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PubMed2004

Miniaturized sample preparation method for determination of amphetamines in urine.

A simple and miniaturized sample preparation method for determination of amphetamines in urine was developed using on-column derivatization and gas chromatography-mass spectrometry (GC-MS). Urine was directly applied to the extraction column that was pre-packed with Extrelut and sodium carbonate. Amphetamine (AP) and methamphetamine (MA) in urine were adsorbed on the surface of Extrelut. AP and MA were then converted to a free base and derivatized to N-propoxycarbonyl derivatives using propylchloroformate on the column. Pentadeuterated MA was used as an internal standard. The recoveries of AP and MA from urine were 100 and 102%, respectively. The calibration curves showed linearity in the range of 0.50-50 microg/mL for AP and MA in urine. When urine samples containing two different concentrations (0.50 and 5.0 microg/mL) of AP and MA were determined, the intra-day and inter-day coefficients of variation were 1.4-7.7%. This method was applied to 14 medico-legal cases of MA intoxication. The results were compared and a good agreement was obtained with a HPLC method.

Amphetamines↗
PubMed2004

Stair climbing detection during daily physical activity using a miniature gyroscope.

A new method of monitoring physical activity that is able to detect walking upstairs using a miniature gyroscope attached to the shank is presented. Wavelet transformation, in conjunction with a simple kinematics model, was used to detect toe-off, heel-strike and foot-flat, as well cycles corresponding to stair ascent. To evaluate the system, three studies were performed. The method was first tested on 10 healthy young volunteer subjects in a gait laboratory where an ultrasonic motion system was used as a reference system. In the second study, the system was tested on three hospitalized elderly people to classify walking upstairs from walking downstairs and flat walking. In the third study, monitoring was performed on seven patients with peripheral vascular disease for 60min during their daily physical activity. The first study revealed a close relationship between the ambulatory and the reference systems. Compared to the reference system, the ambulatory system had an overall sensitivity and specificity of 98% and 97%, respectively. In the second study, the ambulatory system also showed a very high sensitivity (>94%) in identifying a 50 stairs ascent from walking on the flat and walking downstairs. Finally, compared with visual surveillance, we observed a relatively high accuracy in identifying 196 walking upstairs cycles through daily physical activity in the third study. Our results demonstrated a reliable technique of measuring walking upstairs during physical activity.

Activities of Daily Living↗
PubMed2005

Expressed sequence tags from the laboratory-grown miniature tomato (Lycopersicon esculentum) cultivar Micro-Tom and mining for single nucleotide polymorphisms and insertions/deletions in tomato cultivars.

Laboratory-grown miniature tomato (Lycopersicon esculentum) cultivar Micro-Tom has attracted attention as a host for functional genomics research. In this study, we generated 35,824 expressed sequence tags (ESTs) from leaves and fruits of Micro-Tom. The ESTs comprised 10,287 unigenes (5007 contigs and 5280 singletons), including 1858 novel tomato unigenes. Of the 18 unigenes that shared strong homology with tobacco chloroplast genome sequences, one unigene was likely derived from polyadenylated transcripts of the atpH gene. Interestingly, ESTs for vacuolar invertase, pectate lyase and alcohol acyl transferase were underrepresented in the Micro-Tom data set. From all of the ESTs, we mined 2039 candidate single nucleotide polymorphisms (SNPs) and 121 candidate insertions and deletions (indels) based on homology with four tomato inbred lines, E6203, R11-13, Rio Grande PtoR and R11-12, and a wild relative, L. pennellii TA56, for which sequence data was publicly available with more than 5000 entries. Direct genome sequencing of several SNP or indel sites in Micro-Tom and L. esculentum E6203 suggested that more than 69% of the candidate sites were truly polymorphic, making them useful for the preparation of DNA markers.

DNA, Complementary↗
PubMed2006

Detection of a mariner-like element and a miniature inverted-repeat transposable element (MITE) associated with the heterochromatin from ants of the genus Messor and their possible involvement for satellite DNA evolution.

The satellite DNA of ants Messor bouvieri, M. barbarus and M. structor, studied in a previous paper, is organized as tandemly repeated 79-bp monomers in the three species showing high sequence similarity. In the present paper, a mariner-like element (Mboumar) and a new MITE (miniature inverted-repeat transposable element) called IRE-130, inserted into satellite DNA from M. bouvieri, are analyzed. The study of Mboumar element, of its transcription and the putative transposase that it would encode, suggests that it could be an active element. Mboumar elements inserted into IRE-130 elements have also been detected. It is the first time, to our knowledge, that a MITE has been described in Hymenoptera and it is also the first time that a mariner-like element inserted into a MITE has been detected. A mariner-like element, inserted into satellite DNA from M. structor and in M. barbarus, also has been found. The results seem to indicate that transposition events have participated in the satellite DNA mobilization and evolution.

Amino Acid Sequence↗
PubMed2004

Structural characterization of melphalan modified 2'-oligodeoxynucleotides by miniaturized LC-ES MS/MS.

In this study a miniaturized LC coupled to electrospray tandem mass spectrometry was used to analyze modifications originating from the interaction between the chemotherapeutic agent melphalan and 2'-oligodeoxynucleotides. Low energy CAD product ion spectra gave information about the specificity of melphalan alkylation with regard to certain DNA sequences. These data can be very useful to estimate the risk in the development of secondary leukaemia as a result of a melphalan cure. In the study of the interaction between melphalan and d(GG), differentiation could be made between alkylation on the 5'-side and alkylation on the 3'-side, because of the presence or absence of the alkylated w1 fragment in the low energy CAD spectra. In the other di-mers alkylation specificity for the different bases could be observed. Melphalan alkylation occurs in the sequence G > A > C > T. The study of the alkylated d(GGGG) revealed the presence of mainly 5'-end alkylation. Furthermore studies were performed which investigated other melphalan treated di-, tetra-, hepta-, and octa-mers.

Alkylation↗
PubMed2004

Analysis of DNA-phosphate adducts in vitro using miniaturized LC-ESI-MS/MS and column switching: phosphotriesters and alkyl cobalamins.

DNA-phosphate adducts are known to be formed by a variety of alkylating agents. Due to little or no repair of DNA-phosphate adducts, these adducts may offer increased possibilities of both identifying and quantifying DNA adducts. The formation of DNA-phosphate adducts leads to a complete esterification of the phosphate group giving rise to a phosphotriester configuration. This work consists of the characterization of ethyl phosphotriesters (Ethyl PTE) using miniaturized LC-ESI-MS/MS and column switching in enzymatic hydrolysate of DNA treated in vitro with the model compound N-ethyl-N-nitrosourea (ENU). In vitro ENU-treated DNA was enzymatically degraded using nuclease P1, phosphodiesterase, and alkaline phosphatase. The use of column switch allowed for large-volume injections, where unmodified nucleosides were discarded in the loading step. The analytes were forward flushed to the analytical column in the eluting step and separated using a linear gradient. Ten different ethyl PTEs (dGpEtdG, dApEtdA, dCpEtdC, TpEtT, dGpEtdA, dGpEtdC, dGpEtT, dApEtdC, dApEtT, and dCpEtT) were characterized by their masses and CAD product ion spectra. Measurements of accurate masses were carried out yielding experimental masses within 5 ppm of the calculated masses for 9 of the 10 ethyl PTEs. For comparison, the enzymatic hydrolysate of ENU-treated DNA was subjected to transalkylation of the DNA-phosphate adducts by cob(I)alamin. Formed ethyl-cobalamins were analyzed according to earlier developed methods. The limit of detection of an alkyl-cobalamin standard and an alkyl PTE standard was 2 fmol and 5 fmol, respectively.

Animals↗
PubMed2006

Miniature toroidal radio frequency ion trap mass analyzer.

A miniature ion trap mass analyzer is reported. The described analyzer is a 1/5-scale version of a previously reported toroidal radio frequency (rf) ion trap mass analyzer. The toroidal ion trap operates with maximum rf trapping voltages about 1 kVp-p or less; however despite the reduced dimensions, it retains roughly the same ion trapping capacity as conventional 3D quadrupole ion traps. The curved geometry provides for a compact mass analyzer. Unit-mass resolved mass spectra for n-butylbenzene, xenon, and naphthalene are reported and preliminary sensitivity data are shown for naphthalene. The expected linear mass scale with rf amplitude scan is obtained when scanned using a conventional mass-selective instability scan mode combined with resonance ejection.

Journal Article↗
PubMed2005

Proteolysis in miniature cheddar-type cheeses manufactured using extracts from the crustacean Munida as coagulant.

Miniature (20 g) Cheddar-type cheeses were manufactured using enzymes extracted from the crustacean Munida or chymosin as coagulant. Cheeses were ripened at 8 degrees C and samples were collected for analysis after 2, 6 and 12 weeks. Proteolysis was assessed by urea-polyacrylamide gel electrophoresis, which showed that cheeses manufactured with the Munida extracts had a higher extent of degradation of beta-casein than cheeses made using chymosin as coagulant. Patterns of proteolysis were also obtained by reverse-phase high-performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption ionisation-time of flight (MALDI-ToF) mass spectrometry. In general, the products of proteolysis were more complex in cheese made using the Munida extracts than in cheese made by chymosin as coagulant. Statistical analysis of results clearly discriminated the cheeses on the basis of coagulant used. Molecular mass of peptides found in cheese made using Munida extracts were similar to those of peptides commonly detected in cheeses made using chymosin as coagulant.

Animals↗
PubMed2004

Miniaturized mass-selective detector with atmospheric pressure chemical ionization.

The miniaturized mass-spectrometric detector with atmospheric pressure chemical ionization (APCI) is described. The analyzer employed in this instrument is the monopole with rod 54 mm in length and 2 mm in radius, which retains its efficiency up to 0.13 Pa ( approximately 10(-3)Torr). Together with the ion source, channeltron ion detector, radio frequency power supply and preamplifier, it is packed into a case with dimension of 185 mm x 100 mm x 70 mm. Mass spectrometer with the whole vacuum system weighs about 20 kg. In spite of low power of the vacuum system, the limit of detection at ppt level is achieved. The "strong" fragmentation mode is suggested for high-specific detection of phosphor-containing substances. The detector conforms to multicapillary column attachment.

Atmospheric Pressure↗
PubMed2005

Interfacial stabilization of organic-aqueous two-phase microflows for a miniaturized DNA extraction module.

Organic-aqueous liquid (phenol) extraction is one of many standard techniques to efficiently purify DNA directly from cells. The cell components naturally distribute themselves into the two fluid phases in order to minimize interaction energies of the biological components with the surrounding solvents. The membrane components and protein partition to the interface between the organic and aqueous phases while the DNA stays in the aqueous phase. The aqueous phase is then removed with a purified DNA sample. This work studies the first steps towards miniaturizing this liquid extraction technique in a microfluidic device. The first step is to understand how the two liquid phases behave in microchannels. Due to the interfacial tension between the two liquid phases, novel approaches must be examined in order to obtain interfacial stability under flow conditions. The stability of the organic-aqueous interface is improved by reducing the interfacial tension between the two phases by incorporating a surfactant into the aqueous phase. The variation of the interfacial tension as a function of surfactant concentration is also quantified in this work. This has led to the ability to create stable stratified microflows in both a dual inlet and three inlet microfluidic systems. Also, the first step in understanding biological interactions at the organic-aqueous interface is investigated using a fluorescently labeled bovine serum albumin protein.

Cell Membrane↗
PubMed2006

Development of a miniature calorimeter for identification and detection of explosives and other energetic compounds.

The development of versatile systems capable of providing rapid, portable, and inexpensive detection of explosives and energetic compounds are critically needed to offer enhanced levels of protection against current and future threats to homeland security, as well as satisfying a wide range of applications in the fields of forensic analysis, emergency response, and industrial hazards analysis. Calorimetric techniques have been largely overlooked in efforts to develop advanced chemical analysis technology, largely because of limitations associated with the physical size of the instruments and the relatively long timescales (>30 min) required to obtain a result. This miniaturized calorimeter circumvents these limitations, thereby creating a first-of-its-kind system allowing thermal analysis to be performed in a portable format that can be configured for use in a variety of field operations with a significantly reduced response time (approximately 2 min). Unlike current explosives detectors, this system is based on calorimetric techniques that are inherently capable of providing direct measurements of energy release potential and therefore do not depend on prior knowledge of familiar compounds.

Calorimetry↗
PubMed2005

A multiplexed and miniaturized serological tuberculosis assay identifies antigens that discriminate maximally between TB and non-TB sera.

We have developed a multiplexed and miniaturized TB serological assay with the aim of identifying (combinations of) antigens that maximally discriminate between TB and non-TB patients. It features a microarray accommodating 54 TB antigens, less than 1 microl serum consumption and an indirect immunofluorescence detection protocol. With a panel of 20 TB and 80 non-TB sera we ranked combinations of TB antigens with respect to sensitivity and specificity of TB detection by means of logistic step-forward regression analysis. The highest-ranking TB antigen combination had an area-under-the-curve of the receiver-operator-characteristics (ROC) of 0.95. We also identified an antigen that on its own provided good specificity and sensitivity of TB detection (Ara6-BSA; area-under-the-ROC curve: 0.90). These area-under-the-ROC curve values are exceptionally high for a serological TB assay. We conclude that TB antigen microarrays permit rapid identification of TB antigens that, either alone or in combination, discriminate maximally between TB and non-TB patients and that such identification provides an excellent starting point for developing point-of-care diagnostic assays.

Antibodies, Bacterial↗
PubMed2005

Miniature telemetry system for the recording of action and field potentials.

A simple miniature telemetry system for neural recording from freely moving rats is described. It weighs only 1% of the body weight of an adult rat and its recordings are devoid of artifacts due to the animal movement. Together with its long recording time (more than 38 h), its isotropic nature, which is essential for working with freely moving animals, offers further advantages. A frequency-modulation receiver with a flat frequency response down to 6 Hz has been designed for wide-spectrum recording of neural signals, allowing field potential recordings. A detailed printed-circuit layout and the lack of a trimming requirement will allow the system to be easily duplicated by other neuroscientists who are not familiar with wireless-transmission technologies.

Action Potentials↗
PubMed2006

Miniature microdrive-headstage assembly for extracellular recording of neuronal activity with high-impedance electrodes in freely moving mice.

The design of a removable miniature microdrive-headstage assembly for extracellular recordings of single unit activity with high-impedance electrodes in freely moving small animals is presented. The advantages of this construction include simple installation and removal of the electrodes, rapid attachment of the assembly to the animal's skull and rapid removal after recording. The microdrive provides precise vertical positioning of the electrode without rotation or lateral shift, stable recordings of single units for several hours and the possibility to change the penetration track 5-10 times in the same animal. The microdrive permits microelectrode penetration to any desired depth. The small size of the microdrive permits installation of several units simultaneously. The assembly weight is less than 120mg.

Animals↗
PubMed2004

Miniature access pectus excavatum repair: Lessons we have learned.

BACKGROUND: Pectus excavatum (PE) is the most common chest well deformity seen in children. In 1997, the Miniature Access Pectus Excavatum repair (MAPER) was presented by Nuss et al, adding a new option for PE repair. This operation entails placing a custom bent metal bar across the chest to mechanically raise the sternum and remodel the cartilage. The authors have added modifications to Nuss' original description of this operation in an attempt to optimize technique, minimize complications, and improve outcomes. METHODS: The authors have performed 52 MAPERs with an average operating time of 106 minutes, average length of stay of 3.9 days, and return to normal activities of 2 to 6 weeks. Modifications to Nuss' original description include preoperative evaluation consisting of an echocardiogram and pulmonary function tests (PFTs; with and without exercise and with and without bronchodilators), abandoning the use of routine preoperative computed tomography (CT) scans, the use of unilateral positive pressure insufflation of the hemithorax to provide visualization, and anesthesia using an epidural pain catheter (intraoperative and postoperative for 3 days). Intraoperatively, we use a 70 degrees thoracoscope for optimal visualization, and we have modified their location for optimal visualization. Additionally, the bars are secured with surgical wire, not absorbable suture, to avoid bar slippage. RESULTS: Postoperatively, we leave our bars in for 3 years and have had no recurrences. Furthermore, these patients require significant support during the time their bars are in place and occasionally require reoperation to fix symptomatic problems with their bar. CONCLUSIONS: Since the first description of the MAPER was presented more than 5 years ago, the operative treatment of PE has changed dramatically. The authors feel that the MAPER is superior to the open technique, and with the modifications they have implemented, complications have been minimized, and long-term results have been improved.

Adolescent↗
PubMed2005

Heart and en-bloc thymus transplantation in miniature swine.

BACKGROUND: Donor-specific tolerance to organ allografts might be induced by cotransplantation of a sufficient amount of vascularized donor thymus. To facilitate donor thymus-induced cardiac allograft tolerance, we have developed a novel technique for heart and en-bloc thymus transplantation in swine. METHODS: Donor heart and en-bloc thymus grafts were prepared by a technique that preserves the entire arterial supply and venous drainage of the right thymic lobe. En-bloc grafts (n = 4) were transplanted heterotopically into the abdomens of major histocompatibility complex-matched miniature swine. Recipients received 12 days of cyclosporine intravenously. Grafts were monitored by palpation, electrocardiographic monitoring, and periodic open biopsy. Engraftment of the donor thymus was demonstrated by measuring the proportion of recipient-type thymocytes in the donor thymus with flow cytometry. RESULTS: All of the heart and en-bloc thymus grafts had normal cardiac contractility and immediate perfusion of the thymus. All en-bloc grafts were accepted for more than 200 days without significant acute cellular rejection or cardiac allograft vasculopathy. Thymic tissue of en-bloc grafts displayed normal architecture and supported thymopoiesis of recipient-type cells. CONCLUSION: We have validated a new technique of donor thymus transplantation that could have utility in human heart transplantation.

Animals↗
PubMed2004

Using rice to understand the origin and amplification of miniature inverted repeat transposable elements (MITEs).

Recent studies of rice miniature inverted repeat transposable elements (MITEs), largely fueled by the availability of genomic sequence, have provided answers to many of the outstanding questions regarding the existence of active MITEs, their source of transposases (TPases) and their chromosomal distribution. Although many questions remain about MITE origins and mode of amplification, data accumulated over the past two years have led to the formulation of testable models.

DNA Transposable Elements↗
PubMed2006

Brainstem auditory evoked potentials and flash visual evoked potentials in Vietnamese miniature pot-bellied pigs.

Brainstem auditory evoked potentials (BAEP) and flash visual evoked potentials (VEP) were recorded from juvenile (5-7 weeks of age) and adult Vietnamese miniature pot-bellied pigs to provide normative data for clinical applications. BAEP responses were collected in response to stimulus intensities of 85, 95, and 105 dB nHL. VEP responses were collected in response to flashes of white light in a darkened room. Left-right ear and left-right eye responses did not differ significantly, and were combined for analysis, with each animal providing two data points for each response. BAEP responses in juvenile subjects were mature, and in all subjects showed the typical pattern of decreasing peak latencies and increasing peak amplitudes with increasing stimulus intensity. VEP responses in juvenile subjects were near to mature values, but the latencies still exceeded those of adults. Differences in response maturation between precocial and altricial species are discussed.

Aging↗