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This 33-color panel delineates immune phenotypes of the major lymphocyte subsets in murine tissues. The panel identifies surface markers for cell activation, differentiation, and exhaustion, and chemokine receptors, in CD4 T cells, CD8 T cells, γδ T cells, NK cells, NKT cells and B cells in spleen and liver. This panel was designed to include only surface markers to avoid the need for fixation and permeabilization steps. Cells were analyzed on a full spectrum flow cytometer, 5-Laser Aurora system (Cytek Biosciences). This panel enables in-depth immunophenotyping of murine lymphocytes in immune and non-immune tissues of mice. It could be a tool for systematic analysis of immune cell response to infectious diseases and immune disorders. [ABSTRACT FROM AUTHOR]

Proteomes are complex and dynamic entities that are still poorly understood, but the application of proteomic technologies has become invaluable in many areas of biology, including parasitology. These technologies can be exploited to identify proteins in both complex or relatively simple samples, that formerly could only be characterized by targeted approaches such as Western blotting. Quantitative proteomic approaches can reveal modulations in protein expression that accompany phenotypes of interest. Proteomic approaches have been exploited to understand some of the molecular basis for host:parasite interactions and to elucidate phenotypes such as virulence, antigenicity and drug resistance. Many of the same technologies can also be more easily applied to targeted sub-proteomes.Examples from several studies on pathogen proteomes and sub-proteomes, from bacteria to helminths, are presented to illustrate the potential and limitations of proteomic technologies. [ABSTRACT FROM AUTHOR]

Abstract: Mesenchymal stem cells have demonstrated their potentiality for therapeutic use in treating diseases or repairing damaged tissues. However, in some cases, the results of clinical trials have been disappointing or have not worked out as well as hoped. These disappointing results can be attributed to an inadequate or insufficient preclinical study. For medical and surgical purposes, the similarities between the anatomy of pig and human make this animal an attractive preclinical model. In this sense, for mesenchymal stem cell-based therapy, it is strongly necessary to have well characterized animal-derived mesenchymal stem cell lines to validate preclinical effectiveness of these cells. In this work, porcine mesenchymal stem cells (pMSCs) were isolated from bone marrow, adipose tissue and peripheral blood and compared in terms of differentiation potential, cell surface markers and gene expression. Our results demonstrated that the isolation and in vitro expansion protocols were feasible and effective. The data presented in this work are relevant because they provide an extensive phenotypic characterization; genetic study and differentiation behavior of the most commonly used stem cell lines for clinical practices. These pMSCs are widely available to scientists and could be a valuable tool to evaluate the safety and efficacy of adoptively transferred cells. [Copyright &y& Elsevier]

The article discusses a research study on the taxonomic position of adult Fasciola spp. that infect cattle and buffalo in the Potohar Region of Punjab in Pakistan. Study subjects include 54 adult gravid flukes gathered from 7 cattle and 153 bufffalo and processed for staining with Gower's carmine then morphological measurements were taken and the biometric parameters for phenotypic analysis were considered including the length cone (CL), maximum width of body (BW) and maximum diameter of oral sucker (OS max). Morphometric analysis showed that measurements of nine paremeters were resembling that of the F. gigantica while 6 parameters resemble Fasciola spp and 5 parameters resembling F. hepatica. Also noted is the use of cluster analysis in the hierarchical approach.

The SnoB study analysed the variability of the integrase (IN) gene of non-B viruses from treatment-naïve patients to determine whether non-B subtypes carry natural resistance mutations to raltegravir (RAL). Plasma viral RNA from 427 patients was gained, and IN sequences were subtyped and screened for subtype-specific highly-variable residues. Seven viruses of different subtypes were phenotypically tested for RAL susceptibility; 359/427 samples could be sequenced. One hundred and seventy samples (47%) were classified as non-B subtypes. No primary RAL resistance-associated mutations (RRAMs) were detected. Certain secondary mutations were found, mostly related to specific non-B subtypes. L74 M was significantly more prevalent in subtype 02_AG, T97A in A and 06_cpx, V151I in 06_cpx, and G163R in 12_BF. Various additional mutations were also detected and could be associated with the subtype too. While K156 N and S230 N were correlated with B subtype, V72I, L74I, T112I, T125A, V201I and T206S were more frequent in certain non-B subtypes. The resistance factors (RF) of 7 viral strains of different subtypes ranged from 1.0 to 1.9. No primary or secondary but subtype-associated additional RRAMs were present. No correlation between RF and additional RRAMs was found. The prevalence of RRAMs was higher in non-B samples. However, the RFs for the analysed non-B subtypes showed lower values to those reported relevant to clinical failure. As the role of baseline secondary and additional mutations on RAL therapy failure is actually not known, baseline IN screening is necessary. [ABSTRACT FROM AUTHOR]

Abstract: Plants exposed to cadmium (Cd) show morphological and physiological disorders. To increase our knowledge regarding Cd-induced signalling, most often the effects of acute exposure are investigated. However, this does not allow in-depth analysis of morphological effects. Therefore, we chronically exposed Arabidopsis thaliana plants to environmentally realistic Cd concentrations (5 or 10 μM) and, using a described phenotypic framework methodology, we determined the impact of Cd on the plant’s ability to complete its life cycle and produce germinative seeds. Visible Cd-induced morphological changes were observed within a short exposure period, with chlorotic and anthocyanous leaf colouring occurring dose-dependently. Although rosette growth was severely reduced in Cd-exposed plants, all plants were able to emerge inflorescences and produce siliques containing germinative seeds, thus confirming the non-lethality of the used Cd concentrations. Although the growth inhibition of Cd-exposed plants was dependent on the dose, both concentrations had similar effects on inflorescence height and silique counts. In conclusion, vegetative growth of plants chronically exposed to Cd is inhibited in a concentration-dependent manner. However, the effect on plant regeneration is clearly stress-determined but independent on the Cd concentration applied. In Arabidopsis thaliana, vegetative and reproductive growth are differentially influenced by Cd. [Copyright &y& Elsevier]

Abstract: Expression profiles of protein phosphatase (PPase) disruptants were analyzed by use of Pearson''s correlation coefficient to find profiles that correlated with those of 316 Reference Gene (RG) disruptants harboring deletions in genes with known functions. Twenty-six Δppase disruptants exhibited either a positive or negative correlation with 94 RG disruptants when the p value for Pearson''s correlation coefficient was >0.2. Some of the predictions that arose from this analysis were tested experimentally and several new Δppase phenotypes were found. Notably, Δsit4 and Δsiw14 disruptants exhibited hygromycin B sensitivity, Δsit4 and Δptc1 disruptants grew slowly on glycerol medium, the Δptc1 disruptant was found to be sensitive to calcofluor white and congo red, while the Δppg1 disruptant was found to be sensitive to congo red. Because on-going analysis of expression profiles of Saccharomyces cerevisiae disruptants is rapidly generating new data, we suggest that the approach used in the present study to explore PPase function is also applicable to other genes. [Copyright &y& Elsevier]

Abstract: Formation of the mammalian secondary palate is a highly regulated and complex process. Impairment of the underlying cellular and molecular programs often results in cleft palate, a common birth defect in mammals. Here we report that Tbx2 and Tbx3, two closely related genes encoding T-box transcription factors, are expressed in the mesenchyme of the mouse palatal structures during development. Mice homozygous mutant for Tbx2 and mice double heterozygous for Tbx2 and Tbx3 exhibit a cleft palate phenotype arguing for an important contribution of Tbx2 and Tbx3 to palatogenesis. In Tbx2-deficient embryos, the bilateral primordial palatal shelves form but are smaller and retarded in the outgrowth process. They do not make contact but retain the potential to fuse. Development of other craniofacial structures appears normal, suggesting that impaired palate formation in Tbx2-mutant mice is caused by a primary defect in the palatal shelf mesenchyme. This is further supported by increased cell proliferation and apoptosis accompanied by increased expression of Bmp4 and CyclinD1 in Tbx2-deficient palatal shelves. Hence, Tbx2 and Tbx3 function overlappingly to control growth of the palatal shelf mesenchyme. [Copyright &y& Elsevier]

Abstract: The cell-biological events that guide early-embryonic development occur with great precision within species but can be quite diverse across species. How these cellular processes evolve and which molecular components underlie evolutionary changes is poorly understood. To begin to address these questions, we systematically investigated early embryogenesis, from the one- to the four-cell embryo, in 34 nematode species related to C. elegans. We found 40 cell-biological characters that captured the phenotypic differences between these species. By tracing the evolutionary changes on a molecular phylogeny, we found that these characters evolved multiple times and independently of one another. Strikingly, all these phenotypes are mimicked by single-gene RNAi experiments in C. elegans. We use these comparisons to hypothesize the molecular mechanisms underlying the evolutionary changes. For example, we predict that a cell polarity module was altered during the evolution of the Protorhabditis group and show that PAR-1, a kinase localized asymmetrically in C. elegans early embryos, is symmetrically localized in the one-cell stage of Protorhabditis group species. Our genome-wide approach identifies candidate molecules—and thereby modules—associated with evolutionary changes in cell-biological phenotypes. [Copyright &y& Elsevier]

Abstract: Classical studies comparing developing embryos have suggested the importance of modified cell biological processes in the evolution of new phenotypes. Here, I revisit this connection focusing on embryonic development, in particular nematode embryogenesis. I compare phenotypic differences in nematode embryogenesis in two basic cell biological processes, the cell cycle and the localization of the first division axis. The analysis of these and other processes shows that, at the cell biological level, exhaustive variation is found that does not necessarily translate into morphological differences. Modern molecular analyses have led to a view in which molecular complexes, made up of groups of proteins, or modules, that are working together, are responsible for the proper execution of cell biological programs. I discuss how this modular architecture could facilitate the phenotypic changes observed in cell biological processes. Ultimately, understanding the connection between cellular behavior and phenotypic outcome will further elucidate the mechanisms responsible for phenotypic evolution. [Copyright &y& Elsevier]

The measure of the quality of a systems biology model is how well it can reproduce and predict the behaviors of a biological system such as a microbial cell. In recent years, these models have been built up in layers, and each layer has been growing in sophistication and accuracy in parallel with a global data set to challenge and validate the models in predicting the content or activities of genes (genomics), proteins (proteomics), metabolites (metabolomics), and ultimately cell phenotypes (phenomics). This review focuses on the latter, the phenotypes of microbial cells. The development of Phenotype MicroArrays, which attempt to give a global view of cellular phenotypes, is described. In addition to their use in fleshing out and validating systems biology models, there are many other uses of this global phenotyping technology in basic and applied microbiology research, which are also described. [ABSTRACT FROM AUTHOR]

Tyrosinase inhibitor-producing fungus of Trichoderma sp. strain H1-7, which was isolated from marine sediment, was investigated by phylogenetic analysis and physiological characteristics. Phylogenetic analyses of the strain were similar to Trichoderma atroviride or T. viride. Physiological characteristics of the strain were similar to T. viride, and based on these results, it was identified as T. viride. Characterization of tyrosinase inhibitory activity of a culture supernatant of the strain was investigated. The inhibitory activity of the supernatant of the strain decreased after cultivation for more than 3 days. Furthermore, sea water was not essential for the production of the tyrosinase inhibitor (TI). When TI production of the strain was compared to T. viride, the strain showed higher activity than T. viride. From this result, it seemed that the strain had characteristic features comparable to T. viride, which was isolated from the terrestrial environment. As TI production of the strain showed higher potential than that of T. viride, it is necessary to elucidate the chemical structure of TI exemplified in the present study. [ABSTRACT FROM AUTHOR]

It has been shown previously that heterologous expression of inwardly rectifying potassium channels (K+-channels) from plants and mammals in K+-transport defective yeast mutants can restore the ability of growth in media with low [K+]. In this study, the functional expression of an outward rectifying mammalian K+-channel in yeast is presented for the first time. The outward-rectifying mammalian neuronal K+-channel rat ether à go-go channel 1 (rEAG1, Kv 10.1) was expressed in yeast ( Saccharomyces cerevisiae) strains lacking the endogenous K+-uptake systems and/or alkali–metal-cation efflux systems. It was found that a truncated channel version, lacking almost the complete intracellular N-terminus (rEAG1Δ190) but not the full-length rEAG1, partially complemented the growth defect of K+-uptake mutant cells ( trk1,2Δ tok1Δ) in media containing low K+ concentrations. The expression of rEAG1Δ190 in a strain lacking the cation efflux systems ( nha1Δ ena1-4Δ) increased the sensitivity to high monovalent cation concentrations. Both phenotypes were observed, when rEAG1Δ190 was expressed in a trk1,2Δ and nha1, ena1-4Δ mutant strain. In the presence of K+-channel blockers (Cs+, Ba2+ and quinidine), the growth advantage of rEAG1Δ190 expressing trk1,2 tok1Δ cells disappeared, indicating its dependence on functional rEAG1 channels. The results demonstrate that S. cerevisiae is a suitable expression system even for voltage-gated outward-rectifying mammalian K+-channels. [ABSTRACT FROM AUTHOR]

Background/Aims: Recent clinical observations reported the occurrence of amino acid substitutions at position 181 of the HBV polymerase, associated with a viral breakthrough under lamivudine or adefovir therapy. In this study, we characterized the main variants harboring the rtA181T/V mutation isolated from 10 consecutive patients who developed lamivudine and/or adefovir resistance. Methods: We performed a clonal analysis of the HBV polymerase gene amplified by PCR from serum samples during viral breakthrough. The main mutants were then tested after transfection of Huh7 cells for their resistance profile to nucleoside analogs. Results: Clonal analysis revealed the co-localization on the same HBV genome of rtA181T/V with rtN236T, but not with rtM204V/I mutations following lamivudine, adefovir or lamivudine+adefovir breakthrough. In cell culture, the rtA181T/V mutation induced a decreased susceptibility to lamivudine (<10-fold), adefovir (2- to 8-fold) and tenofovir (2- to 3-fold). Interestingly, the association of rtA181T with rtN236T on one clinical isolate genome increased the resistance to these three drugs. All the tested mutants remained sensitive to entecavir. Conclusions: Our observations suggest that a single amino acid change at position rt181 may induce cross-resistance to lamivudine and adefovir. These data emphasize the clinical relevance of genotypic and phenotypic analysis in the management of antiviral drug resistance. [Copyright &y& Elsevier]

The TRP1 marker has been commonly used for gene disruption experiments and subsequent phenotypic analysis. However, introduction of the TRP1 gene into a trp1 strain markedly affects growth under many conditions used for phenotypic profiling. Therefore, its use in the past should be revisited and utilization of this marker should be avoided in future analyses. [ABSTRACT FROM AUTHOR]

Abstract: New methods for an extended physiological characterization of yeast at a microtiter plate scale were applied to 27 deletion mutants of Saccharomyces cerevisiae cultivated on glucose and galactose as sole carbon sources. In this way, specific growth rates, specific rates of glucose consumption and ethanol production were determined. Flux distribution, particularly concerning branching into the pentose phosphate pathway was determined using a new 13C-labelling method using MALDI-ToF-mass spectrometry. On glucose, the growth was predominantly fermentative whereas on galactose respiration was more active with correspondingly lower ethanol production. Some deletion strains showed unexpected behavior providing very informative data about the function of the corresponding gene. Deletion of malic enzyme gene, MAE1, did not show any significant phenotype when grown on glucose but a drastically increased branching from glucose 6-phosphate into the pentose phosphate pathway when grown on galactose. This allows the conclusion that MAE1 is important for the supply of NADPH during aerobic growth on galactose. [Copyright &y& Elsevier]

Yeasts of the genera Candida and Malassezia can be found as commensal micro-organisms in animals. The main species of importance in veterinary medicine are Malassezia pachydermatis and Candida albicans. The objectives of this study were to conduct a phenotypic characterization and to evaluate the in vitro antifungal sensitivity of strains of C. albicans (n = 5), C. tropicalis (n = 3) and M. paehydermatis (n = 32) isolated from dogs. The phenotyping was based on macro and micromorphological features as well as biochemical analysis. The techniques of microdilution in broth and dilution in agar were used to evaluate the in vitro sensitivity of Candida spp. and M. pachydermatis, respectively. The tested drugs were ketoconazole (KTC), itraconazole (1TC), fluconazole (FLC) and amphotericin B (AMB). The morphological analysis of the strains of Candida spp. and M. pachydermatis did not show any noteworthy alterations when compared to standard strains. On the other hand, in the biochemical tests, 34.4% of the strains of M. pachydermatis were negative for the urease test. Four strains of C. albicans were resistant to FLC with a minimum inhibitory concentration (MIC) >64 μg/mL and all were resistant to KTC and ITC (MIC > 16 μpg/mL). The MIC for two strains of C. tropicalis were > 16 μ/mL for KTC and ITC, and >64 μg/mL for FLC. It is worth highlighting that all of the strains tested were sensitive to AMB with the MIC varying from 0.25-1.0 μg/mL. All strains of M. pachydermatis were sensitive to ITC with a minimum fungistatic concentration (MFC) ≤0.0075 μg/mL The MIC for 29 strains was the same (MFC ≤ 0.0075 μ/mL) for KTC. The MFCs for FLC varied from ≤1 to ≤16 μg/mL and for AMB, the MFC interval was 0.125-8 μg/mL. There were no alterations in the classic phenotypic features of the strains of Candida spp. and MI paehydermatis isolated from dogs but, unlike M. pachydermatis, Candida spp. were much more resistant to azole antifungal agents. [ABSTRACT FROM AUTHOR]

• Using a novel setup, we assessed how fast growth of Nicotiana tabacum seedlings responds to alterations in the light regime and investigated whether starch-free mutants of Arabidopsis thaliana show decreased growth potential at an early developmental stage. • Leaf area and relative growth rate were measured based on pictures from a camera automatically placed above an array of 120 seedlings. Detection of total seedling leaf area was performed via global segmentation of colour images for preset thresholds of the parameters hue, saturation and value. • Dynamic acclimation of relative growth rate towards altered light conditions occurred within 1 d in N. tabacum exposed to high nutrient availability, but not in plants exposed to low nutrient availability. Increased leaf area was correlated with an increase in shoot fresh and dry weight as well as root growth in N. tabacum. Relative growth rate was shown to be a more appropriate parameter than leaf area for detection of dynamic growth acclimation. Clear differences in leaf growth activity were also observed for A. thaliana. • As growth responses are generally most flexible in early developmental stages, the procedure described here is an important step towards standardized protocols for rapid detection of the effects of changes in internal (genetic) and external (environmental) parameters regulating plant growth. [ABSTRACT FROM AUTHOR]

Genetic variability in the plasticity of leaf area expansion in response to water deficit has been reported in Arabidopsis thaliana. Here, the objective was to identify the underlying dynamic and cellular processes involved in this variability. Twenty-five accessions were subjected to identical soil water deficit treatments. In all accessions, the plasticity of leaf production was low compared with that of individual leaf expansion. A subset of accessions was selected for further dissection of individual leaf expansion into its underlying variables: the rate and duration of leaf expansion and epidermal cell number and area. In all accessions, water deficit had opposite effects on the rate and duration of leaf expansion. The accumulation of these effects was reflected in changes in final leaf area. At the cellular level, moderate water deficits had opposite effects on cell number and cell size, but more severe ones reduced both variables. The importance of these opposing effects is highlighted by the behaviour of the accession An-1, for which the compensation between the decrease in leaf expansion rate and the increase in the duration of expansion is total. This dynamic plasticity in response to water deficit is not detectable when only final measurements are done. [ABSTRACT FROM AUTHOR]

Abstract: Twenty-seven deletion mutants of Saccharomyces cerevisiae were investigated using a novel method for high-content stoichiometric and kinetic metabolic profiling. Cultivation on glucose and galactose in 96-well microtiter plates with on-line optical sensing of dissolved oxygen was reproducible and identical to shake flask cultivation. At high shaking rate (1020rpm) there was no oxygen limitation throughout all cultivations whereas at low shaking rate (540rpm) oxygen uptake rate could be measured during declining dissolved oxygen concentration. This method allows the determination of specific growth rate, biomass yield on carbon substrate and oxygen and ethanol yield on carbon substrate. Maximum specific growth rates were in the same range for both sugars. On glucose the growth was predominantly fermentative with high yield of ethanol, low yield of biomass and low oxygen consumption rate. On galactose respiration was more active with correspondingly lower ethanol yields, higher biomass yields and higher rates of oxygen consumption. Some strains showed unexpectedly high or low growth rates and rates of ethanol production and respiration. Overall the four parameters determined for each mutant on two different carbon sources allowed statistically significant discrimination of all mutants studied. [Copyright &y& Elsevier]

Abstract: The tumour microenvironment down-modulates antigen-presentation by dendritic cells (DC), presumably due to inhibition of DC maturation. Here, we sought to examine (1) whether monocyte-derived cells cultivated with tumour-conditioned media under conditions that are conducive to DC generation (APCTCM) resemble immature DC (iDC), IL-10-induced regulatory DC (DCIL10) or display other distinctive features; (2) whether APCTCM are convertible to immunostimulatory DC (DCims) upon proper activation and (3) whether APCTCM and activated APCTCM are functionally defective. Four tumour cell lines expressing different cytokines were used to mimic different tumour microenvironments. As compared to iDC, DCims or DCIL10, APCTCM exhibited the highest levels of expression for CD14, CD16 and CD4. These markers and a high phagocytic capacity were unique features of these cells. When APCTCM were activated by a maturation cocktail, CD83, CD86, HLA-DR and CD25 were up-regulated to levels considerably higher than in DCIL10 and comparable to DCims while CD14, CD16, CD4 and dextran-uptake were down-modulated. Activated APCTCM induced 50–60% of the proliferative response of DCims in the allogeneic T-cell proliferation assay while DCIL10 mounted a 20–30% response (iDC elicited ∼10%). Activated APCTCM induced secretion of almost equal amounts of IFN-γ, TNF-α and IL-2 as DCims indicating induction of Th1 differentiation. When mature DCims were exposed to TCM, their immunostimulatory function was not significantly altered. However, when TCM were added to the co-cultures of DCims and CD4 T-cells the proliferative outcome was dependent on the TCM. In summary, APCTCM display special features but can mature into DCims-like cells. [Copyright &y& Elsevier]

The use of Caenorhabditis elegans as a model system for understanding animal development and human disease has long been recognized as an efficient tool of discovery. Recent developments, particularly in our understanding of RNA-mediated interference and its ability to modify gene activity, have facilitated the use of C. elegans in determining gene function via high-throughput analysis. These new strategies have provided a framework that allows investigators to analyse gene function globally at the genomic level and will likely become a prototypic model for biological analysis in the post-genome era. [ABSTRACT FROM AUTHOR]

• The high-throughput phenotypic analysis of Arabidopsis thaliana collections requires methodological progress and automation. Methods to impose stable and reproducible soil water deficits are presented and were used to analyse plant responses to water stress. • Several potential complications and methodological difficulties were identified, including the spatial and temporal variability of micrometeorological conditions within a growth chamber, the difference in soil water depletion rates between accessions and the differences in developmental stage of accessions the same time after sowing. Solutions were found. • Nine accessions were grown in four experiments in a rigorously controlled growth-chamber equipped with an automated system to control soil water content and take pictures of individual plants. One accession, An1, was unaffected by water deficit in terms of leaf number, leaf area, root growth and transpiration rate per unit leaf area. • Methods developed here will help identify quantitative trait loci and genes involved in plant tolerance to water deficit. [ABSTRACT FROM AUTHOR]

Abstract: Background:: Genotypic and phenotypic analysis of HIV-1 resistance mutations constitute one important point for providing guidelines in the choice of antiretroviral regimens and to design lines of rescue treatment for patients holding HIV-1 drug-resistant variants. However, some levels of discordance among them has been described. Objectives:: (i) To compare the genotypic analysis of resistance mutations to reverse transcriptase (RT) and protease (PR) inhibitors by Stanford HIVdb program (http:/hivdb.stanford.edu) (St-HIVdb), and genotype with quantitative phenotypic analysis (Virtual Phenotype™, VircoNET). (ii) To identify drug resistance mutations associated with discrepant results. Study design:: Five hundred HIV-1 infected patients were included in this study. RNA was extracted from plasma. RT and PR regions were amplified and sequenced using ABI-Prism DNA sequencing system. Sequences were corrected and assembled with Seqman and Bioedit computer programs. The corrected sequences were submitted to the Stanford HIV-Seq program (http:/hivdb.stanford.edu) and to Virtual Phenotype™ (VircoNET). Results:: Discrepant cases were considered if results were high or intermediate resistant by Stanford HIV-Seq program and susceptible by Virtual Phenotype™, being detected as follows: (i) nucleoside RT inhibitors (NRT): 31.7% (ABC), 31% (d4T), 29.5% (ddC), 27.6% (ddI), 14.3% (TDF) and 11.3% (ZDV) and to PR inhibitors: 8.8% (SQV), 5% (APV), 3.8% (NFV) and 3.2% (IDV). These discrepant results were related to the presence of thymidine analogue mutations (TAMs) and also to key resistance mutations to NRT inhibitors: 65R, 69D/N, 74V/I, 184V/I and 215Y/F. (ii) PR inhibitors: 82A/F/T/S, 84I and 90M. Concordant results were considered when the interpretations by both programs were coincident, being higher than 96.7% for non-NRT inhibitors. Conclusions:: The detection of discrepant results to NRT inhibitors and PR inhibitors, including the analysis of sequences with key resistant mutations to some drugs, means that further investigation is necessary in order to establish which is the best interpretation system as antiretroviral therapy guide. [Copyright &y& Elsevier]

Abstract: Potassium uptake defective Saccharomyces cerevisiae strains (Δtrk1,2 and Δtrk1,2 Δtok1) were used for the phenotypic analysis of the mouse inward rectifying Kir2.1 channel by growth analysis. Functional expression of both, multi-copy plasmid and chromosomally expressed GFP-mKir2.1 fusion constructs complemented the potassium uptake deficient phenotype in a pHout dependent manner. Upon application of Hygromycin B to chromosomally mKir2.1 expressing cells, significantly lower toxin sensitivity (EC50 15.4μM) compared to Δtrk1,2 Δtok1 cells (EC50 2.6μM) was observed. Growth determination of mKir2.1 expressing strains upon application of Ag+, Cs+ and Ba2+ as known blockers of mKir2.1 channels revealed significantly decreased channel function. Cells with mKir2.1 were about double sensitive to AgNO3, 350-fold more sensitive to CsCl and 1500-fold more sensitive to BaCl2 in comparison to the respective controls indicating functional expression and correct pharmacology. [Copyright &y& Elsevier]

The phenotype is the observable and measurable characteristic (or biochemical trait) of an organism that results from the interaction of the environment with the expression of gene alleles (i.e. the genotype). Accurate phenotyping demands that findings are not missed, anticipated, or biased; a mixture of assays that characterize distinct, but interrelated aspects of the cardiovascular phenotype are often necessary to meet these requirements. Such stringent conditions are crucial because it is phenotypic analysis that ultimately determines the utility of genetically altered and spontaneous mutant mice for biomedical research. [Copyright &y& Elsevier]