Prediction of cytochrome P450 isoform responsible for metabolizing a drug molecule

Background

ATP-binding cassette (ABC) transporters protect cells against unrelated (toxic) substances by pumping them across cell membranes. Earlier we showed that many ABC transporters are highly expressed in hematopoietic stem cells (HSCs) compared to more committed progenitor cells. The ABC transporter expression signature may guarantee lifelong protection of HSCs but may also preserve stem cell integrity by extrusion of agents that trigger their differentiation. Here we have studied whether non-hematopoietic stem cells (non-HSCs) exhibit a similar ABC transporter expression signature as HSCs.

Results

ABC transporter expression profiles were determined in non-hematopoietic stem cells (non-HSCs) from embryonic, neonatal and adult origin as well as in various mature blood cell types. Over 11,000 individual ABC transporter expression values were generated by Taqman Low Density Arrays (TLDA) to obtain a sensitivity comparable with quantitative real-time polymerase chain reactions. We found that the vast majority of transporters are significantly higher expressed in HSCs compared to non-HSCs. Furthermore, regardless their origin, non-HSCs exhibited strikingly similar ABC transporter expression profiles that were distinct from those in HSCs. Yet, sets of transporters characteristic for different stem cell types could be identified, suggesting restricted functions in stem cell physiology. Remarkably, in HSCs we could not pinpoint any single transporter expressed at an evidently elevated level when compared to all the mature blood cell types studied.

Conclusions

These findings challenge the concept that individual ABC transporters are implicated in maintaining stem cell integrity. Instead, a distinct ABC transporter expression signature may be essential for stem cell function. The high expression of specific transporters in non-HSCs and mature blood cells suggests a specialized, cell type dependent function and warrants further functional experiments to determine their exact roles in cellular (patho)physiology.     

Secondary and tertiary structure modeling reveals effects of novel mutations in polycystic liver disease genes PRKCSH and SEC63

Waanders E, Venselaar H, te Morsche RHM, de Koning DB, Kamath PS, Torres VE, Somlo S, Drenth JPH. Secondary and tertiary structure modeling reveals effects of novel mutations in polycystic liver disease genes PRKCSH and SEC63. Polycystic liver disease (PCLD) is characterized by intralobular bile duct cysts in the liver. It is caused by mutations in PRKCSH, encoding hepatocystin, and SEC63, encoding Sec63p. The main goals of this study were to screen for novel mutations and to analyze mutations for effects on protein structure and function. We screened 464 subjects including 76 probands by direct sequencing or conformation‐sensitive capillary electrophoresis. We analyzed the effects of all known and novel mutations using a combination of splice site recognition, evolutionary conservation, secondary and tertiary structure predictions, Poly Phen , and p Mut and sift . We identified a total of 26 novel mutations in PRKCSH (n = 14) and SEC63 (n = 12), including four splice site mutations, eight insertions/ deletions, six non‐sense mutations, and eight missense mutations. Out of 48 PCLD mutations, 13 were predicted to affect splicing. Most mutations were located in highly conserved regions and homology modeling for two domains of Sec63p showed severe effects of the residue substitutions. In conclusion, we identified 26 novel mutations associated with PCLD and we provide in silico analysis in order to delineate the role of these mutations.     

Identification of Clinically Important Anaerobic Bacteria by an Oligonucleotide Array

Anaerobic bacteria can cause a wide variety of infections, and some of these infections can be serious. Conventional identification methods based on biochemical tests are often lengthy and can produce inconclusive results. An oligonucleotide array based on the 16S-23S rRNA intergenic spacer (ITS) sequences was developed to identify 28 species of anaerobic bacteria and Veillonella. The method consisted of PCR amplification of the ITS regions with universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of 35 oligonucleotide probes (17- to 30-mers) immobilized on a nylon membrane. The performance of the array was determined by testing 310 target strains (strains which we aimed to identify), including 122 reference strains and 188 clinical isolates. In addition, 98 nontarget strains were used for specificity testing. The sensitivity and the specificity of the array for the identification of pure cultures were 99.7 and 97.1%, respectively. The array was further assessed for its ability to detect anaerobic bacteria in 49 clinical specimens. Two species (Finegoldia magna and Bacteroides vulgatus) were detected in two specimens by the array, and the results were in accordance with those obtained by culture. The whole procedure of array hybridization took about 8 h, starting with the isolated colonies. The array can be used as an accurate alternative to conventional methods for the identification of clinically important anaerobes.Anaerobic bacteria are important human pathogens, and infections caused by these bacteria can be serious and life-threatening (6). A recent report from the Mayo Clinic (Rochester, MN) revealed an overall increase in the incidence of anaerobic bacteremias of 74% from 2001 to 2004 compared to that from 1993 to 1996 (20), although the same trend was not found in community hospitals or in an European countries (2, 11). The commonly isolated anaerobic bacteria are the members of the Bacteroides fragilis group and Peptostreptococcus, Clostridium, and Fusobacterium species (3, 6, 20).Most clinical laboratories use differential biochemical tests for the identification of anaerobic microorganisms (35). However, Simmon et al. (31) found that 24% of the isolates of anaerobic bacteria recovered from blood cultures were misidentified and that 10% isolates were not identified to the species level by phenotypic characteristics. A rapid commercial kit, the Rapid ID 32A kit (bioMérieux, Marcy l''Etoile, France), was evaluated for its ability to identify strains in the Bacteroides fragilis group. The results showed that only 78.4% of the strains were correctly identified to the species level without supplemental tests (15). The success of the Rapid ID 32A system for species identification varied with different taxa (10), and a low identification rate (50%) was observed for fusobacteria (16). Veillonella isolates are relatively easily identified to the genus level, but the differentiation of Veillonella isolates at the species level remains difficult and inconclusive due to the lack of discriminatory tests (14). In recent years, increasing antimicrobial resistance for some anaerobic bacteria (1, 13, 33) were noted, especially for species in the B. fragilis group (40). The rapid identification of anaerobic bacteria and the administration of appropriate antimicrobials play crucial roles in preventing mortality and morbidity in patients (6).Molecular methods have emerged as accurate alternatives for the identification of anaerobic bacteria (21, 22, 34, 36). Approximately 9% isolates of bacteremic anaerobes could not be identified to the species level by 16S rRNA gene sequencing, although all isolates were correctly assigned to the genus level (31). Other molecular identification methods targeting the rRNA operon include PCR (32), real-time PCR (26), PCR-restriction fragment length polymorphism analysis (39), and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (37).The intergenic spacer (ITS) region separating the 16S and 23S rRNA genes has been suggested to be a good candidate for use for the identification of aerobic and anaerobic bacteria (8, 19, 42). Moreover, the DNA array technology has been applied to the identification of a variety of microorganisms (12, 17, 41). The aim of the study described here was to develop an oligonucleotide array based on the ITS sequences to identify 28 clinically important species of anaerobes and Veillonella.     

Array-based identification of species of the genera Abiotrophia, Enterococcus, Granulicatella, and Streptococcus

Some species of enterococci and streptococci are difficult to differentiate by phenotypic traits. The feasibility of using an oligonucleotide array for identification of 11 viridans group streptococci was previously established. The aim of this study was to expand the array to identify species of Abiotrophia (1 species), Enterococcus (18 species), Granulicatella (3 species), and Streptococcus (31 species and 6 subspecies). The method consisted of PCR amplification of the ribosomal DNA intergenic spacer (ITS) regions, followed by hybridization of the digoxigenin-labeled PCR products to a panel of oligonucleotide probes (16- to 30-mers) immobilized on a nylon membrane. Probes could be divided into three categories: species specific, group specific, and supplemental probes. All probes were designed either from the ITS regions or from the 3' ends of the 16S rRNA genes. A collection of 312 target strains (162 reference strains and 150 clinical isolates) and 73 nontarget strains was identified by the array. Most clinical isolates were isolated from blood cultures or deep abscesses, and only those strains having excellent species identification with the Rapid ID 32 STREP system (bioMérieux Vitek, Taipei, Taiwan) were used for array testing. The test sensitivity and specificity of the array were 100% (312/312) and 98.6% (72/73), respectively. The whole procedure of array hybridization took about 8 h, starting from isolated colonies, and the hybridization patterns could be read by the naked eye. The oligonucleotide array is accurate for identification of the above microorganisms and could be used as a reliable alternative to phenotypic identification methods.     

Excimer laser surface ablation: a review of recent literature

The aim was to review the recently published literature on excimer laser surface ablation procedures, including photorefractive keratectomy (PRK), laser sub‐epithelial keratomileusis (LASEK), microkeratome‐assisted PRK (epi‐LASIK) and trans‐epithelial (laser‐assisted) PRK, to help elucidate where and how surface ablation may best fit into current refractive surgical practice. The emphasis was on publications within the last three years and included systemic reviews, meta‐analyses and randomised controlled trials. Where such evidence did not exist, selective large series cohort studies, case‐controlled studies and case series with follow‐up preferably greater than six months were examined and included. Refractive and visual outcomes are excellent and comparable to those after LASIK even in complex cases after previous corneal surgery. Indeed, surface ablation combined with corneal collagen cross‐linking may be used in selected eyes with biomechanical instability, where LASIK is contraindicated. In addition, there is evidence to suggest that there may be less induction of higher order aberrations with surface techniques. Long‐term stability and safety appear to be extremely satisfactory. The literature supports the use of modern excimer laser surface treatments, with outcomes comparable to those after LASIK and evidence of less induction of higher‐order aberrations. Follow‐up studies at 10 to 20 years indicate excellent stability and safety.     

EARLY CARCINOMA TONGUE – SURGICAL OPTIONS

A retrospective analysis of 127 surgically treated cases of T-1, T-2 carcinoma of oral tongue during the period 1987-1990 was undertaken. 68.5 per cent (87) underwent hemiglossectomy and 31.5 per cent (40) underwent wide excision. There were loco-regional recurrences in 22 per cent (27). In the hemiglossectomy group 9 per cent (8 of 87) had local recurrences compared to 25 per cent (10 of 40) of wide excision group, (P = 0.01). Mean disease free survival was 40 months and 33 months for hemiglossectomy group and wide excision group respectively, (P = 0.006). It is seen that local recurrences are significantly less for the hemiglossectomy group compared to the wide excision group.KEY WORDS: Disease free survival, Early cancer, Recurrence, Surgery, Tongue     

Molecular characterization of U937-dependent T-cell co-stimulation

U937 cells provide a co-stimulatory signal for CD3-mediated T-cell activation which is independent of the CD28/CD80/CD86 interaction. This study set out to identify which molecules contribute to this co-stimulatory activity. Monoclonal antibodies (mAb) to the known accessory molecules CD11a, CD18, CD54 and CD45, all inhibited T-cell proliferation. Although CD11a/18 mAb inhibited U937/T-cell cluster formation as well as proliferation, CD45 enhanced the size of the clusters formed, suggesting that this was not the only mechanism of inhibition. The alternative co-stimulatory pathway provided by U937 cells preferentially stimulated a response in the CD18+ T-cell population, and this reflected the reduced sensitivity of CD8+ T cells to CD28-mediated activation. Monoclonal antibodies to three molecules, CD53, CD98 and CD147, also inhibited U937-dependent T-cell proliferation. The mAb to CD98 and CD147 were inhibitory when prepulsed on to the U937 cells, suggesting an effect mediated by these molecules on the antigen-presenting cell.