A nonradioisotope biomedical assay for intact oligonucleotide and its chain-shortened metabolites used for determination of exposure and elimination half-life of antisense drugs in tissue.

Rigorous extraction methods coupled with capillary gel electrophoresis (CGE) provide a basis for a nonradiolabel assay for quantitation of intact antisense drug and its numerous chain-shortened metabolites. As part of the validation of the CGE method, we compared the quantitation of unlabeled ISIS 3521 (ISI 641A) and its chain-shortened metabolites with total radioactivity of [(35)S]-ISIS 3521. ISIS 3521 was labeled on the fifth nucleotide linkage from the 5'-end with (35)S by well-established methods. Multiple tissues collected from rats after administration of [(35)S]-ISIS 3521 were assayed by both radiolabel (liquid scintillation spectroscopy) and CGE methods. The CGE method provided accurate quantitation of the drug and its metabolites in kidney cortex and liver tissues. The correlation between methods for multiple tissues over time was excellent with 88.5% of the measurements being statistically equivalent. These data suggest that CGE is an accurate means of quantitating oligonucleotide in tissue and that it compares favorably with traditional radiochemical techniques. Clearance half-lives for total measurable oligonucleotides were equivalent to clearance of total radioactivity in both liver and kidney with the longest clearance half-life associated with the kidney.     

Putative Phage Hyperparasite in the Rickettsial Pathogen of Abalone, “Candidatus Xenohaliotis californiensis”

Studies on the ecology of microbial parasites and their hosts are predicated on understanding the assemblage of and relationship among the species present. Changes in organismal morphology and physiology can have profound effects on host–parasite interactions and associated microbial community structure. The marine rickettsial organism, “Candidatus Xenohaliotis californiensis” (WS-RLO), that causes withering syndrome of abalones has had a consistent morphology based on light and electron microscopy. However, a morphological variant of the WS-RLO has recently been observed infecting red abalone from California. We used light and electron microscopy, in situ hybridization and16S rDNA sequence analysis to compare the WS-RLO and the morphologically distinct RLO variant (RLOv). The WS-RLO forms oblong inclusions within the abalone posterior esophagus (PE) and digestive gland (DG) tissues that contain small rod-shaped bacteria; individual bacteria within the light purple inclusions upon hematoxylin and eosin staining cannot be discerned by light microscopy. Like the WS-RLO, the RLOv forms oblong inclusions in the PE and DG but contain large, pleomorphic bacteria that stain dark navy blue with hematoxylin and eosin. Transmission electron microscopy (TEM) examination revealed that the large pleomorphic bacteria within RLOv inclusions were infected with a spherical to icosahedral-shaped putative phage hyperparasite. TEM also revealed the presence of rod-shaped bacteria along the periphery of the RLOv inclusions that were morphologically indistinguishable from the WS-RLO. Binding of the WS-RLO-specific in situ hybridization probe to the RLOv inclusions demonstrated sequence similarity between these RLOs. In addition, sequence analysis revealed 98.9–99.4 % similarity between 16S rDNA sequences of the WS-RLO and RLOv. Collectively, these data suggest that both of these RLOs infecting California abalone are “Candidatus Xenohaliotis californiensis,” and that the novel variant is infected by a putative phage hyperparasite that induced morphological variation of its RLO host.     

Language,Reading, and Math Learning Profiles in an Epidemiological Sample of School Age Children

Dyscalculia, dyslexia, and specific language impairment (SLI) are relatively specific developmental learning disabilities in math, reading, and oral language, respectively, that occur in the context of average intellectual capacity and adequate environmental opportunities. Past research has been dominated by studies focused on single impairments despite the widespread recognition that overlapping and comorbid deficits are common. The present study took an epidemiological approach to study the learning profiles of a large school age sample in language, reading, and math. Both general learning profiles reflecting good or poor performance across measures and specific learning profiles involving either weak language, weak reading, weak math, or weak math and reading were observed. These latter four profiles characterized 70% of children with some evidence of a learning disability. Low scores in phonological short-term memory characterized clusters with a language-based weakness whereas low or variable phonological awareness was associated with the reading (but not language-based) weaknesses. The low math only group did not show these phonological deficits. These findings may suggest different etiologies for language-based deficits in language, reading, and math, reading-related impairments in reading and math, and isolated math disabilities.     

Isolation and characterization of the plasma membrane from the yeast Pichia pastoris

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.     

Disparity in Physician Perception of Patients' Adherence to Medications by Obesity Status

Physician perception of medication adherence may alter prescribing patterns. Perception of patients has been linked to readily observable factors, such as race and age. Obesity shares a similar stigma to these factors in society. We hypothesized that physicians would perceive patients with a higher BMI as nonadherent to medication. Data were collected from the baseline visit of a randomized clinical trial of patient–physician communication (240 patients and 40 physicians). Physician perception of patient medication adherence was measured on a Likert scale and dichotomized as fully adherent or not fully adherent. BMI was the predictor of interest. We performed Poisson regression analyses with robust variance estimates, adjusting for clustering of patients within physicians, to examine the association between BMI and physician perception of medication adherence. The mean (s.d.) BMI was 32.6 (7.7) kg/m². Forty‐five percent of patients were perceived as nonadherent to medications by their physicians. Higher BMI was significantly and negatively associated with being perceived as adherent to medication (prevalence ratio (PrR) 0.76, 95% confidence interval (CI): 0.64–0.90; P = 0.002; per 10 kg/m² increase in BMI). BMI remained significantly and negatively associated with physician perception of medication adherence after adjustment for patient and physician characteristics (PrR 0.80, 95% CI: 0.66–0.96; P = 0.020). In this study, patients with higher BMI were less likely to be perceived as adherent to medications by their providers. Physician perception of medication adherence has been shown to affect prescribing patterns in other studies. More work is needed to understand how this perception may affect the care of patients with obesity.     

Molecular Serotyping of Escherichia coli O26:H11

Serotyping is the foundation of pathogenic Escherichia coli diagnostics; however, few laboratories have this capacity. We developed a molecular serotyping protocol that targets, genetically, the same somatic and flagellar antigens of E. coli O26:H11 used in traditional serotyping. It correctly serotypes strains untypeable by traditional methods, affording primary laboratories serotyping capabilities.     

Dimorphism in methane seep-dwelling ecotypes of the largest known bacteria

We present evidence for a dimorphic life cycle in the vacuolate sulfide-oxidizing bacteria that appears to involve the attachment of a spherical Thiomargarita-like cell to the exteriors of invertebrate integuments and other benthic substrates at methane seeps. The attached cell elongates to produce a stalk-like form before budding off spherical daughter cells resembling free-living Thiomargarita that are abundant in surrounding sulfidic seep sediments. The relationship between the attached parent cell and free-living daughter cell is reminiscent of the dimorphic life modes of the prosthecate Alphaproteobacteria, but on a grand scale, with individual elongate cells reaching nearly a millimeter in length. Abundant growth of attached Thiomargarita-like bacteria on the integuments of gastropods and other seep fauna provides not only a novel ecological niche for these giant bacteria, but also for animals that may benefit from epibiont colonization.     

The disposition of ethiofos (WR-2721) in the isolated perfused rat liver

We have investigated the disposition of ethiofos (20 mg, 4 microCi [14C]ethiofos) in the isolated perfused rat liver preparation to determine the hepatic contribution to the poor oral bioavailability of the drug. Ethiofos clearance (10.6 +/- 3.3 ml h-1) was only a small fraction (1.2 +/- 0.03%) of the perfusate flow rate. The elimination half-life was calculated at 7.1 +/- 1.9 h. The area under curve, AUC0-4 h, for ethiofos (2858 +/- 314 nM h ml-1) was not significantly different from that of 14C (3038 +/- 692 nM h ml-1) or total material convertible to WR-1065 (total WR-1065, 3324 +/- 612 nM h ml-1), indicating a low level of metabolism. The AUC0-4 h for free WR-1065 (37.5 +/- 23.3 nM h ml-1) was less than 2% of ethiofos. Biliary elimination of ethiofos, WR-1065, and 14C was below 1%. At 4 h postdose, 7.9 +/- 1.9% of the dose of radioactivity remained in the liver. Less than 1.5% could be identified as ethiofos (0.12 +/- 0.09%) or total WR-1065 (1.09 +/- 0.05%). Ethiofos, 14C, and total WR-1065 were approximately evenly distributed between the 10,000-g pellet and supernatant. However, significantly more ethiofos, WR-1065, and 14C were recovered from the 105,000-g supernatant compared with the pellet. In summary, both the metabolism and biliary elimination of ethiofos and its derivatives were sparing. Hence it is likely that in the rat, the contribution of the liver to the presystemic biotransformation and poor bioavailability of ethiofos is relatively minor.