EDTA-Modified Carbapenem Inactivation Method: a Phenotypic Method for Detecting Metallo-ß-Lactamase-Producing Enterobacteriaceae.

The increase in the prevalence and impact of infections caused by carbapenemase-producing Enterobacteriaceae is a global health concern. Therefore, rapid and accurate methods to detect these organisms in any clinical microbiology laboratory, including those in resource-limited settings, are essential to prevent and contain their spread. It is also important to differentiate between serine- and metal-dependent carbapenemases elaborated by carbapenemase-producing isolates for epidemiologic, infection control and prevention, and therapeutic purposes. Here, we describe the development and evaluation of the EDTA-modified carbapenem inactivation method (eCIM), an assay for discriminating between serine- and metal-dependent (i.e., metallo-ß-lactamases [MBLs]) carbapenemases when used in conjunction with the modified carbapenem inactivation method (mCIM). The eCIM had an overall sensitivity and specificity of 100% and was adopted by the Clinical and Laboratory Standards Institute as a method to use in combination with the mCIM to identify MBL-producing Enterobacteriaceae.

Looking back: a genetic retrospective study of Brazilian Orf virus isolates.

Orf virus (ORFV), the prototype of the genus Parapoxvirus, is the aetiological agent of contagious ecthyma (CE), a pustular dermatitis that afflicts domestic and wild small ruminants. CE is one of the most widespread poxvirus diseases in the world, causing public health impacts. Outbreaks of ORFV have been observed in all geographical regions of Brazil, affecting ovine and caprine herds. The origins, epidemiology and identity of Brazilian ORFVs are unknown, and no comparative or phylogenetic studies of these viruses have been performed. In the present study, we revisited CE outbreaks which occurred until 32 years ago, and we assessed, genetically, five viral isolates. We performed the sequencing and analysis of the three ORFV molecular markers: B2L gene, virus interferon resistance gene (VIR) and the vascular endothelial growth factor gene. Nucleotide and amino acid analysis of the analysed genes demonstrated that Brazilian ORFVs do not form a unique cluster, and presented more similarity to other worldwide ORFV samples than with each other. These data raise the questions of whether there are different worldwide ORFVs circulating in Brazil, or if all the Brazilian ORFV samples are of the same virus taken at distinct time points.

Varying ratios of wavelengths in dual wavelength LED photomodulation alters gene expression profiles in human skin fibroblasts.

BACKGROUND AND OBJECTIVE: LED photomodulation has been shown to profoundly influence cellular behavior. A variety of parameters with LED photomodulation can alter cellular response in vitro. The effects of one visible and one infrared wavelength were evaluated to determine the optimal ratio to produce a net increase in dermal collagen by altering the ratio of total energy output of each wavelength. The ratio between the two wavelengths (590 and 870 nm) was shifted in 25% increments. STUDY DESIGN/MATERIALS AND METHODS: Human skin fibroblasts in culture were exposed to a 590/870 nm LED array with total combined energy density fixed at 4.0 mW/cm.. The ratio of 590/870 nm tested parameters were: 100/0%, 75/25%, 50/50%, 25/75%, and 0/100%. These ratios were delivered using pulsed duty cycle of exposure (250 milliseconds "on" time/100 milliseconds "off" time/100 pulses) for a total energy fluence of 0.1 J/cm.. Gene expression was examined using commercially available extra cellular matrix and adhesion molecule RT PCR Arrays (SA Biosciences, Frederick, MD) at 24 hours post-exposure. RESULTS: Different expression profiles were noticed for each of the ratios studied. Overall, there was an average (in an 80 gene array) of 6% expression difference in up or downregulation between the arrays. The greatest increase in collagen I and decrease in collagenase (MMP-1) was observed with 75/25% ratio of 590/870 nm. The addition of increasing proportions of IR wavelengths causes alteration in gene expression profile. The ratios of the wavelengths caused variation in magnitude of expression. CONCLUSIONS: Cell metabolism and gene expression can be altered by simultaneous exposure to multiple wavelengths of low energy light. Varying the ratios of specific wavelength intensity in both visible and near infrared light therapy can strongly influence resulting fibroblast gene expression patterns.

Histamine H3 receptor antagonists: from target identification to drug leads.

The successful cloning and functional expression of the histamine H(3) receptor in the late 1990 s has greatly facilitated our efforts to identify small molecule, non-imidazole based compounds to permit the evaluation of H(3) antagonists in models of CNS disorders. High-throughput screening identified several series of lead compounds, including a series of imidazopyridines, which led to JNJ-6379490, a compound with high affinity for the human H(3) receptor. Analysis of structural features common to several series of non-imidazole H(3) receptor ligands resulted in a pharmacophore model. This model led to the design of JNJ-5207852, a diamine-based H(3) antagonist with good in vitro and in vivo efficacy but with an undesirable long half-life. However, further modifications of the template provided an understanding of the effect of structural modifications on pharmacokinetic properties, ultimately affording several additional series of compounds including JNJ-10181457, a compound with an improved pharmacokinetic profile. These compounds allowed in vivo pharmacological evaluation to show that H(3) antagonists promote wakefulness, but unlike modafinil and classical psychostimultants, they do not increase locomotor activity or produce any alteration of the EEG power spectral activity in rats. H(3) antagonists also increase extracellular acetylcholine and norepinephrine but not dopamine in rat frontal cortex and show efficacy in various models of learning-memory deficit. In addition, cFos immunoreactivity studies show H(3) antagonists activate neuronal cells in restricted rat brain regions in contrast to widespread activation after modafinil or amphetamine treatment. Therefore, H(3) antagonists are promising clinical candidates for the treatment of excessive day time sleepiness and/or cognitive disorders.

Acute wake-promoting actions of JNJ-5207852, a novel, diamine-based H3 antagonist.

1 1-[4-(3-piperidin-1-yl-propoxy)-benzyl]-piperidine (JNJ-5207852) is a novel, non-imidazole histamine H3 receptor antagonist, with high affinity at the rat (pKi=8.9) and human (pKi=9.24) H3 receptor. JNJ-5207852 is selective for the H3 receptor, with negligible binding to other receptors, transporters and ion channels at 1 microm. 2 JNJ-5207852 readily penetrates the brain tissue after subcutaneous (s.c.) administration, as determined by ex vivo autoradiography (ED50 of 0.13 mg kg(-1) in mice). In vitro autoradiography with 3H-JNJ-5207852 in mouse brain slices shows a binding pattern identical to that of 3H-R-alpha-methylhistamine, with high specific binding in the cortex, striatum and hypothalamus. No specific binding of 3H-JNJ-5207852 was observed in brains of H3 receptor knockout mice. 3 In mice and rats, JNJ-5207852 (1-10 mg kg(-1) s.c.) increases time spent awake and decreases REM sleep and slow-wave sleep, but fails to have an effect on wakefulness or sleep in H3 receptor knockout mice. No rebound hypersomnolence, as measured by slow-wave delta power, is observed. The wake-promoting effects of this H3 receptor antagonist are not associated with hypermotility. 4 A 4-week daily treatment of mice with JNJ-5207852 (10 mg kg(-1) i.p.) did not lead to a change in body weight, possibly due to the compound being a neutral antagonist at the H3 receptor. 5 JNJ-5207852 is extensively absorbed after oral administration and reaches high brain levels. 6 The data indicate that JNJ-5207852 is a novel, potent and selective H3 antagonist with good in vitro and in vivo efficacy, and confirm the wake-promoting effects of H3 receptor antagonists.

Avaliaçäo soroepidemiológica do vírus influenza em aves domésticas e silvestres no Estado do Rio de Janeiro / Serological survey on influenza virus in domestic and wild birds from Rio de Janeiro State, Brazil

Estudou-se a presença de anticorpos para o vírus da influenza aviária, subtipos H1N1 e H3N2, por meio da técnica de inibiçäo da hemaglutinaçäo no plasma de 225 aves da Fundaçäo RIO-ZOO, do Bwana Park e de pequenas criaçöes do Estado do Rio de Janeiro. Entre as aves estudadas 60 (26,6 por cento) foram soropositivas, sendo 22 (9,8 por cento) para o subtipo H1N1, 28 (12,4 por cento) para o subtipo H3N2 e 10 (4,4 por cento) para os dois subtipos. Esses resultados indicam a ocorrência dos subtipos do vírus da influenza aviária investigados no Rio de Janeiro e apontam para o risco potencial de sua transmissäo para a avicultura industrial e para pessoas

Hydrogen concentrations in sulfate-reducing estuarine sediments during PCE dehalogenation.

Despite recent progress made evaluating the role of hydrogen (H2) as a key electron donor in the anaerobic remediation of chloroethenes, few studies have focused on the evaluation of hydrogen thresholds relative to reductive dehalogenation in sulfidogenic environments. Competition for hydrogen exists among microbial populations in anaerobic sediments, and direct evidence indicates that lower hydrogen thresholds are observed with more energetically favorable electron-accepting processes. This study examined aqueous hydrogen concentrations associated with sulfate reduction and perchloroethylene (PCE) dehalogenation in anoxic estuarine sediment slurry microcosms and evaluated the competition for H2-reducing equivalents within these systems. After an initial lag period of 13 days, PCE was reductively transformed to trichloroethylene (TCE). During the time of continuous PCE dehalogenation, a significantly (P < 0.05) lower hydrogen concentration (0.5 nM) was observed in the sediment slurries amended with PCE as compared to slurries without PCE (0.8 nM). Sulfate reduction to sulfide was observed in all sediment slurries, but in microcosms actively dechlorinating PCE, the amount of reducing equivalents directed to sulfate reduction was approximately half the amount in sediment slurries without PCE. These findings provide evidence that a lower hydrogen threshold exists in anoxic estuarine sediment slurries with PCE as a terminal electron acceptor as compared to sediment slurries in which sulfate reduction was the predominant electron-accepting process. Furthermore, our results utilizing the inhibitor molybdate indicated that H2-utilizing methanogens may have the potential to effectively compete with dechlorinators for hydrogen when sulfate reduction is initially inhibited.

Detection of poxvirus in cattle associated with human cases in the State of Rio de Janeiro: preliminary report.

This preliminary report describes human and cow cases of poxvirus that recently occurred in the State of Rio de Janeiro. The electron microscopic findings were consistent with parapoxviral and orthopoxviral infection. Orthopoxvirus strains were isolated from human and cow cases. Detailed viral characterization by means of genetical techniques is under investigation. Based on these informations, poxviral diseases should be also considered an emerging viral zoonosis that can affect human beings.

Integrin alpha(IIb)beta(3) inhibitor preserves microvascular patency in experimental acute focal cerebral ischemia.

BACKGROUND AND PURPOSE: Platelets become activated and accumulate in brain microvessels of the ischemic microvascular bed after experimental focal cerebral ischemia. The binding of glycoprotein IIb/IIIa (integrin alpha(IIb)beta(3)) on platelets to fibrinogen is the terminal step in platelet adhesion and aggregation. This study tests the hypothesis that inhibition of platelet-fibrin(ogen) interactions may prevent microvascular occlusion after experimental middle cerebral artery occlusion (MCA:O). METHODS: TP9201 is a novel Arg-Gly-Asp (RGD)-containing integrin alpha(IIb)beta(3) inhibitor. Microvascular patency after 3-hour MCA:O and 1-hour reperfusion within the ischemic and nonischemic basal ganglia was compared in adolescent male baboons who received high-dose TP9201 (group A: IC(80) in heparin, n=4), low-dose TP9201 (group B: IC(30) in heparin, n=4), or no treatment (group C: n=4) before MCA:O. RESULTS: After MCA:O, microvascular patency decreased significantly in group C. However, in the ischemic zones of groups A and B compared with group C, patencies were significantly greater in the 4.0- to 7. 5-microm-diameter (capillary) and 7.5- to 30.0-microm-diameter vessels (2P<0.05). A dose-dependent increase in hemorrhagic transformation was seen in group A (3 of 4 animals) compared with group B (1 of 4 animals), and no hemorrhage was visible in group C (chi(2) analysis for trend, P<0.05). CONCLUSIONS: Platelet activation contributes significantly to ischemic microvascular occlusion. Occlusion formation may be prevented by this RGD-integrin alpha(IIb)beta(3) inhibitor at a dose that does not produce clinically significant parenchymal hemorrhage. The effect of microvascular patency on neuron recovery can now be tested.

Molecular characterization of Brazilian isolates of orf virus.

Outbreaks of an epidermic disease suggesting parapox virus infections have been observed in all major herds of sheep and goats from different geographical areas of Brazil. Clinical samples (dried scabs) were collected and orf virus was isolated and characterized by electron microscopy in previous work. In order to characterize these viruses at the molecular level, a modified methodology for genomic DNA extraction directly from scabs was used and such DNA was used to derive the restriction enzyme digestion patterns for clinical samples from three distinct geographic origins. Pulsed field gel electrophoresis was used to separate restriction enzyme DNA fragments and heterogeneity among isolates from different geographic areas could be observed on stained gels. The HindIII-G DNA fragment from orf-A virus genome was cloned and hybridized to DNA of other orf virus isolates. Further heterogeneity was confirmed by these hybridizations.

Uptake and phytotransformation of o,p'-DDT and p,p'-DDT by axenically cultivated aquatic plants.

The uptake and phytotransformation of o,p'-DDT and p,p'-DDT were investigated in vitro using three axenically cultivated aquatic plants: parrot feather (Myriophyllum aquaticum), duckweed (Spirodela oligorrhiza), and elodea (Elodea canadensis). The decay profile of DDT from the aqueous culture medium followed first-order kinetics for all three plants. During the 6-day incubation period, almost all of the DDT was removed from the medium, and most of it accumulated in or was transformed by these plants. Duckweed demonstrated the greatest potential to transform both DDT isomers; 50-66% was degraded or bound in a nonextractable manner with the plant material after the 6-day incubation. Therefore, duckweed also incorporated less extractable DDT (32-49%) after 6 days than did the other plants. The capacity for phytotransformation/binding by elodea is between that of duckweed and parrot feather; approximately 31-48% of the spiked DDT was degraded or bound to the elodea plant material. o,p'-DDD and p,p'-DDD are the major metabolites in these plants; small amounts of p,p'-DDE were also found in duckweed (7.9%) and elodea (4.6%) after 6 days. Apparently, reduction of the aliphatic chlorine atoms of DDT is the major pathway for this transformation. This study, which provides new information on plant biochemistry as related to pollutant accumulation and phytotransformation, should advance the development of phytoremediation processes.

Uptake and phytotransformation of organophosphorus pesticides by axenically cultivated aquatic plants.

The uptake and phytotransformation of organophosphorus (OP) pesticides (malathion, demeton-S-methyl, and crufomate) was investigated in vitro using the axenically aquatic cultivated plants parrot feather (Myriophyllum aquaticum), duckweed (Spirodela oligorrhiza L.), and elodea (Elodea canadensis). The decay profile of these OP pesticides from the aqueous medium adhered to first-order kinetics. However, extent of decay and rate constants depended on both the physicochemical properties of the OP compounds and the nature of the plant species. Malathion and demeton-S-methyl exhibited similar transformation patterns in all three plants: 29-48 and 83-95% phytotransformation, respectively, when calculated by mass recovery balance during an 8-day incubation. No significant disappearance and phytotransformation of crufomate occurred in elodea over 14 days, whereas 17-24% degraded in the other plants over the same incubation period. Using enzyme extracts derived from duckweed, 15-25% of the three pesticides were transformed within 24 h of incubation, which provided evidence for the degradation of the OP compounds by an organophosphorus hydrolase (EC 3.1.8.1) or multiple enzyme systems. The results of this study showed that selected aquatic plants have the potential to accumulate and to metabolize OP compounds; it also provided knowledge for potential use in phytoremediation processes.

Inducible expression of a Porphyromonas gingivalis W83 membrane-associated protease.

The Tpr protease of Porphyromonas gingivalis W83 is a membrane-associated enzyme capable of hydrolyzing a chromogenic bacterial collagenase substrate. An isogenic mutant lacking a functional tpr gene had a greatly reduced ability to hydrolyze the collagenase substrate. Activity was restored to the tpr mutant by introducing a shuttle plasmid containing the tpr gene. Expression of the gene is induced by nutrient limitation, as shown by enzymatic and Northern analyses.

Strategies for school-based response to loss: proactive training and postvention consultation.

The suicide of a student or staff member is one of the most difficult crisis confronting a high school. This article describes the first year of a school-based postvention program to train and consult with crisis personnel in a structured response to this crisis. Responding to Loss (RTL) is one program offered by Community Action for Youth Survival, a project of the Ronald McDonald Children's Charities. Serving a contiguous three county urban area, this program integrates public health, epidemiological, psychiatric, and prevention paradigms to provide a comprehensive approach to the aftermath of adolescent suicide. RTL provides an interdisciplinary model for comprehensive, school-based postvention programs based on the guidelines developed by the Centers for Disease Control and national and local psychological autopsy data. It highlights the significance of responding to increased rates of suicide in minority youth, suicide witnessed by peers, and suicide victims who have dropped out of school. Organizational issues, such as systematic school entry, development of a computerized school database, proactive training, and collaboration with the offices of medical examiners and coroners, are described. Initial evaluation data from year 1 indicate significant gains in participants' knowledge and skills, as well as a high rate of consumer satisfaction.

Selective alpha IIb beta 3 receptor blockage with peptide TP9201 prevents platelet uptake on Dacron vascular grafts without significant effect on bleeding time.

Synthetic vascular prostheses lack the uniquely low thrombogenicity provided by the endothelial cell lining of autogenous saphenous vein or artery grafts. The thrombogenic nature of the synthetic graft surface becomes a major determinant of early prosthetic graft patency. We demonstrate in a baboon ex vivo synthetic graft model that modification of the host's platelet interaction with the graft surface results in inhibition of platelet thrombus formation and thereby, a possible enhancement of early prosthetic graft patency. This was achieved by selective blockage of the platelet alpha IIb beta 3 receptor by the arginine-glycine-aspartic acid-containing synthetic peptide TP9201. Platelet thrombus formation on a Dacron graft indicated by accumulation of indium III-oxine-labeled autologous platelets was measured by gamma camera imaging. After 60 minutes of circulation, TP9201 at a bolus of 125 micrograms/kg; infusion of 3 micrograms/kg/min, bolus of 190 micrograms/kg; infusion of 5 micrograms/kg/min, bolus of 250 micrograms/kg; infusion of 6 micrograms/kg/min, and bolus of 500 micrograms/kg; infusion of 12 micrograms/kg/min decreased platelet uptake on the graft to 50%, 40%, 30%, and 10% of control uptake, respectively. Forelimb template bleeding times were not found to be significantly prolonged at doses that effectively inhibit ex vivo platelet aggregation. As a result of drug treatment, no changes in hemodynamic parameters or hematologic profile, including platelet number and clotting time, were observed. We demonstrate here that the arginine-glycine-aspartic acid-containing peptide TP9201, which competitively inhibits the alpha IIb beta 3 integrin-fibrinogen interaction, significantly decreased the accumulation of platelets on a Dacron vascular graft. Molecules like peptide TP9201, because of its unique activity profile, may represent a superior approach to the control of platelet accumulation on thrombogenic surfaces.

Inhibition of thrombosis by a selective fibrinogen receptor antagonist without effect on bleeding time.

Membrane glycoprotein alpha IIb beta 3 on platelets plays a pivotal role in hemostasis by mediating RGD-(arginine-glycine-aspartic acid)-dependent platelet adhesion and aggregation. Antagonists of alpha IIb beta 3 ligand binding function, such as antibodies, snake venom peptides, or synthetic RGD-containing peptides can completely inhibit platelet aggregation in vitro and cause significant prolongation of bleeding times when injected into experimental animals. The in vitro and in vivo properties of an alpha IIb beta 3 specific RGD-containing peptide 2G (G(Pen)GHRGDLRCA) were compared to two non-specific RGD-containing peptides 1N (G(Pen)GRGDTPCA) and 2H (GRGDSPDG). All three peptides have similar IC50 values in human platelet aggregation (14-22 microM) and ELISA-based alpha IIb beta 3 receptor assays (0.2-0.3 microM) but show different inhibitory activity (IC50 values) in the alpha v beta 5 (2G = 10 microM; 1N = 0.06 microM; 2H = 0.05 microM) and alpha 5 beta 1 receptor assays (2G = 8.3 microM; 1N = 0.06 microM; 2H = 0.04). The alpha IIb beta 3 specific peptide 2G had no effect on monolayers of human saphenous vein endothelial cells while 1N and 2H caused many cells to detach and contract. Peptides 2G and 1N inhibited ADP-stimulated ex vivo platelet aggregation in dogs in a dose dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Utilization of dietary starch and glucose tolerance in juvenile chinook salmon (Oncorhynchus tshawytscha) of different strains in seawater.

Juvenile chinook salmon of three strains responded to inclusion of 28.7% of gelatinized starch in the diet with different degrees of reduction in growth rate and feed efficiency relative to control fish of the respective strains fed a low-starch, high-lipid diet of similar protein (46%) and estimated metabolizable energy content (16 mJ/kg). The productive protein value of the diet was not reduced to the same extent by the high intake of starch. Carcasses of fish fed the high-starch diet contained higher concentrations of protein and lower concentrations of lipid than control fish. The accumulation of liver glycogen in response to the high-starch diet differed among strains. Glucose tolerance curves also varied among strains but were poorly correlated with plasma concentrations of insulin. Tolerance to glucose loading was improved in fish previously fed the high-starch diet.

Molecular analysis of contagious pustular dermatitis virus: a simplified method for viral DNA extraction from scab material.

A new simplified method of DNA extraction of contagious pustular dermatitis virus directly from scab material of natural and experimental infections is described. Scabs are suspended in buffer solution and an enriched core suspension is obtained after treatment with detergent, quelants and centrifugation. DNA is isolated after proteinase digestion and phenolchloroform extraction. Viral DNA and fragments with sizes ranging from 23-25 kb were observed by agarose gel electrophoresis. This DNA was used for digestion with several restriction endonucleases which produced parapoxvirus-specific patterns. Southern blots with the TK gene of vaccinia virus as probe confirmed the virus as being poxvirus-related and allowed a preliminary TK gene location for our isolates. The method was developed in order to allow a quick epidemiological survey of contagious pustular dermatitis virus in Brazil, eliminating the need for time-consuming and expensive viral propagation in cell culture and purification.