Novel Approach to Simulate Sleep Apnea Patients for Evaluating Positive Pressure Therapy Devices

Bench testing is a useful method to characterize the response of different automatic positive airway pressure (APAP) devices under well-controlled conditions. However, previous models did not consider the diversity of obstructive sleep apnea (OSA) patients’ characteristics and phenotypes. The objective of this proof-of-concept study was to design a new bench test for realistically simulating an OSA patient’s night, and to implement a one-night example of a typical female phenotype for comparing responses to several currently-available APAP devices. We developed a novel approach aimed at replicating a typical night of sleep which includes different disturbed breathing events, disease severities, sleep/wake phases, body postures and respiratory artefacts. The simulated female OSA patient example that we implemented included periods of wake, light sleep and deep sleep with positional changes and was connected to ten different APAP devices. Flow and pressure readings were recorded; each device was tested twice. The new approach for simulating female OSA patients effectively combined a wide variety of disturbed breathing patterns to mimic the response of a predefined patient type. There were marked differences in response between devices; only three were able to overcome flow limitation to normalize breathing, and only five devices were associated with a residual apnea-hypopnea index of <5/h. In conclusion, bench tests can be designed to simulate specific patient characteristics, and typical stages of sleep, body position, and wake. Each APAP device behaved differently when exposed to this controlled model of a female OSA patient, and should lead to further understanding of OSA treatment.     

Techniques for the detection of pathogenic Cryptococcus species in wood decay substrata and the evaluation of viability in stored samples

In this study, we evaluated several techniques for the detection of the yeast form of Cryptococcus in decaying wood and measured the viability of these fungi in environmental samples stored in the laboratory. Samples were collected from a tree known to be positive for Cryptococcus and were each inoculated on 10 Niger seed agar (NSA) plates. The conventional technique (CT) yielded a greater number of positive samples and indicated a higher fungal density [in colony forming units per gram of wood (CFU.g^-1) ] compared to the humid swab technique (ST). However, the difference in positive and false negative results between the CT-ST was not significant. The threshold of detection for the CT was 0.05.10³ CFU.g^-1, while the threshold for the ST was greater than 0.1.10³ CFU^-1. No colonies were recovered using the dry swab technique. We also determined the viability of Cryptococcus in wood samples stored for 45 days at 25ºC using the CT and ST and found that samples not only continued to yield a positive response, but also exhibited an increase in CFU.g^-1, suggesting that Cryptococcus is able to grow in stored environmental samples. The ST.1, in which samples collected with swabs were immediately plated on NSA medium, was more efficient and less laborious than either the CT or ST and required approximately 10 min to perform; however, additional studies are needed to validate this technique.     

Structural and functional relationships between prokaryotic and eukaryotic DNA polymerases.

The Bacillus subtilis phage luminal diameter 29 DNA polymerase, involved in protein-primed viral DNA replication, was inhibited by phosphonoacetic acid (PAA), a known inhibitor of alpha-like DNA polymerases, by decreasing the rate of elongation. Three highly conserved regions of amino acid homology, found in several viral alpha-like DNA polymerases and in the luminal diameter 29 DNA polymerase, one of them proposed to be the PAA binding site, were also found in the T4 DNA polymerase. This prokaryotic enzyme was highly sensitive to the drugs aphidicolin and the nucleotide analogues butylanilino dATP (BuAdATP) and butylphenyl dGTP (BuPdGTP), known to be specific inhibitors of eukaryotic alpha-like DNA polymerases. Two potential DNA polymerases from the linear plasmid pGKL1 from yeast and the S1 mitochondrial DNA from maize have been identified, based on the fact that they contain the three conserved regions of amino acid homology. Comparison of DNA polymerases from prokaryotic and eukaryotic origin showed extensive amino acid homology in addition to highly conserved domains. These findings reflect evolutionary relationships between hypothetically unrelated DNA polymerases.     

Transition from initiation to elongation in protein-primed phi 29 DNA replication: salt-dependent stimulation by the viral protein p6.

The transition step from the p3-dAMP initiation complex to the first elongated products, p3-(dAMP)2 and p3-(dAMP)3, requires a dATP concentration higher than that needed for the initiation reaction or for the further elongation of the p3-(dAMP)3 complex. The elongation in phi 29 DNA-protein p3 replication in vitro was strongly inhibited by salt. Under inhibitory salt concentration, the viral protein p6 greatly stimulated phi 29 DNA-protein p3 replication. The effect of protein p6 was not on the rate of elongation but on the amount of elongated product, stimulating the transition from initiation to formation of the first elongation products.     

Relationship between cellular RecA protein concentration and untargeted mutagenesis in Escherichia coli

We measured the production of untargeted mutations in the cI and cII genes of untreated λ phage undergoing a lytic cycle in UV-irradiated bacterial hosts. As previously shown, treatment with 4 μg/ml of rifampicin during post-irradiation incubation inhibited amplification of the RecA protein in these cells. In addition, we observed a decreased mutation rate compared to the untreated, irradiated bacteria. Treatment with 4 μg/ml or 8 μg/ml rifampicin did not prevent the UV induction of the umuDC operon, as judged by assay of β-galactosidase activity in a umuC-lacZ fusion strain. In contrast, the UV-induction of β-galactosidase in the sulA-lacZ fusion strain was decreased by 4 μg/ml rifampicin. The inhibition of untargeted mutagenesis by this drug treatment was also observed in a strain constitutive for SOS functions (lexA (Def)) as well as ina RecA-overproducing plasmid strain, that blocks induction of heat-shock proteins, factor(s) in wild-type recA⁺ cells. An htpR165-carrying strain, that blocks induction of heat-shock proteins, exhibited normal UV-promoted mutagenesis. A correlation was observed between the cellular concentration of RecA protein, increased spontaneously by a temperature shift in a lexA(Ts) strain, and the extent of UV-promoted untargeted mutagenesis. These results suggest a mechanistic role of RecA protein in this process.     

Identification of Endothelin Receptor Subtype (ETB) in Human Cerebral Cortex Using Subtype-Selective Ligands

Abstract: Specific endothelin (ET) binding sites were characterized in membranes prepared from human cerebral cortices using binding assay and cross-linking analysis. The presence of immunoreactive (IR) ET-1 was studied by radioimmunoassay. Saturation binding experiments revealed that the K _D and B _max for ¹²⁵I-ET-1 and ¹²⁵l-ET-3 to membranes from gray matter were 25 ± 6 pM and 115 ± 15 fmol/mg of protein and 24 ± 5 p M and 108 ± 13 fmol/mg of protein, respectively. Similar results were obtained for white matter. In the presence of 10 n M sarafotoxin-6c, which is selective for ET_B receptors, ¹²⁵I-ET-1 and ¹²⁵l-ET-3 binding was totally abolished. However, in the presence of 1 μ M BQ123, which is selective for ET_Areceptors, both bindings were not affected. These results suggest that the human cerebral cortex contains only ET_Breceptors. Cross-linking of ¹²⁵I-ET-1 and ¹²⁵l-ET-3 to membranes with disuccinimidyl suberate resulted in the labeling of two bands of 48 and 31 kDa. Concentrations of IR-ET-1 in the gray and white matter were 7.0 ± 3.2 and 2.5 ± 1.7 fmol/g wet weight, respectively. The demonstration of high-affinity ET_B receptors and the presence of IRET-1 suggest that the peptide may act as a neurotransmitter or neuromodulator in the human cerebral cortex.     

Sandhoff disease in Argentina: high frequency of a splice site mutation in the HEXB gene and correlation between enzyme and DNA-based tests for heterozygote detection

The level of -hexosaminidase activity in plasma and leukocytes and the frequency of three known HEXB mutations were studied in an Argentinean deme with high incidence of infantile Sandhoff disease. Two mutations were previously identified in one of two Sandhoff patients from the region, a splice mutation, IVS-2+1 GA, and a 4-bp deletion, CTTT_782–785. These mutations, and a 16kb deletion from the 5' end of the HEXB gene common in non-Argentineans, were screened in 9 Sandhoff patients (all unrelated), 24 obligate heterozygotes, 33 additional individuals belonging to families with affected members, and 64 randomly ascertained individuals from the high risk region. Of 31 independent alleles examined, including those of the two patients previously reported, 30 had the IVS-2 splice mutation and only the originally reported patient had the CTTT deletion. The 16-kb deletion was not observed. Further, among the 57 unaffected members of families with a previous history of Sandhoff disease, and absolute correlation was found between carrier diagnosis by enzyme assay of leukocytes and the DNA-based tests for mutation. One of the 64 controls was classified as a carrier by enzyme assay but did not have one of the three mutations screened. We conclude that a single mutation predominates in this Argentinean population and that the DNA-based test can be an effective supplement or alternative to enzyme-based testing.