The application of response surface methodology to sterilization process development and validation of a water cascade autoclave.

The application of Response Surface Methodology (RSM) to the performance validation of a water cascade sterilizer is described. The methodology was successfully used to 1) identify the cold and hot zones within maximum load configurations through three dimensional thermal mapping, 2) demonstrate the consistency of consecutive sterilization cycles in companion sterilizers, and 3) set target sterilizing values (F0) to achieve a high statistical assurance that any location will be above a minimum F0 (sterility assurance) and below a maximum F0 (for stability considerations).

Neocentromere activity of structurally acentric mini-chromosomes in Drosophila.

Chromosome fragments that lack centromeric DNA (structurally acentric chromosomes) are usually not inherited in mitosis and meiosis. We previously described the isolation, after irradiation of a Drosophila melanogaster mini-chromosome, of structurally acentric mini-chromosomes that display efficient mitotic and meiotic transmission despite their small size (under 300 kb) and lack of centromeric DNA. Here we report that these acentric mini-chromosomes bind the centromere-specific protein ZW10 and associate with the spindle poles in anaphase. The sequences in these acentric mini-chromosomes were derived from the tip of the X chromosome, which does not display centromere activity or localize ZW10, even when separated from the rest of the X. We conclude that the normally non-centromeric DNAs present in these acentric mini-chromosomes have acquired centromere function, and suggest that this example of 'neocentromere' formation involves appropriation of a self-propagating centromeric chromatin structure. The potential relevance of these observations to the identity, propagation and function of normal centromeres is discussed.

Identification of trans-acting genes necessary for centromere function in Drosophila melanogaster using centromere-defective minichromosomes.

Deletions in the Drosophila minichromosome Dp1187 were used to investigate the genetic interactions of trans-acting genes with the centromere. Mutations in several genes known to have a role in chromosome inheritance were shown to have dominant effects on the stability of minichromosomes with partially defective centromeres. Heterozygous mutations in the ncd and klp3A kinesin-like protein genes strongly reduced the transmission of minichromosomes missing portions of the genetically defined centromere but had little effect on the transmission of minichromosomes with intact centromeres. Using this approach, ncd and klp3A were shown to require only the centromeric region of the chromosome for their roles in chromosome segregation. Increased gene dosage also affected minichromosome transmission and was used to demonstrate that the nod kinesin-like protein gene interacts genetically with the centro mere, in addition to interacting with extracentromeric regions as demonstrated previously. The results presented in this study strongly suggest that dominant genetic interactions between mutations and centromere-defective minichromosomes could be used effectively to identify novel genes necessary for centromere function.

Localization of centromere function in a Drosophila minichromosome.

The DNA elements responsible for centromere activity in a metazoan have been localized using the Drosophila minichromosome Dp1187. Deleted minichromosomes were generated by irradiation mutagenesis, and their molecular structures were determined by pulsed-field Southern blot analysis. Analyses of the transmission behavior of Dp1187 derivatives localized sequences necessary for chromosome inheritance within the centric heterochromatin. The essential core of the centromere is contained within a 220 kb region that includes significant amounts of complex DNA. Completely normal inheritance also requires approximately 200 kb on either side of the essential core. This flanking DNA predominantly contains highly repeated sequences, and the amount required for normal transmission differs among division types and between the sexes. We propose that the essential core is the site of kinetochore formation and that flanking DNA provides two functions: sister chromatid cohesion and indirect assistance in kinetochore formation or function.

Validity of self-assessment of sexual maturation in adolescent male patients with cystic fibrosis.

PURPOSE: Self-assessment of sexual maturity by healthy male adolescents has been found to correlate closely with physician ratings. Data are lacking, however, in adolescents with cystic fibrosis (CF), where an altered body image may affect self-assessment. Delayed sexual maturation occurs in many patients with CF, particularly those with severe disease. We hypothesized that self-assessment of sexual maturation by adolescents with CF would agree with physician ratings. METHODS: Using Tanner's standard photographs for pubic hair (PH) and genital (G) development, we compared self-assessment of sexual maturation to physician rating in 34 adolescent male patients with CF and 27 healthy male controls (C). RESULTS: The two groups did not differ in age. All subjects were initially examined and Tanner-staged by a physician (SRB), and instructed in self-assessment using the Tanner photographs; they then performed a self-assessment. Scores by physician and subjects were assessed for inter-observer agreement by Kappa analysis. For the CF group, 29 of 34 PH assessments and 21 of 34 G assessments demonstrated exact inter-observer agreement between physician and subject ratings. The Kappa coefficient, kappa, (weighted for the degree of closeness between two observers) was 0.946 for PH and 0.840 for G and the C group, kappa was 0.905 for PH and 0.737 for G. Repeat analysis combining stages 3 and 4 PH and G development yielded higher inter-observer agreement in the CF group (33 of 34 PH assessments and 26 of 34 G assessments) and in the C group (24 of 27 PH assessments and 18 of 27 G assessments). CONCLUSIONS: Self-assessment is a valid method to assess sexual maturity in clinical evaluation and as a research tool in the study of patients with CF.

Interactions between the nod+ kinesin-like gene and extracentromeric sequences are required for transmission of a Drosophila minichromosome.

In this study, we demonstrate a role for extracentromeric sequences in chromosome inheritance. Genetic analyses indicate that transmission of the Drosophila minichromosome Dp1187 is sensitive to the dosage of nod+, a kinesin-like gene required for the meiotic transmission of achiasmate chromosomes. Minichromosome deletions displayed increased loss rates in females heterozygous for a loss-of-function allele of nod (nod/+). We have analyzed the structures of nod-sensitive deletions and conclude that multiple regions of Dp1187 interact genetically with nod+ to promote normal chromosome transmission. Most nod+ interactions are observed with regions that are not essential for centromere function. We propose that normal chromosome transmission requires forces generated outside the kinetochore, perhaps to maintain tension on kinetochore microtubules and stabilize the attachment of achiasmate chromosomes to the metaphase spindle.

Diaphragmatic failure during group B streptococcal sepsis in piglets: the role of thromboxane A2.

Group B Streptococcus (GBS) causes an impairment of diaphragmatic pressure generation (Pdi) in 2-wk-old piglets, whereas 4-wk-old piglets are unaffected. In this study, we examined the effect on 4-wk-old piglets of a higher dose of GBS than previously utilized. We sought to determine whether an eicosanoid product of arachidonic acid metabolism accounted for the decrease in Pdi during GBS infusion and whether thromboxane A2 (TxA2) is the putative eicosanoid mediator of decreased Pdi during GBS infusion. Measuring Pdi during phrenic nerve stimulation, we studied four groups of anesthetized spontaneously breathing 4-wk-old piglets. Group 1 (GBS) was infused with live GBS, which caused a decrease in Pdi by 1 h at 20-, 30-, 50-, and 100-Hz stimulation frequencies. Group 2 [GBS + indomethacin (Indo)] was pretreated with Indo before GBS infusion. In the GBS + Indo group, Pdi did not decrease throughout 4 h of GBS infusion. Because Indo proved to be protective of Pdi during GBS infusion, we examined the role of TxA2, the only eicosanoid present at 1 h in the serum of GBS-infused piglets. Group 3 was infused with the TxA2 analogue U-46619 only for 1 h. Group 4 was treated with the TxA2-receptor antagonist SQ-29548 before and concomitant with GBS infusion for 1 h; the SQ-29548 was then discontinued, and GBS was continued for 1 h more. In the U-46619-infused group, Pdi decreased at 1 h, and in the SQ-29548-treated group, Pdi did not decrease during GBS infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of group B streptococcal sepsis on diaphragmatic function in young piglets.

Recent studies indicate that diaphragmatic pressure generation (Pdi) is impaired by bacterial infection. However, group B streptococcus (GBS) had no effect on Pdi when infused into 4-wk-old piglets. As responsiveness to GBS is age-dependent, we therefore studied the acute effect of GBS infusion on Pdi, using a younger, 2-wk-old piglet model. Using trans-Pdi with phrenic nerve stimulation, we studied the effect of continuous GBS infusion in seven anesthetized, spontaneously breathing 2-wk-old piglets. Pdi was measured under baseline conditions (50% O2/50% N2) and at 1, 2, and 4 h of GBS infusion. GBS was infused at a rate which caused a doubling of the pulmonary artery pressure but which avoided hypotension or acidosis--both of which can decrease Pdi. In addition, the piglets were kept hyperoxic [PaO2 > 13.3 kPa (100 torr)], and no piglet with hypercapnia [PaCO2 > 8.7 kPa (65 torr)] was included, as hypoxia and hypercapnia can also cause respiratory muscle dysfunction. For the GBS group, diaphragmatic contractility declined significantly by 1 h at 30-, 50-, and 100-Hz stimulation frequency and, by 2 h, was significantly decreased at all frequencies. We conclude that 2-wk-old piglets, in contrast to 4-wk-old piglets, demonstrate a decline in Pdi during GBS infusion. These data demonstrate an age-related response to GBS in the piglet.(ABSTRACT TRUNCATED AT 250 WORDS)

Group B streptococcus has no effect on piglet diaphragmatic force generation.

Recent studies indicate that diaphragmatic contractility is adversely affected by bacterial infection. Using transdiaphragmatic pressure (Pdi) with phrenic nerve stimulation, the effect of continuous Group B Streptococcus (GBS) infusion on diaphragmatic force output was studied in seven anesthetized, spontaneously breathing 1-month old piglets. Pdi was measured under baseline condition (50% O2/50% N2) and at 1, 2, and 4 h of GBS infusion. The GBS was infused at a level that caused a doubling of the pulmonary artery pressure and a 32% decrease in cardiac output but which avoided hypotension or acidosis--both of which can decrease diaphragmatic contractility. In addition, the piglets were kept hyperoxic (PaO2 greater than 100) and no piglet with hypercapnia (PaCO2 greater than 65) was studied, as hypoxia and hypercapnia also can cause respiratory muscle dysfunction. Pdi in response to phrenic nerve stimulation did not change during GBS infusion. We conclude that GBS infusion, in the absence of hypotension, hypercapnia, hypoxia, or acidosis, has no effect on diaphragmatic force generation in the piglet.

Diaphragmatic force and substrate response to resistive loaded breathing in the piglet.

Inspiratory resistive loaded (IRL) breathing results in hypoventilation and diaphragmatic fatigue in the piglet. We studied the effects of 6 h of IRL on ten 1-mo-old piglets. The load was adjusted to increase spontaneously generated transdiaphragmatic pressure five to six times baseline. Six 1-mo-old piglets acted as controls and were identically instrumented but were not subjected to IRL. Measurements of ventilation, blood gases and pH, diaphragmatic electromyogram, force-frequency curve, blood flow, and end-expiratory lung volume were obtained hourly. Diaphragmatic muscle samples were obtained after 6 h for determination of ATP, phosphocreatine, lactate, and glycogen levels. No changes occurred in the control animals. IRL resulted in a significant decrease in ventilation, an increase in diaphragmatic EMG, onset of abdominal expiratory muscle activity, and a fall in end-expiratory lung volume by 1 h. The force-frequency curve adjusted for lung volume change fell by 20% at all frequencies of stimulation at 1 h and by 40% at 6 h. Blood flow to the costal and crural diaphragm increased by 51 and 141%, respectively. No differences were noted in ATP, phosphocreatine, lactate, or glycogen between control and IRL animals. It is concluded that submaximal spontaneous contractions of the piglet diaphragm over a 6-h period cause a substantial decrease in its maximal force-generating capacity that is not related to substrate depletion.

Given versus induced category representations: use of prototype and exemplar information in classification.

Acquisition of category-level information can be based on experience with category members (induced) as well as on direct presentation of prototypical values (given). To investigate the effects of these two types of information, a relational coding model of categorization was developed in which classification is based on a mixture of exemplar and prototype information. In two experiments, subjects learned about two ill-defined categories. Stimuli were geometric shapes varying along four binary-valued dimensions. For three groups of subjects, training consisted of (a) experience with exemplars only, (b) learning prototype values followed by exemplar experience, or (c) learning prototype values concurrently with exemplar experience. Following training, all subjects received classification tests on prototype values as well as on old and new exemplars. By varying the relative use of prototype and exemplar information, the mixture model accurately accounted for category judgements in all three groups. For subjects directly presented with prototype values, classification was based on a mixture of similarity to prototypes and to stored exemplars. In contrast, subjects who only received experience with exemplars appeared to base their category judgements solely on similarity to stored exemplars, even though they could accurately judge the prototype values. The two components of the mixture model are related to subjects' classification strategies and the nature of abstracted, category-level information.