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PubMed2004

Carbohydrate chips for studying high-throughput carbohydrate-protein interactions.

Carbohydrate-protein interactions play important biological roles in living organisms. For the most part, biophysical and biochemical methods have been used for studying these biomolecular interactions. Less attention has been given to the development of high-throughput methods to elucidate recognition events between carbohydrates and proteins. In the current effort to develop a novel high-throughput tool for monitoring carbohydrate-protein interactions, we prepared carbohydrate microarrays by immobilizing maleimide-linked carbohydrates on thiol-derivatized glass slides and carried out lectin binding experiments by using these microarrays. The results showed that carbohydrates with different structural features selectively bound to the corresponding lectins with relative binding affinities that correlated with those obtained from solution-based assays. In addition, binding affinities of lectins to carbohydrates were also quantitatively analyzed by determining IC(50) values of soluble carbohydrates with the carbohydrate microarrays. To fabricate carbohydrate chips that contained more diverse carbohydrate probes, solution-phase parallel and enzymatic glycosylations were performed. Three model disaccharides were in parallel synthesized in solution-phase and used as carbohydrate probes for the fabrication of carbohydrate chips. Three enzymatic glycosylations on glass slides were consecutively performed to generate carbohydrate microarrays that contained the complex oligosaccharide, sialyl Le(x). Overall, these works demonstrated that carbohydrate chips could be efficiently prepared by covalent immobilization of maleimide-linked carbohydrates on the thiol-coated glass slides and applied for the high-throughput analyses of carbohydrate-protein interactions.

Carbohydrate Metabolism↗
PubMed2004

Classification of cereal flours by chemometric analysis of MIR spectra.

Different kinds of cereal flours submitted to various technological treatments were classified on the basis of their mid-infrared spectra by pattern recognition techniques. Classification in the wavelet domain was achieved by using the wavelet packet transform for efficient pattern recognition (WPTER) algorithm, which allowed singling out the most discriminant spectral regions. Principal component analysis (PCA) on the selected features showed an effective clustering of the analyzed flours. Satisfactory classification models were obtained both on training and test samples. Furthermore, mixtures of varying composition of the studied flours were distributed in the PCA space according to their composition.

Algorithms↗
PubMed1999

Alkyl Substituent Effects on Pipecolyl Amide Isomer Equilibrium: Efficient Methodology for Synthesizing Enantiopure 6-Alkylpipecolic Acids and Conformational Analysis of Their N-Acetyl N'-Methylamides.

Enantiopure 6-alkylpipecolic acid hydrochlorides 1a-e were synthesized in five steps and 15-59% overall yields from alpha-tert-butyl beta-methyl N-(PhF)aspartate (3) via an approach featuring selective hydride reduction to the corresponding aspartate beta-aldehyde 2, aldol condensations with the enolates of various methyl alkyl ketones, and diastereoselective intramolecular reductive aminations. The influence of the 6-position substituent on the equilibrium and the energy barrier for isomerization of the amide N-terminal to pipecolate was then explored via the synthesis of N-acetyl N'-methylpipecolinamide (16) and its (2S,6R)-6-tert-butylpipecolinamide counterpart 17, and their conformational analysis by proton NMR spectroscopy and coalescence experiments. The presence of the tert-butyl substituent augmented the population of the amide cis-isomer and lowered the barrier for pipecolyl amide isomerization in water. Compared with the results from our previous examination of N-acetyl-5-tert-butylproline N'-methylamides (Beausoleil, E.; Lubell, W. D. J. Am. Chem. Soc. 1996, 118, 12902), the consequences of the bulky 6-alkyl substituent on the acetamide geometry and isomerization barrier were less pronounced in the pipecolate series relative to the respective proline amides.

Journal Article↗
PubMed2006

Insight into selected reactions in low-temperature dimethyl ether combustion from Born-Oppenheimer molecular dynamics.

Dimethyl ether is under consideration as an alternative diesel fuel. Its combustion chemistry is as yet ill-characterized. Here we use Born-Oppenheimer molecular dynamics (BOMD) based on DFT-B3LYP forces to investigate the short-time dynamics of selected features of the low-temperature dimethyl ether (DME) oxidation potential energy surface. Along the chain propagation pathway, we run BOMD simulations from the transition state involving the decomposition of (*)CH(2)OCH(2)OOH to two CH(2)=O and an (*)OH radical. We predict that formaldehyde C-O stretch overtones are excited, consistent with laser photolysis experiments. We also predict that O-H overtones are excited for the (*)OH formed from (*)CH(2)OCH(2)OOH dissociation. We also investigate short-time dynamics involved in chain branching. First, we examine the isomerization transition state of (*)OOCH(2)OCH(2)OOH --> HOOCH(2)OCHOOH. The latter species is predicted to be a short-lived metastable radical that decomposes within 500 fs to hydroperoxymethyl formate (HPMF; HOOCH(2)OC(=O)H) and the first (*)OH of chain branching. The dissociation of HOOCH(2)OCHOOH exhibits non-RRKM behavior in its lifetime profile, which may be due to conformational constraints or slow intramolecular vibrational energy transfer (IVR) from the nascent H-O bond to the opposite end of the radical, where O-O scission occurs to form HPMF and (*)OH. In a few trajectories, we see HOOCH(2)OCHOOH recross back to (*)OOCH(2)OCH(2)OOH because the isomerization is endothermic, with only an 8 kcal/mol barrier to recrossing. Therefore, some inhibition of chain-branching may be due to recrossing. Second, trajectories run from the transition state leading to the direct decomposition of HPMF (an important source of the second (*)OH radical in chain branching) to HCO, (*)OH, and HC(=O)OH show that these products can recombine to form many other possible products. These products include CH(2)OO + HC(=O)OH, H(2)O + CO + HC(=O)OH, HC(=O)OH + HC(=O)OH, and HC(=O)C(=O)H + H(2)O, which (save CH(2)OO + HC(=O)OH) are all more thermodynamically stable than the original HCO + (*)OH + HC(=O)OH products. Moreover, the multitude of extra products suggest that standard statistical rate theories cannot completely describe the reaction kinetics of significantly oxygenated compounds such as HPMF. These secondary products consume the second (*)OH required for explosive combustion, suggesting an inhibition of DME fuel combustion is likely.

Journal Article↗
PubMed2003

Total syntheses of bioactive oxidized ethanolamine phospholipids.

[reaction: see text] Truncated ethanolamine phospholipids containing aldehyde functionality, e.g. OVPE, and the corresponding acids, are generated by oxidative cleavage of polyunsaturated phospholipids. To confirm their identities and facilitate studies of the chemistry and biological actions of these analogues of biologically active phosphatidylcholines, e.g. OVPC, total syntheses were developed. An efficient general strategy was used that features selective N-protection of 2-lysophosphatidylethanolamine, and generation of the target compounds by mild deprotection of stable precursors.

Ethanolamines↗
PubMedDate not supplied

Correcting common errors in identifying cancer-specific serum peptide signatures.

"Molecular signatures" are the qualitative and quantitative patterns of groups of biomolecules (e.g., mRNA, proteins, peptides, or metabolites) in a cell, tissue, biological fluid, or an entire organism. To apply this concept to biomarker discovery, the measurements should ideally be noninvasive and performed in a single read-out. We have therefore developed a peptidomics platform that couples magnetics-based, automated solid-phase extraction of small peptides with a high-resolution MALDI-TOF mass spectrometric readout (Villanueva, J.; Philip, J.; Entenberg, D.; Chaparro, C. A.; Tanwar, M. K.; Holland, E. C.; Tempst, P. Anal. Chem. 2004, 76, 1560-1570). Since hundreds of peptides can be detected in microliter volumes of serum, it allows to search for disease signatures, for instance in the presence of cancer. We have now evaluated, optimized, and standardized a number of clinical and analytical chemistry variables that are major sources of bias; ranging from blood collection and clotting, to serum storage and handling, automated peptide extraction, crystallization, spectral acquisition, and signal processing. In addition, proper alignment of spectra and user-friendly visualization tools are essential for meaningful, certifiable data mining. We introduce a minimal entropy algorithm, "Entropycal", that simplifies alignment and subsequent statistical analysis and increases the percentage of the highly distinguishing spectral information being retained after feature selection of the datasets. Using the improved analytical platform and tools, and a commercial statistics program, we found that sera from thyroid cancer patients can be distinguished from healthy controls based on an array of 98 discriminant peptides. With adequate technological and computational methods in place, and using rigorously standardized conditions, potential sources of patient related bias (e.g., gender, age, genetics, environmental, dietary, and other factors) may now be addressed.

Algorithms↗
PubMed2003

Development of binary classification of structural chromosome aberrations for a diverse set of organic compounds from molecular structure.

Classification models are generated to predict in vitro cytogenetic results for a diverse set of 383 organic compounds. Both k-nearest neighbor and support vector machine models are developed. They are based on calculated molecular structure descriptors. Endpoints used are the labels clastogenic or nonclastogenic according to an in vitro chromosomal aberration assay with Chinese hamster lung cells. Compounds that were tested with both a 24 and 48 h exposure are included. Each compound is represented by calculated molecular structure descriptors encoding the topological, electronic, geometrical, or polar surface area aspects of the structure. Subsets of informative descriptors are identified with genetic algorithm feature selection coupled to the appropriate classification algorithm. The overall classification success rate for a k-nearest neighbor classifier built with just six topological descriptors is 81.2% for the training set and 86.5% for an external prediction set. The overall classification success rate for a three-descriptor support vector machine model is 99.7% for the training set, 92.1% for the cross-validation set, and 83.8% for an external prediction set.

Algorithms↗
PubMed2006

Classification of a diverse set of Tetrahymena pyriformis toxicity chemical compounds from molecular descriptors by statistical learning methods.

Toxicity of various compounds has been measured in many studies by their toxic effects against Tetrahymena pyriformis. Efforts have also been made to use computational quantitative structure-activity relationship (QSAR) and statistical learning methods (SLMs) for predicting Tetrahymena pyriformis toxicity (TPT) at impressive accuracies. Because of the diversity of compounds and toxicity mechanisms, it is desirable to explore additional methods and to examine if these methods are applicable to more diverse sets of compounds. We tested several SLMs (logistic regression, C4.5 decision tree, k-nearest neighbor, probabilistic neural network, support vector machines) for their capability in predicting TPT by using 1129 compounds (841 TPT and 288 non-TPT agents) which are more diverse than those in other studies. A feature selection method was used for improving prediction performance and selecting molecular descriptors responsible for distinguishing TPT and non-TPT agents. The prediction accuracies are 86.9% approximately 94.2% for TPT and 71.2% approximately 87.5% for non-TPT agents based on 5-fold cross-validation studies, which are comparable to some of earlier studies despite the use of more diverse sets of compounds. The selected molecular descriptors are consistent with those used in other studies and experimental findings. These suggest that SLMs are useful for predicting TPT potential of diverse sets of compounds and for characterizing the molecular descriptors associated with TPT.

Animals↗
PubMed2006

Computational prediction of the chromosome-damaging potential of chemicals.

We report on the generation of computer-based models for the prediction of the chromosome-damaging potential of chemicals as assessed in the in vitro chromosome aberration (CA) test. On the basis of publicly available CA-test results of more than 650 chemical substances, half of which are drug-like compounds, we generated two different computational models. The first model was realized using the (Q)SAR tool MCASE. Results obtained with this model indicate a limited performance (53%) for the assessment of a chromosome-damaging potential (sensitivity), whereas CA-test negative compounds were correctly predicted with a specificity of 75%. The low sensitivity of this model might be explained by the fact that the underlying 2D-structural descriptors only describe part of the molecular mechanism leading to the induction of chromosome aberrations, that is, direct drug-DNA interactions. The second model was constructed with a more sophisticated machine learning approach and generated a classification model based on 14 molecular descriptors, which were obtained after feature selection. The performance of this model was superior to the MCASE model, primarily because of an improved sensitivity, suggesting that the more complex molecular descriptors in combination with statistical learning approaches are better suited to model the complex nature of mechanisms leading to a positive effect in the CA-test. An analysis of misclassified pharmaceuticals by this model showed that a large part of the false-negative predicted compounds were uniquely positive in the CA-test but lacked a genotoxic potential in other mutagenicity tests of the regulatory testing battery, suggesting that biologically nonsignificant mechanisms could be responsible for the observed positive CA-test result. Since such mechanisms are not amenable to modeling approaches it is suggested that a positive prediction made by the model reflects a biologically significant genotoxic potential. An integration of the machine-learning model as a screening tool in early discovery phases of drug development is proposed.

Chromosomes↗
PubMed1999

A multimethod exploration of the friendships of children considered socially withdrawn by their school peers.

Friendships of children considered socially withdrawn by their school peers were investigated within a population of elementary school children. Reciprocal friends were identified by a friendship nomination procedure; social withdrawal was assessed by peer nominations. Trained graduate students rated videotapes of dyads of friends (n = 58 dyads, of which 29 contained at least one withdrawn child) for selected features of friendship quality. In addition, each friend within a dyad provided ratings of the quality of the relationship. The videotaped data showed the withdrawn children to be somewhat restricted in their verbal communication with their friends, and less competitive with their friends, than were friends in a comparison group. In dyads consisting of one withdrawn and one nonwithdrawn child, the withdrawn child perceived the relationship as characterized by greater closeness and helpfulness than did the nonwithdrawn friend. Despite some signs of inhibited behavior within the friendship context, withdrawn children seem to have access to close friendships of high quality.

Child↗
PubMed2005

Encoding social signals in the mouse main olfactory bulb.

Mammalian urine releases complex mixtures of volatile compounds that are used in reproduction, territoriality and conspecific recognition. To understand how such complex mixtures are represented in the main olfactory bulb, we analysed the electrophysiological responses of individual mitral cells to volatile compounds in mouse urine. In both males and females, urine volatile compounds evoke robust responses in a small subset of mitral cells. Fractionation of the volatile compounds using gas chromatography showed that out of the hundreds of compounds present, mitral cells are activated by single compounds. One cohort of mitral cells responded exclusively to male urine; these neurons were activated by (methylthio)methanethiol, a potent, previously unknown semiochemical present only in male urine. When added to urine, synthetic (methylthio)methanethiol significantly enhances urine attractiveness to female mice. We conclude that mitral cells represent natural odorant stimuli by acting as selective feature detectors, and that their activation is largely independent of the presence of other components in the olfactory stimulus.

Animals↗
PubMed2005

Working memory in primate sensory systems.

Sensory working memory consists of the short-term storage of sensory stimuli to guide behaviour. There is increasing evidence that elemental sensory dimensions - such as object motion in the visual system or the frequency of a sound in the auditory system - are stored by segregated feature-selective systems that include not only the prefrontal and parietal cortex, but also areas of sensory cortex that carry out relatively early stages of processing. These circuits seem to have a dual function: precise sensory encoding and short-term storage of this information. New results provide insights into how activity in these circuits represents the remembered sensory stimuli.

Animals↗
PubMed2006

Hypoxia-ischemia preferentially triggers glutamate depletion from oligodendroglia and axons in perinatal cerebral white matter.

Ischemia is implicated in periventricular white matter injury (PWMI), a lesion associated with cerebral palsy. PWMI features selective damage to early cells of the oligodendrocyte lineage, a phenomenon associated with glutamate receptor activation. We have investigated the distribution of glutamate in rat periventricular white matter at post-natal day 7. Immuno-electron microcopy was used to identify O4(+) oligodendroglia in control rats, and a similar approach was employed to stain glutamate in these cells before and after 90 mins of hypoxia-ischemia. This relatively brief period of hypoxia-ischemia produced mild cell injury, corresponding to the early stages of PWMI. Glutamate-like reactivity was higher in oligodendrocytes than in other cell types (2.13+/-0.25 counts/microm(2)), and declined significantly during hypoxia-ischemia (0.93+/-0.15 counts/microm(2): P<0.001). Astrocytes had lower glutamate levels (0.7+/-0.07 counts/microm(2)), and showed a relatively small decline during hypoxia-ischemia. Axonal regions contained high levels of glutamate (1.84+/-0.20 counts/microm(2)), much of which was lost during hypoxia-ischemia (0.72+/-0.20 counts/microm(2): P>0.001). These findings suggest that oligodendroglia and axons are the major source of extracellular glutamate in developing white matter during hypoxia-ischemia, and that astrocytes fail to accumulate the glutamate lost from these sources. We also examined glutamate levels in the choroid plexus. Control glutamate levels were high in both choroid epithelial (1.90+/-0.20 counts/microm(2)), and ependymal cells (2.20+/-0.28 counts/microm(2)), and hypoxia-ischemia produced a large fall in ependymal glutamate (0.97+/-0.08 counts/microm(2): P>0.001). The ependymal cells were damaged by the insult and represent a further potential source of glutamate during ischemia.

Animals↗
PubMed2001

Modulation of apoptosis by procaspase-2 short isoform: selective inhibition of chromatin condensation, apoptotic body formation and phosphatidylserine externalization.

Procaspase-2 is one of the cysteine aspartate proteases involved in apoptotic cell death. Alternative splicing of CASP-2 messenger RNA generates a long isoform, procaspase-2L, whose overexpression induces cell death and a truncated isoform, procaspase-2S, whose function remains poorly defined. The present study explored the consequences of procaspase-2S overexpression in U937 human leukemic cells exposed to the topoisomerase II inhibitor etoposide as an apoptotic stimulus. Overexpression of procaspase-2S in U937 cells partially prevented nuclear changes associated with etoposide-induced cell death, as determined by Hoechst 33342 staining of nuclear chromatin and electron microscopy studies. Procaspase-2S also prevented the maturation of apoptotic bodies, delayed phosphatidylserine externalization on the plasma membrane and prevented the cleavage and activation of procaspase-2L. These effects were not observed when the cysteine 289 in the consensus QACRG motif was mutated into a serine. Wild-type procaspase-2S overexpression did not influence the cleavage of procaspase-3, procaspase-7 and poly(ADP-ribose)polymerase nor the fragmentation of nuclear DNA into nucleosome-sized fragments. Altogether, these results indicate that the short isoform of procaspase-2 negatively interferes with selective features of apoptosis, an activity that is suppressed by mutation of the cysteine 289.

Amino Acid Motifs↗
PubMed1995

Isolation and characterization of distinct domains of sarcolemma and T-tubules from rat skeletal muscle.

1. Several cell-surface domains of sarcolemma and T-tubule from skeletal-muscle fibre were isolated and characterized. 2. A protocol of subcellular fractionation was set up that involved the sequential low- and high-speed homogenization of rat skeletal muscle followed by KCl washing, Ca2+ loading and sucrose-density-gradient centrifugation. This protocol led to the separation of cell-surface membranes from membranes enriched in sarcoplasmic reticulum and intracellular GLUT4-containing vesicles. 3. Agglutination of cell-surface membranes using wheat-germ agglutinin allowed the isolation of three distinct cell-surface membrane domains: sarcolemmal fraction 1 (SM1), sarcolemmal fraction 2 (SM2) and a T-tubule fraction enriched in protein tt28 and the alpha 2-component of dihydropyridine receptor. 4. Fractions SM1 and SM2 represented distinct sarcolemmal subcompartments based on different compositions of biochemical markers: SM2 was characterized by high levels of beta 1-integrin and dystrophin, and SM1 was enriched in beta 1-integrin but lacked dystrophin. 5. The caveolae-associated molecule caveolin was very abundant in SM1, SM2 and T-tubules, suggesting the presence of caveolae or caveolin-rich domains in these cell-surface membrane domains. In contrast, clathrin heavy chain was abundant in SM1 and T-tubules, but only trace levels were detected in SM2. 6. Immunoadsorption of T-tubule vesicles with antibodies against protein tt28 and against GLUT4 revealed the presence of GLUT4 in T-tubules under basal conditions and it also allowed the identification of two distinct pools of T-tubules showing different contents of tt28 and dihydropyridine receptors. 7. Our data on distribution of clathrin and dystrophin reveal the existence of subcompartments in sarcolemma from muscle fibre, featuring selective mutually exclusive components. T-tubules contain caveolin and clathrin suggesting that they contain caveolin- and clathrin-rich domains. Furthermore, evidence for the heterogeneous distribution of membrane proteins in T-tubules is also presented.

Animals↗
PubMed2003

Aldose reductase and the importance of experimental design.

Kinetic studies on the AR (aldose reductase) protein have shown that it does not behave as a classical enzyme in relation to ring aldose sugars. As with non-enzymatic glycation reactions, there is probably a free-radical element involved derived from monosaccharide autoxidation. In the case of AR, there is free radical oxidation of NADPH by autoxidizing monosaccharides, which is enhanced in the presence of the NADPH-binding protein. Thus any assay for AR based on the oxidation of NADPH in the presence of autoxidizing monosaccharides is invalid, and tissue AR measurements based on this method are also invalid, and should be reassessed. AR exhibits broad specificity for both hydrophilic and hydrophobic aldehydes that suggests that the protein may be involved in detoxification. The last thing we would want to do is to inhibit it. ARIs (AR inhibitors) have a number of actions in the cell which are not specific, and which do not involve them binding to AR. These include peroxy-radical scavenging and effects of metal ion chelation. The AR/ARI story emphasizes the importance of correct experimental design in all biocatalytic experiments. Developing the use of Bayesian utility functions, we have used a systematic method to identify the optimum experimental designs for a number of kinetic model data sets. This has led to the identification of trends between kinetic model types, sets of design rules and the key conclusion that such designs should be based on some prior knowledge of K (m) and/or the kinetic model. We suggest an optimal and iterative method for selecting features of the design such as the substrate range, number of measurements and choice of intermediate points. The final design collects data suitable for accurate modelling and analysis and minimizes the error in the parameters estimated, and is suitable for simple or complex steady-state models.

Aldehyde Reductase↗
PubMed2000

Expression of MUM1/IRF4 selectively clusters with primary effusion lymphoma among lymphomatous effusions: implications for disease histogenesis and pathogenesis.

Primary effusion lymphoma (PEL) is a peculiar B-cell lymphoma characterized by infection by human herpesvirus type-8/Kaposi sarcoma-associated herpesvirus (HHV-8/KSHV) and by preferential growth in the serous body cavities. Histogenetic studies have suggested that PEL originates from B cells at a late stage of differentiation. In this study, we have investigated PEL for the expression status of MUM1/IRF4 (multiple myeloma 1/interferon regulatory factor 4) protein, which is involved in physiological B-cell maturation and represents a histogenetic marker of late B-cell differentiation. Using multiple detection assays, all cases of PEL (n = 22) were found to express MUM1/IRF4 molecules. MUM1/IRF4 expression was a selective feature of PEL among lymphomas involving the serous body cavities as secondary lymphomatous effusions generally failed to express the protein. In reactive lymphoid tissues, MUM1/ IRF4 expression clustered with advanced stages of B-cell differentiation. Comparison of MUM1/IRF4 expression with that of other histogenetic markers defined two phenotypic variants of PEL, i.e. MUM1/IRF4+, CD138/syndecan-1+, B-cell antigen- (20 out of 22 cases) and MUM1/IRF4+, CD138/syndecan-1-, B-cell antigen+ (2 out of 22 cases), suggesting a certain degree of heterogeneity in the disease histogenesis. The implications of these data are threefold. First, MUM1/IRF4 expression corroborates the notion that PEL originates from post-germinal centre, preterminally differentiated B-cells. Second, MUM1/IRF4 may help in the differential diagnosis of PEL among other lymphomas involving the serous body cavities. Finally, MUM1/IRF4 may interact with HHV-8/KSHV-encoded interferon regulatory factors (IRFs) and thus contribute to PEL escape from interferon-mediated control of viral infection.

Antigens, Differentiation, B-Lymphocyte↗
PubMed2003

The randomised controlled trial design: unrecognized opportunities for health sciences librarianship.

OBJECTIVE: to describe the essential components of the Randomised Controlled Trial (RCT) and its major variations; to describe less conventional applications of the RCT design found in the health sciences literature with potential relevance to health sciences librarianship; to discuss the limited number of RCTs within health sciences librarianship. METHODS: narrative review supported to a limited extent with PubMed and Library Literature database searches consistent with specific search parameters. In addition, more systematic methods, including handsearching of specific journals, to identify health sciences librarianship RCTs. RESULTS: While many RCTs within the health sciences follow more conventional patterns, some RCTs assume certain unique features. Selected examples illustrate the adaptations of this experimental design to answering questions of possible relevance to health sciences librarians. The author offers several strategies for controlling bias in library and informatics applications of the RCT and acknowledges the potential of the electronic era in providing many opportunities to utilize the blinding aspects of RCTs. RCTs within health sciences librarianship inhabit a limited number of subject domains such as education. This limited scope offers both advantages and disadvantages for making Evidence-Based Librarianship (EBL) a reality. CONCLUSIONS: The RCT design offers the potential to answer far more EBL questions than have been addressed by the design to date. Librarians need only extend their horizons through use of the versatile RCT design into new subject domains to facilitate making EBL a reality.

Evidence-Based Medicine↗