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Flood‐type disturbances affect both periphyton and macroinvertebrate communities depending on their frequency, magnitude and duration, but some impacts can be mitigated by geomorphic constraints. We studied four rivers in the Adirondack Mountains; the Indian River experienced regulated flow releases creating bank‐full floods four times/week whereas other study sites provided a continuum of control conditions for comparison. We sampled periphyton and macroinvertebrate communities in each river along with channel hydrology, hydraulics and geomorphology. Periphyton abundance varied seasonally and among rivers depending on nitrate concentrations and scour. Flow releases created a static mosaic of patches consisting of heavily scoured bed sediment in the thalweg (high shear stress areas—HSS) which had very low periphyton abundance; boulders and rocky shoals created low shear stress areas (LSS) supporting mats of filamentous algae. Macroinvertebrate densities were highest in HSS areas whereas LSS areas had lower macroinvertebrate densities that were more similar to reference rivers, although both LSS and HSS areas had the lowest species diversity and richness. Macroinvertebrate composition in HSS areas was dominated by filter‐feeders, whereas LSS areas had more gatherers and scrapers. While all rivers had high boulder densities, the Indian River could be considered geomorphically moribund—its substrate was immobile and the channel experienced no adjustments in spite of high flood frequencies. These physical characteristics help mitigate the severe hydrologic conditions that releases create because the static mosaic of low and HSS patches allow certain macroinvertebrates adapted to this level of disturbance to persist. This static mosaic is different from the shifting mosaic pattern often observed in streams following flood‐type disturbances. Geomorphically moribund rivers may lack ecological sensitivity to hydrologic disturbances, and decisions to limit flow modification (e.g. recreational flow releases, hydropower generation) should consider whether assumed ecological impacts are likely to occur given local geomorphic conditions and constraints. Copyright © 2010 John Wiley & Sons, Ltd.

ABSTRACTBackground:The goals of diabetes management have evolved over the past decade to become the attainment of near-normal glucose and cardiovascular risk factor levels. Improved metabolic control is achieved through optimized medication regimens, but costs specifically associated with such optimization have not been examined.Objective:To estimate the incremental medication cost of providing optimal therapy to reach recommended goals versus actual therapy in patients with type 2 diabetes.Methods:We randomly selected the charts of 601 type 2 diabetes patients receiving care from the outpatient clinics of Massachusetts General Hospital March 1, 1996–August 31, 1997 and abstracted clinical and medication data. We applied treatment algorithms based on 2004 clinical practice guidelines for hyperglycemia, hyperlipidemia, and hypertension to patients’ current medication therapy to determine how current medication regimens could be improved to attain recommended treatment goals. Four clinicians and three pharmacists independently applied the algorithms and reached consensus on recommended therapies. Mean incremental medication costs, the cost differences between current and recommended therapies, per patient (expressed in 2004 dollars) were calculated with 95% bootstrap confidence intervals (CIs).Results:Mean patient age was 65 years old, mean duration of diabetes was 7.7 years, 32% had ideal glucose control, 25% had ideal systolic blood pressure, and 24% had ideal low-density lipoprotein cholesterol. Care for these diabetes patients was similar to that observed in recent national studies. If treatment algorithm recommendations were applied, the average annual medication cost/patient would increase from $1525 to $2164. Annual incremental costs/patient increased by $168 (95% CI $133–$206) for antihyperglycemic medications, $75 ($57–$93) for antihypertensive medications, $392 ($354–$434) for antihyperlipidemic medications, and $3 ($3–$4) for aspirin prophylaxis. Yearly incremental cost of recommended laboratory testing ranged from $77–$189/patient.Limitations:Although baseline data come from the clinics of a single academic institution, collected in 1997, the care of these diabetes patients was remarkably similar to care recently observed nationally. In addition, the data are dependent on the medical record and may not accurately reflect patients’ actual experiences.Conclusion:Average yearly incremental cost of optimizing drug regimens to achieve recommended treatment goals for type 2 diabetes was approximately $600/patient. These results provide valuable input for assessing the cost-effectiveness of improving comprehensive diabetes care.

The hydroamination of styrene with aniline catalyzed by phosphine-ligated palladium triflates exhibits a substantial ¹³C isotope effect at the benzylic carbon. This supports rate-determining nucleophilic attack of amine on a η³-phenethyl palladium complex. Deuterium exchange observations and predicted isotope effects based on DFT calculations support this mechanism. Selectivity in these reactions is determined by the facility of palladium displacement after reversible hydropalladation of the alkene.

Two fields that routinely perform reaction optimization studies are chemical development (prior to scale-up) and medicinal or combinatorial chemistry (prior to analogue synthesis or library production). To date, the use of statistical design of experiments (DoE) in conjunction with automated synthesizers has been applied in process research to a greater extent than in the medicinal or combinatorial laboratories. We have applied DoE in conjunction with an automated synthesizer to optimize the synthesis of amides employing resin-bound N-hydroxybenzotriazole (PS−HOBt) active esters as intermediates. This methodology allowed the rapid development of an improved protocol for the parallel synthesis of amides by conversion of carboxylic acids to PS−HOBt esters followed by treatment with appropriate amines. Product isolation involved only simple filtration and evaporation.

The application of a new automated workstation for conducting solution-phase reactions in parallel during chemical process development is described. The fully automated reactor−analysis system is capable of reagent/reactant addition, temperature and agitation control, and sample extraction/quench/injection for online HPLC analysis. Both precision and accuracy of the fluidic delivery and sampling systems were measured through the use of standard ultraviolet chromophore solutions in conjunction with HPLC analysis. Agitation efficiency was evaluated using a phase-transfer catalysis reaction known to be rate-dependent on stirring frequency. Chemistry cases examined included several single and multistep reactions studied to identify the best reagent/solvent combinations (screening) and optimal levels of continuous variables such as temperature, concentration, and stoichiometry (optimization). A multistep route to a chiral substituted imidazolidin-2-one was developed from initial route scouting through reaction condition screening, optimization, and scale-up. Reactions performed included alkylation, reduction, oxidation, reductive amination, displacement, and cyclization.

The yeast poly(A) binding protein Pab1p mediates the interactions between the 5' cap structure and the 3' poly(A) tail of mRNA, whose structures synergistically activate translation in vivo and in vitro. We found that deletion of the PAT1 (YCR077c) gene suppresses a PAB1 gene deletion and that Pat1p is required for the normal initiation of translation. A fraction of Pat1p cosediments with free 40S ribosomal subunits on sucrose gradients. The PAT1 gene is not essential for viability, although disruption of the gene severely impairs translation initiation in vivo, resulting in the accumulation of 80S ribosomes and in a large decrease in the amounts of heavier polysomes. Pat1p contributes to the efficiency of translation in a yeast cell-free system. However, the synergy between the cap structure and the poly(A) tail is maintained in vitro in the absence of Pat1p. Analysis of translation initiation intermediates on gradients indicates that Pat1p acts at a step before or during the recruitment of the 40S ribosomal subunit by the mRNA, a step which may be independent of that involving Pab1p. We conclude that Pat1p is a new factor involved in protein synthesis and that Pat1p might be required for promoting the formation or the stabilization of the preinitiation translation complexes.

Detergent-solubilized plasma membranes from Bacilus subtilis have been characterized for their cytochrome oxidase content. Triton X-100-solubilized membranes show high O2 turnover with ascorbate plus TMPD. Reduced-oxidized difference spectroscopy of ascorbate-TMPD-reduced membranes reveals the presence of cytochrome c and cytochrome a. An additional, b-type cytochrome appears when the membranes are reduced with dithionite. Time-resolved difference spectra taken during reduction by ascorbate-TMPD reveal two kinetic forms of heme A-containing cytochromes. There is a high-turnover form that is rapidly reduced upon anaerobiosis, and a second type which is only slowly reduced upon anaerobiosis. The slowly reduced oxidase is distinguished by an α-band blue-shifted to 600 nm relative to the 603-nm position observed for high-turnover oxidase. Addition of CO to ascorbate-TMPD-reduced membranes gives a spectrum typical of ferrocytochrome a₃-CO, and the intensity corresponds to the total ferrocytochrome a₃ concentration. Photolysis of ascorbate-TMPD-reduced, CObound membranes indicates that both species are photosensitive with similar rates of recombination. Addition of CO to dithionite-reduced membranes shows an additional CO reactive center that has a spectrum characteristic of cytochrome o. Cyanide blocks complete reduction of high-turnover oxidase by ascorbate plus TMPD, but does not appear to effect slowly reduced oxidase. These results indicate the presence of two different types of cytochrome aa₃ oxidase in plasma membranes of B. subtilis.Copyright 1993, 1999 Academic Press

Fibre optic confocal imaging (FOCI) enabled subsurface fluorescence microscopy of the skin of hairless mice in vivo. Application of acridine orange enabled imaging of the layers of the epidermis. The corneocytes of the stratum corneum, the keratinocytes in the basal layers and redundant hair follicles were visualised at depths greater than 100 μm. Cellular and nuclear membranes of keratinocytes of the skin were visualised by the use of acridine orange and DIOC5(3). Imaging of the skin after injection of FITC‐dextran revealed an extensive network of blood vessels with a size range up to 20 μm. Blood cells could be seen moving through dermal vessels and the blood circulation through the dermal vascular bed was video‐taped. The fluorescent dye 4‐di‐2‐ASP showed the presence of nerves fibres around the hair follicles and subsurface blood vessels. Comparison was made between images obtained in vivo using FOCI and in vitro scanning electron microscopy and conventional histology. FOCI offers the potential to study dynamic events in vivo, such as blood flow, skin growth, nerve regeneration and many pathological processes, in ways which have not previously been possible.

A fluorescence confocal microscopy technique was employed to obtain subsurface images of nerve and microvascular structure in the vas deferens and colon of the living rat. The use of dual labelling with vital dyes and 2‐channel confocal acquisition allowed differentiation of microscopic structure at both low and higher magnification. Characteristic staining patterns of nerves and blood vessels were repeatedly obtained in each tissue, suggesting the potential of this technique for studying morphological changes associated with surgical procedures and/or models of neuronal or vascular pathology.

A series of molecular biology experiments were carried out to identify the catalytic domain of two human alpha1,3/4-fucosyltransferases (fucosyltransferases (FucTs) III and V), and to identify amino acids that function in acceptor substrate binding. Sixty-one and 75 amino acids could be eliminated from the N terminus of FucTs III and V, respectively, without a significant loss of enzyme activity. In contrast, the truncation of one or more amino acids from the C terminus of FucT V resulted in a dramatic or total loss of enzyme activity. Results from the truncation experiments demonstrate that FucT III62-361 (containing amino acids 62-361) and FucT V76-374 (containing amino acids 76-374) are active, whereas shorter forms of the enzymes were inactive. The shortest, active forms of the enzymes are more than 93% identical at the predicted amino acid level, but have distinct acceptor substrate specificities. Thus, FucT III is an alpha1,4-fucosyltransferase, whereas FucT V is an alpha1,3-fucosyltransferase with disaccharide substrates. All but one of the amino acid sequence differences between the two proteins occur near their N terminus. Results obtained from domain swapping experiments demonstrated that the single amino acid sequence difference near the C terminus of these enzymes did not alter the enzyme's substrate specificity. However, swapping a region near the N terminus of the truncated form of FucT III into an homologous region in FucT V produced a protein with both alpha1,3- and alpha1,4-fucosyltransferase activity. This region contains 8 of the amino acid sequence differences that occur between the two proteins.

An analysis of the acceptor substrate specificity of domain swap mutants of human alpha1,3/4-fucosyltransferases (FucTs) III and V has been carried out. The results demonstrate that changing Asp336 of FucT III to Ala (as in FucT V) produced a protein (III/V1) with a reduced activity with a variety of acceptors. An analysis of the kinetic properties of FucT III and the III/V1 mutant demonstrated that III/V1 had a 40-fold reduction in its affinity for the H-type 1 acceptor substrate (Fucalpha1,2Galbeta1,3GlcNAc) and 4-fold reduction in its affinity for GDP-fucose when compared with FucT III. Further, the overall catalytic efficiency of III/V1 was approximately 100-fold lower than that of FucT III with an H-type 1 acceptor substrate. The complementary domain swap resulting from the change of Ala349 of FucT V to Asp (V/III1) produced a FucT that had higher enzyme activity with a range of acceptor substrates and had a higher affinity for an H-type 2 acceptor substrate (Fucalpha1, 2Galbeta1,4GlcNAc) with an 8-fold higher overall catalytic efficiency than that of FucT V. No significant change occurred in the Km for GDP-fucose for this protein when compared with FucT V. Kinetic parameters of two other FucT domain swaps (III8/V and V8/III), resulting in proteins that differed from FucT III and V at the NH2 terminus of their catalytic domain, were not significantly different from those of the parental enzymes when H-type 1 and H-type 2 acceptor substrates were utilized. Thus, substitution of an acidic amino acid for a nonpolar amino acid (i.e. Asp versus Ala) at the COOH terminus of FucTs produces an enzyme with enhanced enzyme activities. These results, together with the results presented in the accompanying papers (Nguyen, A. T., Holmes, E. H., Whitaker, J. M., Ho, S., Shetterly, S., and Macher, B. A. (1998) J. Biol. Chem. 273, 25244-25249; Sherwood, A. L., Nguyen, A. T., Whitaker, J. M., Macher, B. A., and Holmes, E. H. (1998) J. Biol. Chem. 273, 25256-25260), provide new insights into the structure/function relationships of human alpha1,3/4-FucT enzymes.

We have isolated two phenotypically distinct nonfastidious Francisella strains (Fx1 and Fx2) from the blood of compromised patients with pneumonia and compared them with eight other Francisella strains, including Francisella tularensis biovar tularensis, F. tularensis biovar novicida, and F. philomiragia. Our isolates grew well on sheep blood agar, chocolate agar, modified Thayer-Martin agar, and Trypticase soy agar. Fx1 and Fx2 were determined to be within the Francisella genus by cellular fatty acid analysis and by the utilization of glucose, production of H2S and catalase, and lack of motility, oxidase, nitrate reductase, and gelatinase. They were additionally shown to belong to the species F. tularensis by sequencing of two variable regions comprising approximately 500 nucleotides of the 16S rRNA gene. Also, RNA probe hybridization confirmed their belonging to the species F. tularensis. However, the new strains, which are not identical, are distinguished from other F. tularensis strains by growth characteristics, repetitive extragenic palindromic PCR fragment pattern, and some biochemical tests. Key biochemical differences included the findings that Fx1 was positive for beta-galactosidase and arabinose hydrolysis and that both strains were citrulline ureidase positive and glycerol negative. Commercial F. tularensis antiserum agglutinated stock F. tularensis strains but not Fx1, Fx2, F. tularensis biovar novicida, or F. philomiragia; serum from either patient failed to agglutinate or only weakly agglutinated commercial antigen but showed agglutination when tested against each patient's respective isolate. Fx1 and Fx2 produced beta-lactamase. Because of their good growth, negative serology, and biochemical profile, the organisms could be misidentified in the clinical laboratory if standard strategies or commercial identification systems are used.

When grown in aerated shaking culture, Bacillus subtilis expresses two different haem A-containing terminal oxidases: cytochrome aa3-quinol oxidase and cytochrome caa3 oxidase. This paper describes a high-yield conventional procedure for purifying the two haem A-containing oxidases from the same aerobic culture of Bacillus subtilis. Yields of close to 40% of the total haem A are achieved and about 6 mg of each of the purified oxidases is obtained from 4 litres of liquid culture. Both of the purified enzymes have two subunits, with apparent molecular masses of 71.6 kDa and 34.3 kDa for the cytochrome caa3 oxidase, and 67.6 kDa and 37.2 kDa for aa3-quinol oxidase. These features are in agreement with the sequence data for the corresponding structural genes in the aa3 and caa3 operons of B. subtilis. Some spectral and enzymic features of the two purified oxidases are reported that are consistent with the inclusion of both of these enzymes as members of the cytochrome oxidase superfamily.

Experiments were carried out to investigate whether the Soai asymmetric autocatalysis can accomplish true absolute asymmetric synthesis. In 54 reactions, R and S enantiomeric products were obtained 27 times each. Of 25 pairs of side-by-side identical reactions, 12 afforded opposite enantiomers. In a test of the mechanistic viability of a random-chance process, it was found that a very few molecules (approximately 60 000) were sufficient to control the enantiomeric outcome of these reactions. These observations appear most consistent with asymmetric synthesis originating from the chance enantiomeric excess in a racemate.