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Large (10 × 10 cm) sheets of surface-enhanced Raman spectroscopy (SERS) active polymer have been prepared by stabilising metal nanoparticle aggregates within dry hydroxyethylcellulose (HEC) films. In these films the aggregates are protected by the polymer matrix during storage but in use they are released when aqueous analyte droplets cause the films to swell to their gel form. The fact that these "Poly-SERS" films can be prepared in bulk but then cut to size and stored in air before use means that they provide a cost effective and convenient method for routine SERS analysis. Here we have tested both Ag and Au Poly-SERS films for use in point-of-care monitoring of therapeutic drugs, using phenytoin as the test compound. Phenytoin in water could readily be detected using Ag Poly-SERS films but dissolving the compound in phosphate buffered saline (PBS) to mimic body fluid samples caused loss of the drug signal due to competition for metal surface sites from Cl(-) ions in the buffer solution. However, with Au Poly-SERS films there was no detectable interference from Cl(-) and these materials allowed phenytoin to be detected at 1.8 mg L(-1), even in PBS. The target range of detection of phenytoin in therapeutic drug monitoring is 10-20 mg L(-1). With the Au Poly-SERS films, the absolute signal generated by a given concentration of phenytoin was lower for the films than for the parent colloid but the SERS signals were still high enough to be used for therapeutic monitoring, so the cost in sensitivity for moving from simple aqueous colloids to films is not so large that it outweighs the advantages which the films bring for practical applications, in particular their ease of use and long shelf life., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The detection of minimal residual disease (MRD) in myeloma using a 0.01% threshold (10(-4)) after treatment is an independent predictor of progression-free survival (PFS), but not always of overall survival (OS). However, MRD level is a continuous variable, and the predictive value of the depth of tumor depletion was evaluated in 397 patients treated intensively in the Medical Research Council Myeloma IX study. There was a significant improvement in OS for each log depletion in MRD level (median OS was 1 year for ≥10%, 4 years for 1% to <10%, 5.9 years for 0.1% to <1%, 6.8 years for 0.01% to <0.1%, and more than 7.5 years for <0.01% MRD). MRD level as a continuous variable determined by flow cytometry independently predicts both PFS and OS, with approximately 1 year median OS benefit per log depletion. The trial was registered at www.isrctn.com as #68454111., (© 2015 by The American Society of Hematology.)

The Medical Research Council Myeloma IX Trial (ISRCTNG8454111) examined traditional and thalidomide-based induction and maintenance regimens and IV zoledronic acid (ZOL) and oral clodronate (CLO) in 1960 patients with newly diagnosed multiple myeloma. Overall survival (OS) and skeletal-related event (SRE) data have been reported for the overall trial population. The present analysis investigated optimal therapy regimens for different patient populations in Myeloma IX. Patients were assigned to intensive or nonintensive treatment pathways and randomized to induction cyclophosphamide, vincristine, doxorubicin, and dexamethasone (CVAD) versus cyclophosphamide, thalidomide, and dexamethasone (CTD; intensive) or melphalan and prednisolone versus attenuated oral CTD (CTDa; nonintensive). Patients were also randomized to ZOL or CLO. In the nonintensive pathway, CTDa produced better responses and lower SRE rates than melphalan and prednisolone. ZOL improved OS compared with CLO independently of sex, stage, or myeloma subtype, most profoundly in patients with baseline bone disease or other SREs. In patients treated for ≥ 2 years, ZOL improved OS compared with CLO from randomization (median not reached for either; P = .02) and also from first on-study disease progression (median, 34 months for ZOL vs 27 months for CLO; P = .03). Thalidomide-containing regimens had better efficacy than traditional regimens, and ZOL demonstrated greater benefits than CLO.

Raman spectra of a representative range of β-ketophenethylamine (β-KP), the rapidly growing family of cathinone-related "legal high" recreational drugs, have been recorded. These spectra showed characteristic changes that were associated with the pattern of substitution on the aromatic rings, for example, the compounds carrying substituents at the 4- position could be distinguished from 3,4-methylenedioxy "ecstasy" derivatives. They also showed small but detectable changes with differences in substitution on the ethylamine substituent. These features allowed the β-KPs present in seized casework samples to be identified. The seized samples typically contained only small amounts of bulking agents, which meant that the band intensities of these components within averaged data were very small. In contrast, grid sampling normally gave at least some spectra which had a higher than average proportion of the bulking agent(s), which allowed them to also be identified. This study therefore demonstrates that Raman spectroscopy can be used both to provide a rapid, non-destructive technique for identification of this class of drugs in seized samples and to detect minor constituents, giving a composition profile which can be used for drugs intelligence work., (Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.)

Thalidomide maintenance has the potential to modulate residual multiple myeloma (MM) after an initial response. This trial compared the effect of thalidomide maintenance and no maintenance on progression-free survival (PFS) and overall survival (OS) in MM patients. After intensive or nonintensive induction therapy, 820 newly diagnosed MM patients were randomized to open-label thalidomide maintenance until progression, or no maintenance. Interphase FISH (iFISH) analysis was performed at study entry. Median PFS was significantly longer with thalidomide maintenance (log-rank P < .001). Median OS was similar between regimens (log-rank P = .40). Patients with favorable iFISH showed improved PFS (P = .004) and a trend toward a late survival benefit. Patients with adverse iFISH receiving thalidomide showed no significant PFS benefit and worse OS (P = .009). Effective relapse therapy enhanced survival after progression, translating into a significant OS benefit. Meta-analysis of this and other studies show a significant late OS benefit (P < .001, 7-year difference hazard ratio = 12.3; 95% confidence interval, 5.5-19.0). Thalidomide maintenance significantly improves PFS and can be associated with improved OS. iFISH testing is important in assessing the clinical impact of maintenance therapy. Overview analysis demonstrated that thalidomide maintenance was associated with a significant late OS benefit. This trial was registered at www.isrctn.org as #ISRCTN68454111.

As part of the randomized MRC Myeloma IX trial, we compared an attenuated regimen of cyclophosphamide, thalidomide, and dexamethasone (CTDa; n = 426) with melphalan and prednisolone (MP; n = 423) in patients with newly diagnosed multiple myeloma ineligible for autologous stem-cell transplantation. The primary endpoints were overall response rate, progression-free survival, and overall survival (OS). The overall response rate was significantly higher with CTDa than MP (63.8% vs 32.6%; P < .0001), primarily because of increases in the rate of complete responses (13.1% vs 2.4%) and very good partial responses (16.9% vs 1.7%). Progression-free survival and OS were similar between groups. In this population, OS correlated with the depth of response (P < .0001) and favorable interphase fluorescence in situ hybridization profile (P < .001). CTDa was associated with higher rates of thromboembolic events, constipation, infection, and neuropathy than MP. In elderly patients with newly diagnosed multiple myeloma (median age, 73 years), CTDa produced higher response rates than MP but was not associated with improved survival outcomes. We highlight the importance of cytogenetic profiling at diagnosis and effective management of adverse events. This trial was registered at International Standard Randomized Controlled Trials Number as #68454111.

Chemical degradations often induce changes in protein conformation and thus influence protein activity and protein stability in solutions. One difficulty in studying of chemical degradations on protein aqueous properties is to obtain sufficient amount of chemically degraded protein which is well characterized. Chemical degradation protocols that are often used may induce also conformation changes and aggregation of the protein. In this article we studied the effect of methionine oxidation on the conformation of recombinant human growth hormone (r-hGH). In literature it is reported that oxidation of methionine residues induces conformation changes on r-hGH. In our study, oxidation of r-hGH was performed by incubation with hydrogen peroxide under mild conditions. Mass spectrometry and chromatographic analysis revealed that oxidation with hydrogen peroxide resulted in more than 90% of oxidized r-hGH. By extensive spectroscopic characterizations no detectable change in conformation and aggregation of r-hGH after oxidation was found. In conclusion, mild oxidation conditions led to selective oxidation of the two more accessible methionine residues of r-hGH (Met(14) and Met(125)) and did not results in any conformation change of the protein. These findings prove that oxidation of human growth hormone does not influence protein conformation and demonstrate the importance of employing mild conditions during production of oxidized protein., (Copyright © 2010 Wiley-Liss, Inc. and the American Pharmacists Association)

Background: Bisphosphonates reduce the risk of skeletal events in patients with malignant bone disease, and zoledronic acid has shown potential anticancer effects in preclinical and clinical studies. We aimed to establish whether bisphosphonates can affect clinical outcomes in patients with multiple myeloma., Methods: Patients of age 18 years or older with newly diagnosed multiple myeloma were enrolled from 120 centres in the UK. Computer-generated randomisation sequence was used to allocate patients equally, via an automated telephone service, to receive 4 mg zoledronic acid as an infusion every 3-4 weeks or 1600 mg oral clodronic acid daily. Patients also received intensive or non-intensive induction chemotherapy. No investigators, staff, or patients were masked to treatment allocation, and bisphosphonate and maintenance therapy continued at least until disease progression. The primary endpoints were overall survival, progression-free survival, and overall response rate. We assessed between-group differences with Cox proportional hazards models for progression-free survival and overall survival, and with logistic regression models for overall response rate. Analysis was by intention to treat. This trial is registered, number ISRCTN68454111., Findings: 1970 patients were enrolled between May, 2003, and November, 2007, of whom 1960 were eligible for intention-to-treat analysis: 981 in the zoledronic acid group (555 on intensive chemotherapy, 426 on non-intensive chemotherapy); and 979 on clodronic acid (556 on intensive chemotherapy, 423 on non-intensive chemotherapy). The treatment cutoff was Oct 5, 2009, with patients receiving bisphosphonates for a median of 350 days (IQR 137-632) before disease progression, with a median of 3·7 years' follow-up (IQR 2·9-4·7). Zoledronic acid reduced mortality by 16% (95% CI 4-26) versus clodronic acid (hazard ratio [HR] 0·84, 95% CI 0·74-0·96; p=0·0118), and extended median overall survival by 5·5 months (50·0 months, IQR 21·0 to not reached vs 44·5 months, IQR 16·5 to not reached; p=0·04). Zoledronic acid also significantly improved progression-free survival by 12% (95% CI 2-20) versus clodronic acid (HR 0·88, 95% CI 0·80-0·98; p=0·0179), and increased median progression-free survival by 2·0 months (19·5 months, IQR 9·0-38·0 vs 17·5 months, IQR 8·5-34·0; p=0·07). Rates of complete, very good partial, or partial response did not differ significantly between the zoledronic acid and clodronic acid groups for patients receiving intensive induction chemotherapy (432 patients [78%] vs 422 [76%]; p=0·43) or non-intensive induction chemotherapy (215 [50%] vs 195 [46%]; p=0·18). Both bisphosphonates were generally well tolerated, with similar occurrence of acute renal failure and treatment-emergent serious adverse events, but zoledronic acid was associated with higher rates of confirmed osteonecrosis of the jaw (35 [4%]) than was clodronic acid (3 [<1%])., Interpretation: Consistent with the potential anticancer activity of zoledronic acid, overall survival improved independently of prevention of skeletal-related events, showing that zoledronic acid has treatment benefits beyond bone health. These findings support immediate treatment with zoledronic acid in patients with newly diagnosed multiple myeloma, not only for prevention of skeletal-related events, but also for potential antimyeloma benefits., Funding: Medical Research Council (London, UK), with unrestricted educational grants from Novartis, Schering Health Care, Chugai, Pharmion, Celgene, and Ortho Biotech., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A venous thromboembolism (VTE) with the subsequent risk of pulmonary embolism is a major concern in the treatment of patients with multiple myeloma with thalidomide. The susceptibility to developing a VTE in response to thalidomide therapy is likely to be influenced by both genetic and environmental factors. To test genetic variation associated with treatment related VTE in patient peripheral blood DNA, we used a custom-built molecular inversion probe (MIP)-based single nucleotide polymorphism (SNP) chip containing 3404 SNPs. SNPs on the chip were selected in "functional regions" within 964 genes spanning 67 molecular pathways thought to be involved in the pathogenesis, treatment response, and side effects associated with myeloma therapy. Patients and controls were taken from 3 large clinical trials: Medical Research Council (MRC) Myeloma IX, Hovon-50, and Eastern Cooperative Oncology Group (ECOG) EA100, which compared conventional treatments with thalidomide in patients with myeloma. Our analysis showed that the set of SNPs associated with thalidomide-related VTE were enriched in genes and pathways important in drug transport/metabolism, DNA repair, and cytokine balance. The effects of the SNPs associated with thalidomide-related VTE may be functional at the level of the tumor cell, the tumor-related microenvironment, and the endothelium. The clinical trials described in this paper have been registered as follows: MRC Myeloma IX: ISRCTN68454111; Hovon-50: NCT00028886; and ECOG EA100: NCT00033332.

The influence of ageing and cooking on the Raman spectrum of porcine longissimus dorsi was investigated. The rich information contained in the Raman spectrum was highlighted, with numerous changes attributed to changes in the environment and conformations of the myofibrillar proteins. Predictions equations for shear force and cooking loss were developed from the Raman spectra of both raw and cooked pork. Good correlations and standard errors of prediction were obtained for both WB shear force and cooking loss, with the raw and the cooked samples showing almost identical results R(2)=0.77, root mean standard error of prediction (RMSEP)% of mean=12% for shear force; R(2)=0.71, RMSEP% of mean=10% for cooking loss. The Raman spectra were also able to predict the extent of cooking that occurred within the pork (R(2)(val)=0.94, RMSEP% of range=5.5%). Raman spectroscopy has considerable potential as a method for non-destructive and rapid determination of pork quality parameters such as tenderness. Raman spectroscopy may provide a means of determining changes during cooking and the extent to which foods have been cooked.

In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 microM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5'-dithiobis 2-nitrobenzoic acid (DTNB) (100 microM) or inhibiting PDI with bacitracin (5mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry). cGMP accumulation in response to GSNO (1 microM) was inhibited by MEG-01 treatment with bacitracin or DTNB, suggesting a role for PDI and surface thiols in NO delivery. PDI activity was present in MEG-01 conditioned medium, and was inhibited by high concentrations of GSNO (500 microM). A number of cell surface thiol-containing proteins were labelled using the impermeable thiol specific probe 3-(N-maleimido-propionyl) biocytin (MPB). Pretreatment of cells with GSNO resulted in a loss of thiol reactivity on some but not all proteins, suggesting selective cell surface thiol modification. Immunoprecipitation experiments showed that GSNO caused a concentration-dependent loss of thiol reactivity of PDI. Our data indicate that PDI is involved in both rapid metabolism of GSNO and intracellular NO delivery and that during this process PDI is itself altered by thiol modification. In contrast, the relevance of PDI-mediated albSNO metabolism to NO signalling is uncertain.

Phospholipase C-gamma2 (PLC-gamma2) is a key component of signal transduction in leukocytes. In natural killer (NK) cells, PLC-gamma2 is pivotal for cellular cytotoxicity; however, it is not known which steps of the cytolytic machinery it regulates. We found that PLC-gamma2-deficient NK cells formed conjugates with target cells and polarized the microtubule-organizing center, but failed to secrete cytotoxic granules, due to defective calcium mobilization. Consequently, cytotoxicity was completely abrogated in PLC-gamma2-deficient cells, regardless of whether targets expressed NKG2D ligands, missed self major histocompatibility complex (MHC) class I, or whether NK cells were stimulated with IL-2 and antibodies specific for NKR-P1C, CD16, CD244, Ly49D, and Ly49H. Defective secretion was specific to cytotoxic granules because release of IFN-gamma on stimulation with IL-12 was normal. Plcg2-/- mice could not reject MHC class I-deficient lymphoma cells nor could they control CMV infection, but they effectively contained Listeria monocytogenes infection. Our results suggest that exocytosis of cytotoxic granules, but not cellular polarization toward targets, depends on intracellular calcium rise during NK cell cytotoxicity. In vivo, PLC-gamma2 regulates selective facets of innate immunity because it is essential for NK cell responses to malignant and virally infected cells but not to bacterial infections.

This study examined the effects of polymeric components on the physical state of chlorhexidine within bioadhesive, semisolid formulations using Raman spectroscopy. Semisolid formulations were prepared in which chlorhexidine base (CHX, 5%w/w, particle size <63 microm) was dispersed in aqueous (phosphate-buffered saline, pH 6.8) polymer matrices consisting of one or more polymeric components, namely HEC (3%w/w), PVP (3%), and PC (PC, 3%). Raman spectra were recorded using 785-nm excitation and were typically accumulated for 360 s. The Raman spectra were dominated by the presence of CHX. The spectra of CHX in HEC and in HEC/PVP gels were indistinguishable from that for solid CHX as a result of the insolubility of CHX in these formulations. However, in systems containing PC and CHX, there was a shift in the strongest band from 1564 cm(-1) to 1608 cm(-1), which may be accredited to protonation of the basic CHX by the numerous carboxylic acidic groups on PC. Identical shifts in the band positions were observed when this protonation was modeled using ethanoic acid, supporting the view that there was a simple acid base reaction between PC and CHX. However, there were notable differences in the relative intensities of the peaks from these samples, with the spectrum of CHX in the PC matrix displaying properties intermediate between those of CHX dissolved in ethanoic acid and solid CHX diacetate. This may be accredited to the limited solubility of the CHX-PC ion pair. In matrices containing HEC and PC, no peak was observed at 1564 cm(-1), whereas the intensity of the peak at 1608 cm(-1) was increased. Therefore, in these formulations CHX was completely converted to the di-cation as a result of the synergistic effects of PC (which protonated CHX) and HEC (which solubilized the di-cation). In the absence of either HEC or PC, complete protonation was not achieved. It is suggested that this enhancement of solubility of H(2)CHX(2+) may be due to hydrogen bonding, given the hydroxylated nature of HEC. In conclusion, this study has shown the applicability of Raman spectroscopy for both the analysis of opaque, semisolid formulations and, additionally, for the examination of the state of therapeutic agents within such matrices. In particular, using Raman spectroscopy, it was uniquely possible to identify the roles of various polymeric components on both the ionization and solubilization of CHX within aqueous semisolid systems., (Copyright 2000 Wiley-Liss, Inc.)

Mortality data are presented from a 16- to 45-year follow-up study of 71 men with antisocial personality disorder. Death ascertainment was made through both a personal follow-up and use of the National Death Index. Comparisons were made with the mortality experience of the general population of the state of Iowa by using gender and age standardized mortality ratios. Seventeen men died (24%) died during the follow-up. Antisocial men younger than 40 years were at excessive risk for premature death (standardized mortality ratio [SMR] = 33, P < 0.25): men between ages 40 and 60 years also appeared to be at risk for premature death, although the excess was not statistically significant. Three subjects (18% of all deaths) died of complications from diabetes mellitus (SMR = 14, P < 0.05). Deaths were spread out among the four decades of follow-up. The findings and their implications are discussed.