Sample preparation of culture medium from Madin-Darby canine kidney cells

A reproducible, standardized and simple sample preparation methodology is the key to successful two-dimensional gel electrophoresis (2-DE). This chapter describes step-by-step the sample preparation of culture medium from Madin-Darby canine kidney (MDCK) cells. Tips and tricks are given to circumvent possible pitfalls.

Analysis of a novel waste heat recovery mechanism for an I.C. engine

Typical internal combustion engines lose about 75% of the fuel energy through the engine coolant, exhaust and surface radiation. Most of the heat generated comes from converting the chemical energy in the fuel to mechanical energy and in turn thermal energy is produced. In general, the thermal energy is unutilized and thus wasted. This report describes the analysis of a novel waste heat recovery (WHR) system that operates on a Rankine cycle. This novel WHR system consists of a second piston within the existing piston to reduce losses associated with compression and exhaust strokes in a four-cycle engine. The wasted thermal energy recovered from the coolant and exhaust systems generate a high temperature and high pressure working fluid which is used to power the modified piston assembly. Cycle simulation shows that a large, stationary natural gas spark ignition engine produces enough waste heat to operate the novel WHR system. With the use of this system, the stationary gas compression ignition engine running at 900 RPM and full load had a net increase of 177.03 kW (240.7 HP). This increase in power improved the brake fuel conversion efficiency by 4.53%.

Performance evaluation of a novel asymmetric capacitor using a light-weight, carbon foam supported nickel electrode

Electrochemical capacitors have been an important development in recent years in the field of energy storage. Capacitors can be developed by utilizing either double layer capacitance at the electrode/solution interfaces alone or in combination with a battery electrode associated with a faradic redox process in one electrode. An asymmetric capacitor consisting of electrochemically deposited nickel hydroxide, supported on carbon foam as a positive electrode and carbon sheet as a negative electrode has been successfully assembled and cycled. One objective of this study has been to demonstrate the viability of the nickel carbon foam positive electrode, especially in terms of cycle life. Electrochemical characterization shows stable, high cycle performance in 26 wt. % KOH electrolyte with a maximum energy density of 4.1 Wh/Kg and a relaxation time constant of 6.24 s. This cell has demonstrated high cycle life, 14,500 cycles, with efficiency better than 98%. In addition, the cell failure mechanism and self-discharge behavior of the aforesaid capacitor are analyzed.

Preparation of highly-ordered TiO₂ nanotube arrays and their applications in Dye-sensitized solar cells

Titanium oxide is an important semiconductor, which is widely applied for solar cells. In this research, titanium oxide nanotube arrays were synthesized by anodization of Ti foil in the electrolyte composed of ethylene glycol containing 2 vol % H2O and 0.3 wt % NH4F. The voltages of 40V-50V were employed for the anodizing process. Pore diameters and lengths of the TiO2 nanotubes were evaluated by field emission scanning electron microscope (FESEM). The obtained highly-ordered titanium nanotube arrays were exploited to fabricate photoelectrode for the Dye-sensitized solar cells (DSSCS). The TiO2 nanotubes based DSSCS exhibited an excellent performance with a high short circuit current and open circuit voltage as well as a good power conversion efficiency. Those can be attributed to the high surface area and one dimensional structure of TiO2 nanotubes, which could hold a large amount of dyes to absorb light and help electron percolation process to hinder the recombination during the electrons diffusion in the electrolyte.

Structural analysis of B-Box 2 from MuRF1: identification of a novel self-association pattern in a RING-like fold

The B-box motif is the defining feature of the TRIM family of proteins, characterized by a RING finger-B-box-coiled coil tripartite fold. We have elucidated the crystal structure of B-box 2 (B2) from MuRF1, a TRIM protein that supports a wide variety of protein interactions in the sarcomere and regulates the trophic state of striated muscle tissue. MuRF1 B2 coordinates two zinc ions through a cross-brace alpha/beta-topology typical of members of the RING finger superfamily. However, it self-associates into dimers with high affinity. The dimerization pattern is mediated by the helical component of this fold and is unique among RING-like folds. This B2 reveals a long shallow groove that encircles the C-terminal metal binding site ZnII and appears as the defining protein-protein interaction feature of this domain. A cluster of conserved hydrophobic residues in this groove and, in particular, a highly conserved aromatic residue (Y133 in MuRF1 B2) is likely to be central to this role. We expect these findings to aid the future exploration of the cellular function and therapeutic potential of MuRF1.

Value of a novel Neisseria meningitidis--specific polymerase chain reaction assay in skin biopsy specimens as a diagnostic tool in chronic meningococcemia

BACKGROUND: Chronic meningococcemia (CM) is a diagnostic challenge. Skin lesions are frequent but in most cases nonspecific. Polymerase chain reaction (PCR)-based diagnosis has been validated in blood and cerebrospinal fluid for acute Neisseria meningitidis infection, in patients in whom routine microbiologic tests have failed to isolate the bacteria. In 2 patients with CM, we established the diagnosis by a newly developed PCR-based approach performed on skin biopsy specimens. OBSERVATIONS: Two patients presented with fever together with systemic and cutaneous manifestations suggestive of CM. Although findings from blood cultures remained negative, we were able to identify N meningitidis in the skin lesions by a newly developed PCR assay. In 1 patient, an N meningitidis strain of the same serogroup was also isolated from a throat swab specimen. Both patients rapidly improved after appropriate antibiotherapy. CONCLUSIONS: To our knowledge, we report the first cases of CM diagnosed by PCR testing on skin biopsy specimens. It is noteworthy that, although N meningitidis-specific PCR is highly sensitive in blood and cerebrospinal fluid in acute infections, our observations underscore the usefulness of PCR performed on skin lesions for the diagnosis of chronic N meningitidis infections. Whenever possible, this approach should be systematically employed in patients for whom N meningitidis infection cannot be confirmed by routine microbiologic investigations.

Antiaggregatory and proangiogenic effects of a novel recombinant human dual specificity anti-integrin antibody

BACKGROUND: beta(3)-Integrins are involved in platelet aggregation via alpha(IIb)beta(3) [glycoprotein (GP)IIb-GPIIIa], and in angiogenesis via endothelial alpha(V)beta(3). Cross-reactive ligands with antiaggregatory and proangiogenic effects, both desirable in peripheral vasculopathies, have not yet been described. OBJECTIVES: In vitro and in vivo characterization of antiaggregatory and proangiogenic effects of two recombinant human Fab fragments, with emphasis on beta(3)-integrins. METHODS: Recombinant Fab fragments were obtained by phage display technology. Specificity, affinity and IC(50) were determined by immunodot assays, enzyme-linked immunosorbent assay (ELISA), and Scatchard plot analysis, and by means of human umbilical vein endothelial cells (HUVECs). Functional analyses included ELISA for interaction with fibrinogen binding to GPIIb-GPIIIa, flow cytometry for measurement of activation parameters and competitive inhibition experiments, human platelet aggregometry, and proliferation, tube formation and the chorioallantoic membrane (CAM) assay for measurement of angiogenic effects. RESULTS: We observed specific and high-affinity binding to an intact GPIIb-GPIIIa receptor complex of two human Fab autoantibody fragments, with no platelet activation. Dose-dependent fibrinogen binding to GPIIb-GPIIIa and platelet aggregation were completely inhibited. One Fab fragment was competitively inhibited by abciximab and its murine analog monoclonal antibody (mAb) 7E3, whereas the other Fab fragment bound to cultured HUVECs, suggesting cross-reactivity with alpha(V)beta(3), and also demonstrated proangiogenic effects in tube formation and CAM assays. CONCLUSIONS: These Fab fragments are the first entirely human anti-GPIIb-GPIIIa Fab fragments with full antiaggregatory properties; furthermore, they do not activate platelets. The unique dual-specificity anti-beta(3)-integrin Fab fragment may represent a new tool for the study and management of peripheral arterial vasculopathies.

[Preoperative preparation of patients with cardiovascular disease for noncardiac surgery]

Preoperative preparation of patients with cardiovascular disease is best initiated by the general practitioner. Updated Guidelines on Perioperative Cardiovascular Evaluation and Care for Noncardiac Surgery have been published by the American Heart Association und American College of Cardiology (2007). Individual cardiac evaluation must take into account active cardiac conditions, functional capacity, additional clinical risk factors and surgical risk. Stable, asymptomatic patients with normal functional capacity can proceed to elective anesthesia and surgery without further cardiac evaluation. Active cardiac conditions require evaluation and treatment by a cardiology service prior to elective surgery. In stable patients with poor (<4 metabolic equivalents, MET) or unknown functional capacity and clinical risk factors, who are scheduled for intermediate- or high-risk surgery, further cardiac evaluation and preparation is to be considered. Established indicated beta blocker and statin medication is to be continued; timely institution of beta blocker medication (target heart rate, <65 bpm) may be required depending on the risk of surgery, the presence of coronary heart disease, and the number of clinical risk factors present. Following percutaneous coronary intervention, specific waiting periods are required prior to elective surgery. In patients on antiplatelet therapy, the risk of stopping it should be weighed against the benefit of reduction in bleeding complications from the planned surgery.

Regioselective preparation of 5-hydroxypropranolol and 4'-hydroxydiclofenac with a fungal peroxygenase

An extracellular peroxygenase of Agrocybe aegerita catalyzed the H(2)O(2)-dependent hydroxylation of the multi-function beta-adrenergic blocker propranolol (1-naphthalen-1-yloxy-3-(propan-2-ylamino)propan-2-ol) and the non-steroidal anti-inflammatory drug diclofenac (2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetic acid) to give the human drug metabolites 5-hydroxypropranolol (5-OHP) and 4'-hydroxydiclofenac (4'-OHD). The reactions proceeded regioselectively with high isomeric purity and gave the desired 5-OHP and 4'-OHD in yields up to 20% and 65%, respectively. (18)O-labeling experiments showed that the phenolic hydroxyl groups in 5-OHP and 4'-OHD originated from H(2)O(2), which establishes that the reaction is mechanistically a peroxygenation. Our results raise the possibility that fungal peroxygenases may be useful for versatile, cost-effective, and scalable syntheses of drug metabolites.

Odorless Preparation Of Thioglycosides And Thio Michael Adducts Of Carbohydrate Derivatives

A generalized, odorless, one-pot methodology has been developed for the preparation of 1,2-trans-thioglycosides and thio-Michael addition products of carbohydrate derivatives through triphenyl phosphine mediated cleavage of disulfides and reaction of the thiolate formed in situ with glycosyl bromides and glycosyl conjugated alkenes.

Characterization of a novel picornavirus isolate from a diseased European eel (Anguilla anguilla)

A novel picornavirus was isolated from specimens of a diseased European eel (Anguilla anguilla). This virus induced a cytopathic effect in eel embryonic kidney cells and high mortality in a controlled transmission study using elvers. Eel picornavirus has a genome of 7,496 nucleotides that encodes a polyprotein of 2,259 amino acids. It has a typical picornavirus genome layout, but its low similarity to known viral proteins suggests a novel species in the family Picornaviridae.

Evaluation of a Novel Barrier—Type Teat Dip Product Applied to Ewes Post-Weaning

Teats of 36 ewes (72 udder halves or teats) were dipped with an experimental barrier - type teat dip product to evaluate product persistency post weaning. Persistency was evaluated one to two times/day and scored positive if the teat end orifice was covered and protected. Persistency or the percentage of teats covered/protected at 36, 54, 72, 96, 132, and 156 hours was 100%, 93%, 89%, 63%, 35%, and 24% respectively. Ewes will be dipped again pre-lambing and both persistency and bacteriology (mastitis prevention) will be evaluated compared to 36 control ewes.

Clinical outcomes following subgingival application of a novel erythritol powder by means of air polishing in supportive periodontal therapy: a randomized, controlled clinical study

OBJECTIVES The aim of this prospective, randomized, controlled clinical study was to compare the clinical outcomes of the subgingival treatment with erythritol powder by means of an air-polishing (EPAP) device and of scaling and root planing (SRP) during supportive periodontal therapy (SPT). METHOD AND MATERIALS 40 patients enrolled in SPT were randomly assigned to two groups of equal size. Sites had to show signs of inflammation (bleeding on probing [BOP]-positive) and a probing pocket depth (PPD) of ≥ 4 mm, however, without presence of detectable subgingival calculus. During SPT, these sites were treated with EPAP or SRP, respectively. Full mouth and site-specific plaque indices, BOP, PPD, and clinical attachment level (CAL) were recorded at baseline (BL) and at 3 months, whereas the percentage of study sites positive for BOP (BOP+) was considered as primary outcome variable. Additionally, patient comfort using a visual analog scale (VAS) and the time needed to treat per site was evaluated. RESULTS At 3 months, mean BOP level measured 45.1% at test sites and 50.6% at control sites, respectively, without a statistically significant difference between the groups (P > .05). PPD and CAL slightly improved for both groups with comparable mean values at 3 months. Evaluation of patient tolerance showed statistically significantly better values among patients receiving the test treatment (mean VAS [0-10], 1.51) compared to SRP (mean VAS [0-10], 3.66; P = .0012). The treatment of test sites was set to 5 seconds per site. The treatment of control sites, on the other hand, lasted 85 seconds on average. CONCLUSION The new erythritol powder applied with an air-polishing device can be considered a promising modality for repeated instrumentation of residual pockets during SPT. CLINICAL RELEVANCE With regard to clinical outcomes during SPT, similar results can be expected irrespective of the two treatment approaches of hand instrumentation or subgingival application of erythritol powder with an air-polishing device in sites where only biofilm removal is required.

Metabolism of nitro drugs metronidazole and nitazoxanide in Giardia lamblia: characterization of a novel nitroreductase (GlNR2).

OBJECTIVES The protozoan parasite Giardia lamblia causes giardiasis, a persistent diarrhoea. Nitro drugs such as the nitroimidazole metronidazole and the nitrothiazolide nitazoxanide are used for the treatment of giardiasis. Nitroreductases may play a role in activating these drugs. G. lamblia contains two nitroreductases, GlNR1 and GlNR2. The aim of this work was to elucidate the role of GlNR2. METHODS Expression of GlNR2 was analysed by reverse transcription PCR. Recombinant GlNR2 was overexpressed in G. lamblia and drug susceptibility was analysed. Recombinant GlNR2 was subjected to functional assays. Escherichia coli expressing full-length or truncated GlNR1 and GlNR2 were grown in the presence of nitro compounds. Using E. coli reporter strains for nitric oxide and DNA damage responses, we analysed whether GlNR1 and GlNR2 elicited the respective responses in the presence, or absence, of the drugs. RESULTS G. lamblia trophozoites overexpressing GlNR2 were less susceptible to both nitro drugs as compared with control trophozoites. GlNR2 was a functional nitroreductase when expressed in E. coli. E. coli expressing GlNR1 was more susceptible to metronidazole under aerobic and semi-aerobic and to nitazoxanide under semi-aerobic growth conditions. E. coli expressing GlNR2 was not susceptible to either drug. In reporter strains, GlNR1, but not GlNR2, elicited nitric oxide and DNA repair responses, even in the absence of nitro drugs. CONCLUSIONS These findings suggest that GlNR2 is an active nitroreductase with a mode of action different from that of GlNR1. Thus, susceptibility to nitro drugs may depend not only on activation, but also on inactivation of the drugs by specific nitroreductases.

Evaluation of a novel photography-based home assessment protocol for identification of environmental risk factors for falls in elderly persons

PRINCIPLES To evaluate the validity and feasibility of a novel photography-based home assessment (PhoHA) protocol, as a possible substitute for on-site home assessment (OsHA). METHODS A total of 20 patients aged ≥65 years who were hospitalised in a rehabilitation centre for musculoskeletal disorders affecting mobility participated in this prospective validation study. For PhoHA, occupational therapists rated photographs and measurements of patients' homes provided by patients' confidants. For OsHA, occupational therapists conducted a conventional home visit. RESULTS Information obtained by PhoHA was 79.1% complete (1,120 environmental factors identified by PhoHA vs 1416 by OsHA). Of the 1,120 factors, 749 had dichotomous (potential hazards) and 371 continuous scores (measurements with tape measure). Validity of PhoHA to potential hazards was good (sensitivity 78.9%, specificity 84.9%), except for two subdomains (pathways, slippery surfaces). Pearson's correlation coefficient for the validity of measurements was 0.87 (95% confidence interval [CI 0.80-0.92, p <0.001). Agreement between methods was 0.52 (95%CI 0.34-0.67, p <0.001, Cohen's kappa coefficient) for dichotomous and 0.86 (95%CI 0.79-0.91, p <0.001, intraclass correlation coefficient) for continuous scores. Costs of PhoHA were 53.0% lower than those of OsHA (p <0.001). CONCLUSIONS PhoHA has good concurrent validity for environmental assessment if instructions for confidants are improved. PhoHA is potentially a cost-effective method for environmental assessment.