Chronic ischemic mitral regurgitation (CIMR) is poorly understood and repair operations are often unsatisfactory. This study elucidates the mechanism of CIMR in an ovine model.
Sonomicrometry array ...localization measured the three-dimensional geometry of the mitral annulus and subvalvular apparatus in five sheep before and 8 weeks after a posterior infarction of the left ventricle that produced progressive severe CIMR.
End systolic annular area increased from 647 ± 44 mm
2 to 1,094 ± 173 mm
2 (
p = 0.01). Annular dilatation occurred equally along the anterior (47.0 ± 5.6 mm to 60.2 ± 4.9 mm,
p = 0.001) and posterior (53.8 ± 3.1 mm to 68.5 ± 8.4 mm,
p = 0.005) portions of the annulus. The tip of the anterior papillary muscle moved away from both the anterior and posterior commissures by 5.2 ± 3.2 mm (
p = 0.021) and 7.3 ± 2.2 mm (
p = 0.002), respectively. The distance from the tip of the posterior papillary muscle to the anterior commissure increased by 11.0 ± 5.7 mm (
p = 0.032) while the distance from the tip of the posterior papillary muscle to the posterior commissure remained constant.
Progressive dilatation of both the anterior and posterior mitral annuli, increased annular area, and asymmetric ventricular dilatation combine to cause CIMR by distortion of mitral valve geometry and tethering of leaflet coaptation. Therefore complete ring annuloplasty may be superior to partial annuloplasty in the treatment of CIMR.
Mycobacterium brisbanense strain JK1, a bacterium capable of degrading the herbicide diuron, was isolated from herbicide-exposed soil. A gene/enzyme system with diuron hydrolase activity was isolated ...from this strain and named PUH (phenylurea hydrolase) B (puhB/PuhB) because of its close similarity to the previously characterized PUH A (puhA/PuhA). Both PUHs were heterologously expressed, purified and characterized. The PUHs were found to oligomerize as hexamers in solution, with each monomer containing a mononuclear Zn2+ active site. Sequence analysis showed that these enzymes belong to the metal-dependent amidohydrolase superfamily, although they contain a hitherto unreported Asn-X-His metal-binding motif and appear to form a novel sub-group within this superfamily. The effects of temperature and solvent on the enzymes were characterized. Determination of the kinetic parameters of the PUHs was used alongside Brønsted plots to develop a plausible catalytic mechanism, which is similar to that used by urease. In addition to the primary PUH activity, both enzymes are catalytically promiscuous, efficiently hydrolysing esters, carbamates and phosphotriesters. In fact, an analogue of diuron, in which the C-N bond was replaced by a C-O bond, was found to be turned over as efficiently as diuron, suggesting that the substrate specificity is predominantly determined by steric factors. The discovery of PuhA and PuhB on separate continents, and the absence of any other close homologues in the available sequence databases, poses a challenging question regarding the evolutionary origins of these enzymes.
Abstract
Objectives
To improve clinical decision support (CDS) by allowing users to provide real-time feedback when they interact with CDS tools and by creating processes for responding to and ...acting on this feedback.
Methods
Two organizations implemented similar real-time feedback tools and processes in their electronic health record and gathered data over a 30-month period. At both sites, users could provide feedback by using Likert feedback links embedded in all end-user facing alerts, with results stored outside the electronic health record, and provide feedback as a comment when they overrode an alert. Both systems are monitored daily by clinical informatics teams.
Results
The two sites received 2,639 Likert feedback comments and 623,270 override comments over a 30-month period. Through four case studies, we describe our use of end-user feedback to rapidly respond to build errors, as well as identifying inaccurate knowledge management, user-interface issues, and unique workflows.
Conclusion
Feedback on CDS tools can be solicited in multiple ways, and it contains valuable and actionable suggestions to improve CDS alerts. Additionally, end users appreciate knowing their feedback is being received and may also make other suggestions to improve the electronic health record. Incorporation of end-user feedback into CDS monitoring, evaluation, and remediation is a way to improve CDS.
The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether ...the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged 4-nitrophenolate product of paraoxon hydrolysis. The 8−11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.
F
is a microbial cofactor that mediates a wide range of physiologically important and industrially relevant redox reactions, including in methanogenesis and tetracycline biosynthesis. This ...deazaflavin comprises a redox-active isoalloxazine headgroup conjugated to a lactyloligoglutamyl tail. Here we studied the catalytic significance of the oligoglutamate chain, which differs in length between bacteria and archaea. We purified short-chain F
(two glutamates) from a methanogen isolate and long-chain F
(five to eight glutamates) from a recombinant mycobacterium, confirming their different chain lengths by HPLC and LC/MS analysis. F
purified from both sources was catalytically compatible with purified enzymes from the three major bacterial families of F
-dependent oxidoreductases. However, long-chain F
bound to these enzymes with a six- to ten-fold higher affinity than short-chain F
. The cofactor side chain also significantly modulated the kinetics of the enzymes, with long-chain F
increasing the substrate affinity (lower
) but reducing the turnover rate (lower
) of the enzymes. Molecular dynamics simulations and comparative structural analysis suggest that the oligoglutamate chain of F
makes dynamic electrostatic interactions with conserved surface residues of the oxidoreductases while the headgroup binds the catalytic site. In conjunction with the kinetic data, this suggests that electrostatic interactions made by the oligoglutamate tail result in higher-affinity, lower-turnover catalysis. Physiologically, we propose that bacteria have selected for long-chain F
to better control cellular redox reactions despite tradeoffs in catalytic rate. Conversely, this suggests that industrial use of shorter-length F
will greatly increase the rates of bioremediation and biocatalysis processes relying on purified F
-dependent oxidoreductases.
Understanding bottom-up, top-down, and abiotic factors along with interactions that may influence additive or compensatory effects of predation on ungulate population growth has become increasingly ...important as carnivore assemblages, land management policies, and climate variability change across western North America. Recruitment and population trends of elk (Cervus canadensis) have been downward in the last 4 decades across the northern Rocky Mountains and Pacific Northwest, USA. In Oregon, changes in vegetation composition and land use practices occurred, cougar (Puma concolor) populations recovered from near-extirpation, and black bear (Ursus americanus) populations increased. Our goal was to provide managers with insight into the influence of annual climatic variation, and bottom-up and top-down factors affecting recruitment of elk in Oregon. We conducted our research in southwestern (SW; Toketee and Steamboat) and northeastern (NE; Wenaha and Sled Springs) Oregon, which had similar predator assemblages but differed in patterns of juvenile recruitment, climate, cougar densities, and vegetative characteristics.
We obtained monthly temperature and precipitation measures from Parameter-elevation Regressions on Independent Slopes Model (PRISM) and estimates of normalized difference vegetation index (NDVI) for each study area to assess effects of climate and vegetation growth on elk vital rates. To evaluate the nutritional status of elk in each study area, we captured, aged, and radio-collared adult female elk in SW (n = 69) in 2002–2005 and NE (n = 113) in 2001–2007. We repeatedly captured these elk in autumn (n = 232) and spring (n = 404) and measured ingesta-free body fat (IFBF), mass, and pregnancy and lactation status. We fitted pregnant elk with vaginal implant transmitters (VITs) in spring and captured their neonates in SW (n = 46) and NE (n = 100). We placed expandable radio-collars on these plus an additional 110 neonates in SW and 360 neonates in NE captured by hand or net-gunning via helicopter and estimated their age at capture, birth mass from mass at capture, and sex. We monitored their fates and documented causes of mortality until 1 year of age. We estimated density of cougars by population reconstruction of captured (n = 96) and unmarked cougars killed (n = 27) and of black bears from DNA analysis of hair collected from snares.
We found evidence in lactating females of nutritional limitations on all 4 study areas where IFBFautumn was below 12%, a threshold above which there are few nutritional limitations (9.8% SE = 0.64%, n = 17 at Toketee, 7.9% SE = 0.78%, n = 17 at Steamboat, 7.3% SE = 0.33%, n = 46 at Sled Springs, and 8.9% SE = 0.51%, n = 23 at Wenaha). In spring, of females known to have been lactating the previous autumn, 48% (SE = 3.3%, n = 56) had IFBFspring <2%, a level indicating severe nutritional limitations, compared to 20% (SE = 1.7%, n = 91) of those not lactating the previous autumn. These low levels of IFBFspring of lactating females likely resulted from a carry-over effect of inadequate nutrition during summer and early autumn. We found a positive relationship between summer precipitation and IFBFautumn in NE, and that IFBFautumn of pregnant females was inversely related to birth date of their neonates the following spring (F
1, 52 = 20.37, P < 0.001, R²adj = 0.27). Mean pregnancy rates of lactating females were below 0.90, a threshold indicating nutritional limitations, at Toketee (0.67, SE = 0.12, n = 15), Wenaha (0.70, SE = 0.10, n = 23), and Sled Springs (0.87, SE = 0.05, n = 47) but not Steamboat (0.93, SE = 0.07, n = 14). Of elk where we sampled femur fat during winter in NE, we saw evidence of imminent starvation in 3 of 21 juveniles (12%) with all 3 killed by cougars, and 2 of 12 adult elk (17%) that both died from non-predation events. Birth mass was <13 kg for 6.5% and 2% of VIT neonates in SW and NE, respectively, a mass associated with reduced probability of survival in previous studies. Birth mass of VIT neonates was greater in Sled Springs (x̄ = 18.3 kg, SD = 2.5, n = 59) than Steamboat (x̄ = 16.3 kg, SD = 2.1, n = 21) or Toketee (x̄ = 16.1 kg, SD = 2.8, n = 24) but not Wenaha (x̄ = 17.1 kg, SD = 2.8, n = 36; F
3, 132 = 7.63, P < 0.001). Median and mean birth date (29 May) for VIT neonates did not differ between regions (F
1, 136 = 0.33, P = 0.56), but NE had greater variation around the mean, indicating a longer parturition interval. We documented 293 mortalities of juveniles across study areas and years, and predation was the proximate cause of mortality in 262 cases primarily from cougar (n = 203), black bear (n = 34), and other or unknown predation (n = 25). We also documented causes of mortality as unknown (n = 16), human-caused (n = 8), and disease or starvation (n = 7). We recorded abandonment of 2 (1.4%) and predation mortality of 4 (2.7%) VIT neonates prior to being collared. We found 4-fold differences between regions of subadult female and adult cougar densities (0.90–4.29/100 km²) and 2-fold differences within study areas across years, with cougar density lower in SW than NE. Black bear densities varied from 15–20/100 km² across our study areas.
We estimated survival of neonates to 30 days, 16 weeks, and 12 months using known fates models in Program MARK. Survival of neonates born to females with VITs was associated with cougar density, IFBFspring, and female mass but not female age or neonate birth date or birth mass. Survival was higher for juveniles born to females with lower IFBF and mass in spring, opposite of what we predicted. In a post hoc analysis, we found females successful in raising their neonate to recruitment were more likely to be successful the following year compared to those not successful the previous year, which may explain this unexpected finding. As cougar density increased, survival of juveniles born to females of known nutritional condition declined.
We conducted separate analyses of survival by region for all neonates captured to evaluate effects of climate, bottom-up (but not maternal condition), and top-down factors. In NE, juvenile survival was little affected by annual variation in climate but decreased as cougar densities increased and as birth date became later. For SW, survival was higher with less April–May precipitation and for later born neonates but less affected by cougar density than observed in NE. Across our 4 study areas, survival varied annually from 0.61 (SE = 0.08) to 1.00 during the first 30 days, 0.41 (SE = 0.11) to 0.90 (SE = 0.09) the first 16 weeks, and 0.18 (SE = 0.06) to 0.57 (SE = 0.11) through 12 months (recruitment) with survival higher in SW than NE. Survival of juvenile elk was inversely related to cougar density through 30 days (F
1, 18 = 16.59, R²adj = 0.45, P < 0.001), 16 weeks (F
1, 18 = 21.07, R²adj = 0.51, P < 0.001), and 12 months (F
1, 11 = 18.94, R²adj = 0.60, P = 0.001). We found that as rates of cougar-specific mortality increased, juvenile survival declined (β̂ = −0.63, 95% CI = −0.84 to −0.42) suggesting cougar predation was partially additive mortality because the estimated regression coefficient was significantly less than 0 but greater than −1. We did not observe a similar relationship with rates of black bear-specific mortality because the estimated regression coefficient overlapped 0, suggesting predation by black bears on juvenile elk was compensatory.
Our results suggest that recruitment in NE but not SW was primarily limited by predation from cougars, which was partially additive mortality. Given that we observed nutritional limitations that influenced juvenile survival in all 4 study areas, we were unable to explicitly quantify how much of the cougar predation was additive mortality. Thus, we caution that a reduction in cougar density may not result in an equivalent increase in recruitment, and maintaining or enhancing summer and winter ranges of elk in our study areas is also vitally important for sustaining populations and distributions. In SW, where cougar densities were lower, maintaining, and enhancing existing elk habitat may be the only management option to improve recruitment. Given the differences we found between regions monitored, basing management on an incomplete understanding of causative factors affecting elk population dynamics may result in ineffective actions to address low recruitment.
La comprensión de los factores de abajo hacia arriba, de arriba hacia abajo y abióticos, junto con las interacciones que pueden influir en los efectos aditivos o compensatorios de la depredación sobre el crecimiento de la población ungulada, se ha vuelto cada vez más importante como asociaciones de carnívoros, políticas de manejo de la tierra y cambios en la variabilidad del clima en el oeste de América del Norte. El reclutamiento y las tendencias poblacionales de los alces (Cervus canadensis) han disminuido en las últimas 4 décadas en el norte de las Montañas Rocosas del Norte y el Pacífico Noroeste, EE. UU. En Oregón, se produjeron cambios en la composición de la vegetación y en las prácticas de uso de la tierra, las poblaciones de pumas (Puma concolor) se recuperaron de la casi extirpación y aumentaron las poblaciones de osos negros (Ursus americanus). Nuestro objetivo era proporcionar a los gerentes información sobre la influencia de la variación climática anual y los factores de abajo hacia arriba y de arriba hacia abajo que afectan el reclutamiento de alces en Oregón. Llevamos a cabo nuestra investigación en el suroeste (SW, Toketee y Steamboat) y en el noreste (NE, Wenaha y Sled Springs) Oregon, que tenían ensamblajes de depredadores similares pero diferían en los patrones de reclutamiento juvenil, clima, densidades de puma y características vegetativas.
Obtuvimos medidas mensuales de temperatura y precipitación a partir de Regresiones de Elevación de Parámetros en el Modelo de Pendientes Independientes (PRISM) y estimaciones del Índice de Vegetación de Di
ABSTRACTF420 is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, ...and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study proposed a revised biosynthesis pathway for F420 in mycobacteria; it was suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather than 2-phospholactate as long proposed, but these findings were subsequently challenged. In this work, we combined metabolomic, genetic, and structural analyses to resolve these discrepancies and determine the basis of F420 biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-phospholactate stimulates F420 biosynthesis. Analysis of F420 biosynthesis intermediates present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor compound dehydro-F420-0. To determine the structural basis of dehydro-F420-0 production, we solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo-, substrate-, and product-bound forms. These data show the essential role of a single divalent cation in coordinating the catalytic precomplex of this enzyme and demonstrate that dehydro-F420-0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F420 in mycobacteria and have significant implications for understanding the emergence of antitubercular prodrug resistance.IMPORTANCE Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F420, and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F420 is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F420 in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F420, determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F420.
Here we report specific activities of all seven naturally occurring LinA variants towards three different isomers, α, γ and δ, of a priority persistent pollutant, hexachlorocyclohexane (HCH). ...Sequence-structure-function differences contributing to the differences in their stereospecificity for α-, γ-, and δ-HCH and enantiospecificity for (+)- and (-)-α -HCH are also discussed.
Enzyme kinetic studies were performed with purified LinA variants. Models of LinA2(B90A) A110T, A111C, A110T/A111C and LinA1(B90A) were constructed using the FoldX computer algorithm. Turnover rates (min(-1)) showed that the LinAs exhibited differential substrate affinity amongst the four HCH isomers tested. α-HCH was found to be the most preferred substrate by all LinA's, followed by the γ and then δ isomer.
The kinetic observations suggest that LinA-γ1-7 is the best variant for developing an enzyme-based bioremediation technology for HCH. The majority of the sequence variation in the various linA genes that have been isolated is not neutral, but alters the enantio- and stereoselectivity of the encoded proteins.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Ceramides (CER) are involved in alcohol-induced neuroinflammation. In a mouse model of chronic alcohol exposure, 16 CER and 18 sphingomyelin (SM) concentrations from whole brain lipid extracts were ...measured using electrospray mass spectrometry. All 18 CER concentrations in alcohol exposed adults increased significantly (range: 25–607%); in juveniles, 6 CER decreased (range: −9 to −37%). In contrast, only three SM decreased in adult and one increased significantly in juvenile. Next, regional identification at 50 μm spatial resolution from coronal sections was obtained with matrix implanted laser desorption/ionization mass spectrometry imaging (MILDI-MSI) by implanting silver nanoparticulate matrices followed by focused laser desorption. Most of the CER and SM quantified in whole brain extracts were detected in MILDI images. Coronal sections from three brain levels show qualitative regional changes in CER-SM ion intensities, as a function of group and brain region, in cortex, striatum, accumbens, habenula, and hippocampus. Highly correlated changes in certain white matter CER-SM pairs occur in regions across all groups, including the hippocampus and the lateral (but not medial) cerebellar cortex of adult mice. Our data provide the first microscale MS evidence of regional lipid intensity variations induced by alcohol.
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