A study of skin marker alignment using different diamond-shaped light fields for prone breast external-beam radiation therapy.

For breast cancer patients treated in the prone position with tangential fields, a diamond-shaped light field (DSLF) can be used to align with corresponding skin markers for image-guided radiation therapy (IGRT). This study evaluates and compares the benefits of different DSLF setups. Seventy-one patients who underwent daily tangential kilovoltage (kV) IGRT were categorized retrospectively into four groups: (1) DSLF field size (FS) = 10 × 10 cm2 , gantry angle = 90° (right breast)/270° (left breast), with the same isocenter as treatment tangential beams; (2) same as group 1, except DSLF FS = 4 × 4 cm2 ; (3) DSLF FS = 4 × 4-6 × 8 cm2 , gantry angle = tangential treatment beam, off-isocenter so that the DSLF was at the approximate breast center; and (4) No-DSLF. We compared their total setup time (including any DSLF/marker-based alignment and IGRT) and relative kV-based couch shift corrections. For groups 1-3, DSLF-only dose distributions (excluding kV-based correction) were simulated by reversely shifting the couch positions from the computed tomography plans, which were assumed equivalent to the delivered dose when both DSLF and IGRT were used. For patient groups 1-4, the average daily setup time was 2.6, 2.5, 5.0, and 8.3 min, respectively. Their mean and standard deviations of daily kV-based couch shifts were 0.64 ± 0.4, 0.68 ± 0.3, 0.8 ± 0.6, and 1.0 ± 0.6 cm. The average target dose changes after excluding kV-IGRT for groups 1-3 were-0.2%, -0.1%, and +0.4%, respectively, whereas DSLF-1 was most efficient in sparing heart and chest wall, DSLF-2 had lowest lung Dmax ; and DSLF-3 maintained the highest target coverage at the cost of highest OAR dose. In general, the use of DSLF greatly reduces patient setup time and may result in smaller IGRT corrections. If IGRT is limited, different DSLF setups yield different target coverage and OAR dose sparing. Our findings will help DSLF setup optimization in the prone breast treatment setting.

Hypocalcemia-Induced Reversible Psychosis.

Hypocalcemia can manifest as a variety of presentations, ranging from neuromuscular irritability to seizures, and psychiatric manifestations such as emotional instability, anxiety, and depression. Here, we present a unique case of hypocalcemia-induced acute psychosis. A 24-year-old woman presented to the emergency department (ED) with confusion and agitation for four to five days. The patient was noted by the family to have decreased oral intake and sleep. Auditory and visual hallucinations prompted the family to bring the patient to the ED. The patient was mildly tachycardic. Initially, the patient was agitated, impulsive, and aggressive, exhibiting psychotic features including visual hallucinations, paranoid delusions, thought broadcasting, tangential and perseverative thought processes, and erotomanic delusions. She had mild leukocytosis and elevated procalcitonin on admission. A thorough workup ruled out infectious/inflammatory processes. Cerebrospinal fluid was negative for acute meningitis/encephalitis, autoimmune encephalitis antibodies, and paraneoplastic etiology. Thyroid-stimulating hormone was normal and thyroid antibodies were negative. The CT brain and MRI brain were unremarkable. The patient was severely hypocalcemic (6.7) with low parathyroid hormone (<6) on admission. To note, the patient has multiple endocrine neoplasia, type 2B (MEN2B). She underwent total thyroidectomy five months prior for metastatic medullary thyroid carcinoma complicated by postsurgical hypoparathyroidism. The patient had been non-compliant with calcium and calcitriol supplementation postoperatively. The patient was started on IV calcium gluconate and transitioned to calcitriol with calcium level improvement over the next three days. She experienced marked improvement, with the resolution of her psychosis. The patient's subacute onset psychosis with no personal or family psychiatric history and a rapid response to calcium correction supports hypocalcemia etiology. This case illustrates new-onset acute psychosis in a patient with calcium regulation imbalance. The development of hypocalcemia secondary to thyroidectomy with postsurgical hypoparathyroidism and calcium supplement non-compliance precipitated psychosis. A few similar cases have been reported, and here, we note that treatment of hypocalcemia promptly resolves symptoms. As per our review, this will be the first case of neuropsychiatric symptoms without associated cortical calcifications seen on imaging. It is important to recognize hypocalcemia as a rare cause of psychosis so as to not mistakenly categorize such presentations as primary psychotic disorders and miss a medically treatable illness.

Spatially Fractionated Radiotherapy (GRID) Prior to Standard Neoadjuvant Conventionally Fractionated Radiotherapy for Bulky, High-Risk Soft Tissue and Osteosarcomas: Feasibility, Safety, and Promising Pathologic Response Rates.

Spatially fractionated radiotherapy (GRID) has been utilized primarily in the palliative and definitive treatment of bulky tumors. Delivered in the modern era primarily with megavoltage photon therapy, this technique offers the promise of safe dose escalation with potential immunogenic, bystander and microvasculature effects that can augment a conventionally fractionated course of radiotherapy. At the University of Maryland, an institutional standard has arisen to incorporate a single fraction of GRID radiation in large (>8 cm), high-risk soft tissue and osteosarcomas prior to a standard fractionated course. Herein, we report on the excellent pathologic responses and apparent safety of this regimen in 26 consecutive patients.

Delivery of TRAIL-expressing plasmid DNA to cancer cells in vitro and in vivo using aminoglycoside-derived polymers.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand that can preferentially induce apoptosis in cancer cells over normal cells. The transmembrane form of TRAIL has been shown to elicit much stronger activity than its soluble counterpart but delivery is a potential challenge. Here, we investigated the potential of aminoglycoside-derived polymers to enhance delivery of a plasmid (pEF-TRAIL) that expresses the transmembrane form of TRAIL in order to determine the effect on cell death in vitro and tumor growth in vivo. Transgene delivery efficacy and toxicity of aminoglycoside-derived polymers was first evaluated using a GFP-expressing plasmid (pEF-GFP) at different plasmid amounts and plasmid : polymer ratios in UMUC3 bladder cancer and HeLa cervical cancer cells. Delivery of the TRAIL plasmid using aminoglycoside-derived polymers resulted in up to 60% cell death in UMUC3 and HeLa cells; TRAIL protein expression was confirmed using Western blots. TRAIL plasmid delivery resulted in a decrease in cellular procaspase-8 and an increase in TRAIL receptor DR5 levels, suggesting a role for the death receptor and caspase cascade in TRAIL-mediated apoptosis. The TRAIL plasmid did not cause cell death in normal human or mouse fibroblasts. The in vivo delivery of the TRAIL plasmid using a paromomycin-derived polymer resulted in significant reduction in tumor burden and increased survival in tumor-bearing live mice.

Projected Improvements in Accelerated Partial Breast Irradiation Using a Novel Breast Stereotactic Radiotherapy Device: A Dosimetric Analysis.

Accelerated partial breast irradiation has caused higher than expected rates of poor cosmesis. At our institution, a novel breast stereotactic radiotherapy device has demonstrated dosimetric distributions similar to those in brachytherapy. This study analyzed comparative dose distributions achieved with the device and intensity-modulated radiation therapy accelerated partial breast irradiation. Nine patients underwent computed tomography simulation in the prone position using device-specific immobilization on an institutional review board-approved protocol. Accelerated partial breast irradiation target volumes (planning target volume_10mm) were created per the National Surgical Adjuvant Breast and Bowel Project B-39 protocol. Additional breast stereotactic radiotherapy volumes using smaller margins (planning target volume_3mm) were created based on improved immobilization. Intensity-modulated radiation therapy and breast stereotactic radiotherapy accelerated partial breast irradiation plans were separately generated for appropriate volumes. Plans were evaluated based on established dosimetric surrogates of poor cosmetic outcomes. Wilcoxon rank sum tests were utilized to contrast volumes of critical structures receiving a percentage of total dose (Vx). The breast stereotactic radiotherapy device consistently reduced dose to all normal structures with equivalent target coverage. The ipsilateral breast V20-100 was significantly reduced (P < .05) using planning target volume_10mm, with substantial further reductions when targeting planning target volume_3mm. Doses to the chest wall, ipsilateral lung, and breast skin were also significantly lessened. The breast stereotactic radiotherapy device's uniform dosimetric improvements over intensity-modulated accelerated partial breast irradiation in this series indicate a potential to improve outcomes. Clinical trials investigating this benefit have begun accrual.

Dosimetric Improvements with a Novel Breast Stereotactic Radiotherapy Device for Delivery of Preoperative Partial-Breast Irradiation.

OBJECTIVE: Partial-breast irradiation (PBI) with external-beam radiotherapy has produced higher than expected rates of fair-to-poor cosmesis. Worsened outcomes have been correlated with larger volumes of breast tissue exposed to radiation. A novel breast-specific stereotactic radiotherapy (BSRT) device (BSRTD) has been developed at our institution and has shown promise in delivering highly conformal dose distributions. We compared normal tissue sparing with this device with that achieved with intensity-modulated radiation therapy (IMRT)-PBI. METHODS: Fifteen women previously treated with breast conservation therapy were enrolled on an institutional review board-approved protocol. Each of them underwent CT simulation in the prone position using the BSRTD-specific immobilization system. Simulated postoperative and preoperative treatment volumes were generated based on surgical bed/clip position. Blinded planners generated IMRT-PBI plans and BSRT plans for each set of volumes. These plans were compared based on clinically validated markers for cosmetic outcome and toxicity using a Wilcoxon rank-sum test. RESULTS: The BSRT plans consistently reduced the volumes receiving each of several dose levels (Vx) to breast tissue, the chest wall, the lung, the heart, and the skin in both preoperative and postoperative settings (p < 0.05). Preoperative BSRT yielded particularly dramatic improvements. CONCLUSION: The novel BSRTD has demonstrated significant dosimetric benefits over IMRT-PBI. Further investigation is currently proceeding through initial clinical trials.

Adjuvant pelvic irradiation for cervical cancer in the setting of a transplanted pelvic kidney.

Postoperative radiation therapy is often needed following resection for gynecological cancers. A pelvic kidney, whether ectopic or transplanted, is considered an absolute contraindication for radiation if the organ is left in place. A 45-year-old, immunosuppressed patient with FIGO IB1 cervical adenocarcinoma was treated with intensity-modulated radiation therapy (IMRT) to 45 Gy to the modified whole pelvis with a boost to 59.4 Gy to high-risk areas despite having a transplanted kidney in the right iliac fossa. The irradiation prevented further local failure in the pelvis at 36-month follow-up with no decrement in renal function. Radiation to the modified pelvis using IMRT while avoiding the renal allograft is technically feasible and should be offered to more high-risk patients.

The crystal structure of the extracellular 11-heme cytochrome UndA reveals a conserved 10-heme motif and defined binding site for soluble iron chelates.

Members of the genus Shewanella translocate deca- or undeca-heme cytochromes to the external cell surface thus enabling respiration using extracellular minerals and polynuclear Fe(III) chelates. The high resolution structure of the first undeca-heme outer membrane cytochrome, UndA, reveals a crossed heme chain with four potential electron ingress/egress sites arranged within four domains. Sequence and structural alignment of UndA and the deca-heme MtrF reveals the extra heme of UndA is inserted between MtrF hemes 6 and 7. The remaining UndA hemes can be superposed over the heme chain of the decaheme MtrF, suggesting that a ten heme core is conserved between outer membrane cytochromes. The UndA structure has also been crystallographically resolved in complex with substrates, an Fe(III)-nitrilotriacetate dimer or an Fe(III)-citrate trimer. The structural resolution of these UndA-Fe(III)-chelate complexes provides a rationale for previous kinetic measurements on UndA and other outer membrane cytochromes.

Overview of peroxiredoxins in oxidant defense and redox regulation.

Peroxiredoxins are important hydroperoxide detoxification enzymes, yet have only come to the fore in recent years relative to the other major players in peroxide detoxification, heme-containing catalases and peroxidases and glutathione peroxidases. These cysteine-dependent peroxidases exhibit high reactivity with hydrogen peroxide, organic hydroperoxides, and peroxynitrite and play major roles not only in peroxide defense, but also in regulating peroxide-mediated cell signaling. This overview focuses on important peroxiredoxin features that have emerged over the past several decades with an emphasis on catalytic mechanism, regulation, and biological function.

Dual pro-drugs of 2'-C-methyl guanosine monophosphate as potent and selective inhibitors of hepatitis C virus.

We have previously reported the power of combining a 5'-phosphoramidate ProTide, phosphate pro-drug, motif with a 6-methoxy purine pro-drug entity to generate highly potent anti-HCV agents, leading to agents in clinical trial. We herein extend this work with the disclosure that a variety of alternative 6-substituents are tolerated. Several compounds exceed the potency of the prior 6-methoxy leads, and in almost every case the ProTide is several orders of magnitude more potent than the parent nucleoside. We also demonstrate that these agents act as pro-drugs of 2'-C-methyl guanosine monophosphate. We have also reported the novel use of hepatocyte cell lysate as an ex vivo model for ProTide metabolism.

Conformational dynamics promote binding diversity of dynein light chain LC8.

A highly conserved and ubiquitous protein known as LC8 binds over twenty different partners, characteristic of a molecular hub (Barbar, 2008 Biochemistry, 47, 503-508). Structural studies of LC8 complexes with binding partners having diverse recognition sequences show that the same binding groove of LC8 accommodates the various binding motifs. Here we use thermodynamics and dynamics measurements of LC8 complexes to group LC8 binding partners in two categories: those whose binding is enthalpically driven and those that are entropically favored. Peptides that are enthalpically driven completely silence the millisecond-microsecond relaxation signal, suggesting a significant rigidifying of the binding groove, while peptides in the entropically favored group exhibit the same conformational dynamics as the free protein, suggesting that the peptide sits loosely in the binding groove and so retains flexibility of the groove, and presumably of the bound peptide. The inherent disorder in the LC8 binding groove and in LC8 binding partners allows both types of binding, accounts for the lack of a conserved recognition consensus motif and underlies the binding specificity and broad selectivity observed in LC8 binding.

Structure of a bacterial cell surface decaheme electron conduit.

Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves decaheme cytochromes that are located on the bacterial cell surface at the termini of trans-outer-membrane electron transfer conduits. The cell surface cytochromes can potentially play multiple roles in mediating electron transfer directly to insoluble electron sinks, catalyzing electron exchange with flavin electron shuttles or participating in extracellular intercytochrome electron exchange along "nanowire" appendages. We present a 3.2-Å crystal structure of one of these decaheme cytochromes, MtrF, that allows the spatial organization of the 10 hemes to be visualized for the first time. The hemes are organized across four domains in a unique crossed conformation, in which a staggered 65-Å octaheme chain transects the length of the protein and is bisected by a planar 45-Å tetraheme chain that connects two extended Greek key split ß-barrel domains. The structure provides molecular insight into how reduction of insoluble substrate (e.g., minerals), soluble substrates (e.g., flavins), and cytochrome redox partners might be possible in tandem at different termini of a trifurcated electron transport chain on the cell surface.

INX-08189, a phosphoramidate prodrug of 6-O-methyl-2'-C-methyl guanosine, is a potent inhibitor of hepatitis C virus replication with excellent pharmacokinetic and pharmacodynamic properties.

INX-08189 is an aryl-phosphoramidate of 6-O-methyl-2'-C-methyl guanosine. INX-08189 was highly potent in replicon assays, with a 50% effective concentration of 10±6 nM against hepatitis C genotype 1b at 72 h. The inhibitory effect on viral replication was rapid, with a 50% effective concentration (EC50) of 35±8 nM at 24 h. An intracellular 2'-C-methyl guanosine triphosphate (2'-C-MeGTP) concentration of 2.43±0.42 pmol/10(6) cells was sufficient to achieve 90% inhibition of viral replication. In vitro resistance studies confirmed that the S282T mutation in the NS5b gene conferred an approximately 10-fold reduction in sensitivity to INX-08189. However, the complete inhibition of S282T mutant replicons still could be achieved with an EC90 of 344±170 nM. Drug combination studies of INX-08189 and ribavirin indicated significant synergy in antiviral potency both in wild-type and S282T-expressing replicons. Genotype 1b replicons could be cleared after 14 days of culture when exposed to as little as 20 nM INX-08189. No evidence of mitochondrial toxicity was observed after 14 days of INX-08189 exposure in both HepG2 and CEM human cell lines. In vivo studies of rats and cynomolgus monkeys demonstrated that 2'-C-MeGTP concentrations in liver equivalent to the EC90 could be attained after a single oral dose of INX-08189. Rat liver 2'-C-MeGTP concentrations were proportional to dose, sustained for greater than 24 h, and correlated with plasma concentrations of the nucleoside metabolite 2'-C-methyl guanosine. The characteristics displayed by INX-08189 support its continued development as a clinical candidate for the treatment of chronic HCV infection.

Structure-based insights into the catalytic power and conformational dexterity of peroxiredoxins.

Peroxiredoxins (Prxs), some of nature's dominant peroxidases, use a conserved Cys residue to reduce peroxides. They are highly expressed in organisms from all kingdoms, and in eukaryotes they participate in hydrogen peroxide signaling. Seventy-two Prx structures have been determined that cover much of the diversity of the family. We review here the current knowledge and show that Prxs can be effectively classified by a structural/evolutionary organization into six subfamilies followed by specification of a 1-Cys or 2-Cys mechanism, and for 2-Cys Prxs, the structural location of the resolving Cys. We visualize the varied catalytic structural transitions and highlight how they differ depending on the location of the resolving Cys. We also review new insights into the question of how Prxs are such effective catalysts: the enzyme activates not only the conserved Cys thiolate but also the peroxide substrate. Moreover, the hydrogen-bonding network created by the four residues conserved in all Prx active sites stabilizes the transition state of the peroxidatic S(N)2 displacement reaction. Strict conservation of the peroxidatic active site along with the variation in structural transitions provides a fascinating picture of how the diverse Prxs function to break down peroxide substrates rapidly.

Structural evidence that peroxiredoxin catalytic power is based on transition-state stabilization.

Peroxiredoxins (Prxs) are important peroxidases associated with both antioxidant protection and redox signaling. They use a conserved Cys residue to reduce peroxide substrates. The Prxs have a remarkably high catalytic efficiency that makes them a dominant player in cell-wide peroxide reduction, but the origins of their high activity have been mysterious. We present here a novel structure of human PrxV at 1.45 A resolution that has a dithiothreitol bound in the active site with its diol moiety mimicking the two oxygens of a peroxide substrate. This suggests diols and similar di-oxygen compounds as a novel class of competitive inhibitors for the Prxs. Common features of this and other structures containing peroxide, peroxide-mimicking ligands, or peroxide-mimicking water molecules reveal hydrogen bonding and steric factors that promote its high reactivity by creating an oxygen track along which the peroxide oxygens move as the reaction proceeds. Key insights include how the active-site microenvironment activates both the peroxidatic cysteine side chain and the peroxide substrate and how it is exquisitely well suited to stabilize the transition state of the in-line S(N)2 substitution reaction that is peroxidation.

Phosphoramidate ProTides of 2'-C-methylguanosine as highly potent inhibitors of hepatitis C virus. Study of their in vitro and in vivo properties.

Hepatitis C virus infection constitutes a serious health problem in need of more effective therapies. Nucleoside analogues with improved exposure, efficacy, and selectivity are recognized as likely key components of future HCV therapy. 2'-C-Methylguanosine triphosphate has been known as a potent inhibitor of HCV RNA polymerase for some time, but the parent nucleoside is only moderately active due to poor intracellular phosphorylation. We herein report the application of phosphoramidate ProTide technology to bypass the rate-limiting initial phosphorylation of this nucleoside. Over 30 novel ProTides are reported, with variations in the aryl, ester, and amino acid regions. l-Alanine compounds are recognized as potent and selective inhibitors of HCV in replicon assay but lack rodent plasma stability despite considerable ester variation. Amino acid variation retaining the lead benzyl ester moiety gives an increase in rodent stability but at the cost of potency. Finally l-valine esters with ester variation lead to potent, stable compounds. Pharmacokinetic studies on these agents in the mouse reveal liver exposure to the bioactive triphosphate species following single oral dosing. Systemic exposure of the ProTide and parent nucleoside are low, indicating possible low toxicity in vivo, while liver concentrations of the active species may be predictive of efficacy in the clinic. This represents one of the most thorough cross-species studies of ProTides to date.

Structural changes common to catalysis in the Tpx peroxiredoxin subfamily.

Thiol peroxidases (Tpxs) are dimeric 2-Cys peroxiredoxins from bacteria that preferentially reduce alkyl hydroperoxides. Catalysis requires two conserved residues, the peroxidatic cysteine and the resolving cysteine, which are located in helix alpha(2) and helix alpha(3), respectively. The partial unraveling of helices alpha(2) and alpha(3) during catalysis allows for the formation of an intramolecular disulfide between these two residues. Here, we present three structures of Escherichia coli Tpx representing the fully folded (peroxide binding site intact), locally unfolded (disulfide bond), and partially locally unfolded (transitional state) conformations. We also compare known Tpx crystal structures and analyze the sequence-conservation patterns among nearly 300 Tpx sequences. Twelve fully conserved Tpx-specific residues cluster at the active site and dimer interface, and an additional 37 highly conserved residues are mostly located in a cradle providing the environment for helix alpha(2). Using the structures determined here as representative fully folded, transitional, and locally unfolded Tpx conformations, we describe in detail the structural changes associated with catalysis in the Tpx subfamily. Key insights include the description of a conserved hydrophobic collar around the active site, a set of conserved packing interactions between helices alpha(2) and alpha(3) that allow the local unfolding of alpha(2) to trigger the partial unfolding of alpha(3), a conserved dimer interface that anchors the ends of helices alpha(2) and alpha(3) to stabilize the active site during structural transitions, and a conserved set of residues constituting a cradle that stabilizes the two discrete conformations of helix alpha(2) involved in catalysis. The involvement of the dimer interface in stabilizing active-site folding and in forming the hydrophobic collar implies that Tpx is an obligate homodimer and explains the high conservation of interface residues.

Typical 2-Cys peroxiredoxins--structures, mechanisms and functions.

Peroxiredoxins are abundant cellular antioxidant proteins that help to control intracellular peroxide levels. These proteins may also function, in part, through an evolved sensitivity of some peroxiredoxins towards peroxide-mediated inactivation in hydrogen peroxide signaling in eukaryotes. This review summarizes recent progress in our understanding of the catalytic and regulatory mechanisms of 'typical 2-Cys' peroxiredoxins and of the biological roles played by these important enzymes in oxidative stress and nonstress-related cellular signaling. New evidence suggests localized peroxide buildup plays a role in nonstress-related signaling.

Cysteine pK(a) values for the bacterial peroxiredoxin AhpC.

Salmonella typhimurium AhpC is a founding member of the peroxiredoxin family, a ubiquitous group of cysteine-based peroxidases with high reactivity toward hydrogen peroxide, organic hydroperoxides, and peroxynitrite. For all of the peroxiredoxins, the catalytic cysteine, referred to as the peroxidatic cysteine (C(P)), acts as a nucleophile in attacking the peroxide substrate, forming a cysteine sulfenic acid at the active site. Because thiolates are far stronger nucleophiles than thiol groups, it is generally accepted that cysteine-based peroxidases should exhibit pK(a) values lower than an unperturbed value of 8.3-8.5. In this investigation, several independent approaches were used to assess the pK(a) of the two cysteinyl residues of AhpC. Methods using two different iodoacetamide derivatives yielded unperturbed pK(a) values (7.9-8.7) for both cysteines, apparently due to reactivity with the wrong conformation of C(P) (i.e., locally unfolded and flipped out of the active site), as supported by X-ray crystallographic analyses. A functional pK(a) of 5.94 +/- 0.10 presumably reflecting the titration of C(P) within the fully folded active site was obtained by measuring AhpC competition with horseradish peroxidase for hydrogen peroxide; this value is quite similar to that obtained by analyzing the pH dependence of the epsilon(240) of wild-type AhpC (5.84 +/- 0.02) and similar to those obtained for two typical 2-cysteine peroxiredoxins from Saccharomyces cerevisiae (5.4 and 6.0). Thus, the pK(a) value of AhpC balances the need for a deprotonated thiol (at pH 7, approximately 90% of the C(P) would be deprotonated) with the fact that thiolates with higher pK(a) values are stronger nucleophiles.