 was 48.3% vs. 28.3% (p < 0.05) and the mean rectal %V50 was 27.8% vs. 17.0% (p < 0.05) for the IB and XB methods, respectively., Conclusions: The IB and XB methods of vaginal brachytherapy offer equivalent target volume coverage; however, the XB method allows increased sparing of the bladder and rectum.

Partial breast irradiation (PBI) was designed in part to decrease overall treatment times associated with whole breast radiation therapy (WBRT). WBRT treats the entire breast and usually portions of the axilla. The goal of PBI is to treat a smaller volume of breast tissue in less time, focusing the dose around the lumpectomy cavity. The following is a case of a 64-year-old woman with early-stage breast cancer treated with PBI who failed regionally in the ipsilateral axilla. With our dosimetric analysis, we found that the entire area of this axillary failure would have likely received at least 45 Gy if WBRT had been used, enough to sterilize microscopic disease. With PBI, this area received a mean dose of only 2.8 Gy, which raises the possibility that this regional failure may have been prevented had WBRT been used instead of PBI.

Background: Suboptimal adherence to prescribed oral anticancer agents has been demonstrated in several studies. Understanding the barriers to treatment compliance is becoming increasingly important because of the growing number of oral agents available to treat women with breast cancer., Patients and Methods: To identify barriers that affect patient adherence to oral hormonal medications, a self-reported, 30-question Internet survey was posted on the Y-ME National Breast Cancer Organization Web site, www.y-me.org, from June 30, 2005, through October 31, 2005. Of the 542 respondents who began the survey, 328 completed it., Results: Of those who answered the question related to whether healthcare professionals encouraged adherence, only 44.2% reported receiving instruction on the importance of taking oral medication as directed at every office visit. For 33%, the topic was reportedly discussed only once, and this occurred before treatment. Although 83.6% of patients indicated that they were taking their oral therapy as directed, only 57.4% rated their ability to adhere to therapy as excellent (not missing a single dose in the past month). The most frequently cited factors for increasing compliance to oral medications were knowing adherence could improve clinical outcomes (88.7%) and better management of treatment-related side effects (60.2%)., Conclusion: The results of this survey suggest that treatment adherence could be improved if healthcare professionals discuss and emphasize (1) the importance of taking oral medications as directed and (2) the effect of adherence on clinical outcomes. Additional research is needed to evaluate the optimal methods for improving compliance with oral therapy.

Putative N-acetylmuramyl-l-alanine amidase genes from LambdaSa1 and LambdaSa2 prophages of Streptococcus agalactiae were cloned and expressed in Escherichia coli. The purified enzymes lysed the cell walls of Streptococcus agalactiae, Streptococcus pneumoniae, and Staphylococcus aureus. The peptidoglycan digestion products in the cell wall lysates were not consistent with amidase activity. Instead, the structure of the muropeptide digestion fragments indicated that both the LambdaSa1 and LambdaSa2 lysins exhibited gamma-d-glutaminyl-l-lysine endopeptidase activity. The endopeptidase cleavage specificity of the lysins was confirmed using a synthetic peptide substrate corresponding to a portion of the stem peptide and cross bridge of Streptococcus agalactiae peptidoglycan. The LambdaSa2 lysin also displayed beta-d-N-acetylglucosaminidase activity.

Purpose: Over 20,000 patients have been treated with partial breast irradiation (PBI) using the MammoSite balloon brachytherapy applicator (IBB). Recently, a new form of balloon-based PBI, Xoft Axxent electronic brachytherapy (KVB), which uses a 50-kV x-ray source, has been introduced. This analysis was undertaken to dosimetrically compare the results of treatment using these two methods of PBI., Methods and Materials: The study population consisted of 15 patients previously treated with IBB. The planning CT scans from these 15 patients were used to construct hypothetical treatment plans using the source characteristics of the KVB device. The plans were then compared using the dosimetric parameters discussed below., Results: The mean %V(90) was 99.6% vs. 99.0% (p=nonsignificant [ns]), the mean %V(100) was 96.5% vs. 96.5%, the mean %V(150) was 41.8% vs. 59.4% (p<0.05), the mean %V(200) was 11.3% vs. 32.0% (p<0.05), and the mean %V(300) was 0.4% vs. 6.7% (p<0.05) for the IBB and KVB methods, respectively. The mean ipsilateral breast %V(50) was 19.8% vs. 13.0% (p<0.05), the mean ipsilateral lung %V(30) was 3.7% vs. 1.1% (p<0.05), and the mean heart %V(5) was 59.2% vs. 9.4% (p<0.05) for the IBB and KVB methods, respectively., Conclusions: The IBB and KVB methods of PBI offer comparable target volume coverage; however, the KVB method is associated with an increased volume of breast tissue in the high-dose regions and a decreased dose to the adjacent normal tissues.

Purpose: Limited information is available comparing target volume and normal tissue dosimetry with the different techniques of partial breast irradiation (PBI). We present results of a dosimetric comparison of single catheter, balloon-based brachytherapy using the MammoSite catheter (BRT), 3D conformal radiation therapy (3DCRT), and intensity-modulated radiation therapy (IMRT)., Methods and Materials: Fifteen patients were treated using the BRT device. With the use of CT scans with balloons in inflated and deflated states, plans were developed for each patient using each of the methods of PBI, for a total of 45 plans. The plans were then compared using the below dosimetric parameters., Results: The mean V100 was 95%, 92%, and 94% for the BRT, 3DCRT, and IMRT techniques, respectively. The mean ipsilateral breast V50 was 29%, 56%, and 46% (p < 0.0001) and the mean ipsilateral lung V30 was 5%, 7%, and 2% (p < 0.001 for IMRT vs. others) for the BRT, 3DCRT, and IMRT methods, respectively. For the 10 patients with left-sided breast tumors, the mean heart V5 was 12%, 4%, and 1% for the BRT, 3DCRT, and IMRT methods, respectively (p < 0.01)., Conclusions: With increasing interest in PBI, our data may help clinicians individualize patient treatment decisions.

The Varian 120 multileaf collimator (MLC) has a leaf thickness of 5 mm projected at the isocenter plane and can deliver a radiation beam of large field size (up to 30 cm) to be used in intensity-modulated radiotherapy (IMRT). Often the dose must be delivered to depths greater than 20 cm. Therefore, during the commissioning of the BrainSCAN v5.21 or any radiation treatment-planning (RTP) systems, extensive testing of dose and monitor unit calculations must encompass the field sizes (1 cm to 30 cm) and the prescription depths (1 cm to 20 cm). Accordingly, the central-axis percent depth doses (PDDs) and off-axis percentage profiles must be measured at several depths for various field sizes. The data for this study were acquired with a 6-MV X-ray beam from a Varian 2100EX LINAC with a water phantom at a source-to-surface distance (SSD) of 100 cm. These measurements were also used to generate a photon beam module, based on a photon pencil beam dose-calculation algorithm with a fast-Fourier transform method. To commission the photon beam module used in our BrainSCAN RTP system, we performed a quantitative comparison of measured and calculated central-axis depth doses and off-axis profiles. Utilizing the principles of dose difference and distance-to-agreement introduced by Van Dyk et al. [Commissioning and quality assurance of treatment planning computers. Int J Radiat Oncol Biol Phys. 1993; 26:261-273], agreements between calculated and measured doses are <2% and <2 mm for the regions of low- and high-dose gradients, respectively. However, large errors (up to approximately 5% and approximately 7% for 20-cm and 30-cm fields, respectively, at the depth 20 cm) were observed for monitor unit calculations. For a given field size, the disagreement increased with the depth. Similarly, for a given depth the disagreement also increase with the field size. These large systematic errors were caused by using the tissue maximum ratio (TMR) in BrainSCAN v5.21 without considering increased field size as depth increased. These errors have been reported to BrainLAB.

The MammoSite breast brachytherapy device was designed to overcome the potential scheduling problems associated with external beam radiotherapy (EBRT) and the technical difficulties of multi-catheter-based interstitial brachytherapy. The device consists of a silicone balloon connected to a catheter which contains an inflation channel and a port for passage of a high-dose-rate brachytherapy source. The American Brachytherapy Society and American Society of Breast Surgeons have published partial breast irradiation (PBI) patient selection guidelines. The MammoSite applicator has been shown in two dosimetric studies to treat a comparable volume to multicatheter-based interstitial implants. The MammoSite catheter can be placed at the time of lumpectomy or in a separate procedure using ultrasound guidance. Four optimization methods have been described: the single point method, the six prescription point method (RUSH Technique), the University of Southern California Norris Cancer Center Method, and the Surface Optimization Technique. An excellent or good cosmetic outcome has been reported in 80% to 93% of patients at 1 year in most studies. Cosmetic results appear highly related to skin spacing. The MammoSite applicator has been associated with early side effects comparable with traditional breast conserving therapy. A NSABP trial will randomize patients to either whole breast irradiation or PBI consisting of interstitial brachytherapy, MammoSite brachytherapy, or 3D conformal radiation.

The nucletron Leipzig applicator is designed for (HDR) 192Ir brachy radiotherapy of surface lesions. The dosimetric characteristics of this applicator were investigated using simulation method based on Monte Carlo N-particle (MCNP) code and phantom measurements. The simulation method was validated by comparing calculated dose rate distributions of nucletron microSelectron HDR 192Ir source against published data. Radiochromic films and metal-oxide-semiconductor field-effect transistor (MOSFET) detectors were used for phantom measurements. The double exposure technique, correcting the nonuniform film sensitivity, was applied in the film dosimetry. The linear fit of multiple readings with different irradiation times performed for each MOSFET detector measurement was used to obtain the dose rate of each measurement and to correct the source transit-time error. The film and MOSFET measurements have uncertainties of 3%-7% and 3%-5%, respectively. The dose rate distributions of the Leipzig applicator with 30 mm opening calculated by the validated MC method were verified by measurements of film and MOSFET detectors. Calculated two-dimensional planar dose rate distributions show similar patterns as the film measurement. MC calculated dose rate at a reference point defined at depth 5 mm on the applicator's central axis is 7% lower than the film and 3% higher than the MOSFET measurements. The dose rate of a Leipzig applicator with 30 mm opening at reference point is 0.241+/-3% cGy h(-1) U(-1). The MC calculated depth dose rates and profiles were tabulated for clinic use.

Phantom measurements and Monte Carlo calculations have been performed for the purpose of characterizing the dose perturbation caused by radiographic contrast inside the MammoSite breast brachytherapy applicator. Specifically, the dose perturbation is quantified as a heterogeneity correction factor (HCF) for various balloon radii and contrast concentration levels. The dose perturbation is larger for larger balloon radii and higher contrast concentrations. Based on a validated Monte Carlo simulation, the calculated HCF values are 0.99 for a 2 cm radius balloon and 0.98 for a 3 cm radius balloon at 6% contrast concentration levels, and 0.89 and 0.87 for 2 and 3 cm radius balloons, respectively, at 100% contrast concentrations. For a typical implanted balloon radius of 2.4 cm, the HCF values decrease from 0.99 at 6% contrast concentration to 0.90 at 100% contrast concentration. For balloons implanted in patients at our institution, the mean HCF is 0.99, corresponding to a dose reduction of approximately 1%. The contrast effect results in a systematic reduction in the delivered dose, therefore the minimal amount of radiographic contrast necessary should be used.

The purpose of this study was to characterize covalent multimers with molecular mass of >90 kDa in the water-insoluble (WI) proteins of aging human lenses. The experimental approach was to first separate the multimers (molecular mass >90 kDa) as individual spots by two-dimensional gel electrophoresis and next analyze compositions of each multimers by matrix-assisted laser desorption ionization-time of flight and electrospray ionization-tandem mass spectrometric (ES-MS/MS) methods. The WI proteins from lenses of 25- and 41-year-old subjects showed distinct 5- and 16-multimer spots on two-dimensional gels, respectively, but the spots from 52- and 72-year-old lenses were non-descript and diffused. ES-MS/MS analyses showed two types of covalent multimers in 25- and 41-year-old lenses, i.e. the first type composed of fragments of eight different crystallins (i.e. alphaA, alphaB, betaA3, betaA4, betaB1, betaB2, gammaS, and gammaD), and the second type of alpha-, beta-, and gamma-crystallins (possibly fragments) and two beaded filament proteins (phakinin and filensin). The most commonly identified species in the complexes of 41-year-old lenses were: alphaA-fragment (C-terminally truncated, residues 1-157), alphaB-fragment (residues 83-90), betaB1-crystallin (residues 60-71), betaA3 (residues 33-44), betaA4 (residues 106-117), filensin (residues 78-90), and phakinin (residues 77-89). Three post-translational modifications (i.e. oxidation of Met and Trp, conversion of Ser to dehydroalanine, and formylation of His) were observed in alphaA-crystallin fragment, and the first two modifications could cross-link proteins. Together, the results suggested that covalent multimers appeared early in life (i.e. 25 years of age) and increased in number with aging, and the two beaded filament proteins form covalent complexes with crystallin fragments in vivo.

We examined the effects of reactive oxygen-nitrogen intermediates on chloride (Cl-) currents across murine tracheal epithelial (MTE) cells isolated from CD-1 mice. MTE cells were cultured on permeable supports until they formed water-tight monolayers with transepithelial resistances (Rt)>500 Omega/cm2 and then were mounted in Ussing chambers. Baseline short-circuit current (ISC) values, prior to and following the addition of 10 microM amiloride (an inhibitor of sodium-transport pathways) into the apical side, were 65 +/- 1.9 microA/cm2 and 7.6 +/- 0.51 microA/cm2, respectively (X +/- 1 SE, n=32). The addition of 3-morpholinosydnominine (SIN-1, 1 mM), which generates both superoxide and nitric oxide anions, to amiloride-treated monolayers resulted in a transient increase of ISC to a peak value of 35 +/- 1.3 microA/cm2 (X +/- SE, n=14) within the next 30-60 min. After this, the ISC decreased gradually and returned to its pre-SIN-1 value. These changes were blocked by glibenclamide (200 microM), an inhibitor of cystic fibrosis transmembrane regulator, or reduced by glutathione (GSH, 5 mM), a scavenger of peroxynitrite. Forskolin (10 microM) augmented the SIN-1 effect when added at the peak of the SIN-1 response but not when ISC had returned to its baseline value. Perfusion of MTE cells with SIN-1 also increased whole cell Cl- currents 4-fold and the open probability of CFTR-type single-channel currents from 0.041 to 0.92 in a transient fashion. Decomposed SIN-1, but not pure SIN-1c (the stable decomposition product of SIN-1), also increased ISC with an EC50 of 5 microM. Electrospray mass spectroscopy revealed several unique and uncharacterized compounds formed during the decomposition of SIN-1 as well as the reaction of SIN-1c with peroxynitrite. Formation of these compounds was inhibited by GSH. We conclude that compounds formed by the reaction of peroxynitrite with by-products of SIN-1, rather than reactive oxygen-nitrogen species per se, were responsible for the modulation of Cl- secretion across primary cultures of MTE cells.

Human serum albumin (HSA), the most abundant protein in plasma, has been proposed to have an antioxidant role. The main feature responsible for this property is its only thiol, Cys34, which comprises approximately 80% of the total free thiols in plasma and reacts preferentially with reactive oxygen and nitrogen species. Herein, we show that the thiol in HSA reacted with hydrogen peroxide with a second-order rate constant of 2.26 M(-1) s(-1) at pH 7.4 and 37 degrees C and a 1:1 stoichiometry. The formation of intermolecular disulfide dimers was not observed, suggesting that the thiol was being oxidized beyond the disulfide. With the reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl), we were able to detect the formation of sulfenic acid (HSA-SOH) from the UV-vis spectra of its adduct. The formation of sulfenic acid in Cys34 was confirmed by mass spectrometry using 5,5-dimethyl-1,3-cyclohexanedione (dimedone). Sulfenic acid was also formed from exposure of HSA to peroxynitrite, the product of the reaction between nitric oxide and superoxide radicals, in the absence or in the presence of carbon dioxide. The latter suggests that sulfenic acid can also be formed through free radical pathways since following reaction with carbon dioxide, peroxynitrite yields carbonate radical anion and nitrogen dioxide. Sulfenic acid in HSA was remarkably stable, with approximately 15% decaying after 2 h at 37 degrees C under aerobic conditions. The formation of glutathione disulfide and mixed HSA-glutathione disulfide was determined upon reaction of hydrogen peroxide-treated HSA with glutathione. Thus, HSA-SOH is proposed to serve as an intermediate in the formation of low molecular weight disulfides, which are the predominant plasma form of low molecular weight thiols, and in the formation of mixed HSA disulfides, which are present in approximately 25% of circulating HSA.

The intermittent vascular occlusion occurring in sickle cell disease (SCD) leads to ischemia-reperfusion injury and activation of inflammatory processes including enhanced production of reactive oxygen species and increased expression of inducible nitric-oxide synthase (NOS2). Appreciating that impaired nitric oxide-dependent vascular function and the concomitant formation of oxidizing and nitrating species occur in concert with increased rates of tissue reactive oxygen species production, liver and kidney NOS2 expression, tissue 3-nitrotyrosine (NO(2)Tyr) formation and apoptosis were evaluated in human SCD tissues and a murine model of SCD. Liver and kidney NOS2 expression and NO(2)Tyr immunoreactivity were significantly increased in SCD mice and humans, but not in nondiseased tissues. TdT-mediated nick end-label (TUNEL) staining showed apoptotic cells in regions expressing elevated levels of NOS2 and NO(2)Tyr in all SCD tissues. Gas chromatography mass spectrometry analysis revealed increased plasma protein NO(2)Tyr content and increased levels of hepatic and renal protein NO(2)Tyr derivatives in SCD (21.4 +/- 2.6 and 37.5 +/- 7.8 ng/mg) versus wild type mice (8.2 +/- 2.2 and 10 +/- 1.2 ng/mg), respectively. Western blot analysis and immunoprecipitation of SCD mouse liver and kidney proteins revealed one principal NO(2)Tyr-containing protein of 42 kDa, compared with controls. Enzymatic in-gel digestion and MALDI-TOF mass spectrometry identified this nitrated protein as actin. Electrospray ionization and fragment analysis by tandem mass spectrometry revealed that 3 of 15 actin tyrosine residues are nitrated (Tyr(91), Tyr(198), and Tyr(240)) at positions that significantly modify actin assembly. Confocal microscopy of SCD human and mouse tissues revealed that nitration led to morphologically distinct disorganization of filamentous actin. In aggregate, we have observed that the hemoglobin point mutation of sickle cell disease that mediates hemoglobin polymerization defects is translated, via inflammatory oxidant reactions, into defective cytoskeletal polymerization.

Hexane extracts of epicuticular wax from cv. Gala apples were noted to have an unusual, broad absorbance maximum at approximately 258 nm, which led us to isolate and identify the primary UV-absorbing compounds. Column and thin-layer chromatography yielded a fraction that gave a series of paired, 260-nm-absorbing peaks on C(18) HPLC. These were shown to be a family of phenolic fatty acid esters, for which retention times increased with increasing fatty acid chain length, and paired peaks were esters of two related phenolics with the same fatty acid moiety. Alkaline hydrolysis of the esters released two water-soluble phenolics separable by C(18) HPLC. Electrospray ionization mass spectrometry gave a molecular mass of 150 for both, and (1)H NMR plus UV absorbance spectra identified them as E and Z isomers of p-coumaryl alcohol. Alkaline cleavage of the fatty acid esters in the presence of methanol or ethanol resulted in partial derivatization of E-p-coumaryl alcohol to the corresponding gamma-O-methyl or O-ethyl ether. Gradient HMQC NMR of the HPLC-purified stearate ester of E-p-coumaryl alcohol indicated that fatty acid esterification occurs at the gamma-OH rather than at the 4-OH on the phenyl ring. This is the first report of fatty acid esters of monolignols as a natural plant product.

The dynamics of proteins within large cellular assemblies are important in the molecular transformations that are required for macromolecular synthesis, transport, and metabolism. The capsid expansion (maturation) accompanying DNA packaging in the dsDNA bacteriophage P22 represents an experimentally accessible case of such a transformation. A novel method, based on hydrogen-deuterium exchange was devised to investigate the dynamics of capsid expansion. Mass spectrometric detection of deuterium incorporation allows for a sensitive and quantitative determination of hydrogen-deuterium exchange dynamics irrespective of the size of the assembly. Partial digestion of the exchanged protein with pepsin allows for region-specific assignment of the exchange. Procapsids and mature capsids were probed under native and slightly denaturing conditions. These experiments revealed regions that exhibit different degrees of flexibility in the procapsid and in the mature capsid. In addition, exchange and deuterium trapping during the process of expansion itself was observed and allowed for the identification of segments of the protein subunit that become buried or stabilized as a result of expansion. This approach may help to identify residues participating in macromolecular transformations and uncover novel patterns and hierarchies of interactions that determine functional movements within molecular machines.

Multiple sclerosis (MS) is clinically heterogeneous and has an uncertain natural history. A high priority for more effective treatment of MS is an objective and feasible laboratory test for predicting the disease's course and response to treatments. Urinary myelin basic protein (MBP)-like material (MBPLM), so designated because it is immunoreactive as a cryptic epitope in peptide 83-89 of the human MBP molecule of 170 amino acids, is present in normal adults, remains normal in relapsing-remitting, but increases in progressive MS. In the present investigation, MBPLM was purified from urine and characterized. p-Cresol sulfate is the major component of urinary MBPLM. This conclusion is based on the following: (1) MBPLM and p-cresol sulfate both have a mass of 187 on negative scans by electrospray ionization mass spectrometry, the same fragments on tandem mass spectrometry of 80 (SO(-)(3)) and 107 (methylphenol), and similar profiles on multiple reaction monitoring; (2) (1)H and (13)C nuclear magnetic resonance spectroscopy revealed identical spectra for MBPLM and p-cresol sulfate; (3) purified p-cresol sulfate reacted in parallel with MBP peptide 83-89 in the same radioimmunoassay for MBPLM; and (4) p-cresol sulfate has the same behavior on preparative HPLC columns as urinary MBPLM. The unexpected immunochemical degeneracy permitting a cross-reaction between p-cresol sulfate and a peptide of an encephalitogenic myelin protein is postulated to be based on shared conformational features. The mechanisms by which urinary p-cresol sulfate, possibly derived from tyrosine-SO(4), reflects progressive worsening that is disabling in MS are unknown., (Copyright 2000 Academic Press.)

The haloethylnitrosoureas form a cytotoxic DNA cross-link in a series of reactions which involves initial alkylation of the O6 position of guanine and rearrangement to the intermediate, 1,O6-ethanoguanine; 1,O6-ethanoguanine then reacts with a neighboring cytosine base. O6-Alkylguanine-DNA alkyltransferase can interrupt this process after the initial alkylation step by removing the alkyl group from the O6 position of guanine. Recent evidence suggests that the O6-alkylguanine-DNA alkyltransferase also recognizes 1,O6-ethanoguanine as a substrate, becoming bound to DNA when it interacts with that intermediate. It has also been shown that glutathione becomes bound to haloethylnitrosourea-treated DNA, apparently through chemical interaction with 1,O6-ethanoguanine. Since both of these reactions involve the thiol group of cysteine, we have examined the reaction of cysteine with 1,O6-ethanoguanine, characterizing the prototype DNA-protein cross-link, 1-(3-cytosinyl),2-(1-guanyl)ethane, which is formed in this reaction. These results establish a competitive reaction with 1,O6-ethanoguanine as a likely route to protein-DNA cross-linking.

Cardiovascular risk factors identified in major studies of children include a family history of heart disease, smoking, blood pressure, dietary factors, exercise or fitness level, and stress. The purpose of this descriptive study was to identify differences in children's physical measurements with regard to six cardiovascular risk factors. A descriptive design using three questionnaires and physical measurements was used. The questionnaires included the Bloomsday Cardiovascular Fitness Questionnaire, the Coronary Risk Profile, and the Diet Habit Survey. Physical measurements included blood pressure readings, weight, total serum cholesterol, and high-density lipoprotein levels. A convenience sample of 78, predominantly Caucasian children between the ages of 7 and 18 years (M = 13.13, SD = 2.79) participated. Results indicated that differences in physical measurements were significantly for children with respect to the cardiovascular risk factors, including dietary factors, blood pressure, exercise, and stress. Nursing implications for thorough assessment and intervention through education are discussed.

Purified bacterial 3-methyladenine DNA glycosylase II releases four 7-alkylguanines from [3H]N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA: 7-(2-hydroxyethyl)guanine,1,2-bis(7-guanyl)ethane, 7-(2-chloroethyl)guanine, and 7-(2-ethoxyethyl)guanine. 7-(2-Ethoxyethyl)guanine, a new compound, is formed as a result of an interaction with ethanol, a common solvent for the 2-haloethylnitrosoureas. Of the four 7-alkylguanines which are released from [3H]N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA, 7-(2-hydroxyethyl)guanine is released at a rate very much slower than the other three. As shown by a study of the spontaneous decomposition of the corresponding 7-alkyl-deoxyguanines, differences in chemical stability do not appear to explain the slow release of 7-(2-hydroxyethyl)guanine. In view of previous results showing a difference in the distribution of alkylation products between sensitive and resistant glial cell lines, the broad specificity of this enzyme suggests that glycosylase activity could play a role in cellular resistance to 2-haloethylnitrosoureas.

HPLC analyses of the bases released by acid from N-(2-chloroethyl)-N-nitrosourea-treated DNA and N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-treated DNA show the presence of a new guanine adduct, N2,3-ethanoguanine. This derivative can be synthesized at the monomer level by treating 2-hydroxyethylguanine with thionyl chloride. The product of this reaction, purified by HPLC, has been shown to have a mol. wt corresponding to ethanoguanine by mass spectrometry; NMR spectrometry also supports this structural assignment. The UV and fluorescence spectra are very similar to those of N2,3-ethenoguanine, providing evidence that the ethano bridge is attached between N2 and 3 positions. Proof that the derivative is N2,3-ethanoguanine comes from the fact that it can be converted to N2,3-ethenoguanine by dehydrogenation on a palladium catalyst. The discovery of this new derivative raises to four the number of tricylic derivatives that have been isolated from DNA treated with 2-haloethylnitrosoureas. The new adduct, N2,3-ethanoguanine, is closely related to an etheno adduct formed by chloroacetaldehyde, a metabolite of the human carcinogen vinyl chloride, and may have relevance to either the therapeutic or carcinogenic actions of the 2-haloethylnitrosoureas.

The 2-haloethylnitrosoureas have been shown to form the cross-linked structure 1-(3-cytosinyl),2-(1-guanyl)ethane in DNA. This cross-link has now been synthesized by the reaction of O6-(2-fluoroethyl)guanosine with deoxycytidine in dimethyl sulfoxide followed by removal of the sugars by acid hydrolysis. This synthetic route supports the mechanism for cross-link formation in DNA that involves an initial attack on the O6-position of guanine, followed by a rearrangement and subsequent reaction with cytosine. It also provides a practical route to the synthesis of 1-(3-cytosinyl),2-(1-guanyl)ethane for studies involving formation of this cross-link in DNA.

1. Studies on the metabolism of 14C-Michler's ketone (4,4'-bis-(dimethylamino)[carbonyl- 14C]benzophenone) in rats have revealed that this carcinogen is subject to demethylation, ring-hydroxylation and N-acetylation after adjacent methyl groups have been removed. 2 As identified by mass spectral analysis, microsomal metabolites are the mono-, di-, tri- and tetra-demethylated derivatives. 3. The major metabolites appearing in the bile are the di- and tri-demethylated derivatives and the N-acetylated tetra-demethylated compound; minor metabolites, tentatively identified, are the ring-hydroxylated derivatives of di- and tri-demethylated Michler's ketone and of N-acetylated tri- and tetra-demethylated Michler's ketone. 4. The major urinary metabolite is tentatively identified as a ring-hydroxylated derivative of N-acetylated, di-demethylated Michler's ketone; a minor urinary metabolite lacks the hydroxyl group. 5. Injection of 14C-Michler's ketone into rats resulted in the irreversible binding of radioactivity to liver proteins. 6. When the rats were pretreated with phenobarbital, this binding was increased and extended to proteins of some other tissues and to DNA and RNA of liver and DNA of kidney.

The chloroethylnitrosoureas are useful antitumor agents which evidently exert a significant part of their cytotoxic action through the formation of a unique 1-(3-deoxycytidyl), 2-(1-deoxyguanosinyl)ethane cross-link in DNA. It has been suggested that this cross-link is formed from O6-(2-chloro-ethyl)guanine, an initial product of DNA alkylation by the chloroethylnitrosoureas; however, O6-(2-chloroethyl)guanine has never been described. We have synthesized this derivative from the reaction of thionyl chloride with O6-(2-hydroxyethyl)guanine, and have found that it decomposes to 1-(2-hydroxyethyl)guanine through an intermediate, presumably 1,O6-ethanoguanine. Its half life at 37 degrees and pH 7.4 is 17.8 min.

Several 13-cis-retinamides were synthesized from 13-cis-retinoic acid via either 13-cis-retinoyl chloride or 13-cis-1-retinoylimidazole. All-trans-retinoylglycine was prepared from all-trans-retinoyl chloride and ethyl glycinate. Detailed procedures were developed for the preparation of other all-trans-retinamides on a large scale for studies of the chemoprevention of cancer.

Field desorption mass spectra of methotrexate and folic acid analogs yield abundant molecular or quasimolecular ions. At emitter currents above the best anode temperature, fragment ions which are useful as further confirmations of structures are observed.

4-Methylcyclophosphamide was synthesized and separated into cis and trans isomers by column chromatography. Isolation of these isomers permitted individual evaluation against murine leukemia L1210 in vivo and assessment of possible differences in antileukemic activity. Results indicate no appreciable difference in activity of the isomers, suggesting essentially equal facility for activation by mouse liver microsomes in vivo.

Thin-layer chromatography and mass spectrometry were used to isolate and identify cyclophosphamide metabolites present in blood of mice. Blood was removed 5, 15, 30 and 45 minutes after intraperitoneal administration and extracted with chloroform followed by methanol. Thin-layer chromatography of the two extracts and the residual solid with or without prior methylation, collection of resulting alkylating components, determination of radioactivity and mass spectral analysis, served to identify cyclophosphamide, 4-ketocyclophosphamide, alcophosphamide, N-dechloroethylcyclophosphamide, carboxyphosphamide, phosphoramide mustard and nor-HN2. The absence of detectable levels of 4-hydroxycyclophosphamide or aldophosphamide in the blood of cyclophosphamide-treated mice suggests that cyclophosphamide is converted rapidly in the liver to carboxyphosphamide, 4-ketocyclophosphamide, phosphoramide mustard and nor-HN2. Direct administration of synthetic 4-hydroxycyclophosphamide to mice and extraction of blood with chloroform demonstrated the recovery of this metabolite in vivo. Analysis of extracts of blood from mice treated with phosphoramide mustard indicated the presence of nor-HN2, 3-(2-chloroethyl)-1,3-oxazolidin-2-one and unchanged drug. Consideration of blood levels, cytotoxicity and alkylating activity of metabolites identified, in or inferred from, this study, implicates phosphoramide mustard as a leading condidate for the biologically active form of cyclophosphamide.

The alkylation of phosphates in DNA by therapeutically active haloethylnitrosoureas was studied by reacting N-chloroethyl-N-nitrosourea (CNU) with dTpdT, separating the products by HPLC, and identifying them by co-chromatography with authentic markers. Both hydroxyethyl and chloroethyl phosphotriesters of dTpdT were identified; a similar reaction between CNU and dTR yielded 3-hydroxyethyl and 3-chloroethyl dTR as the major products of ring alkylation. A DNA-like substrate for repair studies was synthesized by reacting 14C-labelled N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (14C-CCNU) with poly dT and annealing the product to poly dA. An extract of E. coli strain BS21 selectively transferred a chloroethyl group from one of the chloroethyl phosphotriester isomers in this substrate to the bacterial protein; chemical instability of the hydroxyethyl phosphotriesters precluded definite conclusions about the repair of this product.