Blue protein with red fluorescence.

The walleye (Sander vitreus) is a golden yellow fish that inhabits the Northern American lakes. The recent sightings of the blue walleye and the correlation of its sighting to possible increased UV radiation have been proposed earlier. The underlying molecular basis of its adaptation to increased UV radiation is the presence of a protein (Sandercyanin)-ligand complex in the mucus of walleyes. Degradation of heme by UV radiation results in the formation of Biliverdin IXα (BLA), the chromophore bound to Sandercyanin. We show that Sandercyanin is a monomeric protein that forms stable homotetramers on addition of BLA to the protein. A structure of the Sandercyanin-BLA complex, purified from the fish mucus, reveals a glycosylated protein with a lipocalin fold. This protein-ligand complex absorbs light in the UV region (λmax of 375 nm) and upon excitation at this wavelength emits in the red region (λmax of 675 nm). Unlike all other known biliverdin-bound fluorescent proteins, the chromophore is noncovalently bound to the protein. We provide here a molecular rationale for the observed spectral properties of Sandercyanin.

Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis.

During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5'-diphosphoribose have an open protein conformation with the catalytic zinc in one position, tetracoordinated by Cys-46, His-67, Cys-174, and a water molecule. The bidentate chelators 2,2'-bipyridine and 1,10-phenanthroline displace the water and form a pentacoordinated zinc. The enzyme-NADH complex has a closed conformation similar to that of ternary complexes with coenzyme and substrate analogues; the coordination of the catalytic zinc is similar to that found in the apoenzyme, except that a minor, alternative position for the catalytic zinc is ∼1.3 Šfrom the major position and closer to Glu-68, which could form the alternative coordination to the catalytic zinc. Complexes with NADH and N-1-methylhexylformamide or N-benzylformamide (or with NAD+ and fluoro alcohols) have the classical tetracoordinated zinc, and no water is bound to the zinc or the nicotinamide rings. The major forms of the enzyme in the mechanism have a tetracoordinated zinc, where the carboxylate group of Glu-68 could participate in the exchange of water and substrates on the zinc. Hydride transfer in the Michaelis complexes does not involve a nearby water.

Impact of time of day on outcomes after stereotactic radiosurgery for non-small cell lung cancer brain metastases.

BACKGROUND: This study tested the hypothesis that time of day of treatment with stereotactic radiosurgery (SRS) has an effect on local control (LC) and overall survival (OS) in a large cohort of patients with non-small cell lung cancer (NSCLC) brain metastases. METHODS: At Washington University in St. Louis, 437 patients with NSCLC were treated with SRS for NSCLC brain metastases. Receiver operating characteristics analysis was used to identify an optimal cut-point for OS relative to time of day. Kaplan-Meier log-rank statistics, and Cox regression univariate and multivariate analysis were employed to isolate any independent effect of treatment time on OS and LC. Matched-pair analysis was performed to isolate any independent effect of time on OS and LC of day while controlling for confounding variables. RESULTS: Receiver operating characteristics analysis identified a cut-point of 11:41 AM as providing the highest predictive value for OS. On univariate analysis, late SRS was associated with decreased OS, as was age, Karnofsky performance status, risk-stratification schemes, extracranial disease status, and overall burden of brain metastases. On univariate analysis for LC, late SRS was associated with decreased LC, as was burden of brain metastases. On multivariate analysis, only Graded Prognostic Assessment remained predictive of OS, and total number of targets and total tumor volume remained predictive of LC. Matched-pair analysis demonstrated no significant effect of time of day on LC or OS. CONCLUSIONS: Although earlier treatment appears to be associated with improved LC and OS, treatment time fails to remain significant when accounting for confounding variables.

Structures of ubiquitin insertion mutants support site-specific reflex response to insertions hypothesis.

We previously concluded that, judging from NMR chemical shifts, the effects of insertions into ubiquitin on its conformation appear to depend primarily on the site of insertion rather than the sequence of the insertion. To obtain a more complete and atomic-resolution understanding of how these insertions modulate the conformation of ubiquitin, we have solved the crystal structures of four insertional mutants of ubiquitin. Insertions between residues 9 and 10 of ubiquitin are minimally perturbing to the remainder of the protein, while larger alterations occur when the insertion is between residues 35 and 36. Further, the alterations in response to insertions are very similar for each mutant at a given site. Two insertions, one at each site, were designed from structurally homologous proteins. Interestingly, the secondary structure within these five to seven amino acid residue insertions is conserved in the new protein. Overall, the crystal structures support the previous conclusion that the conformational effects of these insertions are determined largely by the site of insertion and only secondarily by the sequence of the insert.

Structural investigations of the ferredoxin and terminal oxygenase components of the biphenyl 2,3-dioxygenase from Sphingobium yanoikuyae B1.

BACKGROUND: The initial step involved in oxidative hydroxylation of monoaromatic and polyaromatic compounds by the microorganism Sphingobium yanoikuyae strain B1 (B1), previously known as Sphingomonas yanoikuyae strain B1 and Beijerinckia sp. strain B1, is performed by a set of multiple terminal Rieske non-heme iron oxygenases. These enzymes share a single electron donor system consisting of a reductase and a ferredoxin (BPDO-FB1). One of the terminal Rieske oxygenases, biphenyl 2,3-dioxygenase (BPDO-OB1), is responsible for B1's ability to dihydroxylate large aromatic compounds, such as chrysene and benzo[a]pyrene. RESULTS: In this study, crystal structures of BPDO-OB1 in both native and biphenyl bound forms are described. Sequence and structural comparisons to other Rieske oxygenases show this enzyme to be most similar, with 43.5 % sequence identity, to naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. While structurally similar to naphthalene 1,2-dioxygenase, the active site entrance is significantly larger than the entrance for naphthalene 1,2-dioxygenase. Differences in active site residues also allow the binding of large aromatic substrates. There are no major structural changes observed upon binding of the substrate. BPDO-FB1 has large sequence identity to other bacterial Rieske ferredoxins whose structures are known and demonstrates a high structural homology; however, differences in side chain composition and conformation around the Rieske cluster binding site are noted. CONCLUSION: This is the first structure of a Rieske oxygenase that oxidizes substrates with five aromatic rings to be reported. This ability to catalyze the oxidation of larger substrates is a result of both a larger entrance to the active site as well as the ability of the active site to accommodate larger substrates. While the biphenyl ferredoxin is structurally similar to other Rieske ferredoxins, there are distinct changes in the amino acids near the iron-sulfur cluster. Because this ferredoxin is used by multiple oxygenases present in the B1 organism, this ferredoxin-oxygenase system provides the structural platform to dissect the balance between promiscuity and selectivity in protein-protein electron transport systems.

One enzyme, many reactions: structural basis for the various reactions catalyzed by naphthalene 1,2-dioxygenase.

Rieske nonheme iron oxygenases (ROs) are a well studied class of enzymes. Naphthalene 1,2-dioxygenase (NDO) is used as a model to study ROs. Previous work has shown how side-on binding of oxygen to the mononuclear iron provides this enzyme with the ability to catalyze stereospecific and regiospecific cis-dihydroxylation reactions. It has been well documented that ROs catalyze a variety of other reactions, including mono-oxygenation, desaturation, O- and N-dealkylation, sulfoxidation etc. NDO itself catalyzes a variety of these reactions. Structures of NDO in complex with a number of different substrates show that the orientation of the substrate in the active site controls not only the regiospecificity and stereospecificity, but also the type of reaction catalyzed. It is proposed that the mononuclear iron-activated dioxygen attacks the atoms of the substrate that are most proximal to it. The promiscuity of delivering two products (apparently by two different reactions) from the same substrate can be explained by the possible binding of the substrate in slightly different orientations aided by the observed flexibility of residues in the binding pocket.

A retrospective analysis of survival and prognostic factors after stereotactic radiosurgery for aggressive meningiomas.

BACKGROUND: While most meningiomas are benign, aggressive meningiomas are associated with high levels of recurrence and mortality. A single institution's Gamma Knife radiosurgical experience with atypical and malignant meningiomas is presented, stratified by the most recent WHO classification. METHODS: Thirty-one patients with atypical and 4 patients with malignant meningiomas treated with Gamma Knife radiosurgery between July 2000 and July 2011 were retrospectively reviewed. All patients underwent prior surgical resection. Overall survival was the primary endpoint and rate of disease recurrence in the brain was a secondary endpoint. Patients who had previous radiotherapy or prior surgical resection were included. Kaplan-Meier and Cox proportional hazards models were used to estimate survival and identify factors predictive of recurrence and survival. RESULTS: Post-Gamma Knife recurrence was identified in 11 patients (31.4%) with a median overall survival of 36 months and progression-free survival of 25.8 months. Nine patients (25.7%) had died. Three-year overall survival (OS) and progression-free survival (PFS) rates were 78.0% and 65.0%, respectively. WHO grade II 3-year OS and PFS were 83.4% and 70.1%, while WHO grade III 3-year OS and PFS were 33.3% and 0%. Recurrence rate was significantly higher in patients with a prior history of benign meningioma, nuclear atypia, high mitotic rate, spontaneous necrosis, and WHO grade III diagnosis on univariate analysis; only WHO grade III diagnosis was significant on multivariate analysis. Overall survival was adversely affected in patients with WHO grade III diagnosis, prior history of benign meningioma, prior fractionated radiotherapy, larger tumor volume, and higher isocenter number on univariate analysis; WHO grade III diagnosis and larger treated tumor volume were significant on multivariate analysis. CONCLUSION: Atypical and anaplastic meningiomas remain difficult tumors to treat. WHO grade III diagnosis and treated tumor volume were significantly predictive of recurrence and survival on multivariate analysis in aggressive meningioma patients treated with radiosurgery. Larger tumor size predicts poor survival, while nuclear atypia, necrosis, and increased mitotic rate are risk factors for recurrence. Clinical and pathologic predictors may help identify patients that are at higher risk for recurrence.

Cytosolic phospholipaseA2 inhibition with PLA-695 radiosensitizes tumors in lung cancer animal models.

Lung cancer remains the leading cause of cancer deaths in the United States and the rest of the world. The advent of molecularly directed therapies holds promise for improvement in therapeutic efficacy. Cytosolic phospholipase A2 (cPLA2) is associated with tumor progression and radioresistance in mouse tumor models. Utilizing the cPLA2 specific inhibitor PLA-695, we determined if cPLA2 inhibition radiosensitizes non small cell lung cancer (NSCLC) cells and tumors. Treatment with PLA-695 attenuated radiation induced increases of phospho-ERK and phospho-Akt in endothelial cells. NSCLC cells (LLC and A549) co-cultured with endothelial cells (bEND3 and HUVEC) and pre-treated with PLA-695 showed radiosensitization. PLA-695 in combination with irradiation (IR) significantly reduced migration and proliferation in endothelial cells (HUVEC & bEND3) and induced cell death and attenuated invasion by tumor cells (LLC &A549). In a heterotopic tumor model, the combination of PLA-695 and radiation delayed growth in both LLC and A549 tumors. LLC and A549 tumors treated with a combination of PLA-695 and radiation displayed reduced tumor vasculature. In a dorsal skin fold model of LLC tumors, inhibition of cPLA2 in combination with radiation led to enhanced destruction of tumor blood vessels. The anti-angiogenic effects of PLA-695 and its enhancement of the efficacy of radiotherapy in mouse models of NSCLC suggest that clinical trials for its capacity to improve radiotherapy outcomes are warranted.

Cosmetic analysis following breast-conserving surgery and adjuvant high-dose-rate interstitial brachytherapy for early-stage breast cancer: a prospective clinical study.

PURPOSE: To prospectively evaluate cosmetic outcomes in women treated with accelerated partial breast irradiation using high-dose-rate interstitial brachytherapy for early-stage breast cancer. METHODS AND MATERIALS: Between 2004 and 2008, 151 patients with early-stage breast cancer were enrolled in a phase 2 prospective clinical trial. Eligible patients had stage Tis-T2 tumors of ≤3 cm that were excised with negative margins and with no nodal involvement. Patients received 3.4 Gy twice daily to a total dose of 34 Gy. Both the patients and the treating radiation oncologist qualitatively rated cosmesis as excellent, good, fair, or poor over time and ascribed a cause for changes in cosmesis. Cosmetic outcome was evaluated quantitatively by percentage of breast retraction assessment (pBRA). Patients also reported their satisfaction with treatment over time. RESULTS: Median follow-up was 55 months. The rates of excellent-to-good cosmesis reported by patients and the treating radiation oncologist were 92% and 97% pretreatment, 91% and 97% at 3 to 4 months' follow-up, 87% and 94% at 2 years, and 92% and 94% at 3 years, respectively. Breast infection and adjuvant chemotherapy were independent predictors of a fair-to-poor cosmetic outcome at 3 years. Compared to pretreatment pBRA (7.35), there was no significant change in pBRA over time. The volume receiving more than 150 Gy (V150) was the only significant predictor of pBRA. The majority of patients (86.6%) were completely satisfied with their treatment. CONCLUSIONS: Patients and the treating physician reported a high rate of excellent-to-good cosmetic outcomes at all follow-up time points. Acute breast infection and chemotherapy were associated with worse cosmetic outcomes. Multicatheter interstitial brachytherapy does not significantly change breast size as measured by pBRA.

Analysis of fat necrosis after adjuvant high-dose-rate interstitial brachytherapy for early stage breast cancer.

PURPOSE: To report the incidence and potential predictors of fat necrosis in women with early stage breast cancer treated with adjuvant high-dose-rate (HDR) multicatheter interstitial brachytherapy. METHODS AND MATERIALS: Between 2003 and 2010, 238 treated breasts in 236 women were treated with accelerated partial breast irradiation using HDR interstitial brachytherapy. Selection criteria included patients with Tis-T2 tumors measuring ≤3cm, without nodal involvement, who underwent breast-conserving surgery. Ninety-nine percent of treatments were to a total dose of 34Gy. The presence and severity of fat necrosis were prospectively recorded during followup. Cosmesis was qualitatively scored in all patients. Cosmesis was quantitatively measured via the percentage breast retraction assessment in 151 cases. RESULTS: Median followup was 56 months. The crude rate of fat necrosis was 17.6%. The rate of symptomatic fat necrosis was 10.1%. In univariate analysis, acute breast infection and anthracycline-based chemotherapy, number of catheters, volume encompassed by the prescription isodose, volume encompassed by the 150% isodose (V150), volume encompassed by the 200% isodose, and integrated reference air kerma were significantly associated with fat necrosis. There was significant collinearity between the brachytherapy-related factors; of these, V150 was most predictive. In multivariate analysis, only V150 was significantly associated with fat necrosis. At 3 years, patients with fat necrosis were more likely to have a fair or poor cosmetic outcome and a larger percentage breast retraction assessment. CONCLUSIONS: Mammary fat necrosis is a common adverse event after breast-conserving surgery and HDR interstitial brachytherapy. Fat necrosis is associated with worse qualitative and quantitative cosmetic outcomes. Minimizing exposure volumes, such as V150, may decrease the incidence of fat necrosis and improve cosmesis.

A prospective longitudinal clinical trial evaluating quality of life after breast-conserving surgery and high-dose-rate interstitial brachytherapy for early-stage breast cancer.

PURPOSE: To prospectively examine quality of life (QOL) of patients with early stage breast cancer treated with accelerated partial breast irradiation (APBI) using high-dose-rate (HDR) interstitial brachytherapy. METHODS AND MATERIALS: Between March 2004 and December 2008, 151 patients with early stage breast cancer were enrolled in a phase 2 prospective clinical trial. Eligible patients included those with Tis-T2 tumors measuring ≤3 cm excised with negative surgical margins and with no nodal involvement. Patients received 3.4 Gy twice daily to a total dose of 34 Gy. QOL was measured using European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30, version 3.0, and QLQ-BR23 questionnaires. The QLQ-C30 and QLQ-BR23 questionnaires were evaluated during pretreatment and then at 6 to 8 weeks, 3 to 4 months, 6 to 8 months, and 1 and 2 years after treatment. RESULTS: The median follow-up was 55 months. Breast symptom scores remained stable in the months after treatment, and they significantly improved 6 to 8 months after treatment. Scores for emotional functioning, social functioning, and future perspective showed significant improvement 2 years after treatment. Symptomatic fat necrosis was associated with several changes in QOL, including increased pain, breast symptoms, systemic treatment side effects, dyspnea, and fatigue, as well as decreased role functioning, emotional functioning, and social functioning. CONCLUSIONS: HDR multicatheter interstitial brachytherapy was well tolerated, with no significant detrimental effect on measured QOL scales/items through 2 years of follow-up. Compared to pretreatment scores, there was improvement in breast symptoms, emotional functioning, social functioning, and future perspective 2 years after treatment.

Autotaxin Inhibition with PF-8380 Enhances the Radiosensitivity of Human and Murine Glioblastoma Cell Lines.

PURPOSE: Glioblastoma multiforme (GBM) is an aggressive primary brain tumor that is radio-resistant and recurs despite aggressive surgery, chemo, and radiotherapy. Autotaxin (ATX) is over expressed in various cancers including GBM and is implicated in tumor progression, invasion, and angiogenesis. Using the ATX specific inhibitor, PF-8380, we studied ATX as a potential target to enhance radiosensitivity in GBM. METHODS AND MATERIALS: Mouse GL261 and Human U87-MG cells were used as GBM cell models. Clonogenic survival assays and tumor transwell invasion assays were performed using PF-8380 to evaluate role of ATX in survival and invasion. Radiation dependent activation of Akt was analyzed by immunoblotting. Tumor induced angiogenesis was studied using the dorsal skin fold model in GL261. Heterotopic mouse GL261 tumors were used to evaluate the efficacy of PF-8380 as a radiosensitizer. RESULTS: Pre-treatment of GL261 and U87-MG cells with 1 µM PF-8380 followed by 4 Gy irradiation resulted in decreased clonogenic survival, decreased migration (33% in GL261; P = 0.002 and 17.9% in U87-MG; P = 0.012), decreased invasion (35.6% in GL261; P = 0.0037 and 31.8% in U87-MG; P = 0.002), and attenuated radiation-induced Akt phosphorylation. In the tumor window model, inhibition of ATX abrogated radiation induced tumor neovascularization (65%; P = 0.011). In a heterotopic mouse GL261 tumors untreated mice took 11.2 days to reach a tumor volume of 7000 mm(3), however combination of PF-8380 (10 mg/kg) with irradiation (five fractions of 2 Gy) took more than 32 days to reach a tumor volume of 7000 mm(3). CONCLUSION: Inhibition of ATX by PF-8380 led to decreased invasion and enhanced radiosensitization of GBM cells. Radiation-induced activation of Akt was abrogated by inhibition of ATX. Furthermore, inhibition of ATX led to diminished tumor vascularity and delayed tumor growth. These results suggest that inhibition of ATX may ameliorate GBM response to radiotherapy.

Survival following gamma knife radiosurgery for brain metastasis from breast cancer.

BACKGROUND: Breast cancer is the second most common cause of brain metastases in the United States. Although breast cancer induced brain metastases represent an incurable condition, some patients experience prolonged survival. In this retrospective study, we examine a cohort of patients with brain metastases from breast cancer treated with Gamma Knife stereotactic radiosurgery to identify factors that predict better outcomes. METHODS: A retrospective database of 100 patients treated for brain metastases due to breast cancer via Gamma Knife radiosurgery (GKS) from July 1998 through March 2009 was reviewed. Patients who received radiosurgery as sole treatment, as a planned boost after whole brain radiotherapy or surgical resection, or as salvage after prior whole brain radiation therapy (WBRT) or surgical resection were included. Prognostic factors identified to be significant for survival in previous brain metastasis studies were analyzed for significance by univariate and multivariate Cox analysis. RESULTS: Overall, the median brain progression-free survival time was 7.1 months and the median survival time was 12.3 months. No prognostic variables were significant for brain progression-free survival. For patients treated with a planned GKS after WBRT, GKS as sole treatment, GKS salvage after WBRT, GKS boost after surgery, or GKS for surgical salvage the median survival times (MSTs) were as follows: 12.2 months, 12.4 months, 9.5 months, 27.6 months and 33.4 months respectively. Differences between the groups were not significant (p = 0.06); however, GKS boost after surgery and GKS for salvage after surgery did have a trend toward better overall survival. CONCLUSION: Stereotactic radiosurgery offers good local control and prolonged survival in selected patients. Age and number of lesions are strong predictors of overall survival.

High-throughput identification of putative receptors for cancer-binding peptides using biopanning and microarray analysis.

Phage-display peptide biopanning has been successfully used to identify cancer-targeting peptides in multiple models. For cancer-binding peptides, identification of the peptide receptor is necessary to demonstrate the mechanism of action and to further optimize specificity and target binding. The process of receptor identification can be slow and some peptides may turn out to bind ubiquitous proteins not suitable for further drug development. In this report, we describe a high-throughput method for screening a large number of peptides in parallel to identify peptide receptors, which we have termed "reverse biopanning." Peptides can then be selected for further development based on their receptor. To demonstrate this method, we screened a library of 39 peptides previously identified in our laboratory to bind specifically to cancers after irradiation. The reverse biopanning process identified 2 peptides, RKFLMTTRYSRV and KTAKKNVFFCSV, as candidate ligands for the protein tax interacting protein 1 (TIP-1), a protein previously identified in our laboratory to be expressed in tumors and upregulated after exposure to ionizing radiation. We used computational modeling as the initial method for rapid validation of peptide-TIP-1 binding. Pseudo-binding energies were calculated to be -360.645 kcal mol(-1), -487.239 kcal mol(-1), and -595.328 kcal mol(-1) for HVGGSSV, TTRYSRV, and NVFFCSV respectively, suggesting that the peptides would have at least similar, if not stronger, binding to TIP-1 compared to the known TIP-1 binding peptide HVGGSSV. We validated peptide binding in vitro using electrophoretic mobility shift assay, which showed strong binding of RKFLMTTRYSRV and the truncated form TTRYSRV. This method allows for the identification of many peptide receptors and subsequent selection of peptides for further drug development based on the peptide receptor.

Trigeminal neuralgia post-styloidectomy in Eagle syndrome: a case report.

INTRODUCTION: Eagle syndrome is a condition characterized by an elongated (>3cm) styloid process with associated symptoms of recurrent facial or throat pain. In this report we present a case of Eagle syndrome exhibiting the typical findings of glossopharyngeal nerve involvement, as well as unusual involvement of the trigeminal nerve. Notably, this patient developed a classical trigeminal neuralgia post-styloidectomy. CASE PRESENTATION: A 68-year-old Caucasian woman presented with a 25-year history of dull pain along the right side of her throat, lateral neck, and jaw. Her symptoms were poorly controlled with medication until 15 years ago when she was diagnosed with Eagle syndrome, and underwent a manual fracture of her styloid process. This provided symptomatic relief until 5 years ago when the pain recurred and progressed. She underwent a styloidectomy via a lateral neck approach, which resolved the pain once again. However, 6 months ago a new onset of triggerable, electric shock-like facial pain began within the right V1 and V2 distributions. CONCLUSIONS: Eagle syndrome is distressing to patients and often difficult to diagnose due to its wide variability in symptoms. It is easily confused with dental pain or temporomandibular joint disorder, leading to missed diagnoses and unnecessary procedures. Pain along the jaw and temple is an unusual but possible consequence of Eagle syndrome. An elongated styloid process should be considered a possible etiology of dull facial pain in the trigeminal distributions, in particular V3.

GSK-3ß: A Bifunctional Role in Cell Death Pathways.

Although glycogen synthase kinase-3 beta (GSK-3ß) was originally named for its ability to phosphorylate glycogen synthase and regulate glucose metabolism, this multifunctional kinase is presently known to be a key regulator of a wide range of cellular functions. GSK-3ß is involved in modulating a variety of functions including cell signaling, growth metabolism, and various transcription factors that determine the survival or death of the organism. Secondary to the role of GSK-3ß in various diseases including Alzheimer's disease, inflammation, diabetes, and cancer, small molecule inhibitors of GSK-3ß are gaining significant attention. This paper is primarily focused on addressing the bifunctional or conflicting roles of GSK-3ß in both the promotion of cell survival and of apoptosis. GSK-3ß has emerged as an important molecular target for drug development.

NDRG4, the N-Myc downstream regulated gene, is important for cell survival, tumor invasion and angiogenesis in meningiomas.

Meningiomas are the second most common brain tumor, and 20-30% of these tumors are aggressive. The aggressive subtypes are characterized by a capacity for invasion of normal brain with frequent and destructive recurrence patterns. Effective local therapies include surgery and radiation, but there is a need for novel molecular targets to improve survival and reduce morbidity for this group or cancer patients. We have recently identified the N-Myc downstream regulated gene 4, NDRG4, protein as being overexpressed in aggressive meningioma, and in this report, demonstrate its role in cell survival, invasion/migration and angiogenesis. Downregulation of NDRG4 mRNA and protein expression in two high-grade meningioma cancer cell lines, IOMM-Lee and CH-157 MN resulted in reduction in cell survival, DNA fragmentation and G2-M cell cycle arrest. NDRG4 downregulation also decreased cellular invasion and migration, as determined by spheroid migration, linear and radial wound healing, Boyden chamber matrigel invasion, and 3D invasion assays. To determine the effect of NDRG4 depletion on angiogenesis, we studied the immortalized brain endothelial cell line, bEnd.3. We treated bEnd.3 cells with conditioned media from NDRG4-depleted IOMM-Lee and CH-157 MN cells and abrogated their ability to elicit bEnd.3 capillary-like tubes, to proliferate, and to invade. NDRG4 is not overexpressed in bEnd.3 cells and direct NDRG4 depletion had no effect on the cells. This study is significant as it is the first to demonstrate the functional role of NDRG4 in various aspects of meningioma tumor biology. NDRG4 is involved in modulating cell proliferation, invasion, migration and angiogenesis in meningioma, and may play a valuable role as a molecular target in its treatment.

Comparison of accelerated partial breast irradiation via multicatheter interstitial brachytherapy versus whole breast radiation.

BACKGROUND: Brachytherapy as adjuvant treatment for early-stage breast cancer has become widely available and offers patients an expedited treatment schedule. Given this, many women are electing to undergo brachytherapy in lieu of standard fractionation radiotherapy. We compare outcomes between patients treated with accelerated partial breast irradiation (APBI) via multicatheter interstitial brachytherapy versus patients who were also eligible for and offered APBI but who chose whole breast radiation (WBI). METHODS: Patients treated from December 2002 through May 2007 were reviewed. Selection criteria included patients with pTis-T2N0 disease, ≤ 3 cm unifocal tumors, and negative margins who underwent breast conservation surgery. Local control (LC), cause-specific (CSS) and overall survival (OS) were analyzed. RESULTS: 202 patients were identified in the APBI cohort and 94 patients in the WBI cohort. Median follow-up for both groups exceeded 60 months. LC was 97.0% for the APBI cohort and 96.2% for the WBI cohort at 5 years (ns). Classification by 2010 ASTRO APBI consensus statement categories did not predict worse outcomes. CONCLUSION: APBI via multicatheter interstitial brachytherapy provides similar local failure rates compared to WBI at 5 years for properly selected patients. Excellent results were seen despite the high fraction of younger patients (< 60 years old) and patients with DCIS.

Recurrent adult-onset hypophyseal Langerhans cell histiocytosis after radiotherapy: a case report.

INTRODUCTION: Langerhans cell histiocytosis is a rare disease within the adult population, with very few cases reported as solitary hypophyseal lesions in adults. Of the reported cases, most have been treated successfully with surgery, radiotherapy, and/or chemotherapy. Radiotherapy has been thought to be curative at the relatively low dose of 20Gy. Here we report a case of recurrent hypophyseal Langerhans cell histiocytosis 9 months after radiotherapy with an interval period of symptomatic and radiographic response to therapy. CASE PRESENTATION: A 50-year-old Caucasian woman who had headaches, memory difficulties, and diabetes insipidus was found to have a 2.5cm suprasellar mass. Langerhans cell histiocytosis was diagnosed following stereotactic brain biopsy. Further workup revealed no other lesions. Initial radiation treatment succeeded in shrinking the tumor and relieving clinical symptoms temporarily; however, growth and recurrence of clinical symptoms was noted at 9 months. Re-irradiation was well tolerated and the patient had no acute side effects. CONCLUSION: Isolated hypophyseal involvement by Langerhans cell histiocytosis in adults is a unique presentation of a rare disease. Although radiotherapy doses as low as 20Gy have been reported to offer control, this case demonstrates that higher doses may be warranted to ensure tumor control. With modern imaging and radiotherapy techniques higher doses should offer little increased more durable risk to surrounding critical structures.

Structural basis for regioselectivity and stereoselectivity of product formation by naphthalene 1,2-dioxygenase.

Rieske oxygenase (RO) systems are two- and three-component enzyme systems that catalyze the formation of cis-dihydrodiols from aromatic substrates. Degradation of pollutants in contaminated soil and generation of chiral synthons have been the major foci of RO research. Substrate specificity and product regio- and stereoselectivity have been shown to vary between individual ROs. While directed evolution methods for altering RO function have been successful in the past, rational engineering of these enzymes still poses a challenge due to the lack of structural understanding. Here we examine the structural changes induced by mutation of Phe-352 in naphthalene 1,2-dioxygenase from Pseudomonas sp. strain NCIB 9816-4 (NDO-O(9816-4)). Structures of the Phe-352-Val mutant in native form and in complex with phenanthrene and anthracene, along with those of wild-type NDO-O(9816-4) in complex with phenanthrene, anthracene, and 3-nitrotoluene, are presented. Phenanthrene was shown to bind in a different orientation in the Phe-352-Val mutant active site from that in the wild type, while anthracene was found to bind in similar positions in both enzymes. Two orientations of 3-nitrotoluene were observed, i.e., a productive and a nonproductive orientation. These orientations help explain why NDO-O(9816-4) forms different products from 3-nitrotoluene than those made from nitrobenzene dioxygenase. Comparison of these structures among themselves and with other known ROs bound to substrates reveals that the orientation of substrate binding at the active site is the primary determinant of product regio- and stereoselectivity.