In search of a counter you can count on: relative efficacy of human visual and automated colony counting.

To evaluate comparative efficiency of traditional vs automated colony counting methods, cultures of Escherichia coli (ATCC 25945), Staphylococcus epidermidis (ATCC 12225), Streptococcus pyogenes (ATCC19615) and Streptococcus pneumoniae (ATCC49619) were prepared as pure cultures and mixed cultures at 0·5 McFarland standard and serial dilutions were performed. Plates were inoculated in triplicate with 50, 125, 250 and 500 colony forming units and counted by four researchers, visually and using each of the automated counters. Colony count and counting time were recorded. The pattern of efficiency for all bacterial species was similar: plates with low counts were accurate and quick to count for all methods, with an increase in time and a decrease in accuracy and precision as counts rose. Higher counts of single round colonies required less time and had greater precision with automated counters than human visual counting counts with no loss of accuracy; however, counts were reduced in accuracy and increased in time for species with less regular morphology or when plates had mixed species. Surprisingly, a free phone application was only slightly less precise and more time consuming than the high-end professional counter indicating that automation may be achievable at lower cost than expected. SIGNIFICANCE AND IMPACT OF THE STUDY: Colony quantification is essential in clinical and research settings as well as pedagogy at the college level. Human visual (HV) counting, the most common method, is time consuming and fraught with errors. The time, accuracy and precision of HV counting were compared to a high-end professional automated counter, an inexpensive phone application and a free phone application. Low cost benefits of increased speed and accuracy with automated counting are maximized when counting single round colonies; but much reduced if colonies have irregular morphology or demonstrate haemolysis.

Misorientation mapping for visualization of plastic deformation via electron back-scattered diffraction.

The ability to map plastic deformation around high strain gradient microstructural features is central in studying phenomena such as fatigue and stress corrosion cracking. A method for the visualization of plastic deformation in electron back-scattered diffraction (EBSD) data has been developed and is described in this article. This technique is based on mapping the intragrain misorientation in polycrystalline metals. The algorithm maps the scalar misorientation between a local minimum misorientation reference pixel and every other pixel within an individual grain. A map around the corner of a Vickers indentation in 304 stainless steel was used as a test case. Several algorithms for EBSD mapping were then applied to the deformation distributions around air fatigue and stress corrosion cracks in 304 stainless steel. Using this technique, clear visualization of a deformation zone around high strain gradient microstructural features (crack tips, indentations, etc.) is possible with standard EBSD data.

Single nucleotide polymorphism markers for genetic mapping in Drosophila melanogaster.

For nearly a century, genetic analysis in Drosophila melanogaster has been a powerful tool for analyzing gene function, yet Drosophila lacks the molecular genetic mapping tools that recently have revolutionized human, mouse, and plant genetics. Here, we describe the systematic characterization of a dense set of molecular markers in Drosophila by using a sequence tagged site-based physical map of the genome. We identify 474 biallelic markers in standard laboratory strains of Drosophila that span the genome. Most of these markers are single nucleotide polymorphisms and sequences for these variants are provided in an accessible format. The average density of the new markers is one per 225 kb on the autosomes and one per megabase on the X chromosome. We include in this survey a set of P-element strains that provide additional use for high-resolution mapping. We show one application of the new markers in a simple set of crosses to map a mutation in the hedgehog gene to an interval of <1 Mb. This new map resource significantly increases the efficiency and resolution of recombination mapping and will be of immediate value to the Drosophila research community.

Drosophila p53 is a structural and functional homolog of the tumor suppressor p53.

The importance of p53 in carcinogenesis stems from its central role in inducing cell cycle arrest or apoptosis in response to cellular stresses. We have identified a Drosophila homolog of p53 ("Dmp53"). Like mammalian p53, Dmp53 binds specifically to human p53 binding sites, and overexpression of Dmp53 induces apoptosis. Importantly, inhibition of Dmp53 function renders cells resistant to X ray-induced apoptosis, suggesting that Dmp53 is required for the apoptotic response to DNA damage. Unlike mammalian p53, Dmp53 appears unable to induce a G1 cell cycle block when overexpressed, and inhibition of Dmp53 activity does not affect X ray-induced cell cycle arrest. These data reveal an ancestral proapoptotic function for p53 and identify Drosophila as an ideal model system for elucidating the p53 apoptotic pathway(s) induced by DNA damage.

Isolation of efficient antivirals: genetic suppressor elements against HIV-1.

The development of general approaches for the isolation of efficient antivirals and the identification and validation of targets for drug screening are becoming increasingly important, due to the emergence of previously unrecognized viral diseases. The genetic suppressor element (GSE) technology is an approach based on the functional expression selection of efficient genetic inhibitors from random fragment libraries derived from a gene or genome of interest. We have applied this technology to isolate potent genetic inhibitors against HIV-1. Two strategies were used to select for GSEs that interfere with latent virus induction and productive HIV-1 infection based on the expression of intracellular and surface antigens. The selected GSEs clustered in seven narrowly defined regions of the HIV-1 genome and were found to be functionally active. These elements are potential candidates for the gene therapy of AIDS. The developed approaches can be applied to other viral pathogens, as well as for the identification of cellular genes supporting the HIV-1 life cycle.

Maintenance of sister-chromatid cohesion at the centromere by the Drosophila MEI-S332 protein.

Sister-chromatid cohesion is essential for the faithful segregation of chromosomes during cell division. Recently biochemical analysis with Xenopus extracts suggests that cohesion is established during S phase by a cohesion complex but that other proteins must maintain it in mitosis. The Drosophila melanogaster MEI-S332 protein is present on centromeres in mitosis and meiosis and is essential for cohesion at the centromeres in meiosis II. Here, we analyze the timing of MEI-S332 assembly onto centromeres and the functional domains of the MEI-S332 protein. We find that MEI-S332 is first detectable on chromosomes during prometaphase, and this localization is independent of microtubules. MEI-S332 contains two separable functional domains, as mutations within these domains show intragenic complementation. The carboxy-terminal basic region is required for chromosomal localization. The amino-terminal coiled-coil domain may facilitate protein-protein interactions between MEI-S332 and male meiotic proteins. MEI-S332 interacts with itself in the yeast two-hybrid assay and in immunoprecipitates from Drosophila oocyte and embryo extracts. Thus it appears that MEI-S332 assembles into a multimeric protein complex that localizes to centromeric regions during prometaphase and is required for the maintenance of sister-chromatid cohesion until anaphase, rather than its establishment in S phase.

The perspective of the community pediatrician.

As the focus of health care moves to the outpatient setting, residency programs are seeking alternative training sites for teaching ambulatory primary care. The community pediatrician's office is an excellent location for residents to learn from preceptors who have been trained to teach them. Residents experience a variety of office and practice management issues not otherwise taught in the traditional hospital-based clinics. Pediatrician preceptors, because of commitment to the communities in which they live and practice, are role models for residents. Academic centers have the resources and expertise to train preceptors to become better teachers. The benefits to both preceptors and academic centers, the problems encountered, and suggestions for creating an exciting and innovative teaching program are discussed.

Patterns of auxin and abscisic acid movement in the tips of gravistimulated primary roots of maize.

Because both abscisic acid (ABA) and auxin (IAA) have been suggested as possible chemical mediators of differential growth during root gravitropism, we compared with redistribution of label from applied 3H-IAA and 3H-ABA during maize root gravitropism and examined the relative basipetal movement of 3H-IAA and 3H-ABA applied to the caps of vertical roots. Lateral movement of 3H-ABA across the tips of vertical roots was non-polar and about 2-fold greater than lateral movement of 3H-IAA (also non-polar). The greater movement of ABA was not due to enhanced uptake since the uptake of 3H-IAA was greater than that of 3H-ABA. Basipetal movement of label from 3H-IAA or 3H-ABA applied to the root cap was determined by measuring radioactivity in successive 1 mm sections behind the tip 90 minutes after application. ABA remained largely in the first mm (point of application) whereas IAA was concentrated in the region 2-4 mm from the tip with substantial levels found 7-8 mm from the tip. Pretreatment with inhibitors of polar auxin transport decreased both gravicurvature and the basipetal movement of IAA. When roots were placed horizontally, the movement of 3H-IAA from top to bottom across the cap was enhanced relative to movement from bottom to top whereas the pattern of movement of label from 3H-ABA was unaffected. These results are consistent with the hypothesis that IAA plays a role in root gravitropism but contrary to the idea that gravi-induced asymmetric distribution of ABA contributes to the response.

The inhibitor of DNA replication encoded by the Drosophila gene plutonium is a small, ankyrin repeat protein.

The plutonium (plu) gene product controls DNA replication early in Drosophila development. plu mutant females lay unfertilized eggs that have undergone extensive DNA synthesis. In fertilized embryos from plu mutant mothers, S-phase is uncoupled from mitosis. The gene is expressed only in ovaries and embryos, null alleles are strict maternal effect mutations, and the phenotype of inappropriate DNA replication is the consequence of loss-of-gene function. plu therefore negatively regulates S-phase at a time in early development when commitment to S-phase does not depend on cyclic transcription. plu encodes a protein with two ankyrin-like repeats, a domain for protein-protein interaction. plu is immediately adjacent to, but distinct from, the PCNA gene.

Managing the patient with liver failure.

Liver failure affects virtually all aspects of body functioning, which challenges medical-surgical nurses to deliver effective comprehensive care. Managing patients with liver failure requires a thorough understanding of hepatic physiology and the pathophysiology of liver disease failure.

Cloning and characterization of human astrovirus immunoreactive epitopes.

We report the cloning of antigenic, protein-coding regions of human astrovirus serotype 1 that appear to be common to most, if not all, serotypes of human astrovirus. Screening of lambda gt11 libraries identified three different but overlapping clones (A43, A35, and A1) and one independent clone (A14) that reacted with serum from a rabbit repeatedly immunized with purified astrovirus particles but not with its preimmunization serum. These clones were shown to be astrovirus specific. Of note, a radiolabeled probe representing the immunoreactive clones A43-A35-A1 hybridized exclusively to the 7.2-kb astrovirus genomic RNA, while a clone A14-specific probe hybridized with both the genomic and the 2.8-kb astrovirus subgenomic RNAs. This suggests that the immunoreactive epitopes, selected by antiserum to purified astrovirus particles, are encoded by the subgenomic RNA as well as other regions of the genomic RNA.

Symptom monitoring in schizophrenia: potential for enhancing self-care.

In this study a symptom self-regulation model was used as a framework to examine the characteristics and stability of indicators of illness identified by individuals with schizophrenia. Subjects were interviewed to determine if they could identify indicators of illness and describe characteristics of their primary indicator. Primary indicators of illness from 51 subjects were categorized as anxiety-based, depressive, or psychotic. Subjects who identified psychotic indicators were more confident that their indicator occurred when they were getting ill than subjects with anxiety-based or depressive indicators, and subjects who identified psychotic and depressive indicators reported that their indicators were more troublesome than those identifying anxiety-based indicators. Anxiety-based indicators were reported by subjects to occur more frequently than indicators from the other two categories. Findings from a follow-up interview of 28 subjects 1 year later showed that approximately half reported either the same primary indicator of illness or identified an indicator in the same category (anxiety-based, depressive, or psychotic) as they had 1 year previously. The implications of the findings for enhancing self-care through monitoring symptoms are discussed.

Characterization of metabolites of xylazine produced in vivo and in vitro by LC/MS/MS and by GC/MS.

The metabolic fate of xylazine, 2-(2,6-dimethylphenylamino)-5,6-dihydro-4H-1,3-thiazine, in horses is described. The major metabolites identified in the hydrolyzed horse urine were 2-(4'-hydroxy-2',6'-dimethylphenylamino)-5,6-dihydro-4H-1,3-thiazi ne, 2-(3'-hydroxy-2',6'-dimethylphenylamino)-5,6-dihydro-4H-1,3-thiazi ne, N-(2,6-dimethylphenyl)thiourea, and 2-(2',6'-dimethylphenylamino)-4-oxo-5,6-dihydro-1,3-thiazine. These metabolites were also produced by incubating xylazine with rat liver microsomes. The major metabolite produced in vitro by rat liver preparations was found to be the ring opened N-(2,6-dimethylphenyl)thiourea. The identities of these metabolites were confirmed by spectroscopic comparisons with synthetic standards. Phenolic metabolic standards were synthesized efficiently by the use of Fenton's reagent. This reagent was used to monohydroxylate multiply substituted aromatic ring systems. LC/MS/MS, with an atmospheric pressure chemical ionization source, was found to be particularly useful in confirming the presence of phenolic metabolites in hydrolyzed equine urine and microsomal extracts. These phenolic metabolites could not be analyzed by GC/MS even after derivatization with silylating agents. The advantage of LC/MS/MS was that no or little sample preparation of urine or microsomal extract was necessary prior to the analysis. A mechanism is also proposed for the formation of the major metabolite, N-(2,6-dimethylphenyl)thiourea, from xylazine.

Effect of calmodulin antagonists on the growth and graviresponsiveness of primary roots of maize.

We examined the effect of calmodulin (CaM) antagonists applied at the root tip on root growth, gravity-induced root curvature, and the movement of calcium across the root tip and auxin (IAA) across the elongation zone of gravistimulated roots. All of the CaM antagonists used in these studies delayed gravity-induced curvature at a concentration (1 micromole) that did not affect root growth. Calmodulin antagonists (> or = 1 micromole) inhibited downward transport of label from 45Ca2+ across the caps of gravistimulated roots relative to the downward transport of 45Ca2+ in gravistimulated roots which were not treated with CaM antagonists. Application of CaM antagonists at the root tip (> or = 1 micromole) also decreased the relative downward movement of label from 3H-IAA applied to the upper side of the elongation zone of gravistimulated roots. In general, tip application of antagonists inhibited neither the upward transport of 45Ca2+ in the root tip nor the upward movement of label from 3H-IAA in the elongation zone of gravistimulated roots. Thus, roots treated with CaM antagonists > or = 1 micromole become less graviresponsive and exhibit reduced or even a reversal of downward polarity of calcium transport across the root tip and IAA transport across the elongation zone. The results indicate that calmodulin-regulated events play a role in root gravitropism.

Hepatitis E virus (HEV): strain variation in the nonstructural gene region encoding consensus motifs for an RNA-dependent RNA polymerase and an ATP/GTP binding site.

Hepatitis is transmitted by a number of infectious agents. The epidemiological characterization of waterborne or enterically transmitted non-A, non-B hepatitis (ET-NANBH) is unique when compared with other known hepatitides. We have reported on the molecular cloning of a cDNA clone derived from the etiologic agent associated with ET-NANBH, the hepatitis E virus (HEV). The complete sequence of these first molecular clones, isolated from an HEV-infected human after passage in Macaca fascicularis (cynomolgus macaques), illustrates a distant relationship to other known positive-strand RNA viruses of plants and animals. The translated major open reading frame (ORF-1) from these clones indicates that this portion of the genome encodes a polyprotein with consensus sequences found in RNA-dependent RNA polymerase and ATP/GTP binding domains. The latter activity has been associated with putative helicases of positive-strand RNA viruses. These viral-encoded enzymatic activities identify this region and ORF-1 as containing at least two different nonstructural genes involved in HEV replication. Molecular clones obtained from two other geographically distinct HEV isolates demonstrated sequence heterogeneity in this nonstructural gene region. Further study will be required to elucidate the pathogenic significance (if any) of this observed divergence in the nonstructural region.

The role of calcium in the regulation of hormone transport in gravistimulated roots.

Prior research has shown that gravistimulation induces preferential movement of calcium toward the lower side of the tips of maize roots and that roots depleted of calcium show impaired gravitropism. To further investigate the role of calcium in root gravitropism, we examined the effects of calcium on auxin movement in both vertical and gravistimulated roots of maize. Longitudinal movement of auxin was basipetally polar in intact roots but acropetally polar in decapped roots. Treatment of the root tip with calcium increased basipetal auxin movement in both intact and decapped roots. Gravistimulation induced asymmetric auxin movement toward the lower side of the root tip. Both asymmetric auxin movement and gravicurvature were inhibited by treatment of the root tip with auxin transport inhibitors or with EGTA. The results indicate that there is a close correlation between curvature and gravity-induced asymmetric auxin movement across the root cap. Since gravistimulation causes calcium movement toward the lower side of the root tip, our observation that calcium promotes basipetal auxin movement supports the idea that gravity-induced calcium asymmetry is a key step linking gravistimulation to the establishment of auxin asymmetry during root gravitropism.

DIC: the insidious killer.

Disseminated intravascular coagulation (DIC) is not a disease but an abnormal syndrome that is always secondary to another process. Because its symptomatology is varied and subtle, frequent assessments of body systems must be conducted. This article and its plan of care are designed to assist the nurse in the effective management of the DIC patient.

Validation of a method for determination of tetracycline antibiotics in salmon muscle tissue.

A method was validated for analysis of oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) in fortified salmon muscle tissue. Recoveries of OTC were 100 +/- 6, 86 +/- 6, and 82 +/- 5% (n = 6) at fortification levels of 1.0, 0.5, and 0.2 ppm, respectively. Recoveries of TC were 68 +/- 4, 65 +/- 6, and 66 +/- 7%; recoveries of CTC were 45 +/- 9, 48 +/- 8, and 0%, respectively. Detection limits for OTC and TC were 0.08 and 0.09 ppm, respectively.