Development and validation of a novel prognostic score to predict survival in patients with metastatic colorectal cancer: the metastatic colorectal cancer score (mCCS).

AIM: Published prognostic scores for metastatic colorectal cancer (mCRC) are based on data from highly selected patient subgroups with specified first-line treatments and may not be applicable to routine practice. We have therefore developed and validated the metastatic colorectal cancer score (mCCS) to predict overall survival (OS) for patients with mCRC. METHOD: A total of 1704 patients from the prospective, multicentre cohort study Tumour Registry Colorectal Cancer were separated into learning (n = 796) and validation (n = 908) samples. Using a multivariate Cox regression model, the six-factor mCCS was established. RESULTS: The six independent prognostic factors for survival are as follows: two or more metastatic sites at the start of first-line treatment, tumour grading ≥ G3 at primary diagnosis, residual tumour classification ≥ R1/unknown, lymph node ratio (of primary tumour) ≥ 0.4, tumour stage ≥ III/unknown at primary diagnosis and KRAS status mutated/unknown. The mCCS clearly separated the learning sample into three risk groups: zero to two factors (low risk), three factors (intermediate risk) and four to six factors (high risk). The prognostic performance of the mCCS was confirmed in the validation sample and additionally stratified a large sample of patients with known (K)RAS mutation status. CONCLUSION: The novel prognostic score, mCCS, clearly defines three prognostic groups for OS at start of first-line therapy. For oncologists, the mCCS represents a simple and easy-to-apply tool for routine clinical use, as it is based on objective tumour characteristics and can assist with treatment decision-making and communication of the prognosis to patients.

A 5-µm pitch charge-coupled device optimized for resonant inelastic soft X-ray scattering.

We have developed a charge-coupled device (CCD) with 5 µm × 45 µm pixels on high-resistivity silicon. The fully depleted 200 µm-thick silicon detector is back-illuminated through a 10 nm-thick in situ doped polysilicon window and is thus highly efficient for soft through >8 keV hard X-rays. The device described here is a 1.5 megapixel CCD with 2496 × 620 pixels. The pixel and camera geometry was optimized for Resonant Inelastic X-ray Scattering (RIXS) and is particularly advantageous for spectrometers with limited arm lengths. In this article, we describe the device architecture, construction and operation, and its performance during tests at the Advance Light Source (ALS) 8.0.1 RIXS beamline. The improved spectroscopic performance, when compared with a current standard commercial camera, is demonstrated with a ∼280 eV (CK) X-ray beam on a graphite sample. Readout noise is typically 3-6 electrons and the point spread function for soft CK X-rays in the 5 µm direction is 4.0 µm ± 0.2 µm. The measured quantum efficiency of the CCD is greater than 75% in the range from 200 eV to 1 keV.

Excellent long-term survival of 170 patients with Waldenström's macroglobulinemia treated in private oncology practices and a university hospital.

The purpose of this study was to compare treatment and outcome of patients with Waldenström's macroglobulinemia (WM) in four private oncology practices (PP) and a university hospital (UH) in southwest Germany. We retrospectively reviewed the charts of all patients with WM of the last two decades of four PP in Mannheim, Heidelberg, Karlsruhe, and Speyer and the Department of Hematology of the University of Heidelberg. One hundred seventy patients could be identified, 74 from PP, 96 from the UH. Median age was 63.3 years. Patients from PP were older (median 65.3 vs. 62.5 years, p = 0.01). Only 54 % of patients from PP have received treatment during the observation time, as compared to 78.1 % of the UH (p < 0.001). In PP, 35 % of treated patients have received rituximab, as compared to 62.6 % of the patients of the UH (p < 0.001). Sixty percent of treated patients of PP have received bendamustine, as compared to only 8 % of the patients of the UH (p < 0.001). Time to first treatment was significantly shorter in patients from the UH compared to PP (median 13.7 vs. 52.9 months, p = 0.05). A trend towards a better overall survival was observed for patients treated with a rituximab-containing first-line regimen. The International Prognostic Scoring System for WM had significant prognostic value. Median overall survival was 25.0 years and did not differ between PP and UH. Despite different treatment strategies between PP and UH today overall survival of patients with WM is excellent, and better than previously reported.

Are routine microbiological investigations indicated in the management of non-perianal cutaneous abscesses?

BACKGROUND: It is common practice to take a specimen of pus for microscopy and bacterial culture during drainage of abscesses. The aim of this study was to determine if routine culture and sensitivity had any therapeutic value in the care of patients with non-perianal cutaneous abscesses. PATIENTS AND METHODS: A retrospective analysis ofall patients undergoing drainage ofa cutaneous abscess during a two year period (June 2003 - June 2005) was performed. Patients were identified from the hospital database and theatre records, and those with perianal, pilonidal or surgical wound sepsis were excluded. Notes were reviewed for clinical details, culture results, subsequent admissions and attendance at follow-up. RESULTS: Of the 239 patients treated during this period, 74 patients had 77 operations to drain abscesses that matched the inclusion criteria. Specimens were sent from 52 (67.5%) procedures. Only 65.4% had an organism identified, of which methicillin-sensitive Staphylococcus aureus (MSSA) was the most commonly isolated organism (36.5%). Forty-one point six per cent of patients received antibiotics as part of their treatment. The results of the bacterial culture and antibiotic sensitivities were not known prior to discharge of any patient. CONCLUSION: This study shows that bacteriology swabs are frequently taken during incision and drainage of non-perianal cutaneous abscesses and had little impact on the subsequent treatment, though these results may not be applicable to immune-compromised patients.

Treatment of prosthetic valve infective endocarditis due to multi-resistant Gram-positive bacteria with linezolid.

OBJECTIVES: Clinical experience with linezolid in the treatment of infective endocarditis either alone or in combination with other agents is limited. We describe our experience in the treatment of two patients with IE due to multi-resistant Gram-positive bacteria. METHODS: One patient with MRSE and one with VRE endocarditis were treated with regimens containing linezolid. The killing kinetics of linezolid in combination with gentamicin or vancomycin against isolates of Staphylococcus epidermidis and Enterococcus faecalis were analysed in vitro. RESULTS: Clinical response and eradication of bacteraemia was achieved with linezolid therapy in both patients. Time-kill curve studies showed that linezolid was bacteriostatic against the MRSE and VRE isolates used. Combination with gentamicin or vancomycin did not produce synergy or antagonism but at best only marginal additive effect. CONCLUSIONS: Although bacteriostatic, linezolid provides an important therapeutic option in IE due to multi-resistant Gram-positive pathogens. It challenges the conventional wisdom that bactericidal synergy is required for the effective treatment of most cases of IE due to Gram-positive organisms.

Structural biochemistry and interaction architecture of the DNA double-strand break repair Mre11 nuclease and Rad50-ATPase.

To clarify functions of the Mre11/Rad50 (MR) complex in DNA double-strand break repair, we report Pyrococcus furiosus Mre11 crystal structures, revealing a protein phosphatase-like, dimanganese binding domain capped by a unique domain controlling active site access. These structures unify Mre11's multiple nuclease activities in a single endo/exonuclease mechanism and reveal eukaryotic macromolecular interaction sites by mapping human and yeast Mre11 mutations. Furthermore, the structure of the P. furiosus Rad50 ABC-ATPase with its adjacent coiled-coil defines a compact Mre11/Rad50-ATPase complex and suggests that Rad50-ATP-driven conformational switching directly controls the Mre11 exonuclease. Electron microscopy, small angle X-ray scattering, and ultracentrifugation data of human and P. furiosus MR reveal a dual functional complex consisting of a (Mre11)2/(Rad50)2 heterotetrameric DNA processing head and a double coiled-coil linker.

Measurement of CP-violating asymmetries in B0 decays to CP eigenstates.

We present measurements of time-dependent CP-violating asymmetries in neutral B decays to several CP eigenstates. The measurement uses a data sample of 23x10(6) Upsilon(4S)-->BbarB decays collected by the BABAR detector at the PEP-II asymmetric B Factory at SLAC. In this sample, we find events in which one neutral B meson is fully reconstructed in a CP eigenstate containing charmonium and the flavor of the other neutral B meson is determined from its decay products. The amplitude of the CP-violating asymmetry, which in the standard model is proportional to sin2beta, is derived from the decay time distributions in such events. The result is sin2beta = 0.34+/-0.20 (stat)+/-0.05 (syst).

Directly observed treatment for multidrug-resistant tuberculosis: an economic evaluation in the United States of America and South Africa.

OBJECTIVE: To estimate the cost-effectiveness of directly observed treatment compared to conventional therapy in reducing the spread of multidrug-resistant tuberculosis, for an industrialised country (represented by the United States of America) and a developing country (South Africa). METHODS: Monte Carlo analysis using published data on probability, cost and health impact. RESULTS: In both countries, directly observed treatment is the dominant strategy, yielding cost savings and improved health outcomes. Cost savings for directly observed treatment relative to conventional therapy become more significant as more expensive second-line drugs are used in treatments. CONCLUSIONS: The cost-effectiveness of directly observed treatment relative to conventional therapy is demonstrated for both the USA and South Africa. Cost savings are more pronounced (especially for South Africa) as the likelihood of multidrug-resistant tuberculosis increases and more expensive second-line therapies are used. Given that health care resources are more severely constrained in developing countries, the data contained in this study are useful in guiding the design of policies for the effective management of multidrug-resistant tuberculosis in settings with limited resources.

Mre11 and Rad50 from Pyrococcus furiosus: cloning and biochemical characterization reveal an evolutionarily conserved multiprotein machine.

The processing of DNA double-strand breaks is a critical event in nucleic acid metabolism. This is evidenced by the severity of phenotypes associated with deficiencies in this process in multiple organisms. The core component involved in double-strand break repair in eukaryotic cells is the Mre11-Rad50 protein complex, which includes a third protein, p95, in humans and Xrs2 in yeasts. Homologues of Mre11 and Rad50 have been identified in all kingdoms of life, while the Nbs1 protein family is found only in eukaryotes. In eukaryotes the Mre11-Rad50 complex has nuclease activity that is modulated by the addition of ATP. We have isolated the Mre11 and Rad50 homologues from the thermophilic archaeon Pyrococcus furiosus and demonstrate that the two proteins exist in a large, heat-stable complex that possesses single-strand endonuclease activity and ATP-dependent double-strand-specific exonuclease activity. These findings verify the identification of the P. furiosus Rad50 and Mre11 homologues and demonstrate that functional homologues with similar biochemical properties exist in all kingdoms of life.

Crystals of the urokinase type plasminogen activator variant beta(c)-uPAin complex with small molecule inhibitors open the way towards structure-based drug design.

Urokinase is a serine protease involved in cancer growth and metastasis. Here we present the first urokinase crystal structure in complex with reversible inhibitors at 2.1 and 2.6 A resolution. These inhibitor complex structures have been obtained from crystals of engineered urokinase type plasminogen activator designed to obtain a crystal form open for inhibitor soaking. The mutant C122S loses its flexible A-chain upon activation cleavage and crystallizes in the presence of benzamidine, which was later displaced by the desired inhibitor. This new soakable crystal form turned out to be of great value in the process of structure-based drug design. The evaluated binding mode of amiloride, and UKI-1D revealed a new subsite of the primary specificity pocket of urokinase that will be employed in the future ligand optimisation process.

Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily.

To clarify the key role of Rad50 in DNA double-strand break repair (DSBR), we biochemically and structurally characterized ATP-bound and ATP-free Rad50 catalytic domain (Rad50cd) from Pyrococcus furiosus. Rad50cd displays ATPase activity plus ATP-controlled dimerization and DNA binding activities. Rad50cd crystal structures identify probable protein and DNA interfaces and reveal an ABC-ATPase fold, linking Rad50 molecular mechanisms to ABC transporters, including P glycoprotein and cystic fibrosis transmembrane conductance regulator. Binding of ATP gamma-phosphates to conserved signature motifs in two opposing Rad50cd molecules promotes dimerization that likely couples ATP hydrolysis to dimer dissociation and DNA release. These results, validated by mutations, suggest unified molecular mechanisms for ATP-driven cooperativity and allosteric control of ABC-ATPases in DSBR, membrane transport, and chromosome condensation by SMC proteins.

Electrically driven microseparation methods for pesticides and metabolites: IV. Effects of the nature of fluorescent labels on the enantioseparation of pesticides and their degradation products by capillary zone electrophoresis with UV and laser-induced fluorescence detection.

Three different fluorescent tags, namely 5-aminonaphthalene-1-sulfonic acid (ANSA), 7-aminonaphthalene-1,3-disulfonic acid (ANDSA), and 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) were evaluated in the precolumn derivatization of some chiral phenoxy acid herbicides, some chiral transformation products of pyrethroid insecticides, and in their subsequent enantiomeric separation by capillary electrophoresis (CE). The electrolyte systems consisted of sodium phosphate buffers containing chiral surfactants such as octylglucoside (OG) or octylmaltoside (OM) at concentrations above the critical micellar concentration (CMC). Among the three different tags investigated, the ANDSA derivatives of the various solutes were more readily enantioseparated than the ANSA and ANTS derivatives. While the tagging with ANSA allowed the enantioseparation of a limited number of the chiral solute-ANSA derivatives investigated, the ANTS derivatization yielded derivatives that could not be enantioseparated. The polarity of the three different tags increases by increasing the number of sulfonic acid groups in the molecule in the following order: ANSA (one sulfonic acid) < ANDSA (two sulfonic acid groups) < ANTS (three sulfonic acid groups). This seems to indicate that the intermediate polarity of the ANDSA tag allowed more equitable nonpolar/polar interactions of the ANDSA-derivatized solutes with the OG or OM micelles, and consequently the enantioseparation of the solute-ANDSA derivatives. Thus, there is a solute polarity window for enantioresolution with alkylglycoside micelle by CE. Solutes of intermediate polarity that undergo more equitable nonpolar/polar interactions with the micelles exhibited chiral separations.

High-performance liquid phase separation of glycosides. 5. Determination of individual glucosinolates in cabbage and rapeseed by laser-induced fluorescene capillary electrophoresis via the enzymatically released isothiocyanate aglycon.

A capillary electrophoresis (CE) method was developed for the profiling and determination of individual glucosinolates (GSs) via their isothiocyanate degradation products upon myrosinase digestion. The resulting isothiocyanates, the structures of which are reflective of the parent GS's, were then converted to their corresponding amines via base hydrolysis or reaction with 1, 2-benzenedithiol. Subsequently, the amines were fluorescently labeled to allow their sensitive detection by laser-induced fluorescence (LIF). The CE method involved the use of in situ charged micelles for the separation of isothiocyanates and their corresponding fluorescently labeled amines by micellar electrokinetic capillary chromatography (MECC). The term "in situ charged micelles" refers to micelles formed by complexing the polar hydroxyl groups of glycosidic surfactants with borate. The MECC method with on-column LIF detection was applied to the determination of GSs in white cabbage, rapeseed leaves, and rapeseed roots.

Capillary electrophoresis of glucosinolates and their degradation products.

Glucosinolates are important natural products occurring mainly in plants of the Cruciferae family. This review article is aimed at describing the recent progress made in capillary electrophoresis of glucosinolates and their degradation products. It describes the various electrophoretic systems and detection schemes introduced to date for the capillary electrophoresis (CE) of glucosinolates and their degradation products. Also included in this review are the applications of CE to the qualitative and quantitative determination of glucosinolates and their degradation products in plant extracts.

Capillary electrophoresis and electrochromatography of pesticides and metabolites.

Synthetic pesticides are important chemicals since they are widely used to control many types of weeds, insects and other pests in a wide variety of agricultural and nonagricultural settings. This review article is aimed at describing the recent progress made in capillary electrophoresis (CE) and capillary electrochromatography (CEC) of pesticides and their metabolites. The various electrophoretic systems and detection schemes that have been introduced so far for the CE and CEC of pesticides are discussed. Also included in this review article are the various approaches for trace enrichment that are involved in the analysis of dilute pesticide samples.

Coagulation factor IXa: the relaxed conformation of Tyr99 blocks substrate binding.

BACKGROUND: Among the S1 family of serine proteinases, the blood coagulation factor IXa (fIXa) is uniquely inefficient against synthetic peptide substrates. Mutagenesis studies show that a loop of residues at the S2-S4 substrate-binding cleft (the 99-loop) contributes to the low efficiency. The crystal structure of porcine fIXa in complex with the inhibitor D-Phe-Pro-Arg-chloromethylketone (PPACK) was unable to directly clarify the role of the 99-loop, as the doubly covalent inhibitor induced an active conformation of fIXa. RESULTS: The crystal structure of a recombinant two-domain construct of human fIXa in complex with p-aminobenzamidine shows that the Tyr99 sidechain adopts an atypical conformation in the absence of substrate interactions. In this conformation, the hydroxyl group occupies the volume corresponding to the mainchain of a canonically bound substrate P2 residue. To accommodate substrate binding, Tyr99 must adopt a higher energy conformation that creates the S2 pocket and restricts the S4 pocket, as in fIXa-PPACK. The energy cost may contribute significantly to the poor K(M) values of fIXa for chromogenic substrates. In homologs, such as factor Xa and tissue plasminogen activator, the different conformation of the 99-loop leaves Tyr99 in low-energy conformations in both bound and unbound states. CONCLUSIONS: Molecular recognition of substrates by fIXa seems to be determined by the action of the 99-loop on Tyr99. This is in contrast to other coagulation enzymes where, in general, the chemical nature of residue 99 determines molecular recognition in S2 and S3-S4. This dominant role on substrate interaction suggests that the 99-loop may be rearranged in the physiological fX activation complex of fIXa, fVIIIa, and fX.