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Properties and secondary structure analysis of BanI endonuclease: identification of putative active site.

Biochemical properties of Type II restriction enzyme BanI were characterized. Kinetic parameters were evaluated and an enhancement of rate was observed when the recognition site was located in a more central position in the substrate, suggesting that BanI locates its recognition site by a sliding mechanism. As BanI has three cysteine residues in its primary sequence, the effect of thiol inhibitors on BanI activity was also studied. Partial inhibition was observed only at a very high concentration of the inhibitor indicating that cysteine residues are not directly involved in catalysis. The gel electrophoretic mobility shift assay demonstrated specific complex formation between BanI and the DNA substrate in the presence of poly dI-dC and Mg(2+). A secondary structure analysis and comparison with EcoRI and BamHI crystal structure revealed a putative active site similar to that seen in BamHI but different in the order in which the catalytic domain (central beta-sheet) and recognition domain (adjacent alpha-helix) were arranged in the protein.

Base Sequence↗

[Enzyme-linked immunosorbent assay biotin/avidin method standardization, for identification of Lutzomyia (Lutzomyia) longipalpis bloodmeals (Lutz & Neiva, 1912)].

Bloodmeals taken by insects constitute an important parameter for clarifying aspects of the transmission of zoonoses, including leishmaniases. Immunological assays can be used to investigate the attraction of vectors to animals, which may be hosts of these parasitoses. The objective of this study was to standardize a sandwich enzyme-linked immunosorbent assay and titer samples with different time periods of digestion, in laboratory-bred Lutzomyia longipalpis fed on rats. In the light of the high sensitivity that the biotin-avidin method permits, the technique provided at least ninety repeat tests for each sample and identified recent bloodmeals taken by these insects. Bloodmeals were detectable up to 12 and 24 h after blood ingestion, and a significant difference between these titers was observed.

Animals↗

Validity of using slide positivity rate (SPR) in identification of high risk malarious segments in rural areas.

The Expert Committee Report-1995, recommended various parameters for identifying worst malaria affected segments in rural areas, which are mainly based on slide positivity rate (SPR). A analysis of the epidemiological data of Uttar Pradesh was carried out in different settings to study the sensitivity of SPR in identifying high risk areas. The validity of using SPR in the entire range in all settings is examined and appropriate corrective measures suggested.

Blood Specimen Collection↗

[The use of capillaroscopy of the skin of the nail wall in personal identification].

Capillaries of the nail wall skin are the most convenient objects for investigation in a live man. Over 2500 investigations were carried out, which demonstrated, that, despite a marked variability of morphometric parameters, the contour of capillary loops on each subject is individual, this recommending capillaroscopy of the skin as a method for personality identification.

Adolescent↗

A computerized system for localization of diabetic lesions from fundus images.

A computerized system for the localization of diabetic lesions from fundus photographs is described. The coordinates for each lesion in relation to reference points are determined from fundus images projected on a digitizing table. The program accepts some variation in coordinates for a single lesion, still to be recorded as the same lesion in a series of photographs, and the magnitude of this variation is adjustable. The data obtained from the localization program are transferred to a register program yielding the following parameters: number of lesions, new lesions, lost lesions, variable lesions, time of presence for lost lesions. These data can be further exported into Microsoft Excel 4, for analysis of large patient materials. An intra-observer error of 0% and an inter-observer error of 1.1% was found in localization of lesions from fundus images. The program was insensitive to small variations in the fundus pictures, but comparisons of 30 degrees and 60 degrees photographs of the same fundus, or 20 degrees deviation of gaze, induced identification errors in 2.9% and 11.9% of localizations, respectively. With this system new parameters on diabetic fundus images can be obtained. The significance of these parameters in the progression of diabetic retinopathy will be analyzed in further studies.

Aneurysm↗

[Identification of asbestos in bulk materials by polarised light microscopy and phase contrast--method and results].

The implementation of the 1997 law on the banned use of asbestos, entailing a successive elimination of asbestos products, is planned for 30 years. Asbestos identification in samples of bulk materials is essential before starting dismantling works. The presented method of asbestos identification by polarized light microscopy and phase contrast involves the study of the following optical parameters of asbestos fibers: color, morphology, refractive index, anisotropy, pleochroism, optic nature and extinguish angle. The major advantages of this method are: small material samples, short time of analysis, high asbestos sample detectability (0.1%) and high reliability of results. This paper presents the results of asbestos identification in over a dozen of samples of bulk materials derived from various buildings and installations.

Asbestos↗

Bladder outlet obstruction index and maximal flow rate during urodynamic study as powerful predictors for the detection of urodynamic obstruction in women.

AIMS: To evaluate whether bladder outlet obstruction index (BOOI), a mathematical index of outlet resistance, in association with maximal flow rate (Qmax) obtained during multichannel urodynamics (UDS) or detrusor pressure at maximal flow rate (PdetQmax) could help the identification of obstruction in women. METHODS: From January 2004 to February 2005, 401 women were assessed in our urodynamic unit. Of these, 133 were neurologically intact women, referred symptoms suggestive of voiding disorders and had an abnormal non-intubated uroflussometry (NIF) (BOO group). A normal NIF was defined as a bell-shaped curve in presence of a Qmax >15 ml/sec and a PVR <50 ml. Symptoms of voiding disorders were ascertained by interview and rated positive if they occurred more than occasionally. Thirty-seven women were enrolled as unobstructed if they presented a normal NIF and symptoms suggestive of voiding disorders less than occasionally (unobstructed group). RESULTS: ROC curve demonstrated similar diagnostic accuracy in the diagnosis of urodynamic obstruction for Qmax obtained during UDS (AUC = 0.908; P < 0.0001; CI99% = 0.831 to 0.985) and BOOI (AUC = 0.879; P < 0.0001; CI99% = 0.794 to 0.918) and the least was seen for PdetQmax (AUC = 0.706; P = 0.001; CI99% = 0.620 to 0.830). A Qmax obtained during UDS cut-off < or =15 ml/sec provides specificity of 85.9% and sensitivity of 78.9%. A BOOI cut-off > or = -8 provides a sensitivity of 80.8% and specificity of 86.1%. The weakest sensitivity (64.2%) and specificity (64.6%) was recorded for PdetQmax > or =28 cmH(2)O. CONCLUSIONS: BOOI and Qmax obtained during UDS appears to be the most discriminating urodynamic parameters of female BOO. The use of these cutoff might facilitate the identification of women with functional and anatomical obstruction.

Aged↗

Simple anthropometric measures identify fasting hyperinsulinemia and clustering of cardiovascular risk factors in Asian Indian adolescents.

Correlations of easily measurable parameters of obesity (body mass index [BMI], waist circumference [WC], and subscapular skinfold thickness) with fasting hyperinsulinemia and cardiovascular risk factors (CRFs) have not been investigated in adolescents. We evaluated the screening performance of 3 anthropometric measurements, BMI, WC, and subscapular skinfold thickness, in identifying fasting hyperinsulinemia and clustering of CRFs in 680 male and 521 female adolescents and young adults aged 14 to 18 years in a cross-sectional population survey. CRFs considered were hypercholesterolemia, hypertriglyceridemia, low levels of high-density lipoprotein cholesterol, impaired fasting blood glucose, hypertension, and fasting hyperinsulinemia. The ability of the anthropometric measurements to identify the clustering of CRFs without (cluster 1) and with fasting hyperinsulinemia (cluster 2), and fasting hyperinsulinemia alone was evaluated. BMI, WC, and subscapular skinfold thickness identified the clustering of CRFs and fasting hyperinsulinemia better in males than in females. Among individual risk factors, WC was better in identifying the presence of 3 or more risk factors in cluster 1 for both males and females, and in cluster 2 in females. Subscapular skinfold thickness was better than BMI and WC in identifying hyperinsulinemia in males, and the presence of 3 or more risk factors in cluster 2 in females. All 3 measurements were more accurate in identifying fasting hyperinsulinemia than presence of 3 or more CRFs in either cluster 1 or cluster 2 with higher odds ratio for males. This study shows gender differences in identification of insulin resistance and clustering of CRFs by using simple anthropometric parameters in Asian Indian adolescents. These simple measurements are useful for preventing and predicting cardiovascular risk and for generating a correct definition of the metabolic syndrome.

Adolescent↗

The use of common continuous monitoring parameters: a quality indicator for critically ill patients with sepsis.

Sepsis is a common source of morbidity and mortality among critically ill patients, and targeting measures to promote early recognition and treatment of sepsis is at the forefront of many critical care initiatives. Starting formally in 1992, with the publication of the definitions of sepsis, continuous monitoring of several common physiologic parameters, including electrocardiogram, blood pressure, and oxygen saturation, have been advocated as important in the early identification and treatment of patients with sepsis. The descriptive study detailed in this article was conducted to assess the perceptions and clinical continuous physiologic monitoring practices of experienced critical care clinicians with regard to their use of common physiologic monitoring parameters in the care of patients with sepsis. A convenience sample of 100 physicians and 517 nurses completed a 20-item survey assessing perceptions and clinical monitoring practices related to the care of patients with sepsis. Results indicated that the basic parameters of electrocardiogram, invasive blood pressure, pulmonary arterial catheter monitoring, and oxygen saturation all have value in the recognition and treatment of patients with sepsis. The majority of clinicians used these parameters routinely and felt they were necessary for patient care. These results indicate that clinical practice is in concordance with current practice recommendations.

Adult↗

Hemodynamic parameters and early intimal thickening in branching blood vessels.

Intimal thickening due to atherosclerotic lesions or intimal hyperplasia in medium to large blood vessels is a major contributor to heart disease, the leading cause of death in the Western World. Balloon angioplasty with stenting, bypass surgery, and endarterectomy (with or without patch reconstruction) are some of the techniques currently applied to occluded blood vessels. On the basis of the preponderance of clinical evidence that disturbed flow patterns play a key role in the onset and progression of atherosclerosis and intimal hyperplasia, it is of interest to analyze suitable hemodynamic wall parameters that indicate susceptible sites of intimal thickening and/or favorable conditions for thrombi formation. These parameters, based on the wall shear stress, wall pressure, or particle deposition, are applied to interpret experimental/clinical observations of intimal thickening. Utilizing the parameters as "indicator" functions, internal branching blood vessel geometries are analyzed and possibly altered for different purposes: early detection of possibly highly stenosed vessel segments, prediction of future disease progression, and vessel redesign to potentially improve long-term patency rates. At the present time, the focus is on the identification of susceptible sites in branching blood vessels and their subsequent redesign, employing hemodynamic wall parameters. Specifically, the time-averaged wall shear stress (WSS), its spatial gradient (WSSG), the oscillatory shear index (OSI), and the wall shear stress angle gradient (WSSAG) are compared with experimental data for an aortoceliac junction. Then, the OSI, wall particle density (WPD), and WSSAG are segmentally averaged for different carotid artery bifurcations and compared with clinical data of intimal thickening. The third branching blood vessel under consideration is the graft-to-vein anastomosis of a vascular access graft. Suggested redesigns reduce several hemodynamic parameters (i.e., the WSSG, WSSAG, and normal pressure gradient [NPG]), thereby reducing the likelihood of restenosis, especially near the critical toe region.

Animals↗

Identification of conserved structural features at sequentially degenerate locations in transcription factor binding sites.

UNLABELLED: Many locations within transcription factor binding sites are not sequentially conserved and appear to be degenerate. We hypothesize that some of these positions contain essential structural codes that are recognized by the transcription factors that bind to them. The structural codes can be defined by base-pair step parameters that describe the relative displacement and orientation of two adjacent base pairs in a nucleic acid structure. We have developed a method, Identification of Conserved Structural Features (ICSF), which uses base-pair step parameters obtained from a collection of high-resolution DNA crystal structures to discover structural conservation that exists in the sequentially degenerate areas within a binding site and produce profiles of the structural features along the entire site. We have focused our study on the transcription factor binding sites in the JASPAR database and have found that one-third (P-value > or = 0.05) of the binding sites contain sequentially degenerate locations with highly conserved structural features as described by the base-pair step parameters. These results will help us to gain a better understanding of the process by which transcription factors recognize their binding sites and possibly lead to an improvement in our ability to find these sites in genomic sequences. AVAILABILITY: ICSF is freely available to academic users at http://zlab.bu.edu/ICSF . SUPPLEMENTARY INFORMATION: http://zlab.bu.edu/ICSF .

Algorithms↗

[Study of ventricular repolarization in an experimental model of arterial hypertension associated with obesity].

UNLABELLED: High fat diet (HFD) in dogs is associated with obesity and hypertension (HTN) but also with a significant and early decrease in heart rate variability (HRV). Decreased HRV has been shown to be a good predictor of sudden cardiac death due mainly to arrhythmic event. The aim of this work was to investigate the changes in ventricular repolarization through 24 hours EKG recordings in dogs with hypertension and rendered obese by 20 weeks of HFD. This was achieved through 24 hour EKG recording analysis of QT parameters. The aims of this work was i) feasibility of this method in dogs and ii) identification of potential arrhythmic risk factors that could explain overmortality during obesity. METHOD: Six dogs received a high fat diet (HFD) ad libitum during 20 weeks. A 24 hour EKG recording was realized just before and after 20 weeks of HFD. The following parameters studying QT interval were collected: QT interval lasting from the beginning of the Q wave to the apex (QTa) and to the end of the T wave (QTe), QT intervals plotted against RR intervals and two regression lines were calculated characterized by their slope and intersection with the Y axis, QT dispersion (longest minus shortest QT interval for each RR value) as well as the difference of QT interval between night and day at a fixed RR value considered as a marker of the sympathovagal balance. Our results show that HFD significantly increased body weight, blood pressure, heart rate, left ventricular mass and insulinemia. QT dispersion was increased in a non-significant manner both during day (+35%) and night (16%) for QTa and only during day for QTe (+27%). This increased dispersion of QT was not associated to any increase of QT interval. There was no effect of HFD on QT dynamicity parameter nor on the night-day difference at any RR interval from 300 to 1,300 ms. CONCLUSION: HFD tend increase QT dispersion without any effect on QT interval. These results are compatible with a heterogeneous repolarization probably related to abnormal autonomic nervous system tone. This study could partly explain occurrence of lethal arrhythmias during obesity which might lead to overmortality of obese patients. These results are different for QTa and QTe, but these two parameters are characterizing different type of ventricular cells. This study confirms the feasibility of this method in an experimental model, but results need to be validated in larger groups and in human.

Animals↗

Development of a bioreactor based on trypsin immobilized on monolithic support for the on-line digestion and identification of proteins.

The preparation and characterization of a new trypsin-based bioreactor is here described for on-line protein digestion and peptide analysis. Trypsin was immobilized on an epoxy-modified silica monolithic support with a single reaction step and the amount of immobilized enzyme was found to be 66.07 mg (+/-11.75 S.D.)/column (n = 6). The bioreactor was coupled through a switching valve to an analytical column for the on-line digestion, peptide separation and identification of test proteins by ESI-MS-MS. The influence of various parameters (flow rate, temperature, buffer pH and molarity, etc.) on enzymatic activity was investigated by an experimental design and the mostly significant factor was found to be the flow rate. The efficacy of the reported on-line bioreactor for tryptic mapping is reported for somatostatin and myoglobin, selected as model compounds. Tryptic peptide maps obtained by on-line digestion of myoglobin were compared to those obtained by traditional off-line digestion. Sequence coverage obtained with the on-line protocol (21 peptides, 75.16% coverage of myoglobin sequence) was found to be comparable to the one obtained with the off-line protocol (18 peptides, 76.47% coverage). Sensitivity for myoglobin digestion and identification was 0.1 mg/ml. The reproducibily of the peptide maps in terms of retention time was from 1.53 to 4.31%, R.S.D.

Amino Acid Sequence↗

[Operations research in group feeding programs: techniques for the identification and analysis of management problems].

The operations of the group feeding programs (PAG--"Programas de Alimentación a Grupos"--) can be viewed as a sequence of decisions and activities that according to their direction, order, relationship and dependency, will influence the quality and quantity of the service's delivery. While searching for the essential characteristics of a PAG: components, relationship, variables, pertinent parameters, restrictions, underlying criteria and objective functions, operations research can be useful for its analysis, the identification of its management problems, and to develop, evaluate and finally, implement solutions. Various operation research techniques are presented in this article, which are geared towards responding to those knowledge needs of the national technicians and program managers, in order to give a solid foundation to solutions for the management problems identified in a PAG. Failures of different quantitative models had been due, mainly, to the tendency to formulate models or investigate problems without users being prepared to implement the solutions. The techniques presented herein take into consideration the interaction between specialists and users.

Food Services↗

[A new diagnostic index of diabetes mellitus based on the 2-dimensional parameter of glucose kinetics].

A new approach to forming a one-part model of glucose kinetics in an i.v. tolerance test (IGTT) made it possible to introduce a two-dimensional parameter of glucose kinetics which reflected, on the one hand, the rate of glucose elimination from the blood and, on the other hand, glucose production by the liver during testing. This parameter serves the basis for a discriminant function which can be used for the identification of patients with diabetes mellitus with 100% reliability (22 patients and 50 healthy persons in a control sampling), overdiagnosis being made in 8% of the cases only. Another function characterizing a degree of carbohydrate metabolic derangement in persons with previously diagnosed diabetes is based on the same principle. Besides, a new highly reliable diagnostic criterion of diabetes mellitus was obtained taking into account a distance and position of the two-dimensional parameter of glucose kinetics with relation to a discriminant curve permitting quantitative characterization of carbohydrate metabolism in an examinee.

Blood Glucose↗

Tumor proliferative activity is predictive of pathological stage in clinical stage A nonseminomatous testicular germ cell tumors.

PURPOSE: Traditional histopathological features have failed to predict accurately the pathological stage of clinical stage A nonseminomatous germ cell tumors of the testis. Based on pilot studies in nonconsecutive patients at our university, we evaluated nontraditional risk factors (cell cycle analysis by flow cytometry, deoxyribonucleic acid analysis by single cell cytophotometry [image analysis] and assessment of proliferative activity by immunohistochemistry) combined with histopathological features in consecutive patients with clinical stage A nonseminomatous testis cancer. MATERIALS AND METHODS: Orchiectomy specimens from 105 consecutive patients with clinical stage A nonseminomatous germ cell tumors who underwent retroperitoneal lymph node dissection (76 with pathological stage A disease and 29 with proved metastasis) were recut, histopathologically reviewed, immunohistochemically stained with proliferation markers (for example Ki-67/MIB-1), and examined by flow cytometry and image analysis. RESULTS: After multiple logistic regression analysis, the G2M+S cell cycle fraction of the aneuploid tumor stemline was the most predictive parameter of pathological stage (p = 0.0004). Using a cutoff of 41%, patients with metastasis were predicted with a sensitivity of 71%. Of 61 patients with a G2M+S value of less than 41%, 53 had pathological stage A cancer (negative predictive value 87%). A low volume of embryonal carcinoma was predominant in patients at low risk for metastasis and MIB-1 immunohistochemical staining identified 23% of patients with pathological stage A tumor who were at extremely low risk for metastatic disease. CONCLUSIONS: Assessment of tumor cell proliferation cannot classify accurately high risk patients at a clinically applicable level. However, identification of patients at low risk for metastasis by flow cytometry, immunohistochemical proliferation markers and volume of embryonal carcinoma may be possible at the 90% level. MIB-1 staining is able to classify patients at extremely low risk for metastasis. These parameters deserve further study, since identification of patients at extremely low risk for metastasis could potentially decrease overall morbidity in the management of clinical stage A nonseminomatous testis cancer.

Cell Division↗

Identification of heterogeneous aquifer transmissivity using an AE-based method.

Determination of hydraulic head, H, as a function of spatial coordinates and time, in ground water flow is the basis for aquifer management and for prediction of contaminant transport. Several computer codes are available for this purpose. Spatial distribution of the transmissivity, T(x,y), is a required input to these codes. In most aquifers, T varies in an erratic manner, and it can be characterized statistically in terms of a few moments: the expected value, the variance, and the variogram. Knowledge of these moments, combined with a few measurements, permits one to estimate T at any point using geostatistical methods. In a review of transmissivity data from 19 unconsolidated aquifers, Hoeksema and Kitanidis (1985) identified two types of the logtransmissivity Y= ln(T) variations: correlated variations with variance sigma2Yc and correlation scale, I(Y), on the order of kilometers, and uncorrelated variations with variance sigma2Yn. Direct identification of the logtransmissivity variogram, Gamma(Y), from measurements is difficult because T data are generally scarce. However, many head measurements are commonly available. The aim of the paper is to introduce a methodology to identify the transmissivity variogram parameters (sigma2Yc, I(Y), and sigma2Yn) using head data in formations characterized by large logtransmissivity variance. The identification methodology uses a combination of precise numerical simulations (carried out using analytic element method) and a theoretical model. The main objective is to demonstrate the application of the methodology to a regional ground water flow in Eagle Valley basin in west-central Nevada for which abundant transmissivity and head measurements are available.

Environmental Monitoring↗

Characterisation of the DNA-binding profile of barley HvCBF1 using an enzymatic method for rapid, quantitative and high-throughput analysis of the DNA-binding activity.

A rapid and quantitative DNA-binding assay was developed based on the translational fusion of a DNA-binding protein (DBP) with a Neocallimastix patriciarum beta-1,4-D-glucanase, CelD. CelD releases a fluorescent 4-methylumbelliferyl product from 4-methylumbelliferyl cellobioside. This hydrolysis activity was used to quantify the amount of DBP-CelD bound to an immobilised biotin-labelled target sequence. The DNA-binding assay can be performed in a 96-well plate format for high- throughput analysis of putative DBPs. This method was applied to analysis of the binding properties and sequence selectivity of a cold-inducible transcription factor HvCBF1 from barley containing an AP2 DNA-binding domain. A base-scanning approach using degenerate oligonucleotide probes was employed for rapid identification of the conserved core motif of the HvCBF1 binding site. Quantitative analysis of the binding site of HvCBF1 using systematic base substitution revealed that a (G/a) (C/t)CGAC sequence was sufficient to constitute a functional motif, where the lower-case letters represent less efficient bases. The method enables us to provide accurate and quantitative data on a comprehensive DNA-binding profile for a cold-inducible AP2 transcription factor as well as information on environmental parameters potentially influencing the DNA-binding activity. The accurate binding sequence data facilitate identification of candidate genes regulated by HvCBF1 from genome sequence databases.

Base Sequence↗