Missense mutation in PFKM associated with muscle-type phosphofructokinase deficiency in the Wachtelhund dog.

Hereditary muscle-type phosphofructokinase (PFK) deficiency causing intermittent hemolytic anemia and exertional myopathy due to a single nonsense mutation in PFKM has been previously described in English Springer and American Cocker Spaniels, Whippets, and mixed breed dogs. We report here on a new missense mutation associated with PFK deficiency in Wachtelhunds. Coding regions of the PFKM gene were amplified from genomic DNA and/or cDNA reverse-transcribed from RNA of EDTA blood of PFK-deficient and clinically healthy Wachtelhunds and control dogs. The amplicons were sequenced and compared to the published canine PFKM sequence. A point mutation (c.550C>T, in the coding sequence of PFKM expressed in blood) was found in all 4 affected Wachtelhunds. This missense mutation results in an amino acid substitution of arginine (Arg) to tryptophan (Trp) at position 184 of the protein expressed in blood (p.Arg184Trp). The mutation is located within an alpha-helix, and based on the SIFT analysis, this amino acid substitution is not tolerated. Amplifying the region around this mutation and digesting the PCR fragment with the restriction enzyme MspI, produces fragments that readily differentiate between PFK-deficient, carrier, and normal animals. Furthermore, we document 2 additional upstream PFKM exons expressed in canine testis but not in blood. Despite their similar phenotypic appearance and use for hunting, Wachtelhunds and English Springer Spaniels are not thought to have common ancestors. Thus, it is not surprising that different mutations are responsible for PFK deficiency in these breeds. Knowledge of the molecular basis of PFK deficiency in Wachtelhunds provides an opportunity to screen and control the spread of this deleterious trait.

Bacteriophages are synergistic with bacterial interference for the prevention of Pseudomonas aeruginosa biofilm formation on urinary catheters.

AIMS: We hypothesized that pretreating urinary catheters with benign Escherichia coli HU2117 plus an antipseudomonal bacteriophage (ΦE2005-A) would prevent Pseudomonas aeruginosa biofilm formation on catheters--a pivotal event in the pathogenesis of catheter-associated urinary tract infection (CAUTI). METHODS AND RESULTS: Silicone catheter segments were exposed to one of four pretreatments (sterile media; E. coli alone; phage alone; E. coli plus phage), inoculated with P. aeruginosa and then incubated up to 72 h in human urine before rinsing and sonicating to recover adherent bacteria. Pseudomonas aeruginosa adherence to catheters was almost 4 log(10) units lower when pretreated with E. coli plus phage compared to no pretreatment (P < 0.001) in 24-h experiments and more than 3 log(10) units lower in 72-h experiments (P < 0.05). Neither E. coli nor phage alone generated significant decreases. CONCLUSIONS: The combination of phages with a pre-established biofilm of E. coli HU2117 was synergistic in preventing catheter colonization by P. aeruginosa. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe a synergistic protection against colonization of urinary catheters by a common uropathogen. Escherichia coli-coated catheters are in clinical trials; adding phage may offer additional benefit.

Novel measurements of periaortic adipose tissue in comparison to anthropometric measures of obesity, and abdominal adipose tissue.

BACKGROUND: Perivascular adipose tissue may be associated with the amount of local atherosclerosis. We developed a novel and reproducible method to standardize volumetric quantification of periaortic adipose tissue by computed tomography (CT) and determined the association with anthropometric measures of obesity, and abdominal adipose tissue. METHODS: Measurements of adipose tissue were performed in a random subset of participants from the Framingham Heart Study (n=100) who underwent multidetector CT of the thorax (ECG triggering, 2.5 mm slice thickness) and the abdomen (helical CT acquisition, 2.5 mm slice thickness). Abdominal periaortic adipose tissue (AAT) was defined by a 5 mm cylindrical region of interest around the aortic wall; thoracic periaortic adipose tissue (TAT) was defined by anatomic landmarks. TAT and AAT were defined as any voxel between -195 and -45 HU and volumes were measured using dedicated semiautomatic software. Measurement reproducibility and association with anthropometric measures of obesity, and abdominal adipose tissue were determined. RESULTS: The intra- and inter-observer reproducibility for both AAT and TAT was excellent (ICC: 0.97 and 0.97; 0.99 and 0.98, respectively). Similarly, the relative intra- and inter-observer difference was small for both AAT (-1.85+/-1.28% and 7.85+/-6.08%; respectively) and TAT (3.56+/-0.83% and -4.56+/-0.85%, respectively). Both AAT and TAT were highly correlated with visceral abdominal fat (r=0.65 and 0.77, P<0.0001 for both) and moderately correlated with subcutaneous abdominal fat (r=0.39 and 0.42, P<0.0001 and P=0.009), waist circumference (r=0.49 and 0.57, P<0.0001 for both) and body mass index (r=0.47 and 0.58, P<0.0001 for both). CONCLUSION: Standardized semiautomatic CT-based volumetric quantification of periaortic adipose tissue is feasible and highly reproducible. Further investigation is warranted regarding associations of periaortic adipose tissue with other body fat deposits, cardiovascular risk factors and clinical outcomes.

Mid-depth circulation of the subpolar north atlantic during the last glacial maximum.

Holocene and glacial carbon isotope data of benthic foraminifera from shallow to mid-depth cores from the northeastern subpolar Atlantic show that this region was strongly stratified, with carbon-13-enriched glacial North Atlantic intermediate water (GNAIW) overlying carbon-13-depleted Southern Ocean water (SOW). The data suggest that GNAIW originated north of the polar front and define GNAIW end-member carbon isotope values for studies of water-mass mixing in the open Atlantic. Identical carbon isotope values in the core of GNAIW and below the subtropical thermocline are consistent with rapid cycling of GNAIW through the northern Atlantic. The high carbon isotope values below the thermocline indicate that enhanced nutrient leakage in response to increased ventilation may have extended into intermediate waters. Geochemical box models show that the atmospheric carbon dioxide response to nutrient leakage that results from an increase in ventilation rate may be greater than the response to nutrient redistribution by conversion of North Atlantic deep water into GNAIW. These results underscore the potential rule of Atlantic Ocean circulation changes in influencing past atmospheric carbon dioxide values.

Glacial surface temperatures of the southeast Atlantic Ocean.

A detailed record of sea surface temperature from sediments of the Cape Basin in the subtropical South Atlantic indicates a previously undocumented progression of marine climate change between 41 and 18 thousand years before the present (ky B.P.), during the last glacial period. Whereas marine records typically indicate a long-term cooling into the Last Glacial Maximum (around 21 ky B.P.) consistent with gradually increasing global ice volume, the Cape Basin record documents an interval of substantial temperate ocean warming from 41 to 25 ky B.P. The pattern is similar to that expected in response to changes in insolation owing to variations in Earth's tilt.

Synchronous radiocarbon and climate shifts during the last deglaciation.

Radiocarbon data from the Cariaco Basin provide calibration of the carbon-14 time scale across the period of deglaciation (15,000 to 10, 000 years ago) with resolution available previously only from Holocene tree rings. Reconstructed changes in atmospheric carbon-14 are larger than previously thought, with the largest change occurring simultaneously with the sudden climatic cooling of the Younger Dryas event. Carbon-14 and published beryllium-10 data together suggest that concurrent climate and carbon-14 changes were predominantly the result of abrupt shifts in deep ocean ventilation.

How animals move: an integrative view.

Recent advances in integrative studies of locomotion have revealed several general principles. Energy storage and exchange mechanisms discovered in walking and running bipeds apply to multilegged locomotion and even to flying and swimming. Nonpropulsive lateral forces can be sizable, but they may benefit stability, maneuverability, or other criteria that become apparent in natural environments. Locomotor control systems combine rapid mechanical preflexes with multimodal sensory feedback and feedforward commands. Muscles have a surprising variety of functions in locomotion, serving as motors, brakes, springs, and struts. Integrative approaches reveal not only how each component within a locomotor system operates but how they function as a collective whole.

Application of ceramic membranes with pre-ozonation for treatment of secondary wastewater effluent.

Membrane fouling is an inevitable problem when microfiltration (MF) and ultrafiltraion (UF) are used to treat wastewater treatment plant (WWTP) effluent. While historically the use of MF/UF for water and wastewater treatment has been almost exclusively focused on polymeric membranes, new generation ceramic membranes were recently introduced in the market and they possess unique advantages over currently available polymeric membranes. Ceramic membranes are mechanically superior and are more resistant to severe chemical and thermal environments. Due to the robustness of ceramic membranes, strong oxidants such as ozone can be used as pretreatment to reduce the membrane fouling. This paper presents results of a pilot study designed to investigate the application of new generation ceramic membranes for WWTP effluent treatment. Ozonation and coagulation pretreatment were evaluated to optimize the membrane operation. The ceramic membrane demonstrated stable performance at a filtration flux of 100 gfd (170LMH) at 20 degrees C with pretreatment using PACl (1mg/L as Al) and ozone (4 mg/L). To understand the effects of ozone and coagulation pretreatment on organic foulants, natural organic matter (NOM) in four waters - raw, ozone treated, coagulation treated, and ozone followed by coagulation treated wastewaters - were characterized using high performance size exclusion chromatography (HPSEC). The HPSEC analysis demonstrated that ozone treatment is effective at degrading colloidal NOMs which are likely responsible for the majority of membrane fouling.

Molecular assessment of salt-tolerant, perchlorate- and nitrate-reducing microbial cultures.

The microbial ecology of enrichment cultures adapted to the removal of perchlorate and nitrate from high salt solutions and ion-exchange brines was examined over a period of four years using denaturing gradient gel electrophoresis and total DNA extraction with cloning and in each case partial sequencing of the 16S rDNA genes. The cultures studied were a result of enrichment from marine sediment inoculum initiated in 2001. The resulting enrichment cultures were fed perchlorate, or perchlorate and nitrate, in a 3% (w/v) NaCl defined medium or ion-exchange brines (5.6% NaCl) containing perchlorate and nitrate with acetate as the electron donor. All of the sequences' closest matches in the NCBI GenBank database were to marine or salt-tolerant organisms. Strains belonging to the genera Halomonas or Marinobacter were found to dominate in cultures that were fed nitrate in addition to perchlorate, but were effectively absent from cultures fed perchlorate alone. The cultures fed perchlorate as the sole electron acceptor were relatively diverse with the dominant sequences belonging to the genera Dechloromarinus and Denitromonas. A study examining the effects of growing the cultures on different electron acceptors to the cultures revealed that Denitromonas may be more dominant than Dechloromarinus as the salt-tolerant, perchlorate-reducing organism.

Perchlorate and nitrate treatment by ion exchange integrated with biological brine treatment.

Groundwater contaminated with perchlorate and nitrate was treated in a pilot plant using a commercially available ion exchange (IX) resin. Regenerant brine concentrate from the IX process, containing high perchlorate and nitrate, was treated biologically and the treated brine was reused in IX resin regeneration. The nitrate concentration of the feed water determined the exhaustion lifetime (i.e., regeneration frequency) of the resin; and the regeneration condition was determined by the perchlorate elution profile from the exhausted resin. The biological brine treatment system, using a salt-tolerant perchlorate- and nitrate-reducing culture, was housed in a sequencing batch reactor (SBR). The biological process consistently reduced perchlorate and nitrate concentrations in the spent brine to below the treatment goals of 500 microg ClO4(-)/L and 0.5mg NO3(-)-N/L determined by equilibrium multicomponent IX modeling. During 20 cycles of regeneration, the system consistently treated the drinking water to below the MCL of nitrate (10 mgNO3(-)-N/L) and the California Department of Health Services (CDHS) notification level of perchlorate (i.e., 6 microg/L). A conceptual cost analysis of the IX process estimated that perchlorate and nitrate treatment using the IX process with biological brine treatment to be approximately 20% less expensive than using the conventional IX with brine disposal.

Fluidized bed reactor for the biological treatment of ion-exchange brine containing perchlorate and nitrate.

The removal of perchlorate and nitrate from contaminated drinking water using regenerable ion-exchange processes produces a high salt brine (3-10% NaCl) laden with high concentrations of perchlorate and nitrate. This bench-scale research describes the operation of acetate-fed granular activated carbon (GAC) based fluidized bed reactors (FBR) for perchlorate-only, and combined nitrate and perchlorate removal from synthetic brine (6% NaCl). The GAC was inoculated with a salt-tolerant culture developed by the authors and used previously in batch systems. An FBR was an effective design for perchlorate reduction and exhibited first-order degradation kinetics with respect to perchlorate concentrations. Nitrate was also removed by the organisms in the column and had no negative effects on the removal of perchlorate using the FBR design. However, at higher concentrations of nitrate the FBR was more difficult to operate due to loss of carbon and biomass from the formation of nitrogen bubbles and the high recycle flow rates needed.

Kinetics of nitrate and perchlorate reduction in ion-exchange brine using the membrane biofilm reactor (MBfR).

Several sources of bacterial inocula were tested for their ability to reduce nitrate and perchlorate in synthetic ion-exchange spent brine (30-45 g/L) using a hydrogen-based membrane biofilm reactor (MBfR). Nitrate and perchlorate removal fluxes reached as high as 5.4 g Nm(-2)d(-1) and 5.0 g ClO(4)m(-2)d(-1), respectively, and these values are similar to values obtained with freshwater MBfRs. Nitrate and perchlorate removal fluxes decreased with increasing salinity. The nitrate fluxes were roughly first order in H(2) pressure, but roughly zero-order with nitrate concentration. Perchlorate reduction rates were higher with lower nitrate loadings, compared to high nitrate loadings; this is a sign of competition for H(2). Nitrate and perchlorate reduction rates depended strongly on the inoculum. An inoculum that was well acclimated (years) to nitrate and perchlorate gave markedly faster removal kinetics than cultures that were acclimated for only a few months. These results underscore that the most successful MBfR bioreduction of nitrate and perchlorate in ion-exchange brine demands a well-acclimated inoculum and sufficient hydrogen availability.

P-Glycoprotein inhibitor valspodar (PSC 833) increases the intracellular concentrations of daunorubicin in vivo in patients with P-glycoprotein-positive acute myeloid leukemia.

PURPOSE: The aim of the present study was to evaluate the effect of the cyclosporine derivative valspodar (PSC 833; Amdray, Novartis Pharma, Basel, Switzerland) on the concentration of daunorubicin (dnr) in leukemic blast cells in vivo during treatment. PATIENTS AND METHODS: Ten patients with acute myeloid leukemia (AML) were included. Leukemic cells from seven of the patients were P-glycoprotein (Pgp)-positive. dnr 100 mg/m(2) was given as a continuous infusion over 72 hours. After 24 hours, a loading dose of valspodar was given, followed by a 36-hour infusion of 10 mg/kg per 24 hours. Blood samples were drawn at regular intervals, and concentrations of dnr and its main metabolite, daunorubicinol, in plasma and isolated leukemic cells were determined by high-pressure liquid chromatography. RESULTS: The mean dnr concentrations in leukemic cells 24 hours after the start of infusion (before valspodar) were 18.8 micromol/L in Pgp-negative samples and 13.5 micromol/L in Pgp-positive samples. After 8 hours of valspodar infusion, these values were 25.8 and 24.0 micromol/L, respectively. The effect of valspodar was evaluated from the ratio of the area under the curve (AUC) for dnr concentration versus time in leukemic cells to the AUC for dnr concentration against time in the plasma. For the seven patients with Pgp-positive leukemia, the mean ratio increased by 52%, from 545 on day 1 to 830 on day 2 (P<.05) when valspodar was given. In the three patients with Pgp-negative leukemia, no significant difference was observed. CONCLUSION: These results strongly suggest that valspodar, by interacting with Pgp, can increase the cellular uptake of dnr in leukemic blasts in vivo.

Promoters of the broad host range plasmid RK2: analysis of transcription (initiation) in five species of gram-negative bacteria.

A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined.

Oculomotor effects of intermittent conduction block in myasthenia gravis and Guillain-Barré syndrome. An oculographic study with computer simulations.

Five abnormal oculographic patterns were identified in eight patients with either myasthenia gravis or Guillain-Barré syndrome (GBS). These could be differentiated into three intrasaccadic and two postsaccadic abnormalities. From our studies of computer simulations, and considering the established pathophysiology of myasthenia gravis and GBS, we believe that our oculographic findings were a consequence of defects in peripheral neural and neuromuscular conduction, together with a simple adaptive increase in duration of the saccadic burst of central innervation. We conclude that the eye movement abnormalities we observed are explained by intermittent block of peripheral conduction, and suggest that any disease causing intermittent blockage of neural signals to extraocular muscles will produce similar abnormalities of eye movement.

Talking while walking: the effect of a dual task in aging and Alzheimer's disease.

We determined the effects of distraction on gait in healthy elderly subjects and Alzheimer's disease (AD) patients. The effects of simultaneous performance of a verbal fluency task (effect of reciting male or female names) on the time and number of steps taken to walk 30 feet were compared using a repeated-measures design with between-group comparison between community-dwelling healthy old old (oOld; n = 20; mean age +/- SD, 86 +/- 4.4), healthy young old (yOld; n = 23; mean age +/- SD, 72 +/- 3.6), and probable AD subjects without parkinsonism (n = 15; mean age +/- SD, 74 +/- 13). AD patients slowed more than the yOld (p = 0.005) and the oOld (p = 0.002). The yOld and oOld did not differ from each other (p = 0.68). Mean (+/-SD) differences in time were as follows: yOld, -2.2 +/- 1.9; oOld, -1.6 +/- 2.0; AD, -7.1 +/- 9.2 seconds. The change in steps did not differ between groups. Walking speed of AD patients slowed more than that of elderly subjects during the dual task. This may contribute to the risk of falls in AD.

Head rotation trajectories compared with eye saccades by main sequence relationships.

A helmet apparatus permitted duration, peak velocity, and peak acceleration measurements as functions of magnitude of horizontal head rotation; these "main sequence" data give evidence for multipulse-step neurological signals appropriate for time optimal control of head rotation similar to those of saccadic eye movements.

Factors associated with brain donation among optimally healthy elderly people.

BACKGROUND: Consent rates for brain donation were examined in 140 healthy elderly participants of the Oregon Brain Aging Study, a longitudinal study of successful aging. Subjects were initially selected for good health. The study population had a relatively high education level, a high socioeconomic status, and were predominantly white. METHODS: At each annual examination, a project physician asked participants to consider brain donation. This analysis examined variables that may affect the rate of brain donation consent: age, gender, education, socioeconomic status, marital status, religiosity, cognitive status, depression, and functional status. RESULTS: Of these variables only age was a meaningful factor. CONCLUSION: The oldest old participants (> or =85 years of age) were more likely to consent to donation than the younger participants (65-84 years of age).

Adaptation to fatigue of long duration in human wrist movements.

Subjects made fast, accurate, consistent wrist flexions under normal conditions and under conditions of low-frequency fatigue. Movements made 1 h after fatiguing exercise were indistinguishable from those made before exercise, even though twitch tensions were only approximately 60% of their fresh values. Electromyograms (EMGs) recorded from the fatigued muscles were, however, different from those recorded before exercise. EMGs during unfatigued movements showed multiple bursts typical for rapid movements. In the presence of low-frequency fatigue, the duration of the first burst was longer than that under normal conditions, and its onset occurred earlier relative to the initiation of movement. The area of the second agonist burst and, in some cases, the antagonist burst, was increased, although changes in their timings were unclear. We conclude that subjects adapted to low-frequency fatigue by changing the neural patterns controlling their muscles and present a simple model of excitation-contraction coupling that demonstrates how the observed changes in excitation can produce the same kinematics.

Increased efficiency of stool culture for the detection of Salmonella and Shigella.

The Wampole Bactigen Salmonella-Shigella Latex Agglutination Test (SSLA) (Wampole Laboratories, Cranbury, New Jersey) was evaluated as a possible substitute for blind subculture of selenite broths from stool cultures. Recovery rates of Salmonella and Shigella from eosin-methylene blue (EMB) agar were reviewed to determine if this medium could be eliminated from primary stool culture. Salmonella was detected in 17 of 822 stools by both SSLA and culture. There were 52 false-positive SSLA for Salmonella (sensitivity 100%, specificity 93%). Of three Shigella isolated on culture, one was SSLA positive, one was SSLA negative, and one was negative by both SSLA and subculture of selenite broth. There were eight false-positive SSLA for Shigella (specificity 99%). Of 50 Salmonella and 11 Shigella isolated from 6200 stools in 1.5 years, two Shigella were isolated on EMB only. The SSLA test is a useful screening test for Salmonella. By eliminating unnecessary subcultures of selenite broth, it reduces turnaround time by 24 hr for negative stool cultures. The combination of primary culture with SSLA screening of enrichment broth should be adequate for the detection of Salmonella and Shigella from stool specimens. Our data suggest that EMB or other differential medium should be retained for primary culture to enhance detection of Shigella.