Oxygen and Bis(3',5')-cyclic Dimeric Guanosine Monophosphate Binding Control Oligomerization State Equilibria of Diguanylate Cyclase-Containing Globin Coupled Sensors.

Bacteria sense their environment to alter phenotypes, including biofilm formation, to survive changing conditions. Heme proteins play important roles in sensing the bacterial gaseous environment and controlling the switch between motile and sessile (biofilm) states. Globin coupled sensors (GCS), a family of heme proteins consisting of a globin domain linked by a central domain to an output domain, are often found with diguanylate cyclase output domains that synthesize c-di-GMP, a major regulator of biofilm formation. Characterization of diguanylate cyclase-containing GCS proteins from Bordetella pertussis and Pectobacterium carotovorum demonstrated that cyclase activity is controlled by ligand binding to the heme within the globin domain. Both O2 binding to the heme within the globin domain and c-di-GMP binding to a product-binding inhibitory site (I-site) within the cyclase domain control oligomerization states of the enzymes. Changes in oligomerization state caused by c-di-GMP binding to the I-site also affect O2 kinetics within the globin domain, suggesting that shifting the oligomer equilibrium leads to broad rearrangements throughout the protein. In addition, mutations within the I-site that eliminate product inhibition result in changes to the accessible oligomerization states and decreased catalytic activity. These studies provide insight into the mechanism by which ligand binding to the heme and I-site controls activity of GCS proteins and suggests a role for oligomerization-dependent activity in vivo.

A versatile internal control for use as DNA in real-time PCR and as RNA in real-time reverse transcription PCR assays.

AIMS: To develop and evaluate a TaqMan-based internal amplification control (IAC) that can be used as DNA in real-time PCR (qPCR) or as RNA in reverse transcription real-time PCR (qRT-PCR) to identify the presence of assay inhibition and to evaluate its incorporation into existing qPCR and qRT-PCR methods for bacterial detection. METHODS AND RESULTS: A DNA IAC was constructed by generating a 198-bp random sequence that was synthesized and inserted into a pZErO-2 vector and transformed into Escherichia coli. The RNA IAC was generated through in vitro transcription of the DNA IAC. Both IAC formats were tested individually in singleplex TaqMan reactions and also included in existing multiplex assays. The DNA IAC was incorporated in a Shigella spp. detection qPCR assay (targeting ipaH). The RNA IAC was successfully evaluated in a Salmonella spp. detection qRT-PCR (using invA mRNA as target). CONCLUSIONS: A highly versatile IAC that can be supplemented to qPCR and qRT-PCR pathogen detection methods was developed, greatly reducing the confounding effects of false negatives because of PCR inhibitors without affecting pathogen detection. SIGNIFICANCE AND IMPACT OF THE STUDY: The frequency of false negatives associated with qPCR analyses is prevalent in certain matrices, particularly those involving complex foods. Hence, the IAC presented here provides a solution to unforeseen false-negative reactions in PCR.

Human immunodeficiency virus type-1 (HIV-1) subtypes in Jamaica.

The subtypes of 141 isolates of human immunodeficiency virus type-1 (HIV-1) from Jamaica were determined by a combination of env and gag heteroduplex mobility analysis (HMA) genotyping. The majority of HIV-1 isolates were subtype B (131/141, 93.0%); one (0.8%) isolate each of subtypes C, D and E was found and 7 (4.9%) were indeterminate. These results and the failure of the sets of primers used to amplify some of the HIV-1 isolates provide strong evidence of genetic diversity of the HIV/AIDS epidemic in Jamaica. Surveillance of the circulating HIV-1 genetic subtypes is a pre-requisite for developing regional vaccine strategies and understanding the transmission patterns of the virus. This is the first study of its kind in Jamaica and the findings complement data from other Caribbean countries. This work supports the view of colleagues from the French and Spanish-speaking Caribbean that an epidemiological network supported by regional laboratories will help track this epidemic accurately with positive outcomes for the public.

20 year follow-up on microinvasive squamous carcinoma of the cervix.

MISC of the cervix is a potentially fatal disease if not properly classified and treated. Strict criteria for diagnosis are required to provide appropriate therapy. It is recommended that a radical approach be considered for those patients in whom the depth of invasion of the carcinoma is greater than 3 mm on cone biopsy and in all cases where vascular invasion is demonstrated. Confluency may not of itself be a reason for more radical therapy but is an indicator of the aggressiveness of the tumor. Vascular invasion is seen more frequently with increased depth of invasion but may be found with minimal infiltration and has a serious connotation. Further refinement of the definition of MISC of the cervix appears to be needed.

A serosurvey of hepatitis B virus, hepatitis C virus, human T lymphotropic virus type-1 and syphilis in HIV-1-infected patients in Jamaica.

The seroprevalences of hepatitis B virus (HBV), hepatitis C virus (HCV), human T lymphotropic virus type-1 (HTLV-1) and syphyilis were determined in 129 HIV-1-infected patients using commercially prepared reagents. The seroprevalences were HCV, 0% (0/129); HBV, 37% (48/129); HTLV-1, 5% (6/129) and syphilis, 20% (26/129). Fifteen per cent (19/129) of the patients had active/chronic HBV infection. The seroprevalence of HBV was statistically significantly higher in HIV-1 infected men (24/49, 50% versus 17/80, 21%; p = 0.005), while the seroprevalence of syphilis was statistically significantly increased in HIV-1 infected patients in the over-40 age group (10/31, 32% versus 6/53, 11%; p = 0.05). These findings throw the spotlight on HBV infection and syphilis and suggest that these two sexually transmitted infections should be carefully surveyed in patients with HIV/AIDS in Jamaica. It is essential for management protocols in Jamaica to include screening for evidence of these co-infections.

Oligomeric state affects oxygen dissociation and diguanylate cyclase activity of globin coupled sensors.

Bacterial biofilm formation is regulated by enzymes, such as diguanylate cyclases, that respond to environmental signals and alter c-di-GMP levels. Diguanylate cyclase activity of two globin coupled sensors is shown to be regulated by gaseous ligands, with cyclase activity and O2 dissociation affected by protein oligomeric state.

Characterization of the zinc-containing metalloprotease encoded by zpx and development of a species-specific detection method for Enterobacter sakazakii.

Enterobacter sakazakii causes a severe form of neonatal meningitis that occurs as sporadic cases as well as outbreaks. The disease has been epidemiologically associated with consumption of reconstituted, dried infant formulas. Very little information is available regarding pathogenicity of the organism and production of virulence factors. Clinical and environmental strains were screened for production of factors which have activity against Chinese hamster ovary (CHO) cells in tissue culture. Polymyxin B lysate and sonicate preparations but not culture supernatants from the strains caused "rounding" of CHO cells. Subsequent studies showed that the CHO cell-rounding factor is a proteolytic enzyme that has activity against azocasein. The cell-bound protease was isolated by using a combination of polymyxin B lysis, followed by sonication of cells harvested from tryptone broth. The protease was purified to homogeneity by sequential ammonium sulfate precipitation, gel filtration chromatography with Sephadex G-100, hydrophobic interaction chromatography with phenyl-Sepharose CL-4B, and a second gel filtration with Sephadex G-100. In addition to activity against azocasein, the purified protease also exhibits activity against azocoll and insoluble casein but not elastin. The protease has a molecular weight of 38,000 and an isoelectric point of 4.4. It is heat labile and for maximal activity against azocasein has an optimum temperature of 37 degrees C and a pH range of 5 to 7. Proteolytic activity is inhibited by ortho-phenanthroline and Zincov but is not affected by phenylmethylsulfonyl fluoride, N-ethylmaleimide, and trypsin inhibitors, which demonstrates that the protease is a zinc-containing metalloprotease. The metalloprotease does not hemagglutinate chicken or sheep erythrocytes. Twenty-three to 27 of the first 42 N-terminal amino acid residues of the metalloprotease are identical to proteases produced by Serratia proteamaculans, Pectobacterium carotovorum, and Anabaena sp. PCR analysis using primers designed from a consensus nucleotide sequence showed that 135 E. sakazakii strains possessed the metalloprotease gene, zpx, and 25 non-E. sakazakii strains did not. The cloned zpx gene of strain 29544 consists of 1,026 nucleotides, and the deduced amino acid sequence of the metalloprotease has 341 amino acid residues, which corresponds to a theoretical protein size of 37,782 with a theoretical pI of 5.23. The sequence possesses three well-characterized zinc-binding and active-site motifs present in other bacterial zinc metalloproteases.

Impact of digital radiography on clinical workflow.

It is commonly accepted that digital radiography (DR) improves workflow and patient throughput compared with traditional film radiography or computed radiography (CR). DR eliminates the film development step and the time to acquire the image from a CR reader. In addition, the wide dynamic range of DR is such that the technologist can perform the quality-control (QC) step directly at the modality in a few seconds, rather than having to transport the newly acquired image to a centralized QC station for review. Furthermore, additional workflow efficiencies can be achieved with DR by employing tight radiology information system (RIS) integration. In the DR imaging environment, this provides for patient demographic information to be automatically downloaded from the RIS to populate the DR Digital Imaging and Communications in Medicine (DICOM) image header. To learn more about this workflow efficiency improvement, we performed a comparative study of workflow steps under three different conditions: traditional film/screen x-ray, DR without RIS integration (ie, manual entry of patient demographics), and DR with RIS integration. This study was performed at the Cleveland Clinic Foundation (Cleveland, OH) using a newly acquired amorphous silicon flat-panel DR system from Canon Medical Systems (Irvine, CA). Our data show that DR without RIS results in substantial workflow savings over traditional film/screen practice. There is an additional 30% reduction in total examination time using DR with RIS integration.

Ten-year prospective study in a management change of vulvar carcinoma.

A prospective study involving 71 cases in which operation for vulvar carcinoma was performed during a 10-year period is presented. Primary closure of the vulvar defect with the use of full-thickness transposition flaps reduced hospitalization without jeopardizing survival. This technique tends to increase the radical nature of the operation. Although complications involving groin breakdown were reduced, such complications remain a formidable challenge for newer techniques. The postoperative results have been superior to those of older methods.

Human immunodeficiency virus type-1 (HIV-1) subtypes in Jamaica / Subtipos del virus tipo-1 de la inmunodeficiencia humano (VIH-1) en Jamaica

The subtypes of 141 isolates of human immunodeficiency virus type-1 (HIV-1) from Jamaica were determined by a combination of env and gag heteroduplex mobility analysis (HMA) genotyping. The majority of HIV-1 isolates were subtype B (131/141, 93.0); one (0.8) isolate each of subtypes C, D and E was found and 7 (4.9) were indeterminate. These results and the failure of the sets of primers used to amplify some of the HIV-1 isolates provide strong evidence of genetic diversity of the HIV/AIDS epidemic in Jamaica. Surveillance of the circulating HIV-1 genetic subtypes is a pre-requisite for developing regional vaccine strategies and understanding the transmission patterns of the virus. This is the first study of its kind in Jamaica and the findings complement data from other Caribbean countries. This work supports the view of colleagues from the French and Spanish-speaking Caribbean that an epidemiological network supported by regional laboratories will help track this epidemic accurately with positive outcomes for the public.

Los subtipos de 141 aislados del virus tipo 1 de la inmunodeficiencia humno (VIH-1) en Jamaica, fueron determinados combinando la genotipificación por análisis de heterodúplex (HMA) en los genes env y gag. La mayor parte de los aislados HIV-1 fueron del subtipo B (131/141, 93.0%), se halló uno (0.8%) aislado para cada uno de los subtipos C, D y E, en tanto que 7 (4.9%) fueron indeterminados. Estos resultados y el fallo de los conjuntos de primers usados para amplificar algunos de los aislados de VIH-1, ofrecen fuerte evidencia de la diversidad epidémica del VIH/SIDA en Jamaica. La vigilancia de los subtipos genéticos de VIH-1 en circulación, constituye un pre-requisito, tanto para desarrollar estrategias de vacunas a nivel regional, como para entender los patrones de transmisión del virus. Este es el primer estudio de este tipo en Jamaica, y nuestros hallazgos complementan los datos obtenidos en otros países del Caribe. Coincidimos con nuestros colegas del Caribe francófono e hispano-parlante en cuanto a que una red epidemiológica apoyada por los laboratorios regionales, nos ayudaría a continuar rastreando esta epidemia con exactitud, y con resultados positivos para el público.

A serosurvey of hepatitis B virus, hepatitis C virus, human T lymphotropic virus type-1 and syphilis in HIV-1-infected patients in Jamaica

The seroprevalences of hepatitis B virus (HBV), hepatitis C virus (HCV), human T lymphotropic virus type-1 (HTLV-1) and syphyilis were determined in 129 HIV-1-infected patients using commercially prepared reagents. The seroprevalences were HCV, 0 (0/129); HBV, 37 (48/129); HTLV-1, 5 (6/129) and syphilis, 20 (26/129). Fifteen per cent (19/129) of the patients had active/chronic HBV infection. The seroprevalence of HBV was statistically significantly higher in HIV-1 infected men (24/49, 50 versus 17/80, 21; p = 0.005), while the seroprevalence of syphilis was statistically significantly increased in HIV-1 infected patients in the over-40 age group (10/31, 32 versus 6/53, 11; p = 0.05). These findings throw the spotlight on HBV infection and syphilis and suggest that these two sexually transmitted infections should be carefully surveyed in patients with HIV/AIDS in Jamaica. It is essential for management protocols in Jamaica to include screening for evidence of these co-infections