Fluorescence Tools Adapted for Real-Time Monitoring of the Behaviors of Streptococcus Species.

Tagging of bacteria with fluorescent proteins has become an essential component of modern microbiology. Fluorescent proteins can be used to monitor gene expression and biofilm growth and to visualize host-pathogen interactions. Here, we developed a collection of fluorescent protein reporter plasmids for Streptococcus mutans UA159 and other oral streptococci. Using superfolder green fluorescent protein (sfGFP) as a reporter for transcriptional activity, we were able to characterize four strong constitutive promoters in S. mutans These promoter-sfgfp fusions worked both for single-copy chromosomal integration and on a multicopy plasmid, with the latter being segregationally stable in the absence of selective pressure under the conditions tested. We successfully labeled S. mutans UA159, Streptococcus gordonii DL1, and Streptococcus sp. strain A12 with sfGFP, DsRed-Express2 (red), and citrine (yellow). To test these plasmids under more challenging conditions, we performed mixed-species biofilm experiments and separated fluorescent populations using fluorescence-activated cell sorting (FACS). This allowed us to visualize two streptococci at a time and quantify the amounts of each species simultaneously. These fluorescent reporter plasmids add to the genetic toolbox available for the study of oral streptococci.IMPORTANCE Oral streptococci are the most abundant bacteria in the mouth and have a major influence on oral health and disease. In this study, we designed and optimized the expression of fluorescent proteins in Streptococcus mutans and other oral streptococci. We monitored the levels of expression and noise (the variability in fluorescence across the population). We then created several fluorescent protein delivery systems (green, yellow, and red) for use in oral streptococci. The data show that we can monitor bacterial growth and interactions in situ, differentiating between different bacteria growing in biofilms, the natural state of the organisms in the human mouth. These new tools will allow researchers to study these bacteria in novel ways to create more effective diagnostic and therapeutic tools for ubiquitous infectious diseases.

Novel phase I trial design to evaluate the addition of cediranib or selumetinib to preoperative chemoradiotherapy for locally advanced rectal cancer: the DREAMtherapy trial.

BACKGROUND: The DREAMtherapy (Dual REctal Angiogenesis MEK inhibition radiotherapy) trial is a novel intertwined design whereby two tyrosine kinase inhibitors (cediranib and selumetinib) were independently evaluated with rectal chemoradiotherapy (CRT) in an efficient manner to limit the extended follow-up period often required for radiotherapy studies. PATIENTS AND METHODS: Cediranib or selumetinib was commenced 10 days before and then continued with RT (45 Gy/25#/5 wks) and capecitabine (825 mg/m2 twice a day (BID)). When three patients in the cediranib 15-mg once daily (OD) cohort were in the surveillance period, recruitment to the selumetinib cohort commenced. This alternating schedule was followed throughout. Three cediranib (15, 20 and 30 mg OD) and two selumetinib cohorts (50 and 75 mg BID) were planned. Circulating and imaging biomarkers of inflammation/angiogenesis were evaluated. RESULTS: In case of cediranib, dose-limiting diarrhoea, fatigue and skin reactions were seen in the 30-mg OD cohort, and therefore, 20 mg OD was defined as the maximum tolerated dose. Forty-one percent patients achieved a clinical or pathological complete response (7/17), and 53% (9/17) had an excellent clinical or pathological response (ECPR). Significantly lower level of pre-treatment plasma tumour necrosis factor alpha (TNFα) was found in patients who had an ECPR. In case of selumetinib, the 50-mg BID cohort was poorly tolerated (fatigue and diarrhoea); a reduced dose cohort of 75-mg OD was opened which was also poorly tolerated, and further recruitment was abandoned. Of the 12 patients treated, two attained an ECPR (17%). CONCLUSIONS: This novel intertwined trial design is an effective way to independently investigate multiple agents with radiotherapy. The combination of cediranib with CRT was well tolerated with encouraging efficacy. TNFα emerged as a potential predictive biomarker of response and warrants further evaluation.

Plasma membrane H+ -ATPase activity and graft success of breadfruit (Artocarpus altilis) onto interspecific rootstocks of marang (A. odoratissimus) and pedalai (A. sericicarpus).

Breadfruit (Artocarpus altilis) is primarily grown as a staple tree crop for food security in Oceania. Significant wind damage has driven interest in developing its dwarfing rootstocks. Due to the predominantly vegetative propagation of the species, grafting onto interspecific seedlings is an approach to identifying dwarfing rootstocks. However, grafting of breadfruit onto unrelated Artocarpus species has not been investigated. Here we first report the success of breadfruit grafting onto interspecific rootstocks, marang (A. odoratissimus) and pedalai (A. sericicarpus). To address the low graft survival, we investigated the relationship of plasma membrane (PM) H+ -ATPase activity to graft success. We provide the first evidence for a positive correlation between PM H+ -ATPase activity and graft survival. The graft unions of successful grafts had higher PM H+ -ATPase activity compared to those of failed grafts. Rootstocks with low PM H+ -ATPase activity in leaf microsomes before grafting had lower graft survival than those with high enzyme activity, with graft success of 10% versus 60% and 0% versus 30% for marang and pedalai rootstocks, respectively. There was a positive correlation between graft success and the PM H+ -ATPase activity measured from the rootstock stem microsomes 2 months after grafting [marang, r(7) = 0.9203, P = 0.0004; pedalai (r(7) = 0. 8820, P = 0.0017]. Removal of scion's own roots decreased the leaf PM H+ -ATPase activity of grafted plants regardless of the final graft outcome. Recovery of the enzyme activity was only found in the successful grafts. The function of PM H+ -ATPase in graft union development and graft success improvement is discussed.

Frequent or complex ventricular ectopy in apparently healthy subjects: a clinical study of 25 cases.

During 18 months, 25 asymptomatic apparently healthy subjects were consecutively studied because of electrocardiographic discovery of frequent (more than 3 beats/min) or complex (multiform, alternating or repetitive patterns) ventricular ectopy of unknown cause. History, physical examination, laboratory data, chest roentgenogram, vectorcardiogram, exercise testing, echocardiogram, His bundle electrogram and cardiac catheterization procedures were performed to detect cardiac abnormalities. Ventricular ectopy was studied with maximal exercise testing, 24 hour Holter recordings and antiarrhythmic drug regimens. The duration of ventricular ectopy ranged from 1 to 30 years (mean 6). Eighteen subjects had an apparently normal cardiac status, and seven had mild or suspected abnormalities. Ventricular ectopy was of predominantly right ventricular origin in 19 of 25 subjects, and disappeared during maximal exercise in 21 of 23 subjects. Characteristic frequent (mean 559 beats/hour) and complex (bigeminal, trigerminal or ventricular couplet) ectopic beats were consistently present in all subjects during a 24 hour diurnal cycle. Antiarrhythmic drug regimens were generally ineffective. Our data suggest that a clinical syndrome of frequent complex ventricular ectopy can occur in apparently healthy subjects. The etiology and natural history of this syndrome are undefined.

Correlation between visual clues, objective architectural features, and interobserver agreement in prostate cancer.

Three pathologists evaluated a number of designated architectural features to assign grades to 41 cases of well- to moderately differentiated adenocarcinoma, and their opinions were compared. The consensus opinion was obtained and evaluated against objective measurements of glandular architecture that were obtained by morphometric techniques. The observers agreed on gland size, gland uniformity, and the number of glands per field in only 49%, 31%, and 39% of cases, respectively. There were significant differences in the Gleason grades assigned by observers. Paired matching of individual Gleason grades showed agreement among observers in 44% (18 of 41), 56% (23 of 41), and 75% (31 of 41) of cases, respectively. This level of interobserver disagreement occurred even though cases with predominant patterns were selected carefully and those with variable patterns were excluded. A direct relationship appears to exist between increasing Gleason grade and increasing glandular variability, and there is an inverse relationship between Gleason grade, gland lumen area, and the number of glandular nuclei, as assessed by a group of pathologists.

The reliability of laboratory data from blood samples collected through pulmonary artery catheters.

Blood samples obtained through quadruple-lumen Swan-Ganz catheters in ten critically ill patients provided reliable results after withdrawal of a total of 2.2 mL (including 1.2 mL of dead space) for hematocrit tests and 3.2 mL (including 1.2 mL of dead space) for plasma sodium tests.

Accurate coagulation studies from heparinized radial artery catheters.

We studied the feasibility of obtaining accurate coagulation studies from indwelling, heparinized radial artery catheters in 28 patients after cardiac surgery. PTT assay was chosen because of its frequent clinical use. Thrombin time assay was chosen because it is a component of the coagulation screening panel and because it is useful in assessing heparin contamination of specimens. We conclude that PTT results are reliable when the dwell volume (0.6 ml for the catheter and extension tubing in our study) and an additional 4.5 ml have been discarded (5.1 ml total discard volume). We recommend the collection of samples for thrombin time assay from a separate venous site because results on samples from heparinized arterial catheters are unpredictable.

Obtaining reliable plasma sodium and glucose determinations from pulmonary artery catheters.

In critically ill patients, blood specimens are frequently withdrawn from intravascular catheters without consideration of possible infusate contamination, of test accuracy, or of undue blood loss. The reliability of sodium (Na+) and glucose determinations withdrawn from the venous infusion port of a pulmonary artery catheter was studied in 30 patients after cardiac surgery. Catheter patency was maintained by a saline and heparin infusate. Venipuncture samples for Na+ and glucose served as controls. A sample containing the dwell volume of the catheter and the stopcock (1 ml) was withdrawn and discarded, followed by fifteen 1 ml samples being taken for analysis. It was concluded that Na+ values are stable after discard of the catheter dwell volume and an additional milliliter of blood. Minimal clinical differences were found in the glucose values after discard of the catheter dwell volume and two additional milliliters of blood.

Surface attachment of nitrifying bacteria and their inhibition by potassium ethyl xanthate.

Ion exchange resins and glass microscope slides were used to investigate factors affecting attachment of nitrifying bacteria to solid surfaces and the effect of attachment on inhibition ofNitrobacter by potassium ethyl xanthate. The ammonium oxidizerNitrosomonas attached preferentially to cation exchange resins while the nitrite oxidizerNitrobacter colonized anion exchange resins more extensively. Colonization was always associated with growth, and the site of substrate (NH4 (+) or NO2 (-)) adsorption was the major factor in attachment and colonization. The specific growth rate of cells colonizing either ion exchange resin beads or glass surfaces was greater than that of freely suspended cells, butNitrobacter populations colonizing glass surfaces were more sensitive to the inhibitor potassium ethyl xanthate. The findings indicate that surface growth alone does not protect soil nitrifying bacteria from inhibition by potassium ethyl xanthate and explain different patterns of inhibition for ammonium and nitrite oxidizers in the soil.

Modification by fentanyl and alfentanil of the intraocular pressure response to suxamethonium and tracheal intubation.

We have measured in a double-blind study the changes in intraocular pressure (IOP) in 40 consecutive patients (pretreated with fentanyl or alfentanil) who received suxamethonium and tracheal intubation. Although IOP increased significantly following administration of suxamethonium, mean IOP in both groups remained significantly less than control values (P less than 0.002). Tracheal intubation caused a further significant increase in IOP in the fentanyl, but not the alfentanil group. There were no significant differences in mean IOP between the fentanyl and alfentanil groups. Both opioids reduced, but did not abolish the haemodynamic responses to tracheal intubation.

Pharmacokinetics of paracetamol after cardiac surgery.

Plasma concentration was measured after rectal and nasogastric administration of paracetamol 15 mg/kg to 28 febrile children aged between 9 days to 7 years who had undergone cardiac surgery. After equivalent doses, rectal administration in neonates and children on the first postoperative day was found to produce plasma concentrations below the therapeutic range with higher concentrations after nasogastric paracetamol on the second postoperative day. There was less variance in plasma paracetamol concentrations in neonates. Both plasma elimination half life and area under the plasma concentration time curve were significantly increased in neonates after suppository dosing compared with older children. There was no difference in antipyretic effect between the two routes of administration, but this was much lower than that previously reported in febrile children.

Methanotrophic attached-film reactor development and biofilm characteristics.

The feasibility of using methanotrophs in an attached-film, fluidized-bed (MAFFB) reactor system has been under investigation since 1987. Mixed culture, methane-utilizing attached biofilms were developed on diatomaceous earth particles and on granular activated carbon. The required feed gases, methane and oxygen, were supplied to the attached biofilm in dissolved form using separate gas-liquid aeration columns. Biofilm growth was steady despite low influent dissolved methane concentrations (1 to 3 mg/L). A breeder MAFFB operated consistently for 4.1 years with attached biofilm concentrations as high as 51.7 g VS/L static-bed with minimal biomass wasting and with minimal buffer and nutrient inputs. The maximum biomass concentration observed was 75.6 g VS/L static-bed in a MAFFB reactor treating trichloroethene. Biofilm thickness reached 160 mum with typical values of 70 mum under methane and oxygen growth-rate-limited conditions. Biofilm densities of 120 to 190 g VS/L film were observed. Growth rates varied from <0.01/d to 0.17/d. Greater than 90% of the biomass concentration in the bed was attached, and effluent total suspended solids ranged from 5 to 74 mg/L, with an average of 24 mg/L over 27 runs in four MAFFB systems at upflow velocities of 11.4 to 25 m/h. Heterotrophic attached-film methanotrophs appear to be stable and useful for applications in toxics treatment, and other product manipulations.

Measurement of lamotrigine under conditions measuring phenobarbitone, phenytoin, and carbamazepine using reversed-phase high-performance liquid chromatography at dual wavelengths.

A reverse-phase high-performance liquid chromatographic (HPLC) method for the measurement of lamotrigine (LTG) simultaneously with phenobarbitone (PB), phenytoin (PHT), and carbamazepine (CBZ) is described using hexobarbital as the internal standard. The method uses the chromophore at 307 nm to detect LTG in the presence of interfering CBZ-10,11-epoxide detected at 220 nm, the wavelength used to measure the other drugs. This method requires < 10 min/sample for completion. Simultaneous monitoring of the chromatographs at 220 and 310 nm with a simple calculation allows LTG to be measured virtually identically to a routine method for monitoring of the other anticonvulsants. Between-batch precisions for LTG at 2 and 6 mg/L were < 5%. Accuracy of LTG estimation was assessed by comparison with known values of samples supplied by an external quality assessment scheme.

Metabolic effects of dobutamine in normal man.

1. Dobutamine in 5% (w/v) D-glucose was infused at sequential doses of 2, 5 and 10 micrograms min-1 kg-1, 45 min at each dose, into eight healthy male subjects, and the effects were compared with those produced by infusion of the corresponding volumes of 5% (w/v) D-glucose alone. 2. The energy expenditure increased and was 33% higher than control (P less than 0.001) at 10 micrograms of dobutamine min-1 kg-1. The respiratory exchange ratio decreased from 0.85 (SEM 0.02) before infusion to 0.80 (SEM 0.01) at 10 micrograms of dobutamine min-1 kg-1, but did not alter during the placebo infusion (P less than 0.001). 3. Plasma noradrenaline concentrations were lower during the dobutamine infusion compared with during the infusion of D-glucose alone (P less than 0.025). Plasma dopamine concentrations remained below 0.1 nmol/l throughout both infusions. 4. Compared with during the placebo infusion, the blood glucose concentration decreased (P less than 0.001), the plasma glycerol and free fatty acid concentrations increased by 150 and 225%, respectively (both P less than 0.001), and the plasma potassium concentration decreased from 3.8 (SEM 0.07) to 3.6 (SEM 0.04) mmol/l (P less than 0.01) during dobutamine infusion. The plasma insulin concentration increased at 2 and 5 micrograms of dobutamine min-1 kg-1 (P less than 0.001) with no further rise at 10 micrograms of dobutamine min-1 kg-1. 5. Compared with during the placebo infusion, the systolic and diastolic blood pressures and the heart rate increased during dobutamine infusion (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

TCE degradation in a methanotrophic attached-film bioreactor.

Trichloroethene was degraded in expanded-bed bioreactors operated with mixed-culture methanotrophic attached films. Biomass concentrations of 8 to 75 g volatile solids (VS) per liter static bed (L(sb)) were observed. Batch TCE degradation rates at 35 degrees C followed the Michaelis-Menten model, and a maximum TCE degradation rate (q(max)) of 10.6 mg TCE/gVS . day and a half velocity coefficient (K(S)) of 2.8 mg TCE/L were predicted. Continuous-flow kinetics also followed the Michaelis-Menten model, but other parameters may be limiting, such as dissolved copper and dissolved methane-q(max) and K(S) were 2.9 mg TCE/gVS . day and 1.5 mg TCE/L, respectively, at low copper concentrations (0.003 to 0.006 mg Cu/L). The maximum rates decreased substantially with small increases in dissolved copper. Methane consumption during continuous-flow operation varied from 23 to 1200 g CH(4)/g TCE degraded. Increasing the influent dissolved methane concentration from 0.01 mg/L to 5.4 mg/L reduced the TCE degradation rate by nearly an order of magnitude at 21 degrees C. Exposure of biofilms to 1.4 mg/L tetrachloroethene (PCE) at 35 degrees C resulted in the loss of methane utilization ability. Tests with methanotrophs grown on granular activated carbon indicated that lower effluent TCE concentrations could be obtained. The low efficiencies of TCE removal and low degradation rates obtained at 35 degrees C suggest that additional improvements will be necessary to make methanotrophic TCE treatment attractive.

Transplantation of fetal neocortex ameliorates sensorimotor and locomotor deficits following neonatal ischemic-hypoxic brain injury in rats.

Ischemic brain injury in neonates can result in the degeneration of cortical and subcortical areas of brain and is associated with neurologic deficits. One approach to restoring function in conditions of ischemic brain injury is the use of neural transplants to repair damaged connections. This approach has been shown to reestablish neural circuitry and to ameliorate associated motor deficits in models of neonatal sensorimotor cortex damage. In this study, we utilized the Rice et al. rodent model of neonatal ischemic-hypoxic (IH) brain injury to assess whether transplantation of fetal neocortical tissue can promote functional recovery in tests of sensorimotor and locomotor ability throughout development and as adults. We show that animals that received neocortical grafts 3 days following the IH injury performed significantly better as adults on two measures of motor ability, the Rota-Rod treadmill and apomorphine-induced rotations, than did control animals that received sham transplants after the IH injury. Transplants were identifiable in 72% of the animals 10-12 weeks after implantation. Histochemical studies revealed that while the transplanted tissue did not establish normal cortical cytoarchitecture, cells and fibers within the grafts stained for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), choline acetyl transferase (ChAT), cholecystokinin (CCK), and glial fibrillary acidic protein (GFAP). These results suggest that transplantation of fetal neocortical tissue following IH injury in the neonatal period is associated with amelioration of motor deficits and that the grafted tissue demonstrated a neurochemical phenotype that resembled normal neocortex. This approach warrants continued investigation in light of potential therapeutic uses.

Septic patients in multiple organ failure can oxidize infused glucose, but non-oxidative disposal (storage) is impaired.

1. Patients suffering trauma and sepsis are insulin resistant, but no studies have specifically been made of patients suffering multiple organ failure. 2. We have studied exogenous glucose utilization in multiple organ failure using a combination of the hyperglycaemic glucose clamp and indirect calorimetry to quantify glucose utilization in multiple organ failure, partitioning it into oxidative and nonoxidative disposal (storage). 3. Fourteen septic patients with multiple organ failure were studied. APACHE II (Acute Physiological and Chronic Health Evaluation Mark II) scores on the day of the study ranged from 11 to 31 (median 16). Twenty percent D-glucose was infused and blood glucose was clamped at 12 mmol/l for 3 h. The results were compared with those obtained on seven healthy control subjects. 4. Glucose utilization and energy expenditure were similar in the two groups for the first 90 min of the clamp, after which glucose utilization and energy expenditure increased steadily in the control subjects but did not change in the patients. Respiratory exchange ratio rose in both groups; considered over the whole of the clamp period, respiratory exchange ratio was slightly lower in the patients than in the control subjects (P < 0.05) but not at any specific time point. Glucose oxidation rose in both groups but non-oxidative glucose disposal (storage) rose only in the control subjects. Glucose oxidation was slightly lower in the patients (P < 0.05) but not at any specific time point and there was no difference between the groups in the amount by which glucose oxidation increased. Non-oxidative disposal in the patients fell significantly (P < 0.01) over the course of the clamp and was significantly lower than in the control subjects (P < 0.01). 5. Growth hormone increased in response to glucose infusion in the patients but not in the control subjects. 6. Like patients suffering uncomplicated sepsis or trauma, patients with multiple organ failure are also insulin resistant. The defect appears to lie in an impairment of the ability to store glucose rather than oxidize it, and this may be due in part to the increase in growth hormone in patients with multiple organ failure.