Human case of bubonic plague resulting from the bite of a wild Gunnison's prairie dog during translocation from a plague-endemic area.

Plague is a zoonotic disease (transmitted mainly by fleas and maintained in nature by rodents) that causes severe acute illness in humans. We present a human plague case who became infected by the bite of a wild Gunnison's prairie dog, and a good practical example of the One Health approach that resulted in a rapid public health response. The exposure occurred while the animal was being transported for relocation to a wildlife refuge after being trapped in a plague enzootic area. This is the first report of a human plague case resulting from the bite of a Gunnison's prairie dog. Additionally, we present an observation of a longer incubation period for plague in captive prairie dogs, leading to a recommendation for a longer quarantine period for prairie dogs during translocation efforts.

Carbonate ooids of the Mesoarchaean Pongola Supergroup, South Africa.

Ooids from the Mesoarchaean Chobeni Formation, Pongola Supergroup, KwaZulu-Natal, South Africa are older than any ooids reported to date. They are made of dolomite and ankerite and show concentric, radial-concentric, micritic, and radial fabrics. Radial ooids are interpreted to have originated from high-Mg-calcite and probably formed by microbial activity in a low-energy regime, while concentric ooids had an aragonite precursor and formed biotically under agitated/high-energy environmental conditions. Micritic ooids formed via the recrystallization of concentric ooids. Ooids and other allochems, such as intraclasts and peloids, contain carbonaceous matter. The close association of carbonaceous matter within ooid cortices with metabolically important elements, such as N, S and P, as identified by nano-scale secondary ion mass spectrometry analysis, allows us to propose a biologically induced origin for some ooids. By analogy with modern examples, a variety of microbial communities probably played a role in carbonate precipitation and ooid formation. Shale-normalized rare earth element (REE) distribution patterns of ooids and other allochems show positive LaSN , GdSN and YSN anomalies, superchondritic Y/Ho ratios and depleted light rare earth elements (LREEs) relative to the heavy rare earth elements (HREEs), which resemble those of seawater. These anomalies are less pronounced than expected for an open marine setting, which is interpreted as evidence for deposition in restricted shallow marine environments. Non-seawater REE patterns in recrystallized matrix and pore- and vein-filling carbonate likely reflect redistribution of rare earth elements during post-depositional alteration and/or reflect differences in the elemental and REE compositions of diagenetic fluids.

The complexity of patient safety reporting systems in UK dentistry.

Since the 'Francis Report', UK regulation focusing on patient safety has significantly changed. Healthcare workers are increasingly involved in NHS England patient safety initiatives aimed at improving reporting and learning from patient safety incidents (PSIs). Unfortunately, dentistry remains 'isolated' from these main events and continues to have a poor record for reporting and learning from PSIs and other events, thus limiting improvement of patient safety in dentistry. The reasons for this situation are complex.This paper provides a review of the complexities of the existing systems and procedures in relation to patient safety in dentistry. It highlights the conflicting advice which is available and which further complicates an overly burdensome process. Recommendations are made to address these problems with systems and procedures supporting patient safety development in dentistry.

MicroRNA expression profiles of seminoma from paraffin-embedded formalin-fixed tissue.

In this study, we used microRNA (miRNA) microarrays in an unbiased screen for aberrantly expressed miRNAs in seminoma, a primitive type of germ cell tumor. Formalin-fixed and paraffin-embedded (FFPE) surgical samples from 11 cases of normal testicular tissue resected for nonneoplastic causes and from 11 cases of seminoma were assessed for miRNA expression. Normal testicular tissue and seminoma were paired by race. We found 112 miRNAs to be differentially expressed between seminoma and normal testicular tissue; 52 miRNAs were overexpressed, and 60, downregulated in seminoma. We did not observe significant differences between black and white populations in our race-paired study. The upregulation of the expression of hsa-mir-21, hsa-mir-372, hsa-mir-373, has-mir-221, and hsa-mir-222 was validated by reverse transcription and real-time PCR. Hsa-mir-372 was upregulated around 1,270-fold (95 % confidence interval (CI) 525.2-3,064.8; p = 8.1e-5 by Mann-Whitney U test). Hsa-mir-373 was upregulated around 1,530-fold (95 % CI 620.5-3,785.6; p = 8.0e-5 by Mann-Whitney U test), consistent with previous reports, indicating that the miRNAs in FFPE are well preserved, and FFPE can be a valuable source for the miRNA study of seminoma. In addition, expression of hsa-mir-21 (12.2-fold, 0.0095), hsa-mir-221 (3.8-fold, 0.014) and hsa-mir-222 (3.8-fold, 0.019) was found elevated in seminoma compared to normal testicular tissue.

Expression of parafibromin in major renal cell tumors.

Parafibromin, encoded by HRPT2 gene, is a recently identified tumor suppressor. Complete and partial loss of its expression have been observed in hyperparathyroidism-jaw tumor (HPT-JT), parathyroid carcinoma, breast carcinoma, lung carcinoma, gastric and colorectal carcinoma. However, little has been known about its expression in renal tumors. In order to study the expression of parafibromin in a series of the 4 major renal cell tumors - clear cell renal cell carcinoma (ccRCC), papillary renal cell carcinoma (pRCC), chromophobe renal cell carcinoma (chRCC) and oncocytoma. One hundred thirty nine renal tumors including 61 ccRCCs, 37 pRCCs, 22 chRCCs and 19 oncocytomas were retrieved and used for the construction of renal tissue microarrays (TMAs). The expression of parafibromin was detected by immunohistochemical method on the constructed TMAs. Positive parafibromin stains are seen in 4 out of 61 ccRCCs (7%), 7 out of 37 pRCCs (19%), 12 out of 23 chRCCs (52%) and all 19 oncocytomas (100%). Parafibromin expression varies significantly (P< 8.8 x10-16) among the four major renal cell tumors and were correlated closely with tumor types. No correlation of parafibromin expression with tumor staging in ccRCCs, pRCCs and chRCCs, and Fuhrman nuclear grading in ccRCCs and pRCCs. In summary, parafibromin expression was strongly correlated with tumor types, which may suggest that it plays a role in the tumorigenesis in renal cell tumors.

Current perspectives in protein array technology.

This article reviews post-2000 trends in the development of two-dimensional protein microarrays and nanoarrays. Progress in array manufacture, assay design and applications are considered, with an emphasis on issues surrounding the implementation of arrays in clinical diagnostics. These include the effect of factors in the pre-analytical phase (quality of the reagents, sample integrity, etc.), and those in the analytical phase that contribute to inaccuracy and imprecision of an array-based assay. Important requirements for the quality control and quality assurance of protein microarray assays as they move from the research environment into routine clinical application are also discussed.

Oxidative stress response genes in Mycobacterium tuberculosis: role of ahpC in resistance to peroxynitrite and stage-specific survival in macrophages.

The Mycobacterium tuberculosis ahpC gene, encoding the mycobacterial orthologue of alkylhydroperoxide reductase, undergoes an unusual regulatory cycle. The levels of AhpC alternate between stages of expression silencing in virulent strains grown as aerated cultures, secondary to a natural loss of the regulatory oxyR function in all strains of the tubercle bacillus, and expression activation in static bacilli by a yet undefined mechanism. The reasons for this unorthodox regulatory cycle controlling expression of an antioxidant factor are currently not known. In this work, M. tuberculosis H37Rv and Mycobacterium smegmatis mc(2)155 ahpC knockout mutants were tested for sensitivity to reactive nitrogen intermediates, in particular peroxynitrite, a highly reactive combinatorial product of reactive nitrogen and oxygen species, and sensitivity to bactericidal mechanisms in resting and activated macrophages. Both M. tuberculosis ahpC::Km(r) and M. smegmatis ahpC::Km(r) showed increased susceptibility to peroxynitrite. In contrast, inactivation of ahpC in M. tuberculosis did not cause increased sensitivity to donors of NO alone. M. tuberculosis ahpC::Km(r) also showed decreased survival in unstimulated macrophages, but the effect was no longer detectable upon IFNgamma activation. These studies establish a specific role for ahpC in antioxidant defences involving peroxynitrite and most likely additional cidal mechanisms in macrophages, with the regulatory cycle likely contributing to survival upon coming out of the stationary phase during dormancy (latent infection) or upon transmission to a new host.

Silencing of oxidative stress response in Mycobacterium tuberculosis: expression patterns of ahpC in virulent and avirulent strains and effect of ahpC inactivation.

Intracellular pathogens such as Mycobacterium tuberculosis are able to survive in the face of antimicrobial products generated by the host cell in response to infection. The product of the alkyl hydroperoxide reductase gene (ahpC) of M. tuberculosis is thought to be involved in protecting the organism against both oxidative and nitrosative stress encountered within the infected macrophage. Here we report that, contrary to expectations, ahpC expression in virulent strains of M. tuberculosis and Mycobacterium bovis grown in vitro is repressed, often below the level of detection, whereas expression in the avirulent vaccine strain M. bovis BCG is constitutively high. The repression of the ahpC gene of the virulent strains is independent of the naturally occurring lesions of central regulator oxyR. Using a green fluorescence protein vector (gfp)-ahpC reporter construct we present data showing that repression of ahpC of virulent M. tuberculosis also occurred during growth inside macrophages, whereas derepression in BCG was again seen under identical conditions. Inactivation of ahpC on the chromosome of M. tuberculosis by homologous recombination had no effect on its growth during acute infection in mice and did not affect in vitro sensitivity to H2O2. However, consistent with AhpC function in detoxifying organic peroxides, sensitivity to cumene hydroperoxide exposure was increased in the ahpC::Km(r) mutant strain. The preservation of a functional ahpC gene in M. tuberculosis in spite of its repression under normal growth conditions suggests that, while AhpC does not play a significant role in establishing infection, it is likely to be important under certain, as yet undefined conditions. This is supported by the observation that repression of ahpC expression in vitro was lifted under conditions of static growth.

Analysis of murine Brca2 reveals conservation of protein-protein interactions but differences in nuclear localization signals.

In this report, we have analyzed the protein encoded by the murine Brca2 locus. We find that murine Brca2 shares multiple properties with human BRCA2 including its regulation during the cell cycle, localization to nuclear foci, and interaction with Brca1 and Rad51. Murine Brca2 stably interacts with human BRCA1, and the amino terminus of Brca2 is sufficient for this interaction. Exon 11 of murine Brca2 is required for its stable association with RAD51, whereas the carboxyl terminus of Brca2 is dispensable for this interaction. Finally, in contrast to human BRCA2, we demonstrate that carboxyl-terminal truncations of murine Brca2 localize to the nucleus. This finding may explain the apparent inconsistency between the cytoplasmic localization of carboxyl-terminal truncations of human BRCA2 and the hypomorphic phenotype of mice homozygous for similar carboxyl-terminal truncating mutations.

Mapping of Mycobacterium tuberculosis katG promoters and their differential expression in infected macrophages.

Intracellular pathogenic bacteria, including Mycobacterium tuberculosis, frequently have multitiered defense mechanisms ensuring their survival in host phagocytic cells. One such defense determinant in M. tuberculosis is the katG gene, which encodes an enzyme with catalase, peroxidase, and peroxynitritase activities. KatG is considered to be important for protection against reactive oxygen and nitrogen intermediates produced by phagocytic cells. However, KatG also activates the front-line antituberculosis drug isoniazid, hence rendering M. tuberculosis exquisitely sensitive to this compound. In this context, katG expression represents a double-edged sword, as it is an important virulence determinant but at the same time its activity levels determine sensitivity to INH. Thus, it is important to delineate the regulation and expression of katG, as this not only can aid understanding of how M. tuberculosis survives and persists in the host but also may provide information of relevance for better management of INH therapy. Here, we report the first extensive analysis of the katG promoter activity examined both in vitro and in vivo. Using S1 nuclease protection analysis, we mapped the katG mRNA 5' ends and demonstrated that two promoters, P(1)furA and P(1)katG, control transcription of katG. The furA and katG genes are cotranscribed from P(1)furA. Both P(1)furA and P(1)katG promoters show induction upon challenge with hydrogen peroxide and cumene hydroperoxide. Studies carried out using the transcriptional fusions P(1)furA-gfp, P(1)katG-gfp, and P(1)furA-P(1)katG-gfp confirmed the existence of two katG promoters. In addition, we showed that both promoters are expressed in vivo during intracellular growth of virulent M. tuberculosis H37Rv. P(1)furA is induced early upon infection, and P(1)katG becomes active only upon extended growth in macrophages. These studies delineate the transcriptional organization of the furA-katG region and indicate differential regulation in vivo of the two katG promoters. These phenomena most likely reflect the differing demands at sequential stages of the infection cycle and may provide information for improved understanding of host-pathogen interactions in tuberculosis and for further optimization of INH chemotherapy.

Impaired DNA damage response in cells expressing an exon 11-deleted murine Brca1 variant that localizes to nuclear foci.

Both human and mouse cells express an alternatively spliced variant of BRCA1, BRCA1-Delta11, which lacks exon 11 in its entirety, including putative nuclear localization signals. Consistent with this, BRCA1-Delta11 has been reported to reside in the cytoplasm, a localization that would ostensibly preclude it from playing a role in the nuclear processes in which its full-length counterpart has been implicated. Nevertheless, the finding that murine embryos bearing homozygous deletions of exon 11 survive longer than embryos that are homozygous for Brca1 null alleles suggests that exon 11-deleted isoforms may perform at least some of the functions of Brca1. We have analyzed both the full-length and the exon 11-deleted isoforms of the murine Brca1 protein. Our results demonstrate that full-length murine Brca1 is identical to human BRCA1 with respect to its cell cycle regulation, DNA damage-induced phosphorylation, nuclear localization, and association with Rad51. Surprisingly, we show that endogenous Brca1-Delta11 localizes to discrete nuclear foci indistinguishable from those found in wild-type cells, despite the fact that Brca1-Delta11 lacks previously defined nuclear localization signals. However, we further show that DNA damage-induced phosphorylation of Brca1-Delta11 is significantly reduced compared to full-length Brca1, and that gamma irradiation-induced Rad51 focus formation is impaired in cells in which only Brca1-Delta11 is expressed. Our results suggest that the increased viability of embryos bearing homozygous deletions of exon 11 may be due to expression of Brca1-Delta11 and suggest an explanation for the genomic instability that accompanies the loss of full-length Brca1.

Analysis of amino acids and catecholamines, 5-hydroxytryptamine and their metabolites in brain areas in the rat using in vivo microdialysis.

In vivo microdialysis, using dialysis probes inserted into discrete brain areas and subsequent analysis of neurotransmitters and related substances in the dialysates (usually with HPLC), has yielded a great deal of important information about the actions of psychotropic drugs and endogenous neurotransmitter systems and about the functional interactions between various brain areas. This paper reviews the principles involved in in vivo microdialysis, its advantages and disadvantages, and recent innovations in methodology and applications. The first section includes brief discussions of principles and applications of dialysis, use of anesthetized versus conscious freely moving animals, and methods used to determine the neural origin of neurotransmitters in the dialysate. The subsequent sections provide detailed descriptions, based largely on our own studies in rats, of stereotaxic surgery, in vivo microdialysis, and dialysate analysis, with an emphasis on amino acids and biogenic amines and their metabolites. A discussion of methodological problems which may be encountered in the analysis of amino acids and biogenic amines is also included.

Cerebral blood flow and the development of ammonia-induced brain edema in rats after portacaval anastomosis.

Two mechanisms may account for brain edema in fulminant hepatic failure: the osmotic effects of brain glutamine, a product of ammonia detoxification, and a change of cerebral blood flow (CBF). We have shown brain edema, a marked increase in brain glutamine, and a selective rise in CBF in rats after portacaval anastomosis receiving an ammonia infusion. In this study, we inhibited the activity of glutamine synthetase with methionine-sulfoximine (MSO) and examined ammonia levels, brain water and CBF. Four groups received either a continuous ammonium acetate or control infusion; half of the animals had been pretreated with MSO or vehicle. The ammonia group exhibited brain edema (79.97 +/- 0.04 vs. 81.11 +/- 0. 13% water), an increase in cerebrospinal fluid (CSF) glutamine (1.29 +/- 0.21 vs. 2.84 +/- 0.39 mmol/L) and CBF (63 +/- 11 vs. 266 +/- 45 mL/min/100 g brain). When MSO was added to the ammonia infusion, ammonia levels rose further (928 +/- 51 vs. 1,293 +/- 145 mmol/L, P <.05) but CSF glutamine decreased (2.84 +/- 0.39 vs. 1.61 +/- 0.2 mmol/L, P <.01). Brain edema (80.48 +/- 0.11%) and cerebral hyperemia (140 +/- 25 mL/min/100 g brain) were significantly ameliorated in the ammonia plus MSO group. Brain output of circulating nitric oxide (NO(x)) was increased in the ammonia-infused group but normalized in the ammonia plus MSO group. In this model, the rise of CBF reflects intracranial events that occur after glutamine synthesis. Activation of nitric oxide synthase in the brain could account for these findings.

Modeling the conscious correlates of recognition memory: reflections on the remember-know paradigm.

Understanding how memory processes contribute to the conscious experience of memory is central to contemporary cognitive psychology. Recently, many investigators (e.g., Gardiner, 1988) have examined the remember-know paradigm to understand the conscious correlates of recognition memory. A variety of studies have demonstrated that variables have different effects on remember and know responses, and these findings have been interpreted in the context of dual-process models of recognition memory. This paper presents a single-process model of the remember-know paradigm, emphasizing the dependence of remember and know judgments on a set of common underlying processes (e.g., criterion setting). We use this model to demonstrate how a single-process model can give rise to the functional dissociations presented in the remember-know literature. We close by detailing procedures for testing our model and describing how those tests may facilitate the development of dual-process models.

A genetic and functional analysis of the unusually large variable region in the M.AluI DNA-(cytosine C5)-methyltransferase.

The M.AluI DNA-(cytosine C5)-methyltransferase (5mC methylase) acts on the sequence 5'-AGCT-3'. The amino acid sequences of known 5mC methylases contain ten conserved motifs, with a variable region between Motifs VIII and IX that contains one or more "target-recognizing domains" (TRDs) responsible for DNA sequence specificity. Monospecific 5mC methylases are believed to have only one TRD, while multispecific 5mC methylases have as many as five. M.AluI has the second-largest variable region of all known 5mC methylases, and sequence analysis reveals five candidate TRDs. In testing whether M.AluI is in fact monospecific it was found that AGCT methylation represents only 80-90% of the methylating activity of this enzyme, while control experiments with the enzyme M.HhaI gave no unexplained activity. Because individual TRDs can be deleted from multispecific methylases without general loss of activity, a series of insertion and deletion mutants of the M.AluI variable region were prepared. All deletions that removed more than single amino acids from the variable region caused significant loss of activity; a sensitive in vivo assay for methylase activity based on McrBC restriction suggested that the central portion of the variable region is particularly important. In some cases, multispecific methylases can accommodate a TRD from another multispecific methylase, thereby acquiring an additional specificity. When TRDs were moved from a multispecific methylase into two different locations in the variable region of M.AluI, all hybrid enzymes had greatly reduced activity and no new specificities. M.AluI thus behaves in most respects as a monospecific methylase despite the remarkable size of its variable region.

Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle.

The muscle myosins and hexomeric proteins consisting of two heavy chains and two pairs of light chains, the latter called essential (ELC) and regulatory (RLC). The light chains stabilize the long alpha helical neck of the myosin head. Their function in striated muscle, however, is only partially understood. We report here the identification of distinct missense mutations in a skeletal/ventricular ELC and RLC, each of which are associated with a rare variant of cardiac hypertrophy as well as abnormal skeletal muscle. We show that myosin containing the mutant ELC has abnormal function, map the mutant residues on the three-dimensional structure of myosin and suggest that the mutations disrupt the stretch activation response of the cardiac papillary muscles.

Parental origin of chromosome 9q22.3-q31 lost in basal cell carcinomas from basal cell nevus syndrome patients.

The basal cell nevus syndrome is an autosomal dominant disease, one of the most prominent phenotypic features of which is a large number of cutaneous basal cell carcinomas. The gene whose mutation underlies this disease has been mapped to chromosome 9q22.3-q31, and basal cell carcinomas frequently have allelic losses including this site. We report here that the chromosome 9q22.3-q31 lost in 24 basal cell carcinomas from basal cell nevus syndrome patients was the one predicted by linkage to contain the wild-type gene. Hence these data are compatible with the exception that the product of the basal cell nevus syndrome gene acts as a tumor suppressor.

Peripheral vision screening for driving in retinitis pigmentosa patients.

The authors evaluated the test protocols used most frequently to screen the peripheral visual field of driving applicants to determine whether they are suitable for detecting peripheral field loss in patients with retinitis pigmentosa (RP). The peripheral vision tests available on the Keystone View Tester and the Titmus Vision Tester were administered to 23 subjects with RP, 3 subjects with Type 2 Usher's syndrome, and 1 subject who was a partially affected carrier of X-linked recessive RP. The subjects had varying degrees and types of visual field loss. Tests were administered using the standard protocol of the State of Illinois, which is a standard procedure used by state licensing bureaus nationwide. Results demonstrate that the screening protocols use stimulus conditions that are primarily sensitive only to appreciable field losses and examine locations that typically lie within an RP patient's remaining visual field rather than at locations that characteristically are scotomatous. The authors suggest that the current test protocols could determine peripheral field impairment more accurately by assessing additional locations in the visual field, and by introducing a background field and/or by reducing the luminance of the test targets.