Quantification of relative proportions of intact cells in microbiological samples using the example of Cryptosporidium parvum oocysts.

UNLABELLED: The fast analysis of relative proportions of live and dead cells can be of great value whether for comparing inactivation efficiencies of different biocidal treatments or for monitoring organisms of interest in environmental samples. We introduce here a straightforward method to determine the percentage of intact cells based on treatment of samples with the viability dye propidium monoazide (PMA). PMA selectively enters membrane-damaged cells and suppresses their PCR detection through modification of their DNA. The study was performed using Cryptosporidium parvum oocysts as a model although the principle should be applicable to other organisms. Validation was performed with defined mixtures of live and heat-killed oocysts and by exposing oocysts to a heat stress gradient. The method correctly indicated increasingly lower proportions of intact cells with increasing temperatures. When comparing the loss of membrane integrity of UV-killed (40 mJ cm(-2) ) oocysts during storage in nonsterile tap water, results suggested that integrity declines slowly (over weeks) and at a rate comparable to non-UV-exposed oocysts. For all experiments, the amplification of longer DNA sequences was found beneficial. In the UV experiment, longer amplicons revealed not only higher sensitivity in excluding membrane-damaged oocysts, but also in excluding DNA with UV-induced damage. SIGNIFICANCE AND IMPACT OF THE STUDY: Whether in the context of microbial ecology or in an industrial context, many questions in microbiology are linked to microbial viability. As cultivation of micro-organisms can be long or may not be possible, fast methods to assess the numbers of live cells are in great demand. We present here a straightforward strategy to determine the relative proportions of intact cells. The PCR-based rapid method is expected to be useful where relative information is sufficient (e.g. for comparing the effect of different antimicrobial treatments on known numbers of micro-organisms) or when the presence of PCR inhibitors does not allow absolute quantification.

Design of peptide imaging agents for whole-body and intraoperative molecular imaging.

Due to the growing toolkit of targeted contrast agents, molecular imaging continues to play a prominent role in the clinical care of cancer. Peptide-based imaging approaches are of particular significance due to their favorable pharmacokinetic properties, established manufacturing infrastructure, and documented clinical success in whole-body imaging. A logical extension of molecular imaging with peptides is to improve surgical outcomes in cancer through highly sensitive and specific probes which can be used intraoperatively. Advances in fluorescent imaging have resulted in various peptide labeling strategies with intraoperative indications. In this review, we focused on the evolving design of peptide imaging agents starting with the clinically used somatostatin targeting peptides. We then review the current synthetic approaches used for dual-labeled agent development and offer perspectives on optimal protection schemes that can be used for multimodal probe development.

Safety and pharmacokinetics of aciclovir in women following release from a silicone elastomer vaginal ring.

OBJECTIVES: Systemic aciclovir and its prodrug valaciclovir are effective in treating and reducing recurrences of genital herpes simplex virus (HSV) and reducing transmission. Local aciclovir delivery, if it can achieve and maintain comparable intracellular genital tract levels, may be equally effective in the treatment and suppression of genital HSV. Intravaginal ring (IVR) delivery of aciclovir may provide pre-exposure prophylaxis against HSV acquisition. METHODS: Tolerability and pharmacokinetics were evaluated in six HIV-negative women with recurrent genital HSV who switched their daily oral valaciclovir suppression to an aciclovir IVR for 7 days (n = 3) or 14 days (n = 3). Blood and cervicovaginal lavage (CVL) were collected after oral and IVR dosing to measure aciclovir concentrations and genital swabs were obtained to quantify HSV shedding by PCR. RESULTS: The rings were well tolerated. Median plasma aciclovir concentrations were 110.2 ng/mL (IQR, 85.9-233.5) 12-18 h after oral valaciclovir. Little or no drug was detected in plasma following IVR dosing. Median (IQR) CVL aciclovir levels were 127.3 ng/mL (21-660.8) 2 h after oral valaciclovir, 154.4 ng/mL (60.7-327.5) 12-18 h after oral valaciclovir and 438 ng/mL (178.5-618.5) after 7 days and 393 ng/mL (31.6-1615) after 14 days of aciclovir ring use. Median CVL aciclovir levels 2 h after oral dosing were similar to levels observed 7 (P = 0.99) and 14 (P = 0.75) days after ring use. HSV DNA was not detected in genital swabs and there was no significant change in inflammatory mediators. CONCLUSIONS: This first-in-human study demonstrated that an IVR could safely deliver mucosal levels of aciclovir similar to oral valaciclovir without systemic absorption. More intensive site-specific pharmacokinetic studies are needed to determine whether higher local concentrations are needed to achieve optimal drug distribution within the genital tract.

Pathogens in Crassostrea ariakensis and other Asian oyster species: implications for non-native oyster introduction to Chesapeake Bay.

With the drastic decline of eastern oyster Crassostrea virginica populations in the Chesapeake Bay due to over-fishing, diseases and habitat destruction, there is interest in Maryland and Virginia in utilizing the non-native oyster species Crassostrea ariakensis for aquaculture, fishery resource enhancement, and ecological restoration. The International Council for the Exploration of the Sea (ICES) recommends that non-native species be examined for ecological, genetic and disease relationships in the native range prior to a deliberate introduction to a new region. Therefore, a pathogen survey of C. ariakensis and other sympatric oyster species was conducted on samples collected in the PR China, Japan and Korea using molecular diagnostics and histopathology. Molecular assays focused on 2 types of pathogens: protistan parasites in the genus Perkinsus and herpesviruses, both with known impacts on commercially important molluscan species around the world, including Asia. PCR amplification and DNA sequence data from the internal transcribed spacer region of the rRNA gene complex revealed the presence of 2 Perkinsus species not currently found in USA waters: P. olseni and an undescribed species. In addition, 3 genetic strains of molluscan herpesviruses were detected in oysters from several potential C. ariakensis broodstock acquisition sites in Asia. Viral gametocytic hypertrophy, Chlamydia-like organisms, a Steinhausia-like microsporidian, Perkinsus sp., Nematopsis sp., ciliates, and cestodes were also detected by histopathology.

Conservation in the first internal transcribed spacer region (ITS1) in Hematodinium species infecting crustacean hosts found in the UK and Newfoundland.

Parasitic dinoflagellates in the genus Hematodinium infect a number of decapod crustaceans in waters off the UK, including the Norway lobster Nephrops norvegicus and the edible crab Cancer pagurus. This study investigated sequence variability in the first internal transcribed spacer (ITS1) region of the ribosomal RNA complex of Hematodinium spp. infecting N. norvegicus, C. pagurus, and Pagurus bernhardus from 4 locations in the UK and from the Hematodinium sp. infecting Chionoecetes opilio from the province of Newfoundland and Labrador, Canada. Phylogenetic analysis of the Hematodinium ITS1 sequences from N. norvegicus, C. pagurus, P. bernhardus and C. opilio suggest that these crustaceans are infected with the same species of Hematodinium. Length variability of the ITS1 region was observed (324 to 345 bp) and attributed to 4 variable microsatellite regions (CATG)n' (GCC)nTCCGC(TG)n' (TA)n' and (GAA)n(GGA)n within the sequenced ITS1 fragment. The observed variation may be due to co-infection of the host crustacean with several different strains of Hematodinium or differences among copies of ITS1 region within the genome of a single parasite cell. The Hematodinium ITS1 sequence from N. norvegicus, C. pagurus, P. bernhardus and C. opilio isolates was sufficiently conserved in primer binding regions targeted by previous molecular diagnostic assays; therefore, we suggest that this assay could be used to screen for Hematodinium infections in these crustacean hosts.

Non-invasive measurement of cardiac output: evaluation of new infrared absorption spectrometer.

The mass spectrometer (MS) traditionally has been the instrument of choice for measuring cardiac output (Q (T)) non-invasively using the foreign gas uptake method. However, the size and cost of the MS has hampered widespread adoption of this technique outside of the laboratory. Here, we present results, from six normal human subjects at rest and during exercise, of simultaneous Q (T) measurements by an MS and a new, portable infrared (IR) device developed in our laboratories. These measurements are made using on the open-circuit acetylene uptake method. The IR device measures inspired and end-tidal concentrations of acetylene, sulfur hexafluoride, and carbon dioxide by IR absorption spectroscopy with a 10-90% response time of 43 ms; accurate measurements were made down to sample flow rates of 50 mL min(-1). Excellent correlation [Q (T)(IR)=0.98 Q (T)(MS), R(2)=0.94] was observed between instruments across the range from rest to heavy exercise. These results suggest that the IR device, which is small, light-weight, and rugged may enable the foreign gas uptake method to be used in clinical, field, and point-of-care settings for Q (T) measurement.

Mapping tertiary interactions in protein folding reactions: a novel mass spectrometry- and chemical synthesis-based approach.

A novel mass spectrometry- and chemical synthesis-based approach for studying protein folding reactions is described, and its initial application to study the folding/unfolding reaction of a homo-hexameric enzyme 4-oxalocrotonate (4OT) is reported. This new approach involves the application of total chemical synthesis to prepare protein analogues that contain a photoreactive amino acid site-specifically incorporated into their primary amino acid sequence. To this end, a photoreactive amino acid-containing analogue of 4OT in which Pro-1 was replaced with p-benzoyl-l-phenylalanine (Bpa) was prepared. This analogue can be used to map structurally specific protein-protein interactions in 4OT's native folded state. These photocrosslinking studies and peptide mapping results with (PlBpa)4OT indicate that this construct is potentially useful for probing the structural properties of equilibrium and kinetic intermediates in 4OT's folding reaction.

Strategy for mutation analysis in the autosomal recessive limb-girdle muscular dystrophies.

We describe a strategy for molecular diagnosis in the autosomal recessive limb-girdle muscular dystrophies, a highly heterogeneous group of inherited muscle-wasting diseases. Genetic mutation analysis is directed by immunoanalysis of muscle biopsies using antibodies against a panel of muscular dystrophy-associated proteins. Performing the molecular analysis in this way greatly increases the chance that mutations will be found in the first gene examined. The use of this strategy can significantly decrease the time involved in determining the genetic fault in a patient with a clinical diagnosis of recessive limb-girdle muscular dystrophy, as well as having a feedback effect, which is useful in helping clinicians to identify subtle clinical differences between the subtypes of the disease. The use of this approach has so far helped us to identify mutations in ten sarcoglycanopathy (limb-girdle muscular dystrophy 2C-2F) patients, and seven calpainopathy (limb-girdle muscular dystrophy 2A) patients.

Secondary reduction in calpain 3 expression in patients with limb girdle muscular dystrophy type 2B and Miyoshi myopathy (primary dysferlinopathies).

Dysferlin is the protein product of the gene (DYSF) that is defective in patients with limb girdle muscular dystrophy type 2B and Miyoshi myopathy. Calpain 3 is the muscle-specific member of the calcium activated neutral protease family and primary mutations in the CAPN3 gene cause limb girdle muscular dystrophy type 2A. The functions of both proteins remain speculative. Here we report a secondary reduction in calpain 3 expression in eight out of 16 patients with a primary dysferlinopathy and clinical features characteristic of limb girdle muscular dystrophy type 2B or Miyoshi myopathy. Previously CAPN3 analysis had been undertaken in three of these patients and two showed seemingly innocuous missense mutations, changing calpain 3 amino acids to those present in the sequences of calpains 1 and 2. These results suggest that there may be an association between dysferlin and calpain 3, and further analysis of both genes may elucidate a novel functional interaction. In addition, an association was found between prominent expression of smaller forms of the 80 kDa fragment of laminin alpha 2 chain (merosin) and dysferlin-deficiency.

Electrocatalysis in photochemically activated electropolymerized thin films.

In reductively electropolymerized thin films of poly-cis-[Ru(vbpy)2(py)2](PF6)2 ("vbpy" is 4-methyl-4'-vinyl-2,2'-bipyridine and "py" is pyridine), on glassy carbon electrodes, photochemical ligand loss with aqueous HClO4 in the external solution occurs to give poly-cis-[Ru(vbpy)2(OH2)2](ClO4)2, but the efficiency of ligand loss is greatly decreased compared to the efficiency in solution. In cyclic voltammograms of films containing the aqua complex, there is evidence for both RuIII/II and higher oxidation state RuIV/III, RuV/IV, and RuVI/V couples. The redox chemistry of the resulting films is dictated by the film structure and can be controlled by varying the electropolymerization conditions and external solution composition. The oxidation of alcohols by these higher oxidation state couples has been investigated electrochemically.

Dysferlin is a plasma membrane protein and is expressed early in human development.

Recently, a single gene, DYSF, has been identified which is mutated in patients with limb-girdle muscular dystrophy type 2B (LGMD2B) and with Miyoshi myopathy (MM). This is of interest because these diseases have been considered as two distinct clinical conditions since different muscle groups are the initial targets. Dysferlin, the protein product of the gene, is a novel molecule without homology to any known mammalian protein. We have now raised a monoclonal antibody to dysferlin and report on the expression of this new protein: immunolabelling with the antibody (designated NCL-hamlet) demonstrated a polypeptide of approximately 230 kDa on western blots of skeletal muscle, with localization to the muscle fibre membrane by microscopy at both the light and electron microscopic level. A specific loss of dysferlin labelling was observed in patients with mutations in the LGMD2B/MM gene. Furthermore, patients with two different frameshifting mutations demonstrated very low levels of immunoreactive protein in a manner reminiscent of the dystrophin expressed in many Duchenne patients. Analysis of human fetal tissue showed that dysferlin was expressed at the earliest stages of development examined, at Carnegie stage 15 or 16 (embryonic age 5-6 weeks). Dysferlin is present, therefore, at a time when the limbs start to show regional differentiation. Lack of dysferlin at this critical time may contribute to the pattern of muscle involvement that develops later, with the onset of a muscular dystrophy primarily affecting proximal or distal muscles.

Identical mutation in patients with limb girdle muscular dystrophy type 2B or Miyoshi myopathy suggests a role for modifier gene(s).

Limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), a distal muscular dystrophy, are both caused by mutations in the recently cloned gene dysferlin, gene symbol DYSF. Two large pedigrees have been described which have both types of patient in the same families. Moreover, in both pedigrees LGMD2B and MM patients are homozygous for haplotypes of the critical region. This suggested that the same mutation in the same gene would lead to both LGMD2B or MM in these families and that additional factors were needed to explain the development of the different clinical phenotypes. In the present paper we show that in one of these families Pro791 of dysferlin is changed to an Arg residue. Both the LGMD2B and MM patients in this kindred are homozygous for this mutation, as are four additional patients from two previously unpublished families. Haplotype analyses suggest a common origin of the mutation in all the patients. On western blots of muscle, LGMD2B and MM patients show a similar abundance in dysferlin staining of 15 and 11%, respectively. Normal tissue sections show that dysferlin localizes to the sarcolemma while tissue sections from MM and LGMD patients show minimal staining which is indistinguishable between the two types. These findings emphasize the role for the dysferlin gene as being responsible for both LGMD2B and MM, but that the distinction between these two clinical phenotypes requires the identification of additional factor(s), such as modifier gene(s).

Characterization of monoclonal antibodies to calpain 3 and protein expression in muscle from patients with limb-girdle muscular dystrophy type 2A.

Monoclonal antibodies were raised to two regions of calpain 3 (muscle-specific calcium-activated neutral protease), which is the product of the gene that is defective in limb-girdle muscular dystrophy type 2A. The antibodies produced characteristic patterns of bands on Western blots: normal calpain 3 protein was represented by bands at 94 kd, plus additional fragments at approximately 60 or 30 kd, according to the antibody used. Specificity was confirmed by the loss of all bands in patients with null gene mutations. The "normal" profile of bands was observed in muscle from 33 control subjects and 70 disease-control patients. Calpain 3 protein was found to be extremely stable in fresh human muscle, with full-size protein being detected 8 hours after the muscle had been removed. Blots of muscle from nine limb-girdle muscular dystrophy type 2A patients with defined mutations showed variation in protein expression, with seven showing a clear reduction in the abundance of protein detected. No simple relationship was found between the abundance and clinical severity. Two patients showed normal expression of the full-size 94 kd band accompanied by a clear reduction in the smaller fragments. This pattern was also observed in one patient with an undefined form of limb-girdle dystrophy. These results indicate that immunodiagnosis is feasible, but caution will need to be exercised with the interpretation of near-normal protein profiles.

Insulin-like growth factor (IGF)-I and IGF-II binding to an IGF binding protein. An investigation using chemical modification of tyrosine residues as a structural probe for the sites of interaction.

We have investigated insulin-like growth factor (IGF)-I and IGF-II binding to bovine insulin-like growth factor binding protein-2 (bIGFBP-2) using chemical modification to locate sites on the IGF involved in the binding interaction. bIGFBP-2 was incubated with either recombinant human (hIGF-I) or purified ovine (oIGF-II) to form a mixture of bound and free IGF. Sites of interaction between the binding protein and IGF were then probed by iodination of the available tyrosine residues. Subsequently, the mixture of free IGF and IGF.bIGFBP-2 complex was resolved by neutral chromatography, and the IGF component of the complex with bIGFBP-2 was recovered by reverse-phase high performance liquid chromatography at pH 2.1. The tyrosine labeling patterns of the two populations of IGF, one iodinated while free and the other iodinated while associated with binding protein, were determined following endoproteinase Glu-C peptide mapping. Binding of hIGF-I or oIGF-II to bIGFBP-2 resulted in reduced iodination of the tyrosines in both hIGF-I and oIGF-II that are near the carboxyl-terminal, Tyr-60 and Tyr-59, respectively. The reduction in labeling of these tyrosine residues was 2-fold and 6-fold for hIGF-I and oIGF-II, respectively. On the other hand, labeling of the other 2 tyrosines in hIGF-I and oIGF-II was not different between the free and complexed growth factors. From these results we conclude that Tyr-60 and Tyr-59 in the carboxyl-terminal regions of hIGF-I and oIGF-II, respectively, are either directly involved in the binding reaction or lie in a region of the IGF molecule encompassed by the association with bIGFBP-2. Conversely, the labeling pattern of the other tyrosines, Tyr-24 and Tyr-31 in hIGF-I and Tyr-2 and Tyr-27 in oIGF-II, implies that they are not involved in binding to bIGFBP-2. To examine the role of IGF tyrosine residues in the association with bIGFBP-2, we prepared nonradioactive 127I-labeled oIGF-II. In bIGFBP-2 competition binding assays, 127I-labeled oIGF-II was 2.5-fold and 5-fold less potent than native oIGF-II when competing for binding of 125I-labeled IGF-I or IGF-II tracers, respectively. We interpret these results as indicating that at least 1 tyrosine in oIGF-II is involved in binding to bIGFBP-2.

Women chiropractors: issues of career and family.

The present study is an examination of the characteristics of female chiropractors in Canada. Of particular interest were their practice patterns, their productivity, their political involvement in the profession and the methods they used for coping with the multiple responsibilities of career and family life. The study was conducted by mailing a questionnaire to all Canadian female chiropractors. The results indicate that the vast majority of respondents are relatively new practitioners, having graduated since 1976. A very high percentage have remained in active practice either full or part-time since graduation and are very satisfied with their choice of career. The majority live in Ontario or Quebec and are politically active as members of their national, provincial and local associations. Childbirth does not appear to have interfered significantly with their career. The majority of mothers returned to work within 3 months of childbirth. The respondents appear to cope with the responsibilities of career and family life, but, a disturbing proportion report feeling chronically drained.

Primary aspergillosis of the larynx simulating carcinoma.

A case of pseudoepitheliomatous hyperplasia of the larynx secondary to fungal infection is presented. The laryngeal lesion resembled carcinoma both clinically and pathologically. Aspergillus species were identified in tissue sections and confirmed by immunofluorescence studies. The lesion responded well to antifungal therapy. This case is reported because of the extreme rarity of primary aspergillosis of the larynx, its occurrence in an otherwise healthy patient, and its marked resemblance both clinically and pathologically to laryngeal carcinoma.

The comparative metabolism of 2,6-dichlorothiobenzamide (Prefix) and 2,6-dichlorobenzonitrile in the dog and rat.

1. A single oral dose of either [(14)C]Prefix or 2,6-dichlorobenzo[(14)C]nitrile to rats is almost entirely eliminated in 4 days: 84.8-100.5% of (14)C from [(14)C]Prefix is excreted, 67.3-79.7% in the urine, and 85.8-97.2% of (14)C from 2,6-dichlorobenzo-[(14)C]nitrile is excreted, 72.3-80.7% in the urine. Only 0.37+/-0.03% of the dose of [(14)C]Prefix and 0.25+/-0.03% of the dose of 2,6-dichlorobenzo[(14)C]nitrile are present in the carcass plus viscera after removal of the gut. Rats do not show sex differences in the pattern of elimination of the respective metabolites of the two herbicides. The rates of elimination of (14)C from the two compounds in the 24hr. and 48hr. urines are not significantly different (P >0.05) from one another. 2. After oral administration to dogs, 85.9-106.1% of (14)C from [(14)C]Prefix is excreted, 66.6-80.9% in the urine, and 86.8-92.5% of (14)C from 2,6-dichlorobenzo[(14)C]nitrile is excreted, 60.0-70.1% in the urine. Dogs do not show sex differences in the pattern of eliminating the metabolites of either Prefix or 2,6-dichlorobenzonitrile. 3. Dogs and rats do not show species differences in the patterns of elimination of the two herbicides. 4. Prefix and 2,6-dichlorobenzonitrile are completely metabolized; unchanged Prefix and 2,6-dichlorobenzonitrile are absent from the urine and faeces, and from the carcasses when elimination is complete. In the hydrolysed urine of rats dosed with either [(14)C]Prefix or 2,6-dichlorobenzo[(14)C]nitrile, 2,6-dichloro-3-hydroxybenzonitrile accounts for approx. 42% of the (14)C, a further 10-11% is accounted for by 2,6-dichlorobenzamide, 2,6-dichlorobenzoic acid, 2,6-dichloro-3- and -4-hydroxybenzoic acid and 2,6-dichloro-4-hydroxybenzonitrile collectively, and 25-30% by six polar constituents, of which two are sulphur-containing amino acids. 5. In the unhydrolysed urines of rats dosed with either [(14)C]Prefix or 2,6-dichlorobenzo[(14)C]nitrile, there are present free 2,6-dichloro-3- and -4-hydroxybenzonitrile, their glucuronide conjugates, ester glucuronides of the principal aromatic acids that are present in the hydrolysed urines, and two sulphur-containing metabolites analogous to mercapturic acids or premercapturic acids. 6. Prefix is thus extensively transformed into 2,6-dichlorobenzonitrile: R.CS.NH(2)-->R.CN+H(2)S, where R=C(6)H(3)Cl(2). However, the competitive reaction: R.CS.NH(2)+H(2)O-->R.CO.NH(2)+H(2)S takes place to a very limited extent.