bluuebunny/crossref_metadata_2025 huggingface dataset:
Early Transjugular Intrahepatic Portosystemic Shunt in Patients With Cirrhosis and Variceal Bleeding
Authors: Rössle Martin , Grandt Daniel
Published: October 2010

Abstract

Background: Patients with cirrhosis in Child–Pugh class C or those in class B who have persistent bleeding at endoscopy are at high risk for treatment failure and a poor prognosis, even if they have undergone rescue treatment with a transjugular intrahepatic portosystemic shunt (TIPS). This study evaluated the earlier use of TIPS in such patients. Methods: We randomly assigned, within 24 hours after admission, a total of 63 patients with cirrhosis and acute variceal bleeding who had been treated with vasoactive drugs plus endoscopic therapy to treatment with a polytetrafluoroethylene-covered stent within 72 hours after randomization (early-TIPS group, 32 patients) or continuation of vasoactive-drug therapy, followed after 3 to 5 days by treatment with propranolol or nadolol and long-term endoscopic band ligation (EBL), with insertion of a TIPS if needed as rescue therapy (pharmacotherapy–EBL group, 31 patients). Results: During a median follow-up of 16 months, rebleeding or failure to control bleeding occurred in 14 patients in the pharmacotherapy–EBL group as compared with 1 patient in the early-TIPS group (P=0.001). The 1-year actuarial probability of remaining free of this composite end point was 50% in the pharmacotherapy–EBL group versus 97% in the early-TIPS group (P<0.001). Sixteen patients died (12 in the pharmacotherapy–EBL group and 4 in the early-TIPS group, P=0.01). The 1-year actuarial survival was 61% in the pharmacotherapy–EBL group versus 86% in the early-TIPS group (P<0.001). Seven patients in the pharmacotherapy–EBL group received TIPS as rescue therapy, but four died. The number of days in the intensive care unit and the percentage of time in the hospital during follow-up were significantly higher in the pharmacotherapy–EBL group than in the early-TIPS group. No significant differences were observed between the two treatment groups with respect to serious adverse events. Conclusions: In these patients with cirrhosis who were hospitalized for acute variceal bleeding and at high risk for treatment failure, the early use of TIPS was associated with significant reductions in treatment failure and in mortality. (Current Controlled Trials number, ISRCTN58150114.)
Defining the Quality Characteristics of Endoscopy for Acute Variceal Hemorrhage in Cirrhosis
Authors: Talwalkar Jayant A.
Published: October 2010

Abstract

Objectives: The optimal timing of endoscopy with acute variceal bleeding (AVB) is unknown. The aim of this study was to evaluate the association between the timing of endoscopy and outcomes of stable AVB patients. Methods: Patients admitted at two tertiary-care centers with hemodynamically stable AVB from 1997 to 2006 were evaluated retrospectively. The primary outcome was mortality. Other recorded outcomes included stigmata at endoscopy, hemostasis, blood transfusions, rebleeding, renal function, hospitalization length, infection, transjugular intrahepatic portosystemic shunt use, and balloon tamponade use. Logistic regression analysis was used to assess the association of time to endoscopy with mortality. Outcome comparisons were also performed for three different urgency times (< or = vs. > 4 h, < or = vs. > 8 h, and < or = vs. > 12 h). Results: There were 210 patients with stable AVB, accounting for 52% of the total number of AVB patients. The mean (+/− s.d.) age was 55 (+/− 12) years. The mean presenting systolic blood pressure and heart rate were 121 (+/− 16) mm Hg and 98 (+/− 20) bpm, respectively. Esophageal varices accounted for 91% (n = 191) of variceal bleeding. The mean time to endoscopy was 12 (+/− 12) h. The overall hemostasis rate after endoscopy was 97% (n = 203). The mortality rate was 9.5% (n = 20). There was no significant association of time to endoscopy with mortality (odds ratio, OR, 1.0; 95% confidence interval, CI, 0.92-1.08; P = 0.91). Significant independent predictors for mortality were lower albumin (OR, 0.82; 95% CI, 0.73-0.93; P = 0.001), infection during admission (OR, 8.9; 95% CI, 2.5-31.6; P < 0.001), and higher model end-stage liver disease (MELD) (OR, 1.17; 95% CI, 1.06-1.29; P = 0.002). There was no difference in outcomes with different urgency times. Conclusions: For patients who present with hemodynamically stable variceal bleeding, hemostasis after endoscopy is high, and the time to endoscopy does not appear to be associated with mortality.
Binding of the Rett syndrome protein, MeCP2, to methylated and unmethylated DNA and chromatin
Authors: Hansen Jeffrey C. , Ghosh Rajarshi P. , Woodcock Christopher L.
Published: October 2010

Methylated CpG Binding Protein 2 (MeCP2) is a nuclear protein named for its ability to selectively recognize methylated DNA. Much attention has been focused on understanding MeCP2 structure and function in the context of its role in Rett syndrome, a severe neurodevelopmental disorder that afflicts one in 10,000–15,000 girls. Early studies suggested a connection between DNA methylation, MeCP2, and establishment of a repressive chromatin structure at specific gene promoters. However, it is now recognized that MeCP2 can both activate and repress specific genes depending on the context. Likewise, in the cell, MeCP2 is bound to unmethylated DNA and chromatin in addition to methylated DNA. Thus, to understand the molecular basis of MeCP2 functionality, it is necessary to unravel the complex interrelationships between MeCP2 binding to unmethylated and methylated regions of the genome. MeCP2 is unusual and interesting in that it is an intrinsically disordered protein, that is, much of its primary sequence fails to fold into secondary structure and yet is functional. The unique structure of MeCP2 is the subject of the first section of this article. We then discuss recent investigations of the in vitro binding of MeCP2 to unmethylated and methylated DNA, and the potential ramifications of this work for in vivo function. We close by focusing on mechanistic studies indicating that the binding of MeCP2 to chromatin results in compaction into local (secondary) and global (tertiary) higher order structures. MeCP2 also competes with histone H1 for nucleosomal binding sites. The recent finding that MeCP2 is found at near stoichiometric levels with nucleosomes in neuronal cells underscores the multiple modes of engagement of MeCP2 with the genome, which include the cooperative tracking of methylation density. © 2010 IUBMB IUBMB Life, 62(10): 732–738, 2010
Metabolic functions of AMPK: Aspects of structure and of natural mutations in the regulatory gamma subunits
Authors: Moffat Cynthia , Ellen Harper Mary
Published: October 2010

AMP‐activated protein kinase, AMPK, is widely accepted as the master regulator of energy levels within the cell. Responding quickly to changing energy demands, AMPK works to restore levels of ATP during times of cellular stress by promoting ATP producing catabolic pathways and inhibiting ATP consuming anabolic ones. As a heterotrimeric protein complex, AMPK's subunits each act in unique and crucial ways to control AMPK function and its localization within the cell. Research in the last decade has identified and begun to characterize the impact of naturally occurring mutations in the gamma regulatory subunits. Mutations in the γ2 subunit have implications for cardiac function and disease, while the R225W mutation in the γ3 subunit have implications for skeletal muscle fuel metabolism and resistance to fatigue. Research focused on structure‐function aspects of AMPK regulatory subunits will lead to a better understanding of the roles of AMPK in health and disease. © 2010 IUBMB IUBMB Life, 62(10): 739–745, 2010
Dose‐dependent pharmacokinetics of toxic metabolites is not related to increased toxicity following high‐dose valproic acid in rats
Authors: Jung Byung Hwa , Kim Bo Jun , Lee Min Sun , Lee Joo Hyun , Oh Ju‐Hee , Lee Young‐Joo
Published: October 2010

It has been reported that urinary excretion of two metabolites of valproic acid (VPA), 4‐ene‐valproic acid (4‐VPA) and 2,4‐diene‐valproic acid (2,4‐VPA), increased exponentially with the administration of high doses of VPA, and this increased formation of toxic metabolites could be related to VPA hepatotoxicity in humans. The aim of this study was to investigate whether the plasma level of 4‐VPA and 2,4‐VPA in rats corresponds to the urinary data for the same metabolites in humans. After the oral administration of VPA at doses of 20, 100 and 500 mg kg−1in rats, the AUC0–24 h, 4‐VPA/AUC0–24 h, VPAratios (0.0399, 0.0120 and 0.0100 for 20, 100 and 500 mg kg−1, respectively) and AUC0–24 h, 2,4‐VPA/AUC0–24 h, VPAratios (0.00104, 0.00201 and 0.00141, respectively) did not increase with increasing doses of VPA in rats. Thus, the plasma exposure of toxic metabolites normalized by dose remained unchanged (for 2,4‐VPA) or even decreased (for 4‐VPA) following high‐dose VPA administration; this contradicts the findings of previous studies. Our results suggest that toxicity induced by high doses of VPA cannot be explained by a nonlinear increase of toxic metabolites in rats. Copyright © 2010 John Wiley & Sons, Ltd.
Characterisation of DNA methylation status using spectroscopy (mid‐IR versus Raman) with multivariate analysis
Authors: Kelly Jemma G. , Najand Ghazal M. , Martin Francis L.
Published: October 2010

Methylation status plays important roles in the regulation of gene expression and significantly influences the dynamics, bending and flexibility of DNA. The aim of this study was to determine whether attenuated total reflection Fourier‐transform infrared (ATR‐FTIR) or Raman spectroscopy with subsequent multivariate analysis could determine methylation patterning in oligonucleotides variously containing 5‐methylcytosine, cytosine and guanine bases. Applied to Low‐E reflective glass slides, 10 independent spectral acquisitions were acquired per oligonucleotide sample. Resultant spectra were baseline‐corrected and vector normalised over the 1750 cm–1–760 cm–1 (for ATR‐FTIR spectroscopy) or the 1750 cm–1–600 cm–1 (for Raman spectroscopy) regions. Data were then analysed using principal component analysis (PCA) coupled with linear discriminant analysis (LDA). Exploiting this approach, biomolecular signatures enabling sensitive and specific discrimination of methylation patterning were derived. For DNA sequence and methylation analysis, this approach has the potential to be an important tool, especially when material is scarce. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Ahead: Deflationary spiral or hyperinflation?
Authors: Wolf Fran , Tackett James A.
Published: October 2010

The 2010 G20 Summit revealed sharply contrasting views among the world's major economic allies. The United States supported another round of government spending to stimulate job growth. But Germany, the United Kingdom, and others advocated fiscal restraint and higher taxes. When the major powers cannot agree on how to fix the world's ailing economy, can we even predict what our economic future holds? And which is the greater threat: fiscal restraint, which could trigger another major economic downturn and a deflationary spiral, or endless deficits, which could lead to hyperinflation? The authors discuss business strategies that treasury managers can use for dealing with each of these difficulties. © 2010 Wiley Periodicals, Inc.
When oil spills, cash flows
Authors: Johnson Leigh Redd , Rudolph Holly R.
Published: October 2010

Nearly seven million businesses—in Alabama, Florida, Louisiana, Mississippi, and Texas—may have been affected by the explosion of BP's Deepwater Horizon oil drilling platform. The total costs to those businesses remain unknown. But losses will surely be staggering, since the annual sales volume for these firms is $5.2 trillion. Some companies may also fall victim to fraudulent scams in connection with the oil spill.

Because of an anticipated interruption in business and the likelihood of scams, companies should plan ahead for sustaining their operations. The authors examine strategies for improving short‐term cash flow and look at long‐term cash problems created by litigation for companies affected by the spill. They also take a close look at scams associated with this disaster—and show how companies can avoid them. © 2010 Wiley Periodicals, Inc.
Is it time for a world currency unit?
Authors: Fanning Kurt , Goldberg Stephen R. , Northway Bethany
Published: October 2010

A supranational currency, the World Currency Unit (Wocu), has recently been introduced. It creates potential opportunities for currency and risk management for businesses involved with international transactions. The authors look at the issues of a world currency, including how corporate treasurers can use it. © 2010 Wiley Periodicals, Inc.
States continue push to tax internet sales
Authors: Sonnier Blaise M.
Published: October 2010

State and local governments have relied on sales tax as a significant source of revenue. But the Internet has made them lose sales tax revenue, as online purchases have soared. So states are continuing a push to tax sales made over the Internet, which can impact companies' cash flow. Here's an update of the situation. © 2010 Wiley Periodicals, Inc.
Time‐driven activity‐based costing: An implementation in a manufacturing company
Authors: Öker Figen , Adigüzel Hümeyra
Published: October 2010

This article demonstrates the implementation of time‐driven activity‐based costing (TDABC) in a manufacturing company, showing how it can provide far more relevant information about product profitability and capacity utilization than standard costing. © 2010 Wiley Periodicals, Inc.
Poly(ADP‐ribose) polymerase 1 is involved in glucose toxicity through SIRT1 modulation in HepG2 hepatocytes
Authors: Pang Jing , Gong Huan , Xi Chao , Fan Wei , Dai Yang , Zhang Tie‐mei
Published: October 2010

Accelerated glucose metabolism leads to oxidative stress and DNA damage in cells; these effects are related to glucose toxicity. The precise mechanisms of glucose toxicity are still unclear. The aim of this work was to investigate the mechanism of poly(ADP‐ribose) polymerase 1 (PARP1), which is a DNA repair enzyme activated by high‐glucose‐induced oxidative stress, and its effect on glucose toxicity in HepG2 hepatocytes. HepG2 cells were cultured under normal (5.5 mM) or high (30 mM) glucose conditions for 4 days. PJ34, which is an inhibitor of PARP1, was used to determine the downstream effects of PARP1 activation. PARP1 activity in 30 mM‐glucose‐treated cells was more than that in 5.5 mM‐glucose‐treated cells, and the activity correlated with the increase in ROS generation and DNA damage. PJ34 suppressed PARP1 activation and prevented the high‐glucose‐induced suppression of SIRT1 and AMP‐activated protein kinase (AMPK) activity, which was similar to its effect on the restoration of intracellular nicotinamide adenine dinucleotide (NAD) content. Further, the phosphorylation of insulin receptor was attenuated in response to insulin stimulation under high glucose conditions, and PJ34 could reverse this effect. The results of transfection of HepG2 cells with PARP1 small interfering RNA were similar to those obtained by treatment of the cells with PARP1 inhibitor PJ34. These data suggest that high‐glucose‐induced PARP1 activation might play a role in glucose toxicity by down‐regulating SIRT1 and AMPK activity through NAD depletion and resulting in insulin insensitivity. J. Cell. Biochem. 112: 299–306, 2011. © 2010 Wiley‐Liss, Inc.
Perfect difference families, perfect difference matrices, and related combinatorial structures
Authors: Ge Gennian , Miao Ying , Sun Xianwei
Published: October 2010

The existence problems of perfect difference families with block size k, k=4,5, and additive sequences of permutations of length n, n=3,4, are two outstanding open problems in combinatorial design theory for more than 30 years. In this article, we mainly investigate perfect difference families with block size k=4 and additive sequences of permutations of length n=3. The necessary condition for the existence of a perfect difference family with block size 4 and order v, or briefly (v, 4,1)‐PDF, is v≡1(mod12), and that of an additive sequence of permutations of length 3 and order m, or briefly ASP (3, m), is m≡1(mod2). So far, (12t+1,4,1)‐PDFs with t<50 are known only for t=1,4−36,41,46 with two definiteexceptions of t=2,3, and ASP (3, m)'s with odd 3<m<200 are known only for m=5,7,13−29,35,45,49,65,75,85,91,95,105,115,119,121,125,133,135,145,147,161,169,175,189,195 with two definite exceptions of m=9,11. In this article, we show that a (12t+1,4,1)‐PDF exists for any t⩽1,000 except for t=2,3, and an ASP (3, m) exists for any odd 3<m<200 except for m=9,11 and possibly for m=59. The main idea of this article is to use perfect difference families and additive sequences of permutations with “holes”. We first introduce the concepts of an incomplete perfect difference matrix with a regular hole and a perfect difference packing with a regular difference leave, respectively. We show that an additive sequence of permutations is in fact equivalent to a perfect difference matrix, then describe an important recursive construction for perfect difference matrices via perfect difference packings with a regular difference leave. Plenty of perfect difference packings with a desirable difference leave are constructed directly. We also provide a general recursive construction for perfect difference packings, and as its applications, we obtain extensive recursive constructions for perfect difference families, some via incomplete perfect difference matrices with a regular hole. Examples of perfect difference packings directly constructed are used as ingredients in these recursive constructions to produce vast numbers of perfect difference families with block size 4. © 2010 Wiley Periodicals, Inc. J Combin Designs 18: 415–449, 2010
Removal of endocrine disrupting chemicals from contaminated industrial groundwater using chitin as a biosorbent
Authors: Krupadam Reddithota J. , Sridevi P. , Sakunthala Sistu
Published: October 2010

BACKGROUND: The removal of endocrine disrupting chemicals (EDCs) from drinking water is of great importance. Chitin isolated from crab shells was used as a biosorbent for removal of three EDCs—benzo(a)antracene, β‐estradiol and bisphenol A—present in contaminated groundwater. Fourier transform infra‐red (FTIR) spectroscopy, porosimetry and scanning electron microscope (SEM) were used for structural elucidation of chitin. Experiments were conducted using batch adsorption mode under different conditions of initial EDCs concentration, solution pH and adsorbent dose. The effect of chitin particle size was also investigated. Batch adsorption data were fitted to Langmuir and Freundlich isotherms and pseudo‐first‐ and second‐order models for kinetic study.

RESULTS: The maximum adsorption capacity of chitin by the three EDCs, calculated using the Langmuir equation, ranged from 42.9 to 84 mg g−1. The adsorption of EDCs by chitin was due primarily to electrostatic forces. The measurement of zeta potential indicated that chitin has higher surface charge in alkaline pH, resulting in reduction in removal of EDCs with increasing pH. Complementary to the experimental results molecular simulations were also performed on HyperChem software to understand the adsorption mechanism between chitin and EDCs in aquesous solutions. Chitin was regenerated after washing with methanol/acetic acid (1/3, v/v) and the regeneration process was repeated for 10 cycles, which showed 90% adsorption capacity retained by chitin.

CONCLUSIONS: The experimental results suggest that chitin could be employed as an adsorbent in the removal of EDCs from aqueous solutions, and the adsorption potential of used chitin can be recovered by methanol/acetic acid solvent washings. The reusable biosorbent chitin would be cost‐effective and a better option for future water remediation endeavours. Copyright © 2010 Society of Chemical Industry
A nano‐Fe0/ACF cathode applied to neutral electro‐Fenton degradation of Orange II
Authors: Li Hualiang , Lei Hengyi , Chen Kai , Yao Chuang , Zhang Xinghong , Leng Qi , Wang Wei
Published: October 2010

BACKGROUND: To extend the working pH range of electro‐Fenton, activated carbon fibre cathode (ACF) supported nano‐Fe0 catalysts were prepared by electo‐deposition and applied to treat a dye effluent in a neutral aqueous medium. The morphology of the Fe0/ACF cathode was characterized by X‐ray diffraction and scanning electron microscopy. The experimental conditions, such as the initial pH, dye concentration and current density of the degradation of the dye effluent were investigated. A pseudo‐first‐order model was used to simulate the experimental results.

RESULTS: Compared with Fe2+ and Fe3+, the degradation of Orange II solution by an Fe0/ACF cathode was shown to achieve much better results at neutral pH values. Additionally, the dissolved iron concentration in the solution was also evaluated. The results showed that 0.79 mg L−1 of total iron was present after 120 min reaction time at neutral pH, suggesting that the Fe0/ACF cathode can be used for a long time and is reusable under neutral pH conditions. A complete description of the possible degradation mechanism in solution is provided.

CONCLUSION: The high catalytic activity of this cathode is probably due to the efficient electrotransfer at the Fe0/iron oxide interface. All this demonstrates that Fe0/ACF is a good and promising cathode for the neutral electro‐Fenton process. Copyright © 2010 Society of Chemical Industry
Gene knockdown with lentiviral vector‐mediated intrathecal RNA interference of protein kinase C gamma reverses chronic morphine tolerance in rats
Authors: Song Zongbin , Zou Wangyuan , Liu Chang , Guo Qulian
Published: October 2010

Background

Although morphine is a widely used opioid analgesic, morphine tolerance (MT) has limited the use of the drug because it creates the necessity for high doses. Protein kinase C (PKC), especially the PKCγ isoform, is considered to play a key role in the development of MT. Because RNA interference provides a powerful method for the investigation of gene function, and lentiviral delivery systems have been approved for human use, this present study examined rats tolerant to morphine to determine whether an intrathecal injection of a lentiviral vector of PKCγ short hairpin RNA (LV‐shPKCγ) down‐regulated the expression of the PKCγ gene and reversed MT.

Methods

MT was induced by intrathecal morphine (10 µg b.i.d.) for six consecutive days. A lentiviral‐mediated short hairpin RNA (shRNA) system was synthesized to deliver the PKCγ shRNAs to the spinal cord of the rats with MT. Mechanical and thermal paw withdrawal threshold were assessed to determine the analgesic effects of morphine. Expression of PKCγ mRNA and protein was determined by reverse transcriptase‐polymerase chain reaction and western blotting analysis, respectively.

Results

The chronic administration of morphine induced a stabilized analgesic tolerance. A single injection of LV‐shPKCγ significantly reversed morphine antinociceptive tolerance. Compared to the control group, PKCγ mRNA and protein levels were dramatically down‐regulated in the LV‐shPKCγ group.

Conclusions

A single injection of LV‐shPKCγ reversed MT by reducing the expression of PKCγ in the spinal cord. These findings indicate that the use of LV‐shPKCγ might be a potential strategy for therapy in MT. Copyright © 2010 John Wiley & Sons, Ltd.
Characterization of a dockerin‐based affinity tag: application for purification of a broad variety of target proteins
Authors: Demishtein Alik , Karpol Alon , Barak Yoav , Lamed Raphael , Bayer Edward A.
Published: October 2010

Cellulose, a major component of plant matter, is degraded by a cell surface multiprotein complex called the cellulosome produced by several anaerobic bacteria. This complex coordinates the assembly of different glycoside hydrolases, via a high‐affinity Ca2+‐dependent interaction between the enzyme‐borne dockerin and the scaffoldin‐borne cohesin modules. In this study, we characterized a new protein affinity tag, ΔDoc, a truncated version (48 residues) of the Clostridium thermocellum Cel48S dockerin. The truncated dockerin tag has a binding affinity (KA) of 7.7 × 108 M−1, calculated by a competitive enzyme‐linked assay system. In order to examine whether the tag can be used for general application in affinity chromatography, it was fused to a range of target proteins, including Aequorea victoria green fluorescent protein (GFP), C. thermocellum β‐glucosidase, Escherichia coli thioesterase/protease I (TEP1), and the antibody‐binding ZZ‐domain from Staphylococcus aureus protein A. The results of this study significantly extend initial studies performed using the Geobacillus stearothermophilus xylanase T‐6 as a model system. In addition, the enzymatic activity of a C. thermocellum β‐glucosidase, purified using this approach, was tested and found to be similar to that of a β‐glucosidase preparation (without the ΔDoc tag) purified using the standard His‐tag. The truncated dockerin derivative functioned as an effective affinity tag through specific interaction with a cognate cohesin, and highly purified target proteins were obtained in a single step directly from crude cell extracts. The relatively inexpensive beaded cellulose‐based affinity column was reusable and maintained high capacity after each cycle. This study demonstrates that deletion into the first Ca2+‐binding loop of the dockerin module results in an efficient and robust affinity tag that can be generally applied for protein purification. Copyright © 2010 John Wiley & Sons, Ltd.
Proteolysis activity of IgM antibodies from rheumatoid arthritis patients' sera: evidence of atypical catalytic site
Authors: Kamalanathan A. S. , Goulvestre C. , Weill B. , Vijayalakshmi M. A.
Published: October 2010

The IgM antibodies from rheumatoid arthritis (RA) patients' sera were screened for peptide hydrolyzing activity. Recovery of structurally intact IgM antibodies (Abs), in a single step, was achieved using a weak anion‐exchange methacrylate monolith disk. The IgM Abs from patients' sera hydrolyzed the Pro‐Phe‐Arg‐4‐methyl‐coumaryl‐7‐amide (PFR‐MCA) substrate appreciably compared to the healthy donors. The apparent Km values of IgM Abs from patients' sera were between 0.4 and 0.7 mM. Furthermore, IgM Abs displayed 5 to 10‐folds greater proteolysis activity than IgG Abs, recovered from the same pathological serum. The proteolysis activity, as a function, was found to be independent of IgM‐RF titer value. Affinity labeling approach targeted at the catalytic site histidine was studied, using a specific irreversible inhibitor, N‐α‐tosyl‐L‐lysine chloromethyl ketone (TLCK). Despite modification of catalytic His, observation of serine protease like activity suggest presence of an atypical catalytic framework in a few pathological IgM Abs. Copyright © 2010 John Wiley & Sons, Ltd.
Bead‐based protein–protein interaction assays for the analysis of Rho GTPase signaling
Authors: Rimmele Stefanie , Gierschik Peter , Joos Thomas O. , Schneiderhan‐Marra Nicole
Published: October 2010

Bead‐based interaction assays are excellently suited to study protein–protein interactions, as they require only minimal amounts of sample material. Miniaturized protein–protein interaction assays were designed to analyze Rho GTPase activation based on its interaction with Rho GDI or p21‐activated kinase (PAK).

Rho GDI plays a key role in the regulation of a variety of cellular functions through its interaction with Rho GTPases. Rho GDI is frequently overexpressed in many human cancers. Therefore, there is a growing and as yet unfulfilled demand for screening assays to identify biologically active compounds that may inhibit the Rho GTPase–Rho GDI interaction. Bead‐based interaction assays provide an interesting alternative that facilitate such assays to be performed faster with only small amounts of material compared to routinely used co‐immunoprecipitation followed by Western Blot analysis.

Bead‐based protein interaction assays for overexpressed HA‐tagged Rho GTPases were established to study the GTPγS‐dependent interaction of five different Rho GTPases with the regulatory protein Rho GDIα and the downstream effector PAK1. In addition, it was demonstrated that the ability of Rho GTPases to interact with Rho GDI in this experimental system was markedly, but differentially sensitive to post‐translational modification of their carboxyl terminus. Importantly, this modification also notably affected the ability of Rac1 and Rac2, but not of Cdc42, to interact with PAK1. Copyright © 2010 John Wiley & Sons, Ltd.
In vitro studies with mammalian cell lines and gum arabic‐coated magnetic nanoparticles
Authors: Bicho Ana , Roque Ana Cecília A. , Cardoso Ana Sofia , Domingos Patrícia , Batalha Íris Luz
Published: October 2010

Iron oxide magnetic nanoparticles (MNPs) were synthesized by the chemical co‐precipitation method and coated with gum arabic (GA) by physical adsorption and covalent attachment. Cultures of mammalian cell lines (HEK293, CHO and TE671) were grown in the presence of uncoated and GA‐coated MNPs. Cellular growth was followed by optical microscopy in order to assess the proportion of cells with particles, alterations in cellular density and the presence of debris. The in vitro assays demonstrated that cells from different origins are affected differently by the presence of the nanoparticles. Also, the methods followed for GA coating of MNPs endow distinct surface characteristics that probably underlie the observed differences when in contact with the cells. In general, the nanoparticles to which the GA was adsorbed had a smaller ability to attach to the cells' surface and to compromise the viability of the cultures. Copyright © 2010 John Wiley & Sons, Ltd.
Capture of human monoclonal antibodies from a clarified cell culture supernatant by phenyl boronate chromatography
Authors: Azevedo Ana M. , Gomes A. G. , Borlido L. , Santos I. F. S. , Prazeres D. M. F. , Aires‐Barros M. R.
Published: October 2010

In this work, we investigated the feasibility of using phenyl boronate (PB) chromatography for the direct capture of monoclonal antibodies from a CHO cell supernatant. Preliminary results, using pure protein solutions have shown that PB media can bind to human antibodies, not only at strong alkaline conditions but also at acidic pH values. In fact, antibodies have been found to bind in the pH range 5.5–8.5. On the other hand, insulin and human serum albumin did not bind at alkaline pH but at lower pH, which reflects the importance of non‐specific interactions with the matrix. Different binding and eluting buffers were evaluated for the capture of immunoglobulin G (IgG) from a CHO cell supernatant and the most promising results were obtained using 20 mM 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid at pH 8.5 as binding buffer and 1.5 M Tris–HCl as eluting buffer. Using a step elution, all IgG was recovered in the elution pool with a maximum purification factor of 56. A gradient elution allowed a further increase of the final purity, yet achieving a slightly lower yield. IgG recovery was around 85% and the purification factor was 76. The highest purity was obtained when the pH of the cell supernatant feed was previously adjusted to 8.5. Starting from an initial protein purity of 1.1% and high‐performance liquid chromatography (HPLC) purity of 2.2%, after PB adsorption, a final protein purity of 85% and a HPLC purity of 88% was achieved. Copyright © 2010 John Wiley & Sons, Ltd.
A new affinity approach to isolateEscherichia coli6S RNA with histidine‐chromatography
Authors: Martins R. , Queiroz J. A. , Sousa F.
Published: October 2010

6S RNA is an abundant non‐coding RNA inEscherichia coli(E.coli), but its function has not been discovered until recently. The first advance on 6S RNA function was the demonstration of its ability to bind the σ70‐holoenzyme form of RNA polymerase, inhibiting its activity and consequently the transcription process. The growing interest in the investigation of non‐coding small RNAs (sRNA) calls for the development of new methods for isolation and purification of RNA. This work presents an optimized RNA extraction procedure and describes a new affinity chromatography method using a histidine support to specifically purify 6S RNA from otherE. colisRNA species. The RNA extraction procedure was optimized, and a high yield was obtained in the separation of sRNA and ribosomal RNA (rRNA) from total RNA (RNAt). This improved method takes advantage of its simplicity and significant cost reduction, since some complex operations have been eliminated. A purification strategy was also developed to separate 6S RNA from an sRNA mixture. Pure RNA can be advantageously obtained using the histidine‐affinity chromatography method, aiming at its application to structural or functional studies. Copyright © 2010 John Wiley & Sons, Ltd.
Solid‐phase preparation of protein complexes
Authors: Pengo Paolo , Veggiani Gianluca , Rattanamanee Kwanchai , Gallotta Andrea , Beneduce Luca , Fassina Giorgio
Published: October 2010

Protein–protein conjugation is usually achieved by solution phase methods requiring concentrated protein solution and post‐synthetic purification steps. In this report we describe a novel continuous‐flow solid‐phase approach enabling the assembly of protein complexes minimizing the amount of material needed and allowing the repeated use of the same solid phase. The method exploits an immunoaffinity matrix as solid support; the matrix reversibly binds the first of the complex components while the other components are sequentially introduced, thus allowing the complex to grow while immobilized. The tethering technique employed relies on the use of the very mild synthetic conditions and fast association rates allowed by the avidin–biotin system. At the end of the assembly, the immobilized complexes can be removed from the solid support and recovered by lowering the pH of the medium. Under the conditions used for the sequential complexation and recovery, the solid phase was not damaged or irreversibly modified and could be reused without loss of binding capacity. The method was specifically designed to prepare protein complexes to be used in immunometric methods of analysis, where the immunoreactivity of each component needs to be preserved. The approach was successfully exploited for the preparation of two different immunoaffinity reagents with immunoreactivity mimicking native squamous cell carcinoma antigen‐immunoglobulin M (SCCA‐IgM) and alphafetoprotein‐immunoglobulin M (AFP‐IgM) immune complexes, which were characterized by dedicated sandwich enzyme‐linked immunosorbent assay (ELISA) and immunoblot. Besides the specific application described in the paper, the method is sufficiently general to be used for the preparation of a broad range of protein assemblies. Copyright © 2010 John Wiley & Sons, Ltd.
Optimization of high‐resolution USPIO magnetic resonance imaging at 4.7 T using novel phantom with minimal structural interference
Authors: Antell Henrik , Numminen Jussi , Abo‐Ramadan Usama , Niemelä Mika R. , Hernesniemi Juha A. , Kangasniemi Marko
Published: October 2010

Purpose:

To characterize the effect of ultrasmall superparamagnetic iron oxides (USPIOs) on magnetic resonance imaging (MRI) signal at 4.7 T, and to find the highest sensitivity pulse sequence for high‐resolution USPIO MRI.

Materials and Methods:

A novel phantom was constructed for optimization of sequence parameters for neuroradiological MR applications, and a wide range of dilutions of the USPIO ferumoxtran‐10 was imaged using T2/T1‐, T1‐, T2‐, T* 2‐, and PD‐weighted sequences. The effect of varying sequence parameters was investigated using phantom measurements and simulations.

Results:

The relaxivities r1, r2, and r*2 of ferumoxtran‐10 at 4.7 T (21°C) were 5.1, 82.2, and 148.4 mmol−1 L s−1, respectively. Gradient echo sequences produced superior susceptibility artifacts at high concentrations; susceptibility artifacts were seen down to a concentration of 137 nmol Fe/mL. A concentration of 17.5 μmol Fe/mL caused a signal void independently of sequence and parameters, and at concentrations ≤273 nmol Fe/mL no signal void was caused. Signal enhancement on T1‐weighted imaging was seen only at concentrations 137–547 nmol Fe/mL. For the same effective echo time T2‐weighted rapid acquisition with relaxation enhancement (RARE) yielded significantly higher contrast‐to‐noise ratio with RARE factor 16 than with RARE factor 8.

Conclusion:

At nanomolar concentrations of USPIO, steady‐state free precession offers an alternative to T2‐ and T* 2‐weighted sequences. Optimum parameters depend highly on USPIO concentration. J. Magn. Reson. Imaging 2010;32:1184–1196. © 2010 Wiley‐Liss, Inc.
Peripheral hyperintense pattern on T2‐weighted magnetic resonance imaging (MRI) in breast carcinoma: Correlation with early peripheral enhancement on dynamic MRI and histopathologic findings
Authors: Kawashima Hiroko , Kobayashi‐Yoshida Miki , Matsui Osamu , Zen Yoh , Suzuki Masayuki , Inokuchi Masafumi
Published: October 2010

Purpose

To investigate the correlation between the peripheral hyperintense pattern of breast carcinoma on T2‐weighted images (T2WI) and the early peripheral enhancement (EPE) on dynamic magnetic resonance imaging (MRI) and to examine the histological characteristics involved in the causes thereof.

Materials and Methods

The signal intensity of 131 breast carcinomas of common type on T2WI was classified into four types and the correlations between the signal intensity and the histopathological subtypes were evaluated. We investigated correlation with the peripheral hyperintense pattern on a T2WI and other findings on MR images including EPE. In addition, we also investigated the histopathological characteristics in the lesions with a peripheral hyperintense pattern.

Results

The signal intensities of the lesions on T2WI demonstrated hyperintensity, isointensity, hypointensity, and a peripheral hyperintense pattern in 26 (20%), 66 (50%), 19 (15%), and 20 (15%) lesions, respectively. A peripheral hyperintense pattern correlated with the appearance of EPE (P < 0.0001) and the degree of central fibrosis (P < 0.0001).

Conclusion

A peripheral hyperintense pattern on T2WI is a characteristic finding observed in breast carcinomas and correlated with the appearance of EPE. The appearance of a peripheral hyperintense pattern was affected by various histopathological features in the same manner as that observed for the formation of EPE. J. Magn. Reson. Imaging 2010;32:1117–1123. © 2010 Wiley‐Liss, Inc.
Real‐time diffusion‐perfusion mismatch analysis in acute stroke
Authors: Straka Matus , Albers Gregory W. , Bammer Roland
Published: October 2010

Diffusion‐perfusion mismatch can be used to identify acute stroke patients that could benefit from reperfusion therapies. Early assessment of the mismatch facilitates necessary diagnosis and treatment decisions in acute stroke. We developed the RApid processing of PerfusIon and Diffusion (RAPID) for unsupervised, fully automated processing of perfusion and diffusion data for the purpose of expedited routine clinical assessment. The RAPID system computes quantitative perfusion maps (cerebral blood volume, CBV; cerebral blood flow, CBF; mean transit time, MTT; and the time until the residue function reaches its peak, Tmax) using deconvolution of tissue and arterial signals. Diffusion‐weighted imaging/perfusion‐weighted imaging (DWI/PWI) mismatch is automatically determined using infarct core segmentation of ADC maps and perfusion deficits segmented from Tmax maps. The performance of RAPID was evaluated on 63 acute stroke cases, in which diffusion and perfusion lesion volumes were outlined by both a human reader and the RAPID system. The correlation of outlined lesion volumes obtained from both methods was r2 = 0.99 for DWI and r2 = 0.96 for PWI. For mismatch identification, RAPID showed 100% sensitivity and 91% specificity. The mismatch information is made available on the hospital's PACS within 5–7 min. Results indicate that the automated system is sufficiently accurate and fast enough to be used for routine care as well as in clinical trials. J. Magn. Reson. Imaging 2010;32:1024–1037. © 2010 Wiley‐Liss, Inc.
Method for simultaneous voxel‐based morphometry of the brain and cervical spinal cord area measurements using 3D‐MDEFT
Authors: Freund Patrick A.B. , Dalton Catherine , Wheeler‐Kingshott Claudia A.M. , Glensman Janice , Bradbury David , Thompson Alan J. , Weiskopf Nikolaus
Published: October 2010

Purpose

To investigate whether a 3D‐modified driven equilibrium Fourier transform (MDEFT)‐based acquisition protocol established for brain morphometry also yields reliable information about the cross‐sectional spinal cord area (SCA).

Materials and Methods

Images of brain and cervical cord of 10 controls and eight subjects with spinal cord injury (SCI) were acquired with the 3D‐MDEFT‐based imaging protocol and an 8‐channel receive head coil. The new protocol was validated by two observers 1) comparing the SCA measured with the standard acquisition protocol (3D magnetization‐prepared rapid acquisition gradient echo [MPRAGE] and dedicated spine coil) and the new protocol; and 2) determining the scan–rescan reproducibility of the new protocol.

Results

Scan–rescan reproducibility of SCA measurements with the MDEFT approach showed a similar precision for both observers with standard deviation (SD) <4.5 mm2 and coefficient of variation (CV) ≤5.1%. Analysis of variance (ANOVA) revealed a main effect of observer and interaction between observer and scan protocol that could be primarily attributed to a small observer bias for MPRAGE (difference in SCA <2.1 mm2). No bias was observed for 3D‐MDEFT vs. 3D‐MPRAGE.

Conclusion

The 3D‐MDEFT method allows for robust unbiased assessment of SCA in addition to brain morphology. J. Magn. Reson. Imaging 2010;32:1242–1247. © 2010 Wiley‐Liss, Inc.
Fast algorithm for calculation of inhomogeneity gradient in magnetic resonance imaging data
Authors: Hui Cheukkai , Zhou Yu Xiang , Narayana Ponnada
Published: October 2010

Purpose

To develop and implement a new approach for correcting the intensity inhomogeneity in magnetic resonance imaging (MRI) data.

Materials and Methods

The algorithm is based on the assumption that intensity inhomogeneity in MR data is multiplicative and smoothly varying. Using a statistically stable method, the algorithm first calculates the partial derivative of the inhomogeneity gradient across the data. The algorithm then solves for the gradient field and fits it to a parametric surface. It was tested on both simulated and real human and animal MRI data.

Results

The algorithm is shown to restore the homogeneity in all images that were tested. On real human brain images the algorithm demonstrated superior or comparable performance relative to some of the commonly used intensity inhomogeneity correction methods such as SPM, BrainSuite, and N3.

Conclusion

The proposed algorithm provides an alternative method for correcting the intensity inhomogeneity in MR images. It is shown to be fast and its performance is superior or comparable to algorithms described in the published literature. Due to its generality, this algorithm is applicable to MR images of both humans and animals. J. Magn. Reson. Imaging 2010;32:1197–1208. © 2010 Wiley‐Liss, Inc.
Local SAR reduction in parallel excitation based on channel‐dependent Tikhonov parameters
Authors: Cloos Martijn Anton , Luong Michel , Ferrand Guillaume , Amadon Alexis , Le Bihan Denis , Boulant Nicolas
Published: October 2010

Purpose

To reduce the local specific absorption rate (SAR) obtained with tailored pulses using parallel transmission while obtaining homogenous flip angle distributions.

Materials and Methods

Finite‐element simulations on a human head model were performed to obtain the individual magnetic and electric field maps for each channel of a parallel transmit array. From those maps, SAR calculations were carried out for “spoke” pulses designed to homogenize the flip angle in an axial slice of a human brain at 7 T. Based on the assumption that the coil element nearest to the maximum local energy deposition is the dominant contributor to the corresponding hot spot, a set of channel‐dependent Tikhonov parameters is optimized. Resulting SAR distributions are compared to the ones obtained when using standard pulse design approaches based on a single Tikhonov parameter.

Results

In both the small‐ and large‐tip‐angle domain, the simulations show local SAR reductions by over a factor of 2 (4) for a well‐centered (off‐centered) head model at the expense of roughly 1% increment in flip‐angle spread over the slice.

Conclusion

Significant SAR reductions can be obtained by optimizing channel‐dependent Tikhonov parameters based on the relation between coil elements and SAR hot spot positions. J. Magn. Reson. Imaging 2010;32:1209–1216. © 2010 Wiley‐Liss, Inc.
Semi‐automatic deformable registration of prostate MR images to pathological slices
Authors: Mazaheri Yousef , Bokacheva Louisa , Kroon Dirk‐Jan , Akin Oguz , Hricak Hedvig , Chamudot Daniel , Fine Samson , Koutcher Jason A.
Published: October 2010

Purpose

To present a semi‐automatic deformable registration algorithm for co‐registering T2‐weighted (T2w) images of the prostate with whole‐mount pathological sections of prostatectomy specimens.

Materials and Methods

Twenty‐four patients underwent 1.5 Tesla (T) endorectal MR imaging before radical prostatectomy with whole‐mount step‐section pathologic analysis of surgical specimens. For each patient, the T2w imaging containing the largest area of tumor was manually matched with the corresponding pathologic slice. The prostate was co‐registered using a free‐form deformation (FFD) algorithm based on B‐splines. Registration quality was assessed through differences between prostate diameters measured in right–left (RL) and anteroposterior (AP) directions on T2w images and pathologic slices and calculation of the Dice similarity coefficient, D, for the whole prostate (WP), the peripheral zone (PZ) and the transition zone (TZ).

Results

The mean differences in diameters measured on pathology and MR imaging in the RL direction and the AP direction were 0.49 cm and −0.63 cm, respectively, before registration and 0.10 cm and −0.11 cm, respectively, after registration. The mean D values for the WP, PZ and TZ, were 0.76, 0.65, and 0.77, respectively, before registration and increased to 0.91, 0.76, and 0.85, respectively, after registration. The improvements in D were significant for all three tissues (P < 0.001 for all).

Conclusion

The proposed semi‐automatic method enabled successful co‐registration of anatomical prostate MR images to pathologic slices. J. Magn. Reson. Imaging 2010;32:1149–1157. © 2010 Wiley‐Liss, Inc.