In Vitro Studies of the Prp9·Prp11·Prp21 Complex Indicate a Pathway for U2 Small Nuclear Ribonucleoprotein Activation

Pre-mRNA splicing takes place on a large ribonucleoprotein particle, the spliceosome which contains the five small nuclear ribonucleoproteins (snRNPs), U1, U2, U4, U5, and U6. In Saccharomyces cerevisiae the mRNA splicing factors, Prp9, Prp11, and Prp21, are necessary for addition of the U2 snRNP to the pre-mRNA in an early step of spliceosome assembly. This paper describes a study of interactions between these proteins and their role in spliceosome assembly. The proteins were expressed in Escherichia coli. Prp9 and Prp11 were purified by metal affinity chromatography. Prp21 was purified using a solubilization/renaturation protocol. We have combined these separately purified proteins and present direct evidence of a Prp9·Prp11·Prp21 protein complex that is functional in in vitro splicing assays. Characteristics of this Prp9·Prp11·Prp21 complex were further investigated using proteins synthesized in vitro. In addition, we found that Prp9, Prp11, and Prp21 influence the structure of the U2 snRNP in a manner that alters the accessibility of the branch point pairing region of the U2 snRNA to oligonucleotide-directed RNaseH cleavage. We present a model, based on the data presented here and in the accompanying paper, for a combined role of Prp9, Prp11, Prp21, and Prp5 in activating the U2 snRNP for assembly into the pre-spliceosome

Synthesis and characterization of Na03RhO206H2O - a semiconductor with a weak ferromagnetic component

We have prepared the oxyhydrate Na03RhO206H2O by extracting Na+ cations from NaRhO2 and intercalating water molecules using an aqueous solution of Na2S2O8. Synchrotron X-ray powder diffraction, thermogravimetric analysis (TGA), and energy-dispersive x-ray analysis (EDX) reveal that a non-stoichiometric Na03(H2O)06 network separates layers of edge-sharing RhO6 octahedra containing Rh3+(4d6, S=0) and Rh4+ (4d5, S=1/2). The resistivities of NaRhO2 and Na03RhO206H2O (T < 300) reveal insulating and semi-conducting behavior with activation gaps of 134 meV and 7.8 meV, respectively. Both Na03RhO206H2O and NaRhO2 show paramagnetism at room temperature, however, the sodium-deficient sample exhibits simultaneously a weak but experimentally reproducible ferromagnetic component. Both samples exhibit a temperature-independent Pauli paramagnetism, for NaRhO2 at T > 50 K and for Na03RhO206H2O at T > 25 K. The relative magnitudes of the temperature-independent magnetic susceptibilities, that of the oxide sample being half that of the oxyhydrate, is consistent with a higher density of thermally accessible electron states at the Fermi level in the hydrated sample. At low temperatures the magnetic moments rise sharply, providing evidence of localized and weakl -ordered electronic spins.Comment: 15 fages 5 figures Solid State Communications in prin

Long-term visual outcomes after ruthenium plaque brachytherapy for posterior choroidal melanoma

BACKGROUND: To assess the long-term visual outcomes in patients with posteriorly located choroidal melanoma treated with ruthenium plaque brachytherapy between January 2013 and December 2015. METHODS: A retrospective review was conducted on consecutive patients treated with ruthenium plaque brachytherapy for post-equatorial choroidal melanoma with available Snellen visual acuity before and after treatment, and the development and treatment of radiation complications. RESULTS: There were 219 patients with posterior choroidal melanoma treated with ruthenium plaque brachytherapy. Median follow up was 56.5 months, range 12-81 months. Final visual acuity was ≥6/12 in 97 (44.3%) patients, 6/12 to 6/60 in 57 (26.0%), <6/60 in 55 (25.1%) and 10 (4.6%) eyes were enucleated. Radiation maculopathy was the most common radiation complication encountered, occurring in 53 (24.2%) patients. Of these, final visual acuity was 6/12 in 10 patients (18.9%), 6/12 to 6/60 in 26 (49.1%), <6/60 in 16 (30.2%) and 1 eye (1.9%) was enucleated. Twenty-five (47%) with radiation maculopathy were treated with intravitreal anti-angiogenic therapy, 27 (51%) were monitored and one (2%) was treated with scatter photocoagulation. Eyes treated with intravitreal anti-angiogenic therapy had better final vision than those observed or treated with retinal laser (chi-square, p = 0.04). On multivariate analysis, close proximity to the optic nerve and fovea, and large or notched plaque type was associated with final vision worse than 6/12. CONCLUSION: Most patients treated with ruthenium plaque brachytherapy for posterior choroidal melanoma retain 6/60 vision, with almost half retaining 6/12 vision at long term follow up

Nanobodies Raised against Monomeric α-Synuclein Distinguish between Fibrils at Different Maturation Stages

AbstractNanobodies are single-domain fragments of camelid antibodies that are emerging as versatile tools in biotechnology. We describe here the interactions of a specific nanobody, NbSyn87, with the monomeric and fibrillar forms of α-synuclein (αSyn), a 140-residue protein whose aggregation is associated with Parkinson's disease. We have characterized these interactions using a range of biophysical techniques, including nuclear magnetic resonance and circular dichroism spectroscopy, isothermal titration calorimetry and quartz crystal microbalance measurements. In addition, we have compared the results with those that we have reported previously for a different nanobody, NbSyn2, also raised against monomeric αSyn. This comparison indicates that NbSyn87 and NbSyn2 bind with nanomolar affinity to distinctive epitopes within the C-terminal domain of soluble αSyn, comprising approximately amino acids 118–131 and 137–140, respectively. The calorimetric and quartz crystal microbalance data indicate that the epitopes of both nanobodies are still accessible when αSyn converts into its fibrillar structure. The apparent affinities and other thermodynamic parameters defining the binding between the nanobody and the fibrils, however, vary significantly with the length of time that the process of fibril formation has been allowed to progress and with the conditions under which formation occurs, indicating that the environment of the C-terminal domain of αSyn changes as fibril assembly takes place. These results demonstrate that nanobodies are able to target forms of potentially pathogenic aggregates that differ from each other in relatively minor details of their structure, such as those associated with fibril maturation

Surface complexation model for strontium sorption to amorphous silica and goethite

Strontium sorption to amorphous silica and goethite was measured as a function of pH and dissolved strontium and carbonate concentrations at 25°C. Strontium sorption gradually increases from 0 to 100% from pH 6 to 10 for both phases and requires multiple outer-sphere surface complexes to fit the data. All data are modeled using the triple layer model and the site-occupancy standard state; unless stated otherwise all strontium complexes are mononuclear. Strontium sorption to amorphous silica in the presence and absence of dissolved carbonate can be fit with tetradentate Sr2+ and SrOH+ complexes on the β-plane and a monodentate Sr2+complex on the diffuse plane to account for strontium sorption at low ionic strength. Strontium sorption to goethite in the absence of dissolved carbonate can be fit with monodentate and tetradentate SrOH+ complexes and a tetradentate binuclear Sr2+ species on the β-plane. The binuclear complex is needed to account for enhanced sorption at hgh strontium surface loadings. In the presence of dissolved carbonate additional monodentate Sr2+ and SrOH+ carbonate surface complexes on the β-plane are needed to fit strontium sorption to goethite. Modeling strontium sorption as outer-sphere complexes is consistent with quantitative analysis of extended X-ray absorption fine structure (EXAFS) on selected sorption samples that show a single first shell of oxygen atoms around strontium indicating hydrated surface complexes at the amorphous silica and goethite surfaces

Shadowing in Inelastic Scattering of Muons on Carbon, Calcium and Lead at Low XBj

Nuclear shadowing is observed in the per-nucleon cross-sections of positive muons on carbon, calcium and lead as compared to deuterium. The data were taken by Fermilab experiment E665 using inelastically scattered muons of mean incident momentum 470 GeV/c. Cross-section ratios are presented in the kinematic region 0.0001 < XBj <0.56 and 0.1 < Q**2 < 80 GeVc. The data are consistent with no significant nu or Q**2 dependence at fixed XBj. As XBj decreases, the size of the shadowing effect, as well as its A dependence, are found to approach the corresponding measurements in photoproduction.Comment: 22 pages, incl. 6 figures, to be published in Z. Phys.

Clinical presentation, histology, and prognoses of malignant melanoma in ethnic Chinese: A study of 522 consecutive cases

<p>Abstract</p> <p>Background</p> <p>Malignant melanoma is a rare disease in Asia, and knowledge on its characteristics and clinical outcome in Asian patients is limited. The purpose of this observational study was to determine the clinical presentation and outcome of patients with melanoma in China.</p> <p>Methods</p> <p>A database was prospectively established for the purpose of this analysis. The elements of the database included basic demographic data of patients and prognosticators previously reported in literature, as well as follow-up data including clinical outcome after treatment. Medical record of all patients with pathologically diagnosed malignant melanoma consulted in our center since 2006 were retrieved and reviewed. No patient was excluded in this study. Statistical analyses including survival and multivariate analyses of factors associated with survival were respectively performed by Kaplan-Meier method and Cox proportional hazard model.</p> <p>Results</p> <p>A total of 522 consecutive and nonselected cases were evaluated. There were 218 cases (41.8%) of acral lentiginous melanoma (ALM), 118 (22.6%) of mucosal melanoma (MCM), 103 (19.7%) of nodular melanoma (NM), 33 (6.3%) of superficial spreading melanoma (SSM), and others were Lentigo maligna melanoma or unclassifiable disease. The proportion of patients with clinical stage I, II, III, and IV diseases were 6.1%, 55.9%, 25.1%, and 12.8%, respectively. Among the 357 cases of cutaneous melanoma, 234 patients (65.5%) had ulceration.</p> <p>The 5-year overall survival rate of all 522 patients was 41.6%, and the median survival time was 3.92 years (95% CI, 3.282 to 4.558). Five-year survival rates of patients with stage I, II, III, and IV diseases were 94.1%, 44.0%, 38.4% and 4.6% respectively (P < 0.001). Multivariate analysis revealed that clinical stage and the ulceration were two significant prognosticators for OS. In addition, extent of surgery and use of adjuvant therapy were significant prognosticators for DFS in patients with non-metastatic disease after definitive treatment. Pathological subtype was not a significant prognostic factor to predict wither OS or DFS.</p> <p>Conclusions</p> <p>Prognoses of patients with malignant melanoma diagnosed in China were suboptimal, and most patients were diagnosed with locally advanced disease (i.e., stage II or above). ALM and MCM are the two most commonly diagnosed pathological subtypes. Clinical staging and presence of ulceration was significantly associated with clinical outcome in terms of OS, while treatment strategy including extent of surgery and use of adjuvant therapy were significant predictors of DFS.</p

Intrathecal treatment of neoplastic meningitis due to breast cancer with a slow-release formulation of cytarabine

DepoCyte is a slow-release formulation of cytarabine designed for intrathecal administration. The goal of this multi-centre cohort study was to determine the safety and efficacy of DepoCyte for the intrathecal treatment of neoplastic meningitis due to breast cancer. DepoCyte 50 mg was injected once every 2 weeks for one month of induction therapy; responding patients were treated with an additional 3 months of consolidation therapy. All patients had metastatic breast cancer and a positive CSF cytology or neurologic findings characteristic of neoplastic meningitis. The median number of DepoCyte doses was 3, and 85% of patients completed the planned 1 month induction. Median follow up is currently 19 months. The primary endpoint was response, defined as conversion of the CSF cytology from positive to negative at all sites known to be positive, and the absence of neurologic progression at the time the cytologic conversion was documented. The response rate among the 43 evaluable patients was 28% (CI 95%: 14–41%); the intent-to-treat response rate was 21% (CI 95%: 12–34%). Median time to neurologic progression was 49 days (range 1–515(+)); median survival was 88 days (range 1–515(+)), and 1 year survival is projected to be 19%. The major adverse events were headache and arachnoiditis. When drug-related, these were largely of low grade, transient and reversible. Headache occurred on 11% of cycles; 90% were grade 1 or 2. Arachnoiditis occurred on 19% of cycles; 88% were grade 1 or 2. DepoCyte demonstrated activity in neoplastic meningitis due to breast cancer that is comparable to results reported with conventional intrathecal agents. However, this activity was achieved with one fourth as many intrathecal injections as typically required in conventional therapy. The every 2 week dose schedule is a major advantage for both patients and physicians. © 2001 Cancer Research Campaign http://www.bjcancer.co

Oncostatin M promotes STAT3 activation, VEGF production, and invasion in osteosarcoma cell lines

<p>Abstract</p> <p>Background</p> <p>We have previously demonstrated that both canine and human OSA cell lines, as well as 8 fresh canine OSA tumor samples, exhibit constitutive phosphorylation of STAT3, and that this correlates with enhanced expression of matrix metalloproteinase-2 (MMP2). While multiple signal transduction pathways can result in phosphorylation of STAT3, stimulation of the cytokine receptor gp130 through either IL-6 or Oncostatin M (OSM) is the most common mechanism through which STAT3 is activated. The purpose of this study was to evaluate the role of IL-6 and OSM stimulation on both canine and human OSA cell lines to begin to determine the role of these cytokines in the biology of OSA.</p> <p>Methods</p> <p>RT-PCR and Western blotting were used to interrogate the consequences of OSM and IL-6 stimulation of OSA cell lines. OSA cells were stimulated with OSM and/or hepatocyte growth factor (HGF) and the effects on MMP2 activity (gel zymography), proliferation (CyQUANT), invasion (Matrigel transwell assay), and VEGF production (Western blotting, ELISA) were assessed. The small molecule STAT3 inhibitor LLL3 was used to investigate the impact of STAT3 inhibition following OSM stimulation of OSA cells.</p> <p>Results</p> <p>Our data demonstrate that the OSM receptor (OSMR), but not IL-6 or its receptor, is expressed by all human and canine OSA cell lines and canine OSA tumor samples; additionally, OSM expression was noted in all tumor samples. Treatment of OSA cell lines with OSM induced phosphorylation of STAT3, Src, and JAK2. OSM stimulation also resulted in a dose dependent increase in MMP2 activity and VEGF expression that was markedly reduced following treatment with the small molecule STAT3 inhibitor LLL3. Lastly, OSM stimulation of OSA cell lines enhanced invasion through Matrigel, particularly in the presence of rhHGF. In contrast, both OSM and HGF stimulation of OSA cell lines did not alter their proliferative capacity.</p> <p>Conclusions</p> <p>These data indicate OSM stimulation of human and canine OSA cells induces STAT3 activation, thereby enhancing the expression/activation of MMP2 and VEGF, ultimately promoting invasive behavior and tumor angiogenesis. As such, OSM and its receptor may represent a novel target for therapeutic intervention in OSA.</p