Characterisation of five missense mutations in the cystathionine beta-synthase gene from three patients with B-6-nonresponsive homocystinuria

Homocystinuria, due to a deficiency of the enzyme cystathionine beta-synthase (CBS), is an inborn error of sulphur-amino acid metabolism, This is an autosomal recessive disease which results in hyperhomocysteinaemia and a wide range of clinical features, including optic lens dislocation, mental retardation, skeletal abnormalities and premature thrombotic events, We report the identification of 5 missense mutations in the protein-coding region of the CBS gene from 3 patients with pyridoxine-nonresponsive homocystinuria. Reverse-transcription PCR was used to amplify CBS cDNA from each patient and the coding region was analysed by direct sequencing, The mutations detected included 3 novel (1058C --> T, 992C --> A and 1316G --> A) and 2 previously identified (430G --> A and 833C --> T) base alterations in the CBS cDNA, Each of these mutations predicts a single amino acid substitution in the CBS polypeptide, Appropriate cassettes of patient CBS cDNA, containing each of the above defined mutations, were used to replace the corresponding cassettes of normal CBS cDNA sequence within the bacterial expression vector pT7-7. These recombinant mutant and normal CBS constructs were expressed in Escherichia coli cells and the catalytic activities of the mutant proteins were compared with normal. All of the mutant proteins exhibited decreased catalytic activity in vitro, which confirmed the association between the individual mutation and CBS dysfunction in each patient.

Acute intermittent porphyria: the in vitro expression of mutant hydroxymethylbilane synthase

Acute intermittent porphyria (AIP) is an inborn error of haem biosynthesis caused by a variety of mutations in the gene coding for hydroxymethylbilane synthase (HMB-S). The entire coding sequence of this gene, from each of three South African AIP patients, was therefore screened for mutations using chemical cleavage mismatch (CCM) analysis and any changes detected characterized by DNA sequencing. Three single base changes were identified; a G(77) to A in exon 3, a C-346 to T in exon 8 and a G(518) to A in exon 10. These missense mutations, previously reported to be present in other populations, are known to be responsible for the structurally deleterious amino acid replacements R26H, R116W and R173Q, respectively. The in vitro expression of the enzymes containing these mutations and the subsequent measurement of their specific activities revealed a reduction to approximately 4% of normal activity. (C) 1997 Academic Press Limited.

Estimating correlations from epidemiological data in the presence of measurement error

Analysis of a major multi-site epidemiologic study of heart disease has required estimation of the pairwise correlation of several measurements across sub-populations. Because the measurements from each sub-population were subject to sampling variability, the Pearson product moment estimator of these correlations produces biased estimates. This paper proposes a model that takes into account within and between sub-population variation, provides algorithms for obtaining maximum likelihood estimates of these correlations and discusses several approaches for obtaining interval estimates. (C) 1997 by John Wiley & Sons, Ltd.

Age-Related Metabolic Profiles in Cognitively Healthy Elders: Results from a Voxel-Based [(18)F]Fluorodeoxyglucose-Positron-Emission Tomography Study with Partial Volume Effects Correction

BACKGROUND AND PURPOSE: Functional brain variability has been scarcely investigated in cognitively healthy elderly subjects, and it is currently debated whether previous findings of regional metabolic variability are artifacts associated with brain atrophy. The primary purpose of this study was to test whether there is regional cerebral age-related hypometabolism specifically in later stages of life. MATERIALS AND METHODS: MR imaging and FDG-PET data were acquired from 55 cognitively healthy elderly subjects, and voxel-based linear correlations between age and GM volume or regional cerebral metabolism were conducted by using SPM5 in images with and without correction for PVE. To investigate sex-specific differences in the pattern of brain aging, we repeated the above voxelwise calculations after dividing our sample by sex. RESULTS: Our analysis revealed 2 large clusters of age-related metabolic decrease in the overall sample, 1 in the left orbitofrontal cortex and the other in the right temporolimbic region, encompassing the hippocampus, the parahippocampal gyrus, and the amygdala. The division of our sample by sex revealed significant sex-specific age-related metabolic decrease in the left temporolimbic region of men and in the left dorsolateral frontal cortex of women. When we applied atrophy correction to our PET data, none of the above-mentioned correlations remained significant. CONCLUSIONS: Our findings suggest that age-related functional brain variability in cognitively healthy elderly individuals is largely secondary to the degree of regional brain atrophy, and the findings provide support to the notion that appropriate PVE correction is a key tool in neuroimaging investigations.

Pleural fluid: Are temperature and storage time critical preanalytical error factors in biochemical analyses?

Background: Biochemical analysis of fluid is the primary laboratory approach hi pleural effusion diagnosis. Standardization of the steps between collection and laboratorial analyses are fundamental to maintain the quality of the results. We evaluated the influence of temperature and storage time on sample stability. Methods: Pleural fluid from 30 patients was submitted to analyses of proteins, albumin, lactic dehydrogenase (LDH), cholesterol, triglycerides, and glucose. Aliquots were stored at 21 degrees, 4 degrees, and-20 degrees C, and concentrations were determined after 1, 2, 3, 4, 7, and 14 days. LDH isoenzymes were quantified in 7 random samples. Results: Due to the instability of isoenzymes 4 and 5, a decrease in LDH was observed in the first 24 h in samples maintained at -20 degrees C and after 2 days when maintained at 4 degrees C. Aside from glucose, all parameters were stable for up to at least day 4 when stored at room temperature or 4 degrees C. Conclusions: Temperature and storage time are potential preanalytical errors in pleural fluid analyses, mainly if we consider the instability of glucose and LDH. The ideal procedure is to execute all the tests immediately after collection. However, most of the tests can be done in refrigerated sample;, excepting LDH analysis. (C) 2010 Elsevier B.V. All rights reserved.

Ultrasonographic Evaluation of Achilles Tendon Repair After Percutaneous Sectioning for the Correction of Congenital Clubfoot Residual Equinus

Background: Most cases of congenital clubfoot treated with the Ponseti technique require percutaneous Achilles tenotomy to correct the residual equinus. Clinical evidence suggests that complete healing occurs between the cut tendon stumps, but there have not yet been any detailed studies investigating this reparative process. This study was performed to assess Achilles tendon repair after percutaneous section to correct the residual equinus of clubfoot treated with the Ponseti method. Method: A prospective study analyzed 37 tenotomies in 26 patients with congenital clubfoot treated with the Ponseti technique, with a minimum follow-up of 1 year after the section. The tenotomy was performed percutaneously with a large-bore needle bevel with patient sedation and local anesthesia. Ultrasonographic scanning was performed after section to ascertain that the tenotomy had been completed and to measure the stump separation. In the follow-up period, the reparative process was followed ultrasonographically and assessed at 3 weeks, 6 months, and 1 year posttenotomy. Results: The ultrasonography performed immediately after the procedure showed that in some cases, residual strands between the tendon ends persisted, and these were completely sectioned under ultrasound control. A mean retraction of 5.65 mm +/- 2.26 mm (range, 2.3 to 11.0 mm) between tendon stumps after section was observed. Unusual bleeding occurred in one case and was controlled by digital pressure, with no interference with the final treatment. After 3 weeks, ultrasonography showed tendon repair with the tendon gap filled with irregular hypoechoic tissue, and also with transmission of muscle motion to the heel. Six months after tenotomy, there was structural filling with a fibrillar aspect, mild or moderate hypoechogenicity, and tendon scar thickening when compared with a normal tendon. One year after tenotomy, ultrasound showed a fibrillar structure and echogenicity at the repair site that was similar to a normal tendon, but with persistent tendon scarring thickness. Conclusions: There is a fast reparative process after Achilles tendon percutaneous section that reestablishes continuity between stumps. The reparative tissue evolved to tendon tissue with a normal ultrasonographic appearance except for mild thickening, suggesting a predominantly intrinsic repair mechanism.

Error in body weight estimation leads to inadequate parenteral anticoagulation

Parenteral anticoagulation is a cornerstone in the management of venous and arterial thrombosis. Unfractionated heparin has a wide dose/response relationship, requiring frequent and troublesome laboratorial follow-up. Because of all these factors, low-molecular-weight heparin use has been increasing. Inadequate dosage has been pointed out as a potential problem because the use of subjectively estimated weight instead of real measured weight is common practice in the emergency department (ED). To evaluate the impact of inadequate weight estimation on enoxaparin dosage, we investigated the adequacy of anticoagulation of patients in a tertiary ED where subjective weight estimation is common practice. We obtained the estimated, informed, and measured weight of 28 patients in need of parenteral anticoagulation. Basal and steady-state (after the second subcutaneous shot of enoxaparin) anti-Xa activity was obtained as a measure of adequate anticoagulation. The patients were divided into 2 groups according the anticoagulation adequacy. From the 28 patients enrolled, 75% (group 1, n = 21) received at least 0.9 mg/kg per dose BID and 25% (group 2, n = 7) received less than 0.9 mg/kg per dose BID of enoxaparin. Only 4 (14.3%) of all patients had anti-Xa activity less than the inferior limit of the therapeutic range (<0.5 UI/mL), all of them from group 2. In conclusion, when weight estimation was used to determine the enoxaparin dosage, 25% of the patients were inadequately anticoagulated (anti-Xa activity <0.5 UI/mL) during the initial crucial phase of treatment. (C) 2011 Elsevier Inc. All rights reserved.

Masticatory muscle function three years after surgical correction of class III dentofacial deformity

Individuals with dentofacial deformities have masticatory muscle changes. The objective of the present study was to determine the effect of interdisciplinary treatment in patients with dentofacial deformities regarding electromyographic activity (EMG) of masticatory muscles three years after surgical correction. Thirteen patients with class III dentofacial deformities were studied, considered as group PI (before surgery) and group P3 (3 years to 3 years and 8 months after surgery). Fifteen individuals with no changes in facial morphology or dental occlusion were studied as controls. The participants underwent EMG examination of the temporal and masseter muscles during mastication and biting. Evaluation of the amplitude interval of EMG activity revealed a difference between P1 and P3 and no difference between P3 and the control group. In contrast, evaluation of root mean square revealed that, in general, P3 values were higher only when compared with PI and differed from the control group. There was an improvement in the EMG activity of the masticatory muscles, mainly observed in the masseter muscle, with values close to those of the control group in one of the analyses.

Masseter muscle thickness three years after surgical correction of class III dentofacial deformity

Objective: To analyse the effect of integrated orthodontic treatment, orthognathic surgery and orofacial myofunctional therapy on masseter muscle thickness in patients with class III dentofacial deformity three years after orthognathic surgery. Design: A longitudinal study was conducted on 13 patients with class III dentofacial deformities, denoted here as group P1 (before surgery) and group P3 (same patients 3 years to 3 years and 8 months after surgery). Fifteen individuals with no changes in facial morphology or dental occlusion were assigned to the control group (CG). Masseter muscle ultrasonography was performed in the resting and biting situations in the three groups. Data were analysed statistically by a mixed-effects linear model considering a level of significance of P < 0.05. Results: Significantly higher values (P < 0.01) of masseter muscle thickness (cm) were detected in group P3 (right rest: 0.82 +/- 0.16, left rest: 0.87 +/- 0.21, right bite: 1 +/- 0.22, left bite: 1.04 +/- 0.28) compared to group P1 (right rest: 0.63 +/- 0.19, left rest: 0.64 +/- 0.15, right bite: 0.87 +/- 0.16, left bite: 0.88 +/- 0.14). Between P3 and CG (right rest: 1.02 +/- 0.19, left rest: 1 +/- 0.19, right bite: 1.18 +/- 0.22, left bite: 1.16 +/- 0.22) there was a significant difference on the right side of the muscle (P < 0.05) in both situations and on the left side at rest. Conclusion: The proposed treatment resulted in improved masseter muscle thickness in patients with class III dentofacial deformity. (C) 2011 Elsevier Ltd. All rights reserved.

Non-coding transcript in T cells (NTT): Antisense transcript activates PKR and NF-kappa B in human lymphocytes

T cell activation is a complex process involving many steps and the role played by the non-protein-coding RNAs (ncRNAs) in this phenomenon is still unclear. The non-coding T cells transcript (NTT) is differentially expressed during human T cells activation, but its function is unknown. Here, we detected a 426 m NTT transcript by RT-PCR using RNA of human lymphocytes activated with a synthetic peptide of HIV-1. After cloning, the sense and antisense 426 nt NTT transcripts were obtained by in vitro transcription and were sequenced. We found that both transcripts are highly structured and are able to activate PKR. A striking observation was that the antisense 426 nt NTT transcript is significantly more effective in activating PKR than the corresponding sense transcript. The transcription factor NF-kappa B is activated by PKR through phosphorylation and subsequent degradation of its inhibitor I-kappa B beta. We also found that the antisense 426 nt NTT transcript induces more efficiently the degradation Of I-kappa B beta than the sense transcript. Thus, this study suggests that the role played by NTT in the activation of lymphocytes can be mediated by PKR through NF-kappa B activation. However, the physiological significance of the activity of the antisense 426 nt NTT transcript remains unknown. (c) 2007 Elsevier Inc. All rights reserved.

Long-term stability of surgical-orthodontic open-bite correction

Introduction: The objective of this study was to evaluate the long-term stability of open-bite surgical-orthodontic correction. Methods: Thirty-nine patients at an initial mean age of 20.83 years were evaluated cephalometrically at pretreatment (T1), immediately after treatment (T2), and at the last recall (T3), with a mean follow-up time of 8.22 years. The surgical protocol included single-jaw or double-jaw surgery. Because the patients had different anteroposterior malocclusions, the sample was divided into a Class I and Class II (I-II) subgroup (3 Class I, 20 Class II malocclusion patients) and a Class III subgroup (16 patients). The dentoskeletal characteristics of the total sample and the subgroups were compared at T1, T2, and T3 with dependent analysis of variance (ANOVA). Results: Overbite relapse in the posttreatment period was statistically significant in the whole sample and the Class I-II subgroup. Fourteen patients of the whole sample (35.9%) had clinically significant open-bite relapse (negative overbite). Conclusions: There was a statistically significant open-bite relapse in the overall sample and in the Class I-II subgroup. The clinically significant values of long-term open-bite correction stability were 64.11%, 47.82%, and 87.50% in the overall sample, the Class I-II subgroup, and the Class III subgroup, respectively. (Am J Orthod Dentofacial Orthop 2010;138:254.e1-254.e10)

Model-based procedure for scale-up of wet, overflow ball mills - Part III: Validation and discussion

A new ball mill scale-up procedure is developed. This procedure has been validated using seven sets of Ml-scale ball mil data. The largest ball mills in these data have diameters (inside liners) of 6.58m. The procedure can predict the 80% passing size of the circuit product to within +/-6% of the measured value, with a precision of +/-11% (one standard deviation); the re-circulating load to within +/-33% of the mass-balanced value (this error margin is within the uncertainty associated with the determination of the re-circulating load); and the mill power to within +/-5% of the measured value. This procedure is applicable for the design of ball mills which are preceded by autogenous (AG) mills, semi-autogenous (SAG) mills, crushers and flotation circuits. The new procedure is more precise and more accurate than Bond's method for ball mill scale-up. This procedure contains no efficiency correction which relates to the mill diameter. This suggests that, within the range of mill diameter studied, milling efficiency does not vary with mill diameter. This is in contrast with Bond's equation-Bond claimed that milling efficiency increases with mill diameter. (C) 2001 Elsevier Science Ltd. All rights reserved.