Effluent from drug manufactures contains extremely high levels of pharmaceuticals

It is generally accepted that the main route for human pharmaceuticals to the aquatic environment is via sewage treatment plants receiving wastewater from households and hospitals. We have analysed pharmaceuticals in the effluent from a wastewater treatment plant serving about 90 bulk drug manufacturers in Patancheru, near Hyderabad, India--a major production site of generic drugs for the world market. The samples contained by far the highest levels of pharmaceuticals reported in any effluent. The high levels of several broad-spectrum antibiotics raise concerns about resistance development. The concentration of the most abundant drug, ciprofloxacin (up to 31,000 microg/L) exceeds levels toxic to some bacteria by over 1000-fold. The results from the present study call for an increased focus on the potential release of active pharmaceutical ingredients from production facilities in different regions.     

Complexation between Amylodextrin Oligomers and Selected Pharmaceuticals Measured through Capillary Electrophoresis

The amylodextrin oligomers, tagged fluorescently at their reducing ends for laser fluorescence detection, were used as model solutes to investigate certain carbohydrate-drug interactions through capillary electrophoresis. The separation patterns of some oligomers were found to be altered significantly by the presence of selected pharmaceuticals used as buffer additives. The selectivity of complexation at a particular size of an oligomer could be judged from alterations in migration times and peak shapes. The formation of complex was influenced substantially by solutes' chemical environment (pH, ionic strength, and the nature and concentration of organic additives). The chemical nature of the guest molecules also played an important role in complexation. Using amylodextrins as chiral selectors, enantiomeric resolution of several pharmaceuticals was achieved electrophoretically. The conclusions drawn from electrophoretic data were found to be in close agreement with the results of a (13)C NMR study.     

Development of polar organic integrative samplers for analysis of pharmaceuticals in aquatic systems

Integrative passive sampling is a new approach developed for environmental monitoring. Nowadays, the evaluations of pollution level are obtained by important sampling campaigns using spot samplings that give a snapshot of the aquatic system contamination state. An alternative way is to achieve a time weighted average concentration using passive samplers. The use of polar organic chemical integrative sampling (POCIS) has been recently documented for the detection of pharmaceuticals in the environment (Alvarez, D.; Petty, J. D.; Huckins, J. N.; Jones-Lepp, T. L.; Getting, D. T.; Goddard, J. P.; Manahan, S. E. Environ. Toxicol. Chem. 2004, 23, 1640-1648 (ref 1). Jones-Lepp, T. L.; Alvarez, D.; Petty, J. D.; Huckins, J. Arch. Environ. Contam. Toxicol. 2004, 47, 427-739 (ref 2). Petty, J. D.; Huckins, J. N.; Alvarez, D.; Brumbaugh, W. G.; Cranor, W. L.; Gale, R. W.; Rastall, A. C.; Jones-Lepp, T. L.; Leiker, T. J.; Rostad, C. E.; Furlong, E. T. Chemosphere 2004, 54, 695-705 (ref 3)). There is a need for laboratory data to extend the use of this type of tool to new compounds. The aim of this study was to determine the sampling rates (Rs; expressed as effective volumes of water extracted daily) of POCIS devices for 14 pharmaceuticals in several conditions of temperature, salinity, and analyte concentration. These values are influenced by significant changes in water temperature and salinity. Overall, POCIS Rs values were independent from aqueous concentrations. After laboratory experiments, an environmental field study has been performed, implementing POCIS devices in the Seine estuary (North Atlantic coast of France) and testing the qualitative and quantitative application of POCIS devices on the contaminated system. The suitability of the devices for monitoring multiple media under a wide range of environmental conditions has also been discussed. The uniformity or reproducibility of the sampling matrix and, on the other hand, the ability to detect compounds at low concentration levels below detection limits of discrete sampling have been highlighted.     

Intramolecular isobaric fragmentation: a curiosity of accurate mass analysis of sulfadimethoxine in pond water

Liquid chromatography with time-of-flight mass spectrometry (TOF-MS) and quadrupole-time-of-flight (Q-TOF) mass spectrometry/mass spectrometry (MS/MS) were used for the accurate mass analysis of sulfadimethoxine in pond water of a fish hatchery. Sulfadimethoxine is the most important sulfa antimicrobial used in aquaculture to treat bacterial disease in a wide variety of fish. Because correct identification is essential to environmental monitoring of antimicrobial pharmaceuticals, accurate mass analyses (TOF and Q-TOF-MS/MS) were compared to nominal mass measurement (quadrupole ion trap). It was known that all six members of the sulfa antimicrobial family gave a common 6-sulfanilamido ion at a nominal mass of m/z 156; thus, this ion was the focus of TOF confirmation (exact mass 156.0119 u) along with the protonated molecule (exact mass 311.0814 u). In the process of accurate mass confirmation of the 156 m/z fragment ion, a second isobaric ion (exact mass m/z 156.0773), was discovered at the same nominal mass, which was not differentiated by quadrupole ion trap. The structure was assigned as 2-4-dimethoxypyridine and is exactly the other protonated half of the sulfadimethoxine molecule. This discovery led to the subsequent use of Q-TOF-MS/MS and high-resolution identification of five other important ion fragments for the identification of sulfadimethoxine in pond water at environmental concentrations. The caveats of using low-resolution mass spectrometry without MS/MS for environmental monitoring are discussed in the light of high profile monitoring of sulfa antimicrobial pharmaceuticals in the aquatic environment.     

Biophysical Approaches to the Pharmaceutical Development of Proteins

The complexity of protein pharmaceuticals necessitates a multifaceted approach to their characterization, stabilization, and development. The combined use of spectroscopic, hydrodynamic, chromatographic and thermodynamic methods to evaluate various levels of macromolecular structure is described. Near future developments in our ability to evaluate proteins at high structural resolution are also considered.     

Biophysical Approaches to the Pharmaceutical Development of Proteins

Abstract

The complexity of protein pharmaceuticals necessitates a multifaceted approach to their characterization, stabilization, and development. The combined use of spectroscopic, hydrodynamic, chromatographic and thermodynamic methods to evaluate various levels of macromolecular structure is described. Near future developments in our ability to evaluate proteins at high structural resolution are also considered.     

Sample depletion of the matrix-assisted laser desorption process monitored using radionuclide detection

To investigate analyte consumption during the laser desorption process, matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is combined with radionuclide detection. Radionuclide detection provides highly sensitive and quantitative information on the amount of radiolabeled analytes in a MALDI MS sample spot. 14C-Labeled cytochrome c is deposited with 2,5-dihydroxybenzoic acid in 10-nL volume spots. By comparing radioactivity levels of the labeled cytochrome c both before and after spectral acquisition, the reduction in labeled analyte molecules on the target allows monitoring of the moles of desorbed sample. Through a depletion study on this sample, the amount of analyte consumed for MALDI time-of-flight spectral acquisition and the average number of molecules desorbed per laser ablation are determined. When [14C]-cytochrome c is no longer detected by MALDI MS, approximately 70% of the original analyte remains in the sample spots. Redissolving the spots produced further desorption, indicating that the analyte before dissolution was in a physical environment that did not facilitate the desorption process. As a technique with a response that does not depend on the environment of the analyte, radionuclide detection allows characterization of mass-limited sampling methods to better understand the MALDI process.     

Novel organic-inorganic glasses

The preparation and properties of novel organic-inorganic glasses with phenyl groups covalently bonded to phosphorus in an inorganic glass are presented. Phenyl phosphonic acid (PPA) can be incorporated into tin and zinc fluorophosphate glasses (MO/MF₂/PO_2.5PPA) at levels up to 72 mol%. The T _g's of these glasses range from below room temperature to near 200°C depending on glass composition. For low melt glasses, these glasses have low moisture absorption and may solubilize dyes, organic photoconductors, NLO molecules, and pharmaceuticals. Several applications have been considered.     

DNA biosensor for the detection of hydrazines

A double-stranded (ds) DNA-coated carbon paste electrode is employed as a remarkably sensitive biosensor for the detection of hydrazine compounds. The sensor relies on monitoring changes in the intrinsic anodic response of the surface-confined DNA resulting from its interaction with hydrazine compounds and requires no label or indicator. Short reaction times (1-10 min) are sufficient for monitoring part-per-billion levels of different hydrazines. Applicability to untreated natural water samples is illustrated. The response mechanism is discussed, along with prospects of using DNA biosensors for quantitaing other important molecules and elucidating DNA interactions and damage.     

Inspection and monitoring planning for RC structures based on minimization of expected damage detection delay

The deterioration mechanism of reinforced concrete (RC) structures under corrosion is highly dependent on environment and material properties. Uncertainties in structural damage occurrence and propagation due to corrosion should be considered in a rational way using a probabilistic approach. In this study, such an approach is proposed to establish a life-cycle optimum inspection plan under uncertainty. This plan leads to cost-effective maintenance interventions, considering uncertainties associated with damage occurrence/propagation and inspection methods. Uncertainties associated with prediction of damage occurrence time are considered by using the Monte Carlo simulation. A damage detectability function is used to assess the quality of inspection method according to damage intensity. The inspection planning is formulated as an optimization problem with the objective of minimizing the expected damage detection delay. This formulation is further used for optimum monitoring planning. Effects of number of inspections and/or monitoring actions, quality of inspection, monitoring duration, and uncertainties associated with damage occurrence/propagation are investigated. The proposed approach is applied to an existing highway bridge. This approach can be used to develop cost-effective management strategies by considering effects of damage detection delay on life-cycle cost and performance of deteriorating structures.     

Opportunities at the skin interface for continuous patient monitoring: a reverse iontophoresis model tested on lactate and glucose

Skin has the potential to provide an important noninvasive route for diagnostic monitoring of human subjects for a wide range of applications. Dimensions of surface features in skin suggest that nanodevices and microdevices could be utilized to monitor molecules and ions extracted from the skin. Methods of enhancing extraction from the skin for diagnostics are being developed including reverse iontophoresis, electroporation and sonophoresis. A model system for the simulation of in vivo extraction of molecules and ions by reverse iontophoresis is described here that displays similar behavior to skin both in terms of molecular flux levels and electrical impedance characteristics. The device has potential for use in the development of complete reverse iontophoresis/sensor systems, allowing sensor and extraction systems to be studied and optimized before being tested in the complex in vivo environment. The system has been tested using glucose and lactate and the results are reported and discussed.     

Electrochemical Monitoring of Paclitaxel‐Induced ROS Release from Mitochondria inside Single Cells

Mitochondria are believed to be the major source of intracellular reactive oxygen species (ROS). However, in situ, real‐time and quantitative monitoring of ROS release from mitochondria that are present in their cytosolic environment remains a great challenge. In this work, a platinized SiC@C nanowire electrode is placed into a single cell for in situ detection of ROS signals from intracellular mitochondria, and antineoplastic agent (paclitaxel) induced ROS production is successfully recorded. Further investigations indicate that complex IV (cytochrome c oxidase, COX) is the principal site for ROS generation, and significantly more ROS are generated from mitochondria in cancer cells than that from normal cells. This work provides an effective approach to directly monitor intracellular mitochondria by nanowire electrodes, and consequently obtains important physiological evidence on antineoplastic agent‐induced ROS generation, which will be of great benefit for better understanding of chemotherapy at subcellular levels.     

Nanopore sensors for nucleic acid analysis

Nanopore analysis is an emerging technique that involves using a voltage to drive molecules through a nanoscale pore in a membrane between two electrolytes, and monitoring how the ionic current through the nanopore changes as single molecules pass through it. This approach allows charged polymers (including single-stranded DNA, double-stranded DNA and RNA) to be analysed with subnanometre resolution and without the need for labels or amplification. Recent advances suggest that nanopore-based sensors could be competitive with other third-generation DNA sequencing technologies, and may be able to rapidly and reliably sequence the human genome for under $1,000. In this article we review the use of nanopore technology in DNA sequencing, genetics and medical diagnostics.     

单元化柔性制造系统集成开发的模块化方法

提出一套用于单元化柔性制造系统（CFMS）开发的结构化方法。主要强调和突出了建筑块（Building Block)概念以及集成开发与控制环境。建筑块是一种迷你型自治单元,它可以执行规划功能、计划执行和执行监控。在开发建筑块的过程中,用到了面向对象的软件工程方法论。基于这种方法论,通过一个集成CFMS开发与运作环境,一系列的FMS（CFMS）可由事先定义好的软件建筑块类库中产生。     

Modulating the performance of carbon nanotube field-effect transistors via Rose Bengal molecular doping

A simple, reliable, and large scale ambient environment doping method for carbon nanotubes is a highly desirable approach for modulating the performance of nanotube based electronics. One of the major challenges is doping carbon nanotubes to simultaneously offer a large shift in threshold voltage and an improved subthreshold swing. In this paper, we report on modulating the performance of carbon nanotube field-effect transistors (CNTFETs) by rationally selecting doping molecules. We demonstrated that Rose Bengal sodium salt (RB-Na) molecular doping can effectively shift the threshold voltage (ΔVth) of CNTFETs up to ～6 V, decrease the subthreshold swing down to 130 mV/decade, and increase the effective field-effect mobility to 5 cm2 V(-1) s(-1). It is also shown that CNTFETs doped with Rose Bengal lactone (RBL) show a smaller variation in ΔVth (～2 V) and subthreshold swing than those doped by RB-Na, which can be attributed to the difference in their molecular structures. The observed right-shift of the threshold voltage is attributed to the positive charge doping of the nanotube conduction channel from Rose Bengal molecules. The resultant lowering of the subthreshold swing is due to the reduced Schottky barrier at the CNT/metal/molecule interface. This room temperature chemical doping approach provides an efficient, simple, and cost-effective method to fabricate highly reliable and high-performance nanotube transistors for future nanotube based electronics.     

Bispecific antibody-mediated detection of the Staphylococcus aureus thermonuclease

We report a novel fluorescence-based immunoassay which enables qualitative detection of the Staphylococcus aureus Thermonuclease (TNase) enzyme, thus providing confirmation of the presence of the S. aureus bacterium in vitro. The biomedical problem of chronic wound healing and the continuing emergence of antibiotic-resistant species is addressed in the development of a detection system capable of the rapid, real-time assessment of bacterial load and diversity. The use of bispecific antibodies (BsAb) provides integration of the molecular detection and signal response components of a standard immunoassay due to steric hindrance-mediated release of prebound fluorescent reporter molecules upon specific binding of TNase to adjacent sites. Rhodamine and fluorescein-labeled hemocyanin from Megathura crenulata (KLH) were prepared as effective immunoconjugates containing a sensitive fluorescent reporter moiety. BsAb that both specifically quenched the fluorescence of the reporter conjugate and bound the TNase target antigen were produced using cell fusion techniques. Assays were then performed to analyze the properties attributable to the steric hindrance-mediated release of the fluorescent reporter molecules upon adjacent TNase binding. This was performed by monitoring the intensity of fluorescence emission of the immunogenic reporter conjugate released into an aqueous environment at 578 and 520 nm, respectively.     

Nucleic acid beacons for long-term real-time intracellular monitoring

Real time intracellular monitoring of biological molecules inside living cells is important in many biomedical studies and reveals valuable information unobtainable by conventional molecular biology techniques. A variety of methods and molecular probes have been developed, but long term (from a few hours to days) intracellular monitoring with high sensitivity and selectivity is impossible and has not been accomplished. We have used locked nucleic acids (LNA) to engineer novel molecular beacons (MBs) for long-term intracellular monitoring. The LNA-MBs were made of a mixed LNA and DNA bases to have extremely high biostability. The new beacons were tested with MDA-MB-231 cancer cells and used effectively to monitor mRNA expression levels in real-time for 5-24 h. After 24 h inside living cells, the LNA-MBs were still functional, demonstrating a greatly enhanced stability enabling the measurement of intracellular gene expression over an extended period of time.     

Probing the radiative transition of single molecules with a tunable microresonator

Using a tunable optical microresonator with subwavelength spacing, we demonstrate controlled modulation of the radiative transition rate of a single molecule, which is measured by monitoring its fluorescence lifetime. Variation of the cavity length changes the local mode structure of the electromagnetic field, which modifies the radiative coupling of an emitting molecule to that field. By comparing the experimental data with a theoretical model, we extract both the pure radiative transition rate as well as the quantum yield of individual molecules. We observe a broad scattering of quantum yield values from molecule to molecule, which reflects the strong variation of the local interaction of the observed molecules with their host environment.     

Label-Free Identification of Exosomes using Raman Spectroscopy and Machine Learning

Exosomes, nano-sized extracellular vesicles (EVs) secreted from cells, carry various cargo molecules reflecting their cells of origin. As EV content, structure, and size are highly heterogeneous, their classification via cargo molecules by determining their origin is challenging. Here, a method is presented combining surface-enhanced Raman spectroscopy (SERS) with machine learning algorithms to employ the classification of EVs derived from five different cell lines to reveal their cellular origins. Using an artificial neural network algorithm, it is shown that the label-free Raman spectroscopy method's prediction ratio correlates with the ratio of HT-1080 exosomes in the mixture. This machine learning-assisted SERS method enables a new direction through label-free investigation of EV preparations by differentiating cancer cell-derived exosomes from those of healthy. This approach will potentially open up new avenues of research for early detection and monitoring of various diseases, including cancer.     

Depth-profiling of environmental pharmaceuticals in biological tissue by solid-phase microextraction

The parallel in vivo measurement of chemicals at various locations in living tissues is an important approach furthering our understanding of biological uptake, transportation, and transformation dynamics. However, from a technical perspective, such measurements are difficult to perform with traditional in vivo sampling techniques, especially in freely moving organisms such as fish. These technical challenges can be well addressed by the proposed depth-profiling solid-phase microextraction (DP-SPME) technique, which utilizes a single soft, flexible fiber with high spatial resolution. The analytical accuracy and depth-profiling capability of DP-SPME was established in vitro within a multilayer gel system and an onion artificially contaminated with pharmaceuticals. In vivo efficacy was demonstrated by monitoring pharmaceutical distribution and accumulation in fish muscle tissue. The DP-SPME method was validated against pre-equilibrium SPME (using multiple small fibers), equilibrium SPME, and liquid extraction methods; results indicated DP-SPME significantly improved precision and data quality due to decreased intersample variation. No significant adverse effects or increases in mortality were observed in comparisons of fish sampled by DP-SPME relative to comparable fish not sampled by this method. Consequently, the simplicity, effectiveness, and improved precision of the technique suggest the potential for widespread application of DP-SPME in the sampling of heterogeneous biotic and abiotic systems.