Molecular phenotype of a human lymphoblastoid cell-line homoplasmic for the np 7445 deafness-associated mitochondrial mutation

We have studied mitochondrial gene expression and metabolic function in a human lymphoblastoid cell-line homoplasmic for the np 7445, deafness- associated mitochondrial DNA mutation. The mutation maps to the 3' termini of the oppositely oriented genes encoding cytochrome oxidase subunit I (COI) and tRNA-ser(UCN). In comparison with control lymphoblastoid cells, we detected a marked depletion (> 60%) of tRNA- ser(UCN). There was, however, no significant impairment of respiratory function, no alteration to the structure or abundance of COI mRNA or its precursors, and no detectable abnormality of mitochondrial protein synthesis. We also found considerable tissue-variation in the abundance of tRNA-ser(UCN). We propose that the tissue-specific phenotype associated with this mutation results from an inherent deficiency in the processing of the mutant pre-tRNA, that becomes limiting for protein synthesis only in a restricted set of cells of the auditory system in which the tRNA is, for other reasons, already at a critically low level.      

Sources and sequelae of bacterial contamination of hematopoietic stem cell components: implications for the safety of hematotherapy and graft engineering

Background: It is important to compare the incidence of bacterial contamination of components collected from the peripheral blood or bone marrow (BM), as well as of components processed with or without cell selection or depletion, and to evaluate the sequelae of such contamination. Study Design and Methods: Bacterial contamination rates were compared in 1380 untreated autologous peripheral blood progenitor cells (PBPCs), 291 untreated autologous BM samples, 916 monoclonal antibody (MoAb)-treated autologous and allogeneic BM samples, and in 45 autologous PBPC components from which the CD34+ cells were selected. Bacterial cultures were performed at sequential time points during the processing of MoAb-treated BM. Results: Bacterial contamination was documented in 44 of 2632 components from 1593 patients (1.67% of components, 2.76% of patients) before cryopreservation. Although only 0.65% of untreated PBPCs were contaminated before cryopreservation, each patient was more likely to have given a contaminated PBPC component than a contaminated BM component (2.41% vs. 0%, p < 0.01). Bacterial contamination of MoAb-treated BM was greater during or after manipulation than it was before (2.33% vs. 0.77%, p < 0.05). At thawing, contamination was documented in 42 (1.97%) of 2136 components cultured. Ten (13.7%) of 73 patients who received hematopoietic progenitor cells that were contaminated before cryopreservation or at thawing developed fever or positive blood cultures within 48 hours of transfusion. Fever was associated with bacteremia in two cases, but no irreversible clinical sequelae were noted. Conclusion: These studies suggest that, despite careful attention to sterile procedures, low-level contamination of hematopoietic stem cell components can be introduced before or during manipulation as well as at thawing, and that standards for monitoring of the procedures for collection, processing, cryopreservation, thawing, and transfusion of hematopoietic progenitor cells are necessary.     

Enhanced passive mixing for paper microfluidics

Imprecise control of fluid flows in paper-based devices is a major challenge in pushing the innovations in this area towards societal implementation. Assays on paper tend to have low reaction yield and reproducibility issues that lead to poor sensitivity and detection limits. Understanding and addressing these issues is key to improving the performance of paper-based devices. In this work, we use colorimetric analysis to observe the mixing behaviour of molecules from two parallel flow streams in unobstructed (on unpatterned paper) and constricted flow (through the gap of a patterned hourglass structure). The model system used for characterization of mixing involved the reaction of Fe³⁺ with SCN⁻ to form the coloured, soluble complex Fe(SCN)²⁺. At all tested concentrations (equal concentrations of 50.0 mM, 25.0 mM or 12.5 mM for KSCN and FeCl₃ in each experiment), the reaction yield increases (higher colorimetric signal) and better mixing is obtained (lower relative standard deviation) as the gap of the flow constriction becomes smaller (4.69–0.32 mm). This indicates enhanced passive mixing of reagents. A transition window of gap widths exhibiting no mixing enhancement (about 2 mm) to gap widths exhibiting complete mixing (0.5 mm) is defined. The implementation of gap sizes that are smaller than 0.5 mm (below the transition window) for passive mixing is suggested as a good strategy to obtain complete mixing and reproducible reaction yields on paper. In addition, the hourglass structure was used to define the ratio of reagents to be mixed (2 : 1, 1 : 1 and 1 : 2 HCl–NaOH) by simply varying the width ratio of the input channels of the paper. This allows easy adaptation of the device to reaction stoichiometry.

Efficient passive mixing can be achieved by contricting the reagent flow using structures having narrow gaps.     

Need for recovery from work in relation to age: a prospective cohort study

Purpose  

To investigate the impact of increasing age on the need for recovery (NFR) over time among day workers     

Effective treatment of painful bone crises in type I gaucher's disease with high dose prednisolone

In type I Gaucher's disease, episodes of severe disabling bone pain, the so called bone crises, may be resistant to all analgesics, including narcotics. The demonstration of subperiosteal oedema on magnetic resonance imaging (MRI) led to an attempt to use steroids to relieve the oedema and thereby the pain. On eight occasions, five patients with documented bone crises received conventional dose steroids (20 mg/m2/day) with considerable shortening of the attacks. On six occasions five further patients received high dose methylprednisolone (30 mg/kg intravenously or 1 g/m2 orally daily for two days), which was followed by oral prednisone for three to five days on the last four occasions. In this later group, pain relief was evident within several hours. Three treatments were given on an ambulatory basis. The MRI scan of one of these patients showed no subperiosteal fluid collection five days after high dose steroids had been started, and on subsequent x ray examination, there was no periosteal elevation. This treatment should be considered in cases of Gaucher's disease with bone crises.     

Keratoprosthesis results in animals: An update

Background: The report presented is an update on continuing development work on modified PHEMA core-and-shirt KPros in animals. Methods: Two variations (improved wet-eye, and dry-eye) of a prototype core-and-skirt Chirila KPro are described. The clinical success rate on implantation of these versions of the Chirila KPro was assessed. Results: It was found that a significant improvement in retention rate was shown in the improved model but that the dry-eye model failed early in two of the three implanted. Conclusions: The significance of the improved strength and the reasons for disappointing results with the early dry-eye KPros are discussed. Ongoing work is briefly outlined.     

Quantitation of apo-, mono-, and diferric transferrin by polyacrylamide gradient gel electrophoresis in patients with disorders of iron metabolism

We have developed a polyacrylamide gradient gel electrophoretic method to quantitate apo-, mono-, and diferric transferrin based upon differences in their molecular size. Purified transferrin saturated to different extents (3% to 98%) with iron showed proportions of the three forms as predicted from an approximately random distribution of iron between the two metal-binding sites. The iron distributions in sera of 14 normal individuals similarly correlated with the predicted values. In contrast, 22 of 43 patients with diseases associated with abnormalities in iron or transferrin metabolism had a disproportionate increase in monoferric transferrin. This abnormality occurred in seven of nine patients who had received bone marrow transplants, seven of 14 with chronic liver disease, and eight of nine menstruating women with probable iron deficiency anemia. Interestingly, 11 patients with malabsorption or chronic renal disease had normal iron distributions. The finding of abnormal distributions of iron on transferrin suggests that gradient gel analysis may be a useful tool for studying the physiologic mechanisms controlling iron utilization.