Comparison of expectorated sputum after manual chest physical therapy and high-frequency chest compression

This study is a quantitative comparison of the sputum produced by 12 subjects with cystic fibrosis (CF) who received high-frequency chest compression (HFCC) and standard chest physical therapy (CPT) in randomized order. Six subjects routinely used manual CPT and six routinely used the HFCC. None had acute infections or hospitalization in the six weeks before the study. Two certified respiratory therapists alternated subjects and CPT vs HFCC order during the two weeks of the matched study. For all sessions, the expectorated sputum was collected in preweighed cups, which were reweighed immediately after collection and again after evaporation to dryness. The wet and dry weights of the sputum produced as a result of the two techniques were significantly different, with HFCC having greater weight. Regardless of the mode of therapy, the sputum produced by the subjects who regularly received HFCC had greater water content than did the sputum produced by those subjects who regularly received CPT. No significant difference was found between the two therapists regarding sputum expectorated by the subjects during CPT. These results show that sputum production by subjects with CF who receive CPT by certified respiratory therapists can be as great as the sputum produced by the same subjects who receive HFCC. The results also suggest that unknown factors attributed to the therapists may produce different levels of effort from time to time that may decrease the respiratory therapists' effectiveness, whereas the HFCC therapy may be more consistently effective because it is entirely machine based.     

Increasing peptide identifications and decreasing search times for ETD spectra by pre-processing and calculation of parent precursor charge

Background

Early diagnosis and treatment of Mycobacterium tuberculosis infection can prevent most deaths resulting from this pathogen; however, multidrug-resistant strains present serious threats to global tuberculosis control and prevention efforts. In this study, we identified antigens that could be used for the serodiagnosis of drug-resistant M. tuberculosis strains, using a proteomics-based analysis.

Results

Serum from patients infected with drug-resistant or drug-susceptible M. tuberculosis strains and healthy controls was subjected to two-dimensional gel electrophoresis using a western blot approach. This procedure identified nine immunoreactive proteins, which were subjected to MALDI-TOF-MS analysis. Six recombinant proteins, namely rRv2031c, rRv0444c, rRv2145c, rRv3692, rRv0859c, and rRv3040, were expressed and used to determine the immuno-reactivity of 100 serum samples. Antibody reactivity against rRv2031c, rRv3692, and rRv0444c was consistently observed. Among them, the best sensitivity and specificity of rRv3692 were 37% and 95% respectively. Furthermore, when rRv2031c and rRv3692 or rRv2031c, rRv3692, and rRv0444c were combined in 2:1 or equal amounts, the assay sensitivity and specificity were improved to 56.7% and 100% respectively.

Conclusions

These results suggest that Rv2031c, Rv3692, and Rv0444c are possible candidate biomarkers for effective use in the serodiagnosis of drug-resistant tuberculosis infections, and a combined formula of these antigens should be considered when designing a subunit assay kit.     

Increasing and decreasing functional area of the dentition (FAD) of Mammuthus primigenius

The occlusal morphology and continuous molar replacement in elephants provide a very effective functional area for grinding the biomass that is more or less abrasive. Parts of two subsequent molars contribute to the “functional area of the dentition” (FAD). The FAD size, measured in cm², is associated with age and body size. The FAD stage is indicated by the specific teeth contributing to the FAD and represents the individual age. This study concentrates on Mammuthus primigenius and compares the FAD stages, as derived from growth series, with the fossil Elephas antiquus, as well as the extant Elephas maximus and Loxodonta africana. During the life history of the taxa studied, the functional area increases simultaneously with an increase in body size, but decreases severely in senile age stages. In some senile individuals, the FAD is only about 20–50 % of the mean area of an adult animal. The reduction of the FAD beyond a specific size does not mean an immediate starvation of the animal. The general constitution of the individual and the resources of fat accumulation earlier may support the animal for some time but certainly not over a longer period. Nevertheless, the highly reduced functional area was sufficient to keep the animal alive despite its full adult body mass. A much larger FAD in all adult stages provides the energy requirements needed for all additional life functions including competition and reproduction.     

Oral atorvastatin therapy increases nitric oxide-dependent cutaneous vasodilation in humans by decreasing ascorbate-sensitive oxidants

Elevated low-density lipoproteins (LDL) are associated with cutaneous microvascular dysfunction partially mediated by increased arginase activity, which is decreased following a systemic atorvastatin therapy. We hypothesized that increased ascorbate-sensitive oxidant stress, partially mediated through uncoupled nitric oxide synthase (NOS) induced by upregulated arginase, contributes to cutaneous microvascular dysfunction in hypercholesterolemic (HC) humans. Four microdialysis fibers were placed in the skin of nine HC (LDL = 177 ± 6 mg/dl) men and women before and after 3 mo of a systemic atorvastatin intervention and at baseline in nine normocholesterolemic (NC) (LDL = 95 ± 4 mg/dl) subjects. Sites served as control, NOS inhibited, L-ascorbate, and arginase-inhibited+L-ascorbate. Skin blood flow was measured while local skin heating (42°C) induced NO-dependent vasodilation. After the established plateau in all sites, 20 mM ?ngname? was infused to quantify NO-dependent vasodilation. Data were normalized to maximum cutaneous vascular conductance (CVC) (sodium nitroprusside + 43°C). The plateau in vasodilation during local heating (HC: 78 ± 4 vs. NC: 96 ± 2% CVC(max), P < 0.01) and NO-dependent vasodilation (HC: 40 ± 4 vs. NC: 54 ± 4% CVC(max), P < 0.01) was reduced in the HC group. Acute L-ascorbate alone (91 ± 5% CVC(max), P < 0.001) or combined with arginase inhibition (96 ± 3% CVC(max), P < 0.001) augmented the plateau in vasodilation in the HC group but not the NC group (ascorbate: 96 ± 2; combo: 93 ± 4% CVC(max), both P > 0.05). After the atorvastatin intervention NO-dependent vasodilation was augmented in the HC group (HC postatorvastatin: 64 ± 4% CVC(max), P < 0.01), and there was no further effect of ascorbate alone (58 ± 4% CVC(max,) P > 0.05) or combined with arginase inhibition (67 ± 4% CVC(max,) P > 0.05). Increased ascorbate-sensitive oxidants contribute to hypercholesteromic associated cutaneous microvascular dysfunction which is partially reversed with atorvastatin therapy.     

Increasing and decreasing protein stability: effects of revertant substitutions on the thermal denaturation of phage lambda repressor

The thermal denaturations of five revertant lambda repressors containing single amino acid substitutions in their N-terminal domains have been studied by differential scanning calorimetry. Two substitutions slightly decrease stability, and the remaining three render the protein more stable than wild type. The Gly48----Asn and Gly48----Ser proteins are 4 degrees C more stable than wild type. These two substitutions replace an alpha helical residue, and in each case a poor helix forming residue, glycine, is replaced by a residue with a higher helical propensity. We also present data showing that one revertant, Tyr22----Phe, has reduced operator DNA binding affinity despite its enhanced stability.     

Hereditary melanoblastoma in miniature pigs and its successful therapy by devitalization technique.

Applying selective breeding for 10 years we established the MeLiM (Melanoblastoma-bearing Libechov Minipigs) strain. Melanoblastoma (MB) in this strain shows a hereditary occurrence. Cutaneous tumours are usually nodular, multiple and distributed on various parts of body. They appear in darkly pigmented animals already at the birth or during two months thereafter (57% of all animals). Numerous organ metastases mainly into the spleen, lymph nodes and lungs are regularly ascertained in animals with cutaneous MB. Tumour cells were surprisingly found also in the inner organs of phenotypically healthy minipigs in which no cutaneous MBs were observed visually (27% of all animals). About 34% of all affected piglets die during the first 2 months of age. These features document a malignancy of this tumour in the MeLiM strain. Original surgical technique was applied in more than 40 affected minipigs at 1-2 months of age. It consists in a devitalization (ischemization) of one of cutaneous tumours by the mattress sutures conducted around the tumour base without any excision of tumour tissue. This simple procedure causes a total destruction of MB cells in all cutaneous tumours as well as in all organ metastases during 4-6 months. Animals treated by this technique were fully healed of tumour cells and no relapses were observed. This technique could bring similar positive results also in therapy of human MB.     

Increasing Himalayan tahr and decreasing chamois densities in the eastern Southern Alps, New Zealand: evidence for interspecific competition

There is anecdotal evidence that increasing densities of Himalayan tahr (Hemitragus jemlahicus) are associated with declining densities of chamois (Rupicapra rupicapra) in the Southern Alps, New Zealand. To examine this phenomenon, densities of tahr and chamois were measured at 53 sites within their sympatric range in the eastern Southern Alps during 1978–1979. In sites where only one species was present, tahr density was significantly higher than chamois density (P=0.032), probably reflecting species differences in sociality. Chamois density was higher in catchments without tahr than in those with tahr (P=0.012). Similarly, tahr density was significantly higher at sites without chamois than at those with chamois (P=0.033). Sites with both species present (termed sympatric) were significantly larger than those with only chamois or tahr (P<0.001). Following the prohibition of aerial hunting of tahr in 1983, 16 of the 17 sites where tahr and chamois were sympatric during 1978–1979 were recounted during 1991–1996. There was a 6-fold increase in the mean density of tahr between the two counts (P=0.001), whereas chamois density had declined significantly (P=0.006). Chamois persisted at only three sites, two of which had the highest chamois densities in 1978–1979. This is evidence that increasing densities of tahr exclude chamois from all but the `best' habitats. We conclude that intensive aerial hunting of tahr during 1967–1983 reduced tahr densities such that chamois could co-exist with tahr. Received: 20 March 1997 / Accepted: 8 September 1997     

Colorimetric determination of inorganic pyrophosphate by a manual or automated method.

A microprocedure for the colorimetric determination of inorganic pyrophosphate (PP_i) in the presence or absence of orthophosphate (P_i) has been developed. PP_i is estimated quantitatively as the amount of chromophore formed with molybdate reagent, 1-amino-2-naphthol-4-sulfonic acid in bisulfite and thiol reagent (monothioglycerol or 2-mercaptoethanol). The latter is obligatory for color formation. P_i is estimated without thiol reagent. The two chromophores differ in absorption spectra, the greatest difference being at 580 nm. For both, color develops fully by 10 min and is stable up to 1 hr. Just less than 0.4 μm PP_i can be detemined. The extinction coefficients are 2.70 × 10⁴ and 8.76 × 10³ for PP_i and P_i, respectively, both with thiol reagent present, and 2.77 × 10³ for P_i with no thiol reagent.A ten-fold excess of P_i does not interfere with the determination of PP_i and in fact can be estimated in the same mixture. A 15-fold excess, however, diminishes the accuracy of PP_i estimations. Trichloroacetic acid and sodium fluoride inhibi color formation, but this inhibition is overcome by the addition of sodium acetate buffer, pH 4.0. Nucleoside triphosphates and adenosine 3′:5′-cyclic monophosphate are stable in the reaction mixture.The method was tested in assays of Escherichia coli DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6). Progress curves measured by either the rate of PP_i formation or the rate of synthesis of labeled RNA were very similar. Product PP_i formed by as little as 0.6 unit of RNA polymerase in a 225-μl incubation medium could be measured.An automated version of the method was devised which allows accurate determination of PP_i down to 1 μm (without range expander attachment) at a sampling rate of 20–40 tubes/hr.     

Increasing the net charge and decreasing the hydrophobicity of bovine carbonic anhydrase decreases the rate of denaturation with sodium dodecyl sulfate

This study compares the rate of denaturation with sodium dodecyl sulfate (SDS) of the individual rungs of protein charge ladders generated by acylation of the lysine epsilon-NH3+ groups of bovine carbonic anhydrase II (BCA). Each acylation decreases the number of positively charged groups, increases the net negative charge, and increases the hydrophobic surface area of BCA. This study reports the kinetics of denaturation in solutions containing SDS of the protein charge ladders generated with acetic and hexanoic anhydrides; plotting these rates of denaturation as a function of the number of modifications yields a U-shaped curve. The proteins with an intermediate number of modifications are the most stable to denaturation by SDS. There are four competing interactions-two resulting from the change in electrostatics and two resulting from the change in exposed hydrophobic surface area-that determine how a modification affects the stability of a rung of a charge ladder of BCA to denaturation with SDS. A model based on assumptions about how these interactions affect the folded and transition states has been developed and fits the experimental results. Modeling indicates that for each additional acylation, the magnitude of the change in the activation energy of denaturation (DeltaDeltaG(double dagger)) due to changes in the electrostatics is much larger than the change in DeltaDeltaG(double dagger) due to changes in the hydrophobicity, but the intermolecular and intramolecular electrostatic effects are opposite in sign. At the high numbers of acylations, hydrophobic interactions cause the hexanoyl-modified BCA to denature nearly three orders of magnitude more rapidly than the acetyl-modified BCA.     

Increasing secretion of a bivalent anti-T-cell immunotoxin by Pichia pastoris

The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G(4)S) was developed for treatment of T-cell leukemia and autoimmune diseases and for tolerance induction for transplantation. This immunotoxin was produced extracellularly in toxin-sensitive Pichia pastoris JW102 (Mut(+)) under control of the AOX1 promoter. There were two major barriers to efficient immunotoxin production, the toxicity of the immunotoxin for P. pastoris and the limited capacity of P. pastoris to secrete the immunotoxin. The immunotoxin toxicity resulted in a decrease in the methanol consumption rate, cessation of cell growth, and low immunotoxin productivity after the first 22 h of methanol induction. Continuous cell growth and continuous immunotoxin secretion after the first 22 h of methanol induction were obtained by adding glycerol to the methanol feed by using a 4:1 methanol-glycerol mixed feed as an energy source and by continuously adding a yeast extract solution during methanol induction. The secretory capacity was increased from 22.5 to 37 mg/liter by lowering the induction temperature. A low temperature reduced the methanol consumption rate and protease activity in the supernatant but not cell growth. The effects of adding glycerol and yeast extract to the methanol feed were synergistic. Adding yeast extract primarily enhanced methanol utilization and cell growth, while adding glycerol primarily enhanced immunotoxin production. The synergy was further enhanced by decreasing the induction temperature from 23 to 15 degrees C, which resulted in a robust process with a yield of 37 mg/liter, which was sevenfold greater than the yield previously reported for a toxin-resistant CHO cell expression system. This methodology should be applicable to other toxin-related recombinant proteins in toxin-sensitive P. pastoris.     

Increasing Secretion of a Bivalent Anti-T-Cell Immunotoxin by Pichia pastoris

The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G₄S) was developed for treatment of T-cell leukemia and autoimmune diseases and for tolerance induction for transplantation. This immunotoxin was produced extracellularly in toxin-sensitive Pichia pastoris JW102 (Mut⁺) under control of the AOX1 promoter. There were two major barriers to efficient immunotoxin production, the toxicity of the immunotoxin for P. pastoris and the limited capacity of P. pastoris to secrete the immunotoxin. The immunotoxin toxicity resulted in a decrease in the methanol consumption rate, cessation of cell growth, and low immunotoxin productivity after the first 22 h of methanol induction. Continuous cell growth and continuous immunotoxin secretion after the first 22 h of methanol induction were obtained by adding glycerol to the methanol feed by using a 4:1 methanol-glycerol mixed feed as an energy source and by continuously adding a yeast extract solution during methanol induction. The secretory capacity was increased from 22.5 to 37 mg/liter by lowering the induction temperature. A low temperature reduced the methanol consumption rate and protease activity in the supernatant but not cell growth. The effects of adding glycerol and yeast extract to the methanol feed were synergistic. Adding yeast extract primarily enhanced methanol utilization and cell growth, while adding glycerol primarily enhanced immunotoxin production. The synergy was further enhanced by decreasing the induction temperature from 23 to 15°C, which resulted in a robust process with a yield of 37 mg/liter, which was sevenfold greater than the yield previously reported for a toxin-resistant CHO cell expression system. This methodology should be applicable to other toxin-related recombinant proteins in toxin-sensitive P. pastoris.     

FP-TDI SNP scoring by manual and statistical procedures: a study of error rates and types

For technologies that are commonly used in ordinary laboratories such as fluorescence-polarization detection with template-directed, dye-terminator incorporation (FP-TDI), SNP genotype scoring is usually done manually. Here we study rates of errors and missing genotypes obtained with this procedure. We also introduce three statistical genotype scoring methods to examine whether they form a viable alternative. Data consisted of eight SNPs typed in about 1400 individuals from 268 pedigrees. The statistical procedures performed better on several internal criteria, such as the number of Mendelian errors, and showed much higher agreement with discrepant genotypes re-scored by two raters. The best results were obtained with the statistical procedure that incorporated information about regularities in the error structure of the FP-TDI data. We estimated that there were about 1.6% more errors if genotypes were scored manually. About 0.6% of these errors could be explained by data manipulation errors, leaving 1% as the result of possible incorrect scoring. There were 3.3% more missing genotypes in the manual scoring due to errors in data manipulation (1.7%) and conservative scoring (1.6%).     

Automated counting of phytoplankton by pattern recognition: a comparison with a manual counting method

Computer-based image analysis and pattern recognition methodswere used to construct a system able automatically to identify,count and measure selected groups of phytoplankton. An imageanalysis algorithm was employed to isolate and measure objectsfrom digitized images of a phytoplankton sample. The measurementsobtained were used to identify selected groups of phytoplanktonby a combination of artificial neural networks and simple rule-basedprocedures. The system was trained and tested using samplesof lake water covering an annual growth cycle from Lough Neaghin Northern Ireland. Total volume estimates were obtained forthe four major phytoplankton species, using both the automatedsystem and a manual counting method. Estimates of total cellvolume obtained from the automated system were within 10% ofthose derived by manual analysis of the same cells. The automatedsystem produced total cell volume estimates close to those obtainedfrom manual analysis of different aliquots of the same watersample. Variation between successive counts of the same watersample was higher with the automated system than with the manualcounting method. Limitations and possible improvements to thetechnology are discussed.