Drug-drug interaction software in clinical practice: a systematic review.

PURPOSE: Several electronic databases which report the prevalence of drug-drug interactions (DDIs) are used as a tool for evaluation of potentially harmful DDIs. The aim of our review was to evaluate the usability and appropriateness of commercially available electronic databases which assess the prevalence of potential DDIs. METHODS: The systematic electronic literature search was conducted with the following search terms: "database" AND "software," and "drug-drug interactions" AND "database," and the inclusion and exclusion criteria were applied in order to identify the publications of interest. RESULTS: A total of 3766 papers were identified by systematic search. After applying inclusion and exclusion criteria, 38 publications were included in the analysis. The most commonly used software in the included studies was Micromedex® Drug-Reax, for which some authors argue to be the most reliable due to highest sensitivity. It gives information about clinical consequences of DDIs, classifies underlying mechanism and onset of the adverse outcome (either rapid, or delayed) as well as severity (such as minor, moderate, or major), and provides the level of evidence which supports this information. This data is also provided by Drug Interaction Facts®, Lexi-Interact®, and Pharmavista®. A small number of studies which compared assessment of DDIs with electronic database and the clinician's assessment showed large discrepancy in number and relevance of detected DDIs. The overlap was in some cases as low as 11 %. CONCLUSION: The deficiency of clinical relevance of detected DDIs should be addressed in the upcoming research as it would provide more relevant information to the prescribers' in clinical practice.

HIV Nef is secreted in exosomes and triggers apoptosis in bystander CD4+ T cells.

The HIV accessory protein negative factor (Nef) is one of the earliest and most abundantly expressed viral proteins. It is also found in the serum of infected individuals (Caby MP, Lankar D, Vincendeau-Scherrer C, Raposo G, Bonnerot C. Exosomal-like vesicles are present in human blood plasma. Int Immunol 2005;17:879-887). Extracellular Nef protein has deleterious effects on CD4(+) T cells (James CO, Huang MB, Khan M, Garcia-Barrio M, Powell MD, Bond VC. Extracellular Nef protein targets CD4(+) T cells for apoptosis by interacting with CXCR4 surface receptors. J Virol 2004;78:3099-3109), the primary targets of HIV, and can suppress immunoglobulin class switching in bystander B cells (Qiao X, He B, Chiu A, Knowles DM, Chadburn A, Cerutti A. Human immunodeficiency virus 1 Nef suppresses CD40-dependent immunoglobulin class switching in bystander B cells. Nat Immunol 2006;7:302-310). Nevertheless, the mode of exit of Nef from infected cells remains a conundrum. We found that Nef stimulates its own export via the release of exosomes from all cells examined. Depending on its intracellular location, these Nef exosomes form at the plasma membrane, late endosomes or both compartments in Jurkat, SupT1 and primary T cells, respectively. Nef release through exosomes is conserved also during HIV-1 infection of peripheral blood lymphocytes (PBLs). Released Nef exosomes cause activation-induced cell death of resting PBLs in vitro. Thus, HIV-infected cells export Nef in bioactive vesicles, which facilitate the depletion of CD4(+) T cells that is a hallmark of acquired immunodeficiency syndrome (AIDS).

Gene expression profiling of rat fetuses exposed to 2-dimensional ultrasound.

OBJECTIVES: This study evaluated the possible effects of ultrasound (US) on gene expression in brain tissue of rat embryos. METHODS: Four groups (n = 5 each) of pregnant Wistar Han rats were exposed to US for different durations (55, 100, 145, and 195 seconds) via a multifrequency transducer in the 2-dimensional imaging mode with a pulse duration of 1.29 microseconds, a pulse repetition frequency of 1 kHz, and a derated spatial-peak pulse-average intensity of 222.4 W/cm(2) on day 5, 9, 7, or 13 of gestation. Gene expression profiling was performed in fetal brain tissue (n = 5 per group) by quantitative reverse transcription-polymerase chain reaction arrays. RESULTS: The results indicated substantial alterations in gene expression. The most differentially expressed genes were Adamts5, Gadd45a, Npy2r, and Chrna1, which are implicated in important developmental signaling pathways. CONCLUSIONS: On the basis of our findings, routine short US examinations for monitoring fetal development are not contraindicated, but prolonged exposures should be used only when needed to obtain important diagnostic information.

Effect of cetirizine, a histamine (H(1)) receptor antagonist, on bone modeling during orthodontic tooth movement in rats.

INTRODUCTION: Histamine (H(1)) receptor antagonists are widely used drugs for treatment of allergic conditions. Although histamine was shown to be involved in bone remodeling, the aim of this study was to determine the effects of cetirizine, an H(1) receptor antagonist, on bone modeling processes during orthodontic tooth movement. METHODS: We used 3 groups of Wistar rats: control group (n = 16), appliance-only group (n = 16) and cetirizine group (n = 16). Each animal of the last 2 groups was fitted with a superelastic closed-coil spring appliance and treated daily with saline solution or cetirizine. Tooth movement was measured weekly from day 0 to day 42. Gene expression levels for bone turnover markers cathepsin K and osteocalcin were determined by means of real-time polymerase chain reaction. Histologic samples were analyzed by using histomorphometry. RESULTS: Cetirizine decreased the amount of tooth movement from day 28 onward (P <0.01), and it also decreased osteoclast volume density (P <0.001). An increase in alveolar bone volume density was observed in the cetirizine group (P <0.01) compared with the appliance-only group. No statistically significant differences were observed in osteoclast activity, osteoblast volume density, and osteoblast activity between the cetirizine and the appliance-only groups. CONCLUSIONS: Cetirizine influences bone modeling, mainly by inhibiting bone resorption. Therefore, H(1) receptor antagonists could interfere with orthodontic treatment.

The endothelin system mediates bone modeling in the late stage of orthodontic tooth movement in rats.

The endothelin system is involved in orthodontic tooth movement (OTM). The aim of the study was to examine the role of ET-1, ET(A) and ET(B) in bone modeling during OTM in rats. Male Wistar rats (n=62) were divided into three groups: control animals (n=10; control group) without appliance, and two groups of experimental animals, which were applied a super-elastic closed-coil spring between the first left maxillary molar and the incisors and were treated daily with either TBC3214 (n=10; TBC3214 group) or with saline (n=42; appliance only group). TBC3214 is a highly selective antagonist on ET(A) receptors. The distance between teeth was measured on days 0 and 42. On days 0, 14, 28 and 42 animals of the appliance only group (n=8) were sacrificed and tissue samples were taken. Total RNA and protein contents were isolated. Gene expression levels of ET-1, ET(A) and ET(B) were assessed by means of relative RT-PCR. Protein levels of ET(A) and ET(B) were examined by immunoblotting. Ten animals of each group were sacrificed on day 42 and tissue samples were prepared for histological analysis. Alveolar bone volume, osteoblast and osteoclast volume were determined histomorphometrically. Gene expression levels of ET-1, ET(A) and ET(B) varied throughout the experiment and were significantly up-regulated on day 42 (p<0.001). The immunoreactivity of ET(A) and ET(B) significantly decreased on day 14 (p<0.001) and increased on day 28 (p<0.001). Alveolar bone volume was significantly higher in the TBC3214 group compared to the appliance only group (p<0.001). Osteoclast volume was significantly lower in the TBC3214 group compared to the appliance only group (p<0.05). Gene and protein expression levels of ET-1, ET(A) and ET(B) varied significantly during OTM, suggesting their different roles in the various stages of OTM. TBC3214 significantly increased alveolar bone volume and significantly decreased osteoclast volume, indicating that it decreased bone resorption in stage three of OTM. These data suggest that ET-1 increases osteoclastic bone resorption via ET(A) in the late stage of OTM.

Mitochondrial mediation of environmental osmolytes discrimination during osmoadaptation in the extremely halotolerant black yeast Hortaea werneckii.

We have investigated the mitochondrial responses to hyperosmotic environments of ionic (4.5 M NaCl) and non-ionic (3.0 M sorbitol) osmolytes in the most halo/osmo-tolerant black yeast, Hortaea werneckii. Adaptation to both types of osmolytes resulted in differential expression of mitochondria-related genes. Live-cell imaging has revealed a condensation of mitochondria in hyperosmotic media that depends on osmolyte type. In the hypersaline medium, this was accompanied by increased ATP synthesis and oxidative damage protection, whereas adaptation to the non-ionic osmolyte resulted in a decrease in ATP synthesis and lipid peroxidation level in mitochondria. A proteomic study of the mitochondria revealed preferential accumulation of energy metabolism enzymes in the hypersaline medium, and accumulation of protein chaperones in the non-ionic osmolyte. The HwBmh1/14-3-3 protein, localized to mitochondria in hypersaline conditions, and not at optimal salinity, suggesting its role in differential perception of ionic and non-ionic osmolytes in H. werneckii.

Differential gene expression and Hog1 interaction with osmoresponsive genes in the extremely halotolerant black yeast Hortaea werneckii.

BACKGROUND: Fluctuations in external salinity force eukaryotic cells to respond by changes in the gene expression of proteins acting in protective biochemical processes, thus counteracting the changing osmotic pressure. The high-osmolarity glycerol (HOG) signaling pathway is essential for the efficient up-regulation of the osmoresponsive genes. In this study, the differential gene expression of the extremely halotolerant black yeast Hortaea werneckii was explored. Furthermore, the interaction of mitogen-activated protein kinase HwHog1 and RNA polymerase II with the chromatin in cells adapted to an extremely hypersaline environment was analyzed. RESULTS: A cDNA subtraction library was constructed for H. werneckii, adapted to moderate salinity or an extremely hypersaline environment of 4.5 M NaCl. An uncommon osmoresponsive set of 95 differentially expressed genes was identified. The majority of these had not previously been connected with the adaptation of salt-sensitive S. cerevisiae to hypersaline conditions. The transcriptional response in hypersaline-adapted and hypersaline-stressed cells showed that only a subset of the identified genes responded to acute salt-stress, whereas all were differentially expressed in adapted cells. Interaction with HwHog1 was shown for 36 of the 95 differentially expressed genes. The majority of the identified osmoresponsive and HwHog1-dependent genes in H. werneckii have not been previously reported as Hog1-dependent genes in the salt-sensitive S. cerevisiae. The study further demonstrated the co-occupancy of HwHog1 and RNA polymerase II on the chromatin of 17 up-regulated and 2 down-regulated genes in 4.5 M NaCl-adapted H. werneckii cells. CONCLUSION: Extremely halotolerant H. werneckii represents a suitable and highly relevant organism to study cellular responses to environmental salinity. In comparison with the salt-sensitive S. cerevisiae, this yeast shows a different set of genes being expressed at high salt concentrations and interacting with HwHog1 MAP kinase, suggesting atypical processes deserving of further study.

Osmoadaptation-dependent activity of microsomal HMG-CoA reductase in the extremely halotolerant black yeast Hortaea werneckii is regulated by ubiquitination.

We have investigated regulation of HMG-CoA reductase (HMGR) in one of the most salt-tolerant fungi, Hortaea werneckii, under different salinities and at the level of protein degradation. Two different HwHMGR isoenzymes were identified, specific to mitochondria and endoplasmic reticulum: HwHmg1 and HwHmg2, respectively. The activity of microsomal HwHmg2 is highest under hypo-saline and extremely hyper-saline conditions, and down-regulated under optimal growth conditions. We show that this is due to intense ubiquitination and proteasomal degradation of HwHmg2. The activity of the truncated mitochondrial HwHmg1 is constant under different growth conditions, suggesting an osmoadaptation-directed fate for mevalonate utilization in H. werneckii.

Prevalence of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay.

BACKGROUND: Hereditary hemochromatosis (HH) is a common genetic disease characterized by excessive iron overload that leads to multi-organ failure. Although the most prevalent genotype in HH is homozygosity for C282Y mutation of the HFE gene, two additional mutations, H63D and S65C, appear to be associated with a milder form of HH. The aim of this study was to develop a high-throughput assay for HFE mutations screening based on TaqMan technology and to determine the frequencies of HFE mutations in the Slovenian population. METHODS: Altogether, 1282 randomly selected blood donors from different Slovenian regions and 21 HH patients were analyzed for the presence of HFE mutations by an in-house developed real-time PCR assay based on TaqMan technology using shorter non-interfering fluorescent single nucleotide polymorphism (SNP)-specific MGB probes. The assay was validated by RFLP analysis and DNA sequencing. RESULTS: The genotyping assay of the H63D, S65C and C282Y mutations in the HFE gene, based on TaqMan technology proved to be fast, reliable, with a high-throughput capability and 100% concordant with genotypes obtained by RFLP and DNA sequencing. The observed frequency of C282Y homozygotes in the group of HH patients was only 48%, others were of the heterogeneous HFE genotype. Among 1282 blood donors tested, the observed H63D, S65C and C282Y allele frequency were 12.8% (95% confidence interval (CI) 11.5-14.2%), 1.8% (95% CI 1.4-2.5%) and 3.6% (95% CI 3.0-4.5%), respectively. Approximately 33% of the tested subjects had at least one of the three HH mutations, and 1% of them were C282Y homozygotes or compound heterozygotes C282Y/H63D or C282Y/S65C, presenting an increased risk for iron overload disease. A significant variation in H63D allele frequency was observed for one of the Slovenian regions. CONCLUSION: The improved real-time PCR assay for H63D, S65C and C282Y mutations detection is accurate, fast, cost-efficient and ready for routine screening and diagnostic procedures. The genotype frequencies in the Slovenian population agree with those reported for the Central European populations although some deviations where observed in comparison with other populations of Slavic origin. Regional distribution of the mutations should be considered when planning population screening.

Novel 3'-phosphoadenosine-5'-phosphatases from extremely halotolerant Hortaea werneckii reveal insight into molecular determinants of salt tolerance of black yeasts.

The 3'-phosphoadenosine-5'-phosphatase encoded by HAL2 gene, is a ubiquitous enzyme required for the removal of the cytotoxic 3'-phosphoadenosine-5'-phosphate produced during sulfur assimilation in eukaryotes. Salt toxicity in yeast and plants results from Hal2 inhibition by sodium or lithium ions. Two novel HAL2-like genes, HwHAL2A and HwHAL2B, have been cloned from saltern-inhabited extremely halotolerant black yeast Hortaea werneckii. Expression of both HwHAL2 isoforms was differentially inducible upon salt. When the HwHAL2 genes were transferred from such a halotolerant species into the salt sensitive Saccharomyces cerevisiae, the resulting organism can tolerate 1.8M NaCl or 0.8M LiCl, the highest reported salt concentrations at which S. cerevisiae can grow. With genetic and biochemical validation we demonstrated the critical HwHal2B sequence motif--the META sequence--common only to Dothideales fungi, with evident effect on the HwHal2B-dependent salt tolerance. These results may have significance for biosaline agriculture in coastal environments.

The MAP kinase HwHog1 from the halophilic black yeast Hortaea werneckii: coping with stresses in solar salterns.

BACKGROUND: Hortaea werneckii is one of the most salt-tolerant species among microorganisms. It has been isolated from hypersaline waters of salterns as one of the predominant species of a group of halophilic and halotolerant melanized yeast-like fungi, arbitrarily named as "black yeasts". It has previously been shown that H. werneckii has distinct mechanisms of adaptation to high salinity environments that are not seen in salt-sensitive and only moderately salt-tolerant fungi. In H. werneckii, the HOG pathway is important for sensing the changes in environmental osmolarity, as demonstrated by identification of three main pathway components: the mitogen-activated protein kinase (MAPK) HwHog1, the MAPK kinase HwPbs2, and the putative histidine kinase osmosensor HwHhk7. RESULTS: In this study, we show that the expression of HwHOG1 in salt-adapted cells depends on the environmental salinity and that HwHOG1 transcription responds rapidly but reciprocally to the acute hyper-saline or hypo-saline stress. Molecular modelling of HwHog1 reveals an overall structural homology with other MAPKs. HwHog1 complements the function of ScHog1 in the Saccharomyces cerevisiae multistress response. We also show that hyper-osmolar, oxidative and high-temperature stresses activate the HwHog1 kinase, although under high-temperature stress the signal is not transmitted via the MAPK kinase Pbs2. Identification of HOG1-like genes from other halotolerant fungi isolated from solar salterns demonstrates a high degree of similarity and excellent phylogenetic clustering with orthologues of fungal origin. CONCLUSION: The HOG signalling pathway has an important role in sensing and responding to hyper-osmolar, oxidative and high-temperature stresses in the halophilic fungi H. werneckii. These findings are an important advance in our understanding of the HOG pathway response to stress in H. werneckii, a proposed model organism for studying the salt tolerance of halophilic and halotolerant eukaryotes.

AIRE recruits P-TEFb for transcriptional elongation of target genes in medullary thymic epithelial cells.

AIRE is a transcriptional activator that directs the ectopic expression of many tissue-specific genes in medullary thymic epithelial cells, which plays an important role in the negative selection of autoreactive T cells. However, its mechanism of action remains poorly understood. In this study, we found that AIRE regulates the step of elongation rather than initiation of RNA polymerase II. For these effects, AIRE bound and recruited P-TEFb to target promoters in medullary thymic epithelial cells. In these cells, AIRE activated the ectopic transcription of insulin and salivary protein 1 genes. Indeed, by chromatin immunoprecipitation, we found that RNA polymerase II was already engaged on these promoters but was unable to elongate in the absence of AIRE. Moreover, the genetic inactivation of cyclin T1 from P-TEFb abolished the transcription of AIRE-responsive genes and led to lymphocytic infiltration of lacrimal and salivary glands in the CycT1-/- mouse. Our findings reveal critical steps by which AIRE regulates the transcription of genes that control central tolerance in the thymus.