Linking eutrophication indicators in eelgrass habitats to nitrogen loading and mitigating site characteristics in eastern New Brunswick, Canada.

Nitrogen loading has been linked to eutrophication and seagrass bed declines worldwide, yet early warning signs and potential mitigating factors are often less clear. Our objective was to use published nitrogen loading model results together with eelgrass habitat surveys from 7 bays in Atlantic Canada to assess linkages between nitrogen loading, tidal flushing and bivalve aquaculture on observed eutrophication indicators in eelgrass habitats. Field surveys revealed significant differences in primary indicators (annual algae, tissue nitrogen) and secondary changes in eelgrass bed structure, yet no large loss of eelgrass cover or biomass. Multivariate analyses found positive correlations between nitrogen loading and eutrophication indicators, with distinct clusters of high- and low-impact sites, and the mitigating effects of flushing time and aquaculture. Our results highlight that combining measures of nitrogen loading, eutrophication indicators and mitigating factors can help detect early warning signs and assess eutrophication risk to inform management and conservation of coastal ecosystems before significant losses of seagrass occurs.

Private sex workers' engagement with sexual health services: an online survey.

This study examined where private sex workers (PSW) present for sexual health services, disclosure, services received, and their satisfaction with care. An online anonymous survey was conducted via SurveyMonkey (surveymonkey.com). Among the 53 participants, 42% attended a sexual health clinic, 24% attended a general practitioner (GP) and 34% attended both. Participants attending GPs were less likely to be offered a throat swab and opportunities for cervical screening, contraception and vaccination were often missed in both service models. Participants attending GPs were less likely to disclose sex work and were less satisfied. Better awareness of the sexual health needs of PSWs is important in GP services.

The thin blue line: a review and discussion of aseptic technique and postprocedural infections in rodents.

A basic tenet of animal welfare philosophy is that pain and distress must be minimized whenever possible without interfering with the goals of the research. Aseptic technique during surgical procedures is essential to prevent pain and distress associated with post-procedural infections. However, many investigators have found that applying the aseptic techniques used for large animal and human surgery is not always practical when performing surgery on small rodents. Furthermore, the efficacy of some of these techniques for preventing post-procedural infections has been questioned. This review examines what is known about the development of postprocedural infections in animals and humans and the methods used to prevent them. Detection of postprocedural infections in rodents can be difficult unless objective measurements of physiologic indices are made. These measurements should be used experimentally to assess the relative benefits of various methods for preventing postprocedural infections. Measures of contamination, such as quantitative bacterial cultures, also can be used; however, they do not reliably predict infection rates. Much of the dogma about decontamination of skin and hair prior to surgery is not supported by valid experimental evidence. Hair removal may not be necessary. Alcohol may in fact be a better disinfectant than is often credited. Draping should be used when it contributes to the maintenance of the sterile field, but when it does not, modification of surgical technique may provide more protection from infection than the drape does. The contribution of surgical technique to the prevention of postprocedural infections is probably equal to that of aseptic technique. Further research needs to be done to assess various aseptic techniques for use in rodent surgery.

Detection of RET proto-oncogene codon 634 mutations using mass spectrometry.

Mutations located in the RET proto-oncogene at codon 634 associated with multiple endocrine neoplasia type 2A and medullary thyroid carcinoma are detected by low-resolution and high-resolution mass spectrometry schemes not requiring labeling or electrophoretic separation of diagnostic products. The former requires measurement by matrix-assisted laser desorption ionization time-of-flight mass spectrometry of 21- to 27-mer oligonucleotides generated by a primer oligo base extension reaction. The latter is based upon direct measurement of artificial products which include the mutation site using matrix-assisted laser desorption ionization Fourier transform mass spectrometry. In this feasibility study a synthetic 25-mer representing the wildtype allele (7660.3 Da) was easily distinguished from G to A (7644.3 Da) and G to T (7635.3 Da) mutant alleles; the mutant alleles, which differed in mass by only 9.0 Da, were easily resolved when analyzed as a mixture. The results of both detection schemes were highly accurate and reliable, indicating mass spectrometry to be a high-quality alternative for future DNA diagnostics performed in clinical laboratories and genetic profiling studies.

High-resolution MALDI Fourier transform mass spectrometry of oligonucleotides.

The matrix-assisted laser desorption/ionization (MALDI) method has been used with an external ion source Fourier transform mass spectrometer (FIMS) to analyze single-stranded, mixed-base oligomers of DNA. It is demonstrated that ultrahigh mass resolution (830 000 fwhm) can be achieved for small oligomers, and high resolution (136 000 fwhm) can be achieved for a 25-mer at m/z 7634. MALDI-FTMS can clearly separate the molecular ion peaks from analyte-matrix adduct peaks and alkali metal-containing species that result from replacement of hydrogen ions with sodium or potassium ions at multiple sites along the phosphate backbone. Previous MALDI-FTMS studies of oligonucleotides had two limitations: (1) low sensitivity due to difficulty in trapping the high kinetic energy ions made by the laser and (2) fragmentation of the ions due to the long delay (tens to hundreds of milliseconds) between their formation and detection. Both of these problems are alleviated in the present study. With the external ion source FTMS instrument, ions made by MALDI are injected at low energy into the analyzer cell by a rf-only quadrupole ion guide, captured by gating the voltage on the trapping plates, and cooled by a 0.5-s pulse of argon gas. Under these conditions, fragmentation is minimized, and DNA ions can be trapped in the FTMS analyzer cell for greater than 50 s. Sensitivity is also improved, as demonstrated by detection of 1 pmol of a single-stranded, mixed-base 20-mer of DNA, with a signal-to-noise ratio greater than 20:1.

Detection limits for matrix-assisted laser desorption of polypeptides with an external ion source Fourier-transform mass spectrometer.

Sensitivity in the low-femtomole range with mass resolution greater than 20,000 is demonstrated for several polypeptides analyzed by a mass spectrometer that pairs matrix-assisted laser desorption/ionization (MALDI) and Fourier-transform mass spectrometry (FTMS). The compounds investigated were substance P, renin substrate, melittin, the B-chain of bovine insulin, and bovine insulin. Standard solutions of the polypeptides were prepared with 30% acetonitrile+water, and micropipettes were used to transfer small amounts (1-20 fmol) to a sample probe. The samples were embedded in a large excess of matrix material (2,5-dihydroxybenzoic acid) and ionized by a pulse from an excimer laser. The FTMS instrument used for these experiments has the MALDI source in a separate chamber outside the magnetic field. Ions are extracted from the source and transported by an RF-only quadrupole ion guide to an FTMS analyzer cell mounted in the homogeneous region of a 6.5 T superconducting magnet. The high sensitivity of MALDI-FTMS is due, in part, to the high transfer efficiency of the ion guide, even for ions with a wide range of kinetic energies. The ion guide is easy to use because there are only two adjustments (RF amplitude and DC offset voltage), and unlike electrostatic ion transport means, alignment of it with the axis of the magnetic field is not critical. The mass resolution and sensitivity of MALDI-FTMS is compared with that of MALDI done with time-of-flight, magnetic sector, and quadrupole ion-trap mass spectrometers.

High-resolution mass spectrometer for protein chemistry.

Matrix-assisted laser desorption/ionization is an accurate and sensitive method for measuring the molecular weights of peptides and proteins. Usually time-of-flight mass spectrometry is used to detect the laser-produced ions, but a new method called Fourier transform mass spectrometry offers greater sensitivity and much higher mass measurement accuracy.

High-accuracy molecular mass determination for peptides and proteins by Fourier transform mass spectrometry.

A new calibration method has been developed for Fourier transform mass spectrometry (FTMS) that is accurate to better than 0.001% (10 ppm) for peptides and proteins up to 5700 Da. The custom-designed FTMS instrument used for this work has a matrix-assisted laser desorption/ionization (MALDI) source located outside of the magnetic field in a differentially pumped chamber, and ions are injected through the fringing fields of the magnet into the FTMS analyzer cell by a long quadrupole ion guide. The mass spectrometer is calibrated with four model compounds ([Arg8]-vasopressin, melittin, bovine insulin B-chain, and bovine insulin) of known molecular mass. The set of measured ion resonance frequencies (f) for these compounds are fit to a three-term calibration equation of the form f = A(z/m) + B(V) + C(V2) (m/z), where m/z is the mass-to-charge ratio of a calibrant peak, V is the trapping voltage, and A, B, and C are calibration coefficients that depend on the magnetic field strength and the dimensions of the analyzer cell. The same set of calibration coefficients can be used for many weeks because the magnet and the electronics of the FTMS instrument are very stable. This method is useful because unknowns can be run separately without the need to add an internal calibration compound in with the sample.

High-resolution laser desorption mass spectrometry of peptides and small proteins.

Matrix-assisted laser desorption/ionization (MALDI) has been used with an external ion source Fourier-transform mass spectrometer to obtain the highest mass resolution ever, to our knowledge, demonstrated for laser-produced ions (m/delta m = 1,100,000 for [Arg8]vasopressin, 228,000 for melittin, and 90,000 for bovine insulin). The peaks in the isotope cluster for bovine insulin are fully resolved, and the mass measurement accuracy is an order of magnitude better than can be achieved with time-of-flight mass spectrometry. With the method described here, analyte is applied to a sample probe and mixed with a solution containing a matrix material (2,5-dihydroxybenzoic acid) that strongly absorbs ultraviolet light. Upon irradiation with a pulse from an excimer laser (353 nm, 2 mJ), a large number of intact protonated molecular ions are produced. The ions are focused by a 117-cm-long quadrupole ion guide and injected into an ion cyclotron resonance analyzer cell located inside the bore of a 6.5-T superconducting magnet. A pulse of argon buffer gas cools the ions prior to detection. One of the principal advantages of an external ion source Fourier-transform mass spectrometer is that the ion formation and ion detection processes are separated and can be independently optimized.

Matrix-assisted laser desorption/ionization with an external ion source Fourier-transform mass spectrometer.

Mass spectra of several model oligopeptides (substance P, [Arg8]-vasopressin, tyrothricin, and the B-chain of bovine insulin) have been obtained with a matrix-assisted laser desorption/ionization (MALDI) source and a custom-designed Fourier-transform mass spectrometer (FTMS). The MALDI source is outside the magnetic field in a separately pumped external chamber, and ions are injected through the fringing fields of the magnet and into the FTMS analyzer cell by a long quadrupole mass filter that is operated in the RF-only mode. A large window on the ion source housing makes it easy to point and focus the laser beam onto the sample probe tip. A good quality mass spectrum is achieved for 0.5 pmol of substance P, and the mass resolution for the B-chain of bovine insulin is 19,000.

Inhibitory effect of disulfiram on rat mammary tumor induction by N-2-fluorenylacetamide and on its metabolic conversion to N-hydroxy-N-2-fluorenylacetamide.

The effect of an antioxidant, disulfiram (DSF), on the carcinogenicities of N-2-fluorenylacetamide (2-FAA) and N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) was examined. DSF given in a diet at a concentration of 0.9% for 1 week before and throughout the carcinogen treatment (0.1 mmol/kg 3 times a week for 4 weeks) reduced the incidence of mammary tumors induced with 2-FAA by 50% and extended the mean latency period of malignant tumors from 5 to 10 months. By contrast, DSF had no effect on mammary carcinogenesis by N-OH-2-FAA. Consistent with these results was the demonstration of the inhibitory effect of DSF on the first step of metabolic activation of 2-FAA, i.e., N-hydroxylation. N-hydroxylation of 2-FAA was significantly inhibited in hepatic microsomes of untreated and 2-FAA-treated male and female rats by DSF given orally. A similar inhibition was shown in vitro after preincubation of hepatic microsomes with DSF. Measurements of cytochrome P450 after pretreatment of rats or microsomes with the inhibition showed no appreciable changes in the hemoprotein content. It was concluded, therefore, that the inhibitory effect of DSF on N-hydroxylation of 2-FAA is accomplished through mechanism(s) other than depression of the cytochrome P450 level. Because both 2-FAA and DSF bind to cytochrome P450 producing a type I spectrum, DSF may interfere with the binding of 2-FAA and thus alter its metabolism.

Pulsed ion cyclotron resonance mass spectrometer for studying ion-molecule reactions.

A pulsed ion cyclotron resonance mass spectrometer utilizes the cyclotron resonance principle for mass analysis of ions trapped at low pressures by electric and magnetic fields. Both mass analysis and ion trapping are accomplished in a one-region device called a trapped ion analyzer cell. A pulsing sequence is described which allows for generation of ions by electron impact, reaction with added gases, and mass analysis of the products of ion-molecule reactions. Methods are described for measuring rate constants and equilibrium constants for ion-molecule reactions. The high ion trapping efficiency and open geometry of the analyzer cell make it well suited for studying the interaction of laser radiation with gaseous ions and may also be useful for high-accuracy isotope ration mass spectrometry.

Identification of carcinogenic acetates of fluorenylhydroxamic acids by high-pressure liquid chromatography.

A method for the identification and quantitation of carcinogeniic O-acetates of fluorenylhydroxamic acids by high-pressure liquid chromatography is described. The adsorbent is Corasil II and a mixture of ethyl acetate-n-hexane (1:1) is used as the solvent. N-Acetoxy-2-fluorenylacetamide or N-acetoxy-3-fluorenylacetamide are separable as single peaks from N-acetoxy-4-fluorenylacetamide and from N-acetoxy-2-fluorenylbenzamide. The peak height traced by the recorder is linearly proportional to the amount of compound in the effluent. The method can be utilized for the detection of 0.1-1.0 mug of compound. The method has been used to identify the products of the decomposition of N-acetoxy-2-fluorenylacetamide in aqueous media.