Evaluation of the impact of arterial wall viscosity in pressure morphology by means of a simplified 1D model.

INTRODUCTION: The interest on computational simulations of biomedical models has increased in the last years. Nevertheless, the viscous properties of vessel all are usually neglected. MATERIALS AND METHODS: A 1D model of blood flow and its interaction with the arterial wall was implemented. Non-viscous modeling and the addition of a wall-viscosity term were considered. The goodness of fit in each case was evaluated. RESULTS: Simulated radial blood pressure was generated using central blood pressure as the input of the proposed 1D tapered model. Results obtained showed an improvement as a consequence of introducing wall viscosity into the model calculations. DISCUSSION: The effect of viscoelasticity has a great significance in the implementation of computational models, since a better fit between simulated and experimental data is achieved.

El frasco humidificador como sistema de humidificación del gas inspirado no cumple con las recomendaciones. Evaluación y comparación de tres sistemas de humidificación. Estudio de laboratorio / The humidifier bottle as humidification system of the inspired gas does not meet with the recommendations. Evaluation and comparison of three humidification systems. Laboratory study

Objetivos. Demostrar que el frasco humidificador (FH), como sistema de humidificación del gas inspirado, no alcanza los valores mínimos de humedad relativa (HR), humedad absoluta (HA) y temperatura (Tº) recomendados por la literatura. Secundariamente, comparar el rendimiento del FH y dos sistemas activos de humidificación (SAH). Materiales y métodos. Las variables principales fueron T° del agua, HR, Tº y HA del gas entregado. Se realizaron mediciones a diferentes niveles de Tº, volumen de agua y flujos. Resultados. El FH no alcanzó los valores recomendados de HR 100%, HA 30 mg/l y Tº 31ºC. El SAH sin circuito calefaccionado alcanzó valores recomendados en el NIVEL III con flujos de 20-60lpm, y en el NIVEL II con flujos de 20-30lpm. El SAH con circuito calefaccionado logró valores sugeridos en los NIVELES II y III (20-60lpm). Se encontró diferencia significativa (p<0,001 Global) para flujo, Tº y tipo de humidificador. El modelo ANOVA arrojó significación estadística (p<0,001) del término interacción de flujo y tipo de humidificador en cada nivel de Tº. Conclusiones. El FH no acondiciona el gas de acuerdo a lo recomendado. El mejor rendimiento fue con 300 ml y flujo de 1 lpm: Tº 23,92(±0,69), HR 74,02%(±6,53) y HA 16,02 mg/l (±1,86), estos valores apenas superan el 50% de lo mínimo sugerido en la literatura. Los SAH acondicionaron el gas adecuadamente. El modelo ANOVA arroja que existen otros factores involucrados en mantener la HA y que la significación varía en cada tipo de humidificador a cada nivel de flujo(AU)

Objetive. To demonstrate that humidifier bottle (HB) as inspired gas humidification system does not attain the minimum values of relative humidity (RH), absolute humidity (AH) and temperature (Tº) as recommended by the literature. Furthermore, to compare the HB performance with two active humidification systems (AHS). Materials and Methods. Main variables were: water Tº, RH, Tº and AH of delivered gas. Measurements were made at different levels of Tº, water and flows volume. Results. Recommended values of RH 100%, AH 30 mg/l and Tº 31º C were not reached by the HB. AHS without heating circuit reached recommended values in LEVEL III with flows of 20-60 lpm, and in LEVEL II with flows of 20-30lpm. AHS with heating circuit obtained recommended values in LEVELS II and III (20-60lpm). A significant difference (p<0.001 Global) for flow, Tº and humidifier type was found. ANOVA model showed statistical evidence (p<0.001) of interaction between flow and type of humidifier in each Tº level. Conclusions. The HB does not condition gas in accordance with recommended values. The best performance was with 300ml and 1lpm flow: Tº 23.92 (±0.69), RH 74.02% (±6.53) and AH 16.02 mg/l (±1.86) and these values hardly exceed the 50% of minimum recommended by literature. AHS conditioned gas in a proper way. ANOVA model shows that there exist other factors involved to maintain AH and that there are important differences between each type of humidifier and each flow level.(AU)

Quality of vision: A consensus building initiative for a new ophthalmologic concept.

PURPOSE: Many studies have addressed the quantification of visual acuity, and the conventional method of measuring it has so far demonstrated serious limitations. Vision testing requires new methods that can more precisely express the quality of vision as perceived by the patient. METHODS: This study employed the Delphi method of consensus building. Concepts associated with quality of vision (QoV) were identified by a board of experts and proposed to participating specialists in two subsequent questionnaires. Upon receipt of the completed questionnaires, the replies were classified to determine the building blocks of a consensus. RESULTS: By analyzing the replies to the two questionnaires, the authors determined the key elements of QoV on which a consensus was found among the respondents. CONCLUSIONS: A consensus was reached on the opinion that the quantification of visual acuity by traditional means is inadequate for investigating QoV. Although visual acuity is still a basic element for testing, the experts believe that contrast sensitivity, reading speed, and microperimetry are additional parameters necessary for quantifying QoV. The use of a psychometric questionnaire on visual function could allow a better interpretation of visual impairment.

Is there any relationship between photodynamic therapy for exudative age-related macular degeneration and choroidal neovascolarization recurrence? A rationale for combined treatments.

PURPOSE: Photodynamic therapy (PDT) is the treatment of choice for subfoveal choroidal neovascularization (CNV) in age-related macular degeneration (ARMD). Interpretation of PDT mechanism of action is not yet fully understood and causes of CNV recurrences are unclear. The authors have conducted a retrospective analysis of fluorescein and indocyanine green angiographies in patients treated with multiple PDT in order to identify risk factors for recurrence. METHODS: A total of 342 eyes of 342 patients (207 women and 135 men) with ARMD and subfoveal CNV were treated with at least two PDT. Angiographic (fluorescein and indocyanine green) features of recurrences were confronted to pretreatment examinations in all patients. RESULTS: Post-PDT angiographies showed in all eyes a dark circle corresponding to the laser spot even 1 year after treatment. Persistence or progressive regrowth of CNV developed in an area adjacent or corresponding to the original lesion, without any specific relationship with the location of fluorescein and indocyanine green late leakage or with presence of abnormal fluorescence due to pigment abnormalities. At the 3-month angiographic follow-up, 23 patients (6.7%) showed a recurrent CNV resembling shape and dimension of the laser spot used for the PDT treatment. CONCLUSIONS: The authors failed to identify angiographic signs helpful to predict the risk of CNV persistence or recurrence. PDT leaves minor but persistent changes in the choroidal vasculature within the treatment area. In some cases, the recurrent CNV seems to be related to the laser spot of the PDT.

Incidence of nosocomial rotavirus infections, symptomatic and asymptomatic, in breast-fed and non-breast-fed infants.

Rotavirus is one of the most important aetiological agents of nosocomial infections in childhood. We studied the incidence of nosocomial rotavirus infections in 420 patients (age range 1-18 months) consecutively admitted from 1 December 1999 to 31 May 2000 to the infant ward of the Department of Paediatrics, University of Turin. We also evaluated the protective effect of breast feeding. Faecal specimens were collected from every child (whether developing diarrhoeic symptoms or not) and tested for rotavirus during hospitalization and 72 h after discharge. The incidence of rotavirus nosocomial infections was 27.7%. The incidence of symptomatic nosocomial infections was 16.8%, and the incidence of asymptomatic infections was 10.9%. The attack rate of the infections that occurred during hospitalization was 11.8%, while for those occurring after discharge, it was 15.9%. Rotavirus infection, on average, prolonged hospital stay from 5.2 to 6.4 days. 10.6% of breast-fed infants and 32.4% of non-breast-fed infants contracted rotavirus infection (P<0.005). None of the breast-fed infants who contracted rotavirus infection developed diarrhoeic symptoms.

Infundibulopelvic stenosis, multicystic kidney, and calyectasis in a kindred: clinical observations and genetic analysis.

Congenital obstructive anomalies of the urinary tract usually occur sporadically. We describe inheritance in a three-generation kindred of a spectrum of kidney anomalies consistent with an autosomal-dominant mode of transmission, with incomplete penetrance, calyectasis (maternal grandmother), infundibulopelvic stenosis (uncle), and multicystic kidney (male proband, age 4 years). The proband's mother, father and half sister had normal renal imaging studies. Inheritance of informative polymorphic markers (3'-HVR, GGG1, GGG9, SM-7, KG8, and CW3) mapping close to the adult polycystic kidney disease type 1 (PKD-1) and tuberous sclerosis (TSC-2) loci on chromosome 16p was evaluated by Southern blot studies and by PCR-based, fluorescent genotyping for linkage to phenotype. The 3 affected individuals, as well as the unaffected mother (obligate carrier) and unaffected half-sister, inherit a common chromosome haplotype linked to the PKD1 locus. Our findings support the hypothesis that these anomalies may be part of a spectrum of obstructive renal dysplasia which are inherited as a simple Mendelian trait exhibiting an autosomal-dominant mode of transmission with variable expression and incomplete penetrance.

Leber's hereditary optic neuropathy (LHON)-related mitochondrial DNA sequence changes in italian patients presenting with sporadic bilateral optic neuritis.

Eighteen Italian patients presenting with sporadic, bilateral, simultaneous, or sequential optic neuritis (ON) were evaluated for 14 base changes in mitochondrial DNA (mtDNA) previously found associated with Leber's hereditary optic neuropathy (LHON), aiming to identify at a molecular level LHON cases with nontypical phenotypes. During a 36-month follow-up, 11 ON patients developed clinical or laboratory features allowing diagnosis of clinically definite multiple sclerosis (MS). None was positive for any of the "primary" LHON-associated mutations. However, single or multiple "secondary" LHON-associated sequence changes at 4216/ND1, 4917/ND2, and 13708/ND5 were detected in ON and ON-MS patients. MS controls without visual failure as well as healthy control subjects harbored the same base changes at similar frequencies. In addition, coexistence of three sequence changes was found in two cases (1 ON-MS patient and 1 MS control patient). We also report finding two new neutral sequence base changes in the ND-4 gene which were identified by SSCP and confirmed by automated DNA sequence analysis. The results suggests that these secondary mutations do not contribute to MS susceptibility in these patients, but rather represent neutral mitochondrial DNA polymorphisms. In addition, whether there are biochemical abnormalities related to single and multiple secondary mtDNA sequence changes remain to be demonstrated.

Growth and water relations of three geographically separate origins of maritime pine (Pinus pinaster) under saline conditions.

Water relations and growth of maritime pine (Pinus pinaster Ait.) were investigated in 2-year-old seedlings of French ('Landes'), Iberian ('Iberian') and Moroccan ('Tamjoute') origin raised for 67 days in a flowing solution culture system containing 0, 50, 150 or 250 mM NaCl. Height growth, and stem, needle and root dry matter were reduced by salinity with minor differences among geographic origins. Predawn needle water potential was decreased by salinity and corresponded approximately to the osmotic potential of the nutrient solution. Stomatal conductance was reduced according to the amount of salinity applied. Whole-plant hydraulic conductance was also reduced, even when expressed on a root dry weight basis. The osmotic potential of xylem sap was five- to sixfold lower than that of the nutrient solution. Seedlings of the most southerly origin (Tamjoute) exhibited a greater ability to decrease osmotic potential under saline conditions than seedlings of more northerly origin (Landes and Iberian) as a result of higher mineral cation transport to the shoot.

Fluorescence-based DNA minisequence analysis for detection of known single-base changes in genomic DNA.

We describe a rapid, automated method for direct detection of known single-base changes in genomic DNA. Fluorescence-based DNA minisequence analysis is employed in a template-dependent reaction which involves a single nucleotide extension of an oligonucleotide primer by the correct fluorescently-tagged dideoxynucleotide chain terminator. Detection following electrophoresis on denaturing acrylamide gels is facilitated by alkaline phosphatase treatment of reaction products after extension followed by isopropanol precipitation of the dye-tagged, single-base-extended primer to remove unincorporated deoxynucleotides. Fluorescence analysis of the incorporated dye tag reveals the identity of the template nucleotide immediately 3' to the primer site. This technique does not require radioactivity or biotinylated PCR product, relies on the incorporation of a single dideoxynucleotide terminator to extend the primer by one nucleotide and takes advantage of the sensitivity of fluorescent terminators developed for automated DNA sequence analysis. As a demonstration, we have applied the assay to human genomic DNA for detection of the sickle mutation in the beta-globin gene, and have also examined feasibility for simultaneous delineation using a multiplex-like strategy in a single gel-lane of some of the most common beta-thalassemia mutations in the Mediterranean basin.

Rapid detection of medium chain acyl-CoA dehydrogenase gene mutations by non-radioactive, single strand conformation polymorphism minigels.

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is a common inherited metabolic disorder affecting fatty acid beta oxidation. Identification of carriers is important since the disease can be fatal and is readily treatable once diagnosed. Twelve molecular defects have been identified in the MCAD gene; however, a single highly prevalent mutation, A985G, accounts for > 90% of mutant alleles in the white population. In order to facilitate the molecular diagnosis of MCAD deficiency, oligonucleotide primers were designed to amplify the exon regions encompassing the 12 mutations enzymatically, and PCR products were then screened with a single strand conformation polymorphism (SSCP) based method. Minigels were used allowing much faster run times, and silver staining was used after gel electrophoresis to eliminate the need for radioisotopic labelling strategies. Our non-radioactive, minigel SSCP approach showed that normals can be readily distinguished from heterozygotes and homozygotes for all three of the 12 known MCAD mutations which were detected in our sampling of 48 persons. In addition, each band pattern is characteristic for a specific mutation, including those mapping in the same PCR product like A985G and T1124C. When necessary, the molecular defect was confirmed using either restriction enzyme digestion of PCR products or by direct DNA sequence analysis or both. This rapid, non-radioactive approach can become routine for molecular diagnosis of MCAD deficiency and other genetic disorders.

Interaction of rare illegitimate recombination event and a poly A addition site mutation resulting in a severe form of alpha thalassemia.

The clinical diversity of thalassemia depends on interaction of diverse genetic defects. We have characterized a severe form of alpha thalassemia caused by coinheritance of a rare alpha-globin gene deletion and a nondeletional defect in a southern Italian family. The proband, a 7-year-old girl, exhibited an abnormal hemoglobin electrophoresis pattern with hemoglobin H and hemoglobin Barts, indicating inheritance of H and hemoglobin Barts, indicating inheritance of a severe form of alpha thalassemia. Southern blot analysis of DNA showed normal as well as aberrant alpha-globin gene fragments indicating heterozygosity for a deletional form of alpha thalassemia in the proband and her mother. The coinheritance of a nondeletional form of alpha thalassemia (alpha alpha T) was suspected because of the severity of the proband's phenotype and the presence of normal alpha-globin gene fragments in the father. Selective polymerase chain reaction of the paternal alpha 1- and alpha 2-globin genes in the proband followed by DNA sequence analysis showed an AATAAA to AATGAA mutation in the polyadenylation signal sequence of the alpha 2-globin gene. Genomic DNA mapping and sequence analysis of a unique polymerase chain reaction product generated across the deletion breakpoint of the maternal allele showed a 5,201-bp deletion extending from 870 nucleotides 5' of the alpha 2-globin gene to nucleotide +519 in the alpha 1-globin gene. This deletion is similar to that previously suggested by blotting studies in a Greek family (Pressley et al, Nucleic Acids Res 8:4889, 1980) and removes the entire alpha 2-globin gene and a portion of the 5' end of the alpha 1-globin gene. Sequence characterization of the resultant aberrant truncated alpha 1-globin gene from the proband showed a 27 nucleotide duplication corresponding to the 3' end of the alpha-globin gene IVS-2 region separated by the insertion of a tetranucleotide (GGTT), suggesting that this deletion is caused by an illegitimate recombination event.

Maple syrup urine disease (MSUD): screening for known mutations in Italian patients.

Maple syrup urine disease (MSUD) is an autosomal recessive disease due to deficiency of the branched-chain alpha-ketoacid dehydrogenase (BCKDH) caused by a large number of mutations. In the present study, DNA from Italian patients and their relatives was examined for three point mutations (Y393N in the E1 alpha gene, T841G and G1031A in the E2 gene) and two deletions (-G at the intron/exon border of exon 8 in the E2 gene and an 11 bp deletion in exon 1 of the E1 beta gene) using the polymerase chain reaction (PCR) followed by allele-specific oligonucleotide (ASO) hybridization, gene-scanning size analysis of fluorescent-tagged PCR products and/or automated DNA sequence analysis. Our results show that two different mutations account for 7 of the 20 mutant MSUD alleles. Two unrelated affected children, two of their parents and one sibling were carriers for the 11 bp deletion in the E1 beta gene, one patient and her mother were heterozygous for Y393N in E1 alpha, while T841G, G1031A and the -G deletion in E2 were not detected. This study is the first attempt to characterize at a nucleic acid level MSUD mutations in Italy. Our results indicate that additional defects are present in the Italian population and that, unlike the Mennonites, a number of different MSUD mutations exist in Italians.

Duchenne/Becker muscular dystrophy carrier detection using quantitative PCR and fluorescence-based strategies.

Dystrophin gene deletions account for up to 68% of all Duchenne (DMD) and Becker (BMD) muscular dystrophy mutations. In affected males, these deletions can be detected easily using multiplex PCR tests which monitor for exon presence. In addition, quantitative dosage screening can discriminate female carriers. We previously analyzed multiplex PCR products by gel electrophoresis and quantitation of fluorescently labeled primers with the Gene Scanner in order to test carrier status. These multiplex PCR protocols detect DMD gene deletions adequately, but require up to 18 pairs of fluorochrome-labeled primers. We previously described two alternative fluorescent labeling strategies, each with approximately 1,000-fold greater sensitivity than ethidium bromide staining, which can be used to quantify the products of multiplex PCR. The first method uses the DNA intercalating thiazole orange dye TOTO-1 to stain PCR products after 20 cycles. In the second method, fluorescein-12,2'-dUTP is incorporated into products during PCR as a fluorescent tag for subsequent quantitative dosage studies. Both methods label all multiplexed exons including the 506 bp exon 48 fragment that is difficult to detect and quantify by standard ethidium bromide staining. Using this approach, we determined DMD/BMD carrier status in 24 unrelated families using a fluorescent fragment analyzer. Analysis of fluorochrome-labeled PCR products facilitates quantitative multiplex PCR for gene-dosage analysis.

Fluorescent approaches to diagnosis of Lesch-Nyhan syndrome and quantitative analysis of carrier status.

Lesch-Nyhan syndrome is an X-linked recessive disorder caused by molecular defects within the HPRT gene. Deletional forms of this syndrome, most of which are inherited, account for 15% of the cases. In addition, a large percentage of cases are due to de novo point mutations. We have used complementary fluorescence-based PCR assays to analyse disease-causing mutations in three unrelated families: (1) inheritance of dye-labelled PCR products of linked polymorphic loci mapping within and flanking the HPRT gene; (2) dye-labelled exon dosage analysis and (3) automated fluorescence-based DNA sequence analysis. Our results using fluorescent, dye-tagged PCR products show that inheritance of two polymorphic small tandem repeats, HPRTB [AGAT]n, mapping within intron 3 of the HPRT gene, and the CA-repeat at DXS294 can be used to establish linkage to the disease. In addition, we modified a previously described PCR protocol to use fluorescent dye-labelled oligoprimers and an ABI Gene Scanner in order to rapidly quantitate deletional forms of Lesch-Nyhan syndrome. Quantitative PCR analysis of individual exons followed by dosage analysis confirmed a deletion encompassing exon 9. A similar approach was used to confirm a previously described HPRT gene duplication involving exons 2 and 3. In this analysis, we co-amplified the HPRTB [AGAT]n and HUMARA [AGC]n repeats and confirmed increased exon dosage in carriers for the duplication. DNA sequence analysis remains the method of choice for delineating new disease-causing mutations, most of which are non-deletional forms of Lesch-Nyhan syndrome. We have also used a cycle-sequencing strategy employing dye-labelled dideoxy terminators and a laser-activated, fluorescence-emission DNA sequencer in order to define carrier status in 10 family members at risk for Lesch-Nyhan syndrome due to a splice donor mutation in intron 7. Our DNA sequence analyses corroborate small tandem repeat (STR) inheritance patterns in this family. Multiple fluorescence-based strategies should facilitate rapid diagnosis of the various Lesch-Nyhan disease-causing mutations.

Hyperphenylalaninemia and pterin metabolism in serum and erythrocytes.

The relationship between blood phenylalanine concentrations and serum and erythrocyte biopterin and neopterin concentrations was investigated in 20 phenylketonuric patients with different dietary compliance. At serum phenylalanine concentrations ranging from 43 to 1004 mumol/l, a good correlation was found with serum biopterin (r = 0.76, P < 0.001) and with red blood cell biopterin (r = 0.62, P < 0.001). A similar correlation was found between serum neopterin and phenylalanine (r = 0.60, P < 0.001). The correlation between red blood cell neopterin and serum phenylalanine was less evident, however (r = 0.47, P < 0.005). After oral loading with phenylalanine (100 mg/kg body weight), serum and red blood cell biopterin concentrations increased in patients with classical phenylketonuria as well as in one patient with dihydropteridine reductase deficiency in response to the induced acute hyperphenylalaninemia. One patient suffering from 6-pyruvoyl tetrahydropterin synthase deficiency was loaded orally with tetrahydrobiopterin (20 mg/kg body weight). The kinetics of administered cofactor confirmed its rapid absorption, with early increase of serum concentrations followed by its transport into the red blood cells. The half-life of biopterin was approximately 7 h in serum and 15 h in red blood cells. Because both values are less than the half-life of phenylalanine (20-30 h) in serum, biopterin measurement offers no advantage in monitoring dietary control in hyperphenylalaninemic patients.

IMAGO: a complete system for acquisition, processing, two/three-dimensional and temporal display of microscopic bio-images.

This work describes IMAGO, an integrated bio-imaging system developed in our laboratory. The whole system consists of a personal computer, a commercially available frame grabber directly plugged into a personal computer, video input/output modules, specific hardware for z-axis movement and light shuttering, and a software package. IMAGO is user-friendly, menu driven and enables one to perform image acquisition with different methods: optical sectioning, flashing epifluorescence, transmitted and phase contrast microscopy. It makes various functions possible, including: image transfer, gray scale processing, conventional and advanced filtering, logical operations, look-up table management, three-dimensional (3D) editing, 3D representation and auto-correlation techniques. More than 100 image processing functions have been implemented and can be easily managed through IMAGO. Examples are given in the area of biophysical research, like 3D representation of nuclei and of electron microscopic images, in situ microscopy of living cells. IMAGO processes information in an x, y, z, t space.