text:
- Research
- Open Access
Expression profiling of the schizont and trophozoite stages of Plasmodium falciparumwith a long-oligonucleotide microarray
© Bozdech et al.; licensee BioMed Central Ltd. 2003
- Received: 23 August 2002
- Accepted: 5 December 2002
- Published: 31 January 2003
The worldwide persistence of drug-resistant Plasmodium falciparum, the most lethal variety of human malaria, is a global health concern. The P. falciparum sequencing project has brought new opportunities for identifying molecular targets for antimalarial drug and vaccine development.
We developed a software package, ArrayOligoSelector, to design an open reading frame (ORF)-specific DNA microarray using the publicly available P. falciparum genome sequence. Each gene was represented by one or more long 70 mer oligonucleotides selected on the basis of uniqueness within the genome, exclusion of low-complexity sequence, balanced base composition and proximity to the 3' end. A first-generation microarray representing approximately 6,000 ORFs of the P. falciparum genome was constructed. Array performance was evaluated through the use of control oligonucleotide sets with increasing levels of introduced mutations, as well as traditional northern blotting. Using this array, we extensively characterized the gene-expression profile of the intraerythrocytic trophozoite and schizont stages of P. falciparum. The results revealed extensive transcriptional regulation of genes specialized for processes specific to these two stages.
DNA microarrays based on long oligonucleotides are powerful tools for the functional annotation and exploration of the P. falciparum genome. Expression profiling of trophozoites and schizonts revealed genes associated with stage-specific processes and may serve as the basis for future drug targets and vaccine development.
- Malaria
- Additional Data File
- Microarray Hybridization
- Plastid Genome
- Apical Membrane Antigen
Plasmodium falciparum, a parasitic protozoan, is the causative agent of the most lethal form of human malaria. It is responsible for 300-500 million infections per year in some 90 countries and regions throughout the tropical and subtropical world. Of these clinical cases, approximately 2.1 million result in death annually [1]. In areas where mosquito abatement has failed, chemotherapy, consisting of a limited selection of antimalarial agents, is the only defense against this disease. The increase in drug resistance throughout the malaria endemic regions is cause for great concern and calls for the development of new antimalarial measures, which would involve a larger variety of drug targets as well as a wider array of vaccine strategies (reviewed in [2,3]).
The study of malaria will be greatly helped by the publicly available complete genome sequence of P. falciparum. The sequencing project, driven by the Sanger Centre, the Institute for Genomic Research (TIGR), and Stanford University is essentially complete [4]. The sequence of the completed chromosomes are available for download from each sequencing center and from the Plasmodium Genome Resource, PlasmoDB [5,6]. Preliminary analysis of the 23 megabase-pair (Mbp) P. falciparum genome indicates the presence of approximately 5,400 genes spread across 14 chromosomes, a circular plastid genome and a mitochondrial genome. Strikingly, more than 60% of the predicted open reading frames (ORFs) lack orthologs in other genomes [4]. This fact underscores the need to elucidate gene function, yet many of the tools that have propelled the study of model organisms remain inefficient or nonexistent in Plasmodium. Despite recent improvements in P. falciparum transformation techniques, [7] the efficiency of stable transfection under a direct drug selection remains approximately 10-6, making knockout and gene replacement experiments difficult, and genetic complementation strategies nearly impossible. Genome-wide expression profiling by microarray technology provides an easy alternative for the functional genomic exploration of P. falciparum.
In organisms ranging from bacteria to humans, expression profiling has proved a powerful tool. Profiling has been used to gain important insights into processes such as development, responses to environmental perturbations, gene mutation, pathogen and host response, and cancer [8,9,10,11,12,13,14]. Expression profiling has already been successfully applied to the partial genome sequence of P. falciparum, and has been used to characterize the role of previously unannotated genes [15,16,17].
Here we present the design and assembly of a long-oligonucleotide P. falciparum gene-specific microarray using the currently available genomic sequence generated by the Malaria Genome Consortium [18,19,20]. During the course of this work, we have developed software, improved by experimental data and an open-source policy, for rapidly selecting unique sequences from predicted ORFs of any genome. Subsequently, we constructed a long-oligonucleotide-based P. falciparum microarray, which we used to evaluate changes in the global expression profile between two distinct stages of P. falciparum erythrocytic-stage asexual development - mid-trophozoite and mid-schizont. The large number of differentially expressed genes detected in this analysis suggests that extensive transcriptional regulation has a major role in the functional specialization of parasite development.
Results and discussion
P. falciparumORF predictions
At the outset of these studies, a total of 27.6 Mbp of P. falciparum genomic sequence was obtained from the publicly available sources presented by the Malaria Genome Consortium [18,19,20] in October 2000. The sequence comprised two completely assembled chromosomes, the complete mitochondrial and plastid genomes, and the sum of all the partial contigs from the remaining chromosomes. ORF predictions were carried out using GlimmerM, a gene-finding tool trained with P. falciparum specific sequences [21,22]. Using default parameters, GlimmerM frequently yielded a large number of overlapping predictions (competing gene models) and thus additional filtering of the initial prediction output was required. As slight overprediction of ORFs is generally desirable for the purpose of expression array building, the post-prediction filtering of the GlimmerM output was modified with respect to the process used by the Malaria Genome Consortium [21]. Briefly, individual predictions that overlapped and were on opposite strands or in different reading frames were retained. For competing predictions within a given GlimmerM gene model, ORFs that were extended downstream by at least 300 bp and were within 300 bp of the total size compared to the size of the largest prediction were chosen. In all other cases, the largest predicted ORF was selected. This selection method resulted in 290 ORF predictions for chromosome 2, whereas the Malaria Genome Consortium selected 210 for the same chromosome [21].
The first round of predictions, carried out on the publicly available genomic sequence as of August 2000, yielded 8,008 putative ORFs. The predicted ORFs are available as additional data with the online version of this paper (see Additional data files) and from [23]. As a first step to annotation, the translation of all predicted ORFs were used to search the Astral, SwissProt, and non-redundant (NR) databases for sequence similarities using the Smith-Waterman algorithm [24]. In addition, all ORF predictions were linked to their counterparts in PlasmoDB [5,6].
ArrayOligoSelector: array element design
An important aspect of oligonucleotide design for microarray hybridization is avoiding secondary structures within the oligonucleotide, as these are likely to be detrimental to hybridization performance. To avoid selecting oligonucleotides with secondary complex structure, ArrayOligoSelector uses the Smith-Waterman algorithm with the PAM47 DNA matrix to calculate the optimal alignment score between the candidate oligonucleotide sequence and the reverse complement of that sequence [24]. A high Smith-Waterman score indicates the potential to create secondary structures (Figure 2b).
The presence of low-complexity sequence could also result in significant nonspecific cross-hybridization. For example, the P. falciparum genome contains a large number of low-complexity sequence elements as a result of a high frequency of continuous stretches of A and T nucleotides in both the non-coding and the coding regions. ArrayOligoSelector automatically detects such sequences by subjecting candidate oligonucleotide sequences to a lossless compression [36]. The compression score, calculated as the difference in bytes between the original sequence and the compressed version, is inversely proportional to complexity (Figure 2c). Using this score, repeats of essentially any nature are detected in a computationally efficient manner.
In addition, in order to avoid specific sequence features, ArrayOligoSelector supports filtering based on user-defined patterns. This feature can be used to implement filtering rules based on empirically derived data. Finally, the melting temperature of an oligonucleotide is largely determined by its GC content. As is the case with most ORFs, there exists a large range of %GC values (< 10 to > 60%) over a 70 bp window (Figure 2d). For this reason, a user-defined %GC target range is used by ArrayOligoSelector such that the majority of the array elements will share a similar base composition and hybridization properties across the array.
Given the above parameters, ArrayOligoSelector evaluates every 70 mer sequence within an ORF and chooses an optimal set on the following criteria. The uniqueness-filter requires oligonucleotides to satisfy two simultaneous threshold criteria based on the calculation of the binding energy to their second-best target (the best target is itself). First, the oligonucleotide must rank among the top 5% of the unique or almost unique 70 mers in the entire ORF. Second, its binding energy must be within 5 kcal/mol of the best candidate for the ORF. In addition, an optional user-defined energy threshold can operate in conjunction with the default threshold. Initial settings for the low-complexity and the self-binding terms allow the top-scoring 33% of 70 mers to pass to the next selection step. Finally, an optional 'user-defined sequence filter' simply eliminates the 70 mer candidates containing the defined sequences. These four filters operate on the entire set of 70 mer candidates for a particular ORF and generate four independent output sets. The intersection of the four outputs is then subjected to the final selection. If no common oligonucleotide is identified in the first intersection, the self-binding and complexity filters are incrementally relaxed until an intersection becomes available. The final selection of candidate oligonucleotides depends upon the %GC filter and 3'-end proximity ranking. Initially, oligonucleotides are allowed to pass if they meet the user-specified %GC. If no oligonucleotide with the desired GC content is found, the target %GC range is relaxed by one percentage point in each direction until one or more oligonucleotides pass. As a final step, a single candidate, closest to the 3' end of the gene is chosen. Finally, ArrayOligoSelector generates an output file containing the oligonucleotide selections for each putative ORF.
Oligonucleotide performance
To measure the extent to which the energy calculation implemented in ArrayOligoSelector matches reality, we have plotted in Figure 3 the calculated energy of the 100 control oligos shown in Figure 4 and their relative intensities of hybridization. The calculated energy and relative intensity of hybridization correlate at |r| = 0.91. For comparison, the relative intensity of hybridization and number of nucleotide identities correlate at |r| = 0.72. This indicates that a calculated binding energy approach may be used to estimate the potential for cross-hybridization for any sequence relative to the rest of the genome. The specificity for each oligonucleotide is uniquely and computationally determined and expressed as a binding energy (kcal/mol).
To further address the question of specificity of oligonucleotide hybridization to their targets in a complex sample we introduced a set of probes targeting a set of 19 non-repetitive sequences from S. cerevisiae to the microarray. To control for the nucleotide bias of the malaria genome relative to yeast, the selection criteria for this set were identical to selection of the plasmodial microarray elements. The average GC content of the S. cerevisiae oligonucleotides was 31.5%, whereas the average GC content of plasmodial oligonucleotides is 32.5%. The average signal-to-background ratios across all hybridizations for these negative control spots was less than twofold, which is well below the conservative fivefold signal-to-background threshold used to filter data (see Materials and methods). In addition, a series of 10 hybridizations was carried out where total RNA from an asynchronous parasite culture was hybridized against PCR products corresponding to the negative control S. cerevisiae sequences. In these hybridizations the yeast PCR fragment hybridized strictly to its cognate sequence, while the average signal to background value for plasmodial elements in the same channel was 1.17 ± 0.05. In no individual case did a plasmodial element yield a signal greater than 2.3% of the target hydridization signal intensity. The results of these microarray hybridizations are available as additional data files [23].
Gene-expression profiling of trophozoites and schizonts
We chose a direct comparison of the trophozoite and schizont stages of the P. falciparum asexual intraerythrocytic life cycle as a first step toward comprehensively profiling all life-cycle stages of this parasite. The trophozoite and schizont represent two distinct developmental stages within the 48-hour plasmodial erythrocytic life cycle. These stages vary greatly in morphology, biochemical properties, and transcriptional activity (reviewed in [15,37]). The mid-trophozoite stage, 18-24 hours post-invasion, contains a highly transcriptionally active nucleus with abundant euchromatin. In addition, trophozoites are characterized by massive hemoglobin ingestion, intake of nutrients from the surrounding medium, increasing concentration of cytoplasmic ribosomes and rapid formation of organelles. In contrast, the mid-schizonts, at 36-42 hours post-invasion, are characterized by DNA replication (16-32 copies) and compaction into newly formed nuclei. In addition, maturation of merozoite cells begins at the schizont stage and is characterized by the appearance of merozoite organelles such as the rhoptry and dense granules. The several trophozoite- and schizont-specific genes identified previously provide an excellent source of positive controls for the experiments described below.
For microarray hybridization, total RNA was prepared from synchronized in vitro P. falciparum cultures representing the trophozoite stage and the schizont stage (see Materials and methods). Six independent hybridizations were carried out; in three, the trophozoite-derived cDNA was labeled with Cy3 and the schizont-derived cDNA with Cy5. In the other three hybridizations, the fluorophore assignment was reversed. Of the genes assayed, 854 features displayed a differential expression greater than twofold (Figure 5): 525 showed higher relative transcript abundance in trophozoites than in schizonts, whereas 326 had greater relative transcript abundance in schizonts. Linear regression ratios were calculated for each possible pair of microarray hybridizations using the filtered dataset. The correlation between hybridizations with the same Cy3/Cy5 assignment was r = 0.94 ± 0.02, while correlation of hybridizations with the opposite Cy3/Cy5 order was r = 0.89 ± 0.03.
Northern blot hybridizations
Biological significance of the gene-expression results
The genome-wide expression data summarized by hierarchical cluster analysis (Figure 5) resulted in two main gene categories, corresponding to genes differentially expressed between trophozoite and schizont stages. Serving as internal positive controls, a number of previously well-characterized plasmodial genes were detected in both categories. In addition, an evaluation of the homology-based gene identities within these categories revealed several functional gene groups. All data from these experiments are available at PlasmoDB and the DeRisi Lab website [23].
Trophozoite genes
The predominant group of features with elevated expression in the trophozoite stage comprised genes encoding various components of the eukaryotic translation machinery. This group contained 24 and 33 proteins of the 40S and 60S ribosomal subunits (RPS and RPL), respectively. In addition, nine orthologs of aminoacyl-tRNA synthetases, and 10 initiation and seven elongation translation factors were detected among trophozoite-specific genes. Several previously identified plasmodial genes were present in this group, including Asp-tRNA synthetase, two plasmodial elongation factors (PfEF1A and PfEF2) and one ribosome-releasing factor, PfRF1 [21,39]. Consistent with our findings, PfEF-1A has been previously shown to have peak expression during the trophozoite stage [40]. Two additional gene groups whose functions are linked to the process of protein synthesis were present among the trophozoite genes: five DEAD-box RNA helicases, including a close homolog of P. cynomolgi RNA helicases-1 [41] and 23 molecular chaperone-like molecules, including two P. falciparum heat-shock proteins such as PfHSP70 (GenBank accession number M19753) and PfHSP86 (accession number L34028), and a homolog of a DnaJ-domain-containing protein family, DNJ1/SIS1 homolog [42]. These data agree with previous studies that found a group of DEAD-box RNA helicases to be overexpressed during the trophozoite stage in P. cynomolgi [41]. Along with the genes for the translation machinery a number of genes involved in various steps of RNA synthesis and processing were located among the 'trophozoite genes', including 16 ORFs belonging to various RNA polymerase complexes and 11 splicing factors (Figure 5). Two previously identified plasmodial RNA polymerase components were found in this group, including the largest subunit of P. falciparum RNA polymerase II, PfRNApolIIA (M73770), and a homolog RNApolK (14 kD) [39]. The expression characteristics revealed are also consistent with several previous studies that suggested that the plasmodial transcription and translation machinery is active through the late ring and early trophozoite stage before decaying during the late schizont stage [15,40].
Another functional group of genes that encode enzymes of cellular biosynthetic pathways was distinguished within the trophozoite category. This gene set includes 16 enzymes of carbohydrate metabolism, 10 ORFs likely to be involved in nucleotide metabolism, and 11 ORFs involved in the biosynthetic pathways of several amino acids. Several well-characterized plasmodial genes were identified in this metabolic collection, including P. falciparum lactate dehydrogenase (PfLDH; 027743), enolase (U00152), triose-phosphate isomerase (PfTPI; L01654), glucose-6-phosphate isomerase (PfG6PI; J05544), hypoxanthine-guanine phosphoribosyl-transferase (PfHGPRT; X16279) and dihydropteroate synthetase (PfDHPS; U07706). In addition, a group of 11 proteolytic enzymes potentially involved in hemoglobin degradation was detected among the trophozoite genes; these include a cysteine protease, falcipain-2 (AF251193), a metalloprotease falcilysine, (AF123458), and a member of an aspartic protease family, plasmepsin-2 (L10740). Falcipain-2 and plasmepsin-2 have been the targets of recent drug discovery research [43,44].
Overall, the emergent gene clusters suggest that the trophozoite stage, a central phase of plasmodial intraerythrocytic development, is characterized by the activation of general cellular growth functions such as transcription, translation and hemoglobin degradation and biosynthesis of basic metabolites.
Schizont genes
A large number of ORFs found in the schizont-expressed category correspond to genes previously associated with the various steps by which newly released merozoites invade new host cells. The initial step of this process, adhesion of the merozoite to the surface of an erythrocyte, is facilitated by several classes of proteins exposed on the surface of the parasite. Eighteen ORFs, identical or homologous to proteins associated with the merozoite surface, were present among the schizont-enriched genes. This group included four merozoite surface proteins (MSP): MSP1 (M19753), MSP4, MSP5, (AF033037) and MSP6 (AY007721). Additional members of this group include two ORFs containing Duffy-like binding domains, erythrocyte-binding antigen, EBA 175 (L07755), a putative erythrocyte-binding protein, EBL1 (AF131999), and proteins known to be delivered to the surface from apical organelles, including apical membrane antigen, AMA1 (U65407), and finally two rhoptry-associated proteins (RAP1 (U20985) and RAP2).
Initial attachment of the merozoite is followed by reorientation of the parasite cell with its apical part toward the erythrocyte membrane followed by invagination of the membrane. Previous studies suggested that both steps are facilitated by the action of actomyosin, which requires ATP hydrolysis [45]. Consistent with these findings, we found five proteins previously associated with this process, pf-actinI (M19146), pf-myoA (AF255909), and merozoite cap protein-1 (U14189), and two subtilisin-like proteases (PfSUB1 and PfSUB2 (AJ132422)) differentially enriched in schizonts. Interestingly, one additional homolog of PfSUB1 was identified among the schizont genes. Moreover, the expression levels of a set of plasmodial protein kinases were previously found to be augmented during the late stages of the malarial erythrocytic life cycle [15]. Our findings confirm and extend this report: 26 unique ORFs sharing a high to medium level of homology with protein kinases and phosphorylases had elevated mRNA levels during the schizont stage (Figure 5). Two previously identified representatives were present in this set: a cAMP-dependent protein kinase, PfPKAc (AF126719), and a plasmodial serine/threonine protein phosphatase, PfPPJ (AF126719).
A second functional group of genes with increased expression in schizonts encodes proteins that are thought to function on the periphery of a newly infected erythrocyte at the early stages of asexual development. Representatives include: the genes for ring-infected erythrocyte surface antigen (RESA) (X04572) and several close RESA homologs, CLAG9 (AF055476) the related gene CLAG3.1, and two members of the serine-repeat rich protein (SERA) family [21]. In addition to these well-characterized surface proteins, the schizont-enriched set of transcripts contained a number of ORFs identical or homologous to proteins recognized by antibodies present in plasmodium immune sera obtained either from model organisms [46] or from acute and/or convalescent patients [47]. In summary, the schizont stage of plasmodial development featured genes predominantly occupied with the process of merozoite function as well as the advance synthesis of transcripts for proteins that facilitate parasite establishment within the newly infected erythrocyte.
Taken together, these results suggest that the parasite cell in the trophozoite stage is dedicated to cell growth, and the predominant function of the mid-late schizont stage is maturation of the next generation of merozoites. Of particular interest is the large number of ORFs within both categories (39% in trophozoite and 61% in schizonts) with no putative functions assigned. These ORFs have little to no homology to any other known genes and may possibly represent highly specialized functions not likely to be shared outside this family of parasites.
In this study, we present a P. falciparum ORF-specific microarray utilizing 70 mer oligonucleotides as individual microarray elements. This approach helped to overcome potential problems originating from low PCR amplification and allowed us to select probes with a high specificity, thereby minimizing potential cross-hybridization. Moreover, the oligonucleotide-selection algorithm allowed a balanced GC content (around 28%) across the entire microarray set, which is significantly higher than the plasmodial genome average, which is 19.4% with 23.7% in coding regions [4].
Application of the ArrayOligoSelector is not restricted to the P. falciparum genome, but is broadly useful for the automated selection of hybridization probes for a range of species. The flexibility of the selection parameters controlling stringency of uniqueness, self-binding, complexity, user-defined filters and GC content, allows the selection of oligonucleotides appropriate for any genome.
Evaluation of results from derivative control oligonucleotides showed that long oligonucleotides could tolerate 10% mismatches; however, alteration of the target sequence by more then 20% eliminated most of the hybridization signal. Therefore, small sequencing errors and natural variation among isolates are not likely to impact on sensitivity. These performance characteristics imply that the array design for this effort can accommodate the study of essentially any P. falciparum strain with a high degree of specificity.
At present, the P. falciparum microarray used in this study consists of approximately 6,000 gene-specific elements corresponding to the majority of the total coding content predicted for the P. falciparum genome. As new sequence and improved gene predictions arise, additional elements will be added to this evolving platform. Moreover, the present oligonucleotide representation could be further extended for investigation of several unusual P. falciparum genetic and transcriptional phenomena, including antisense mRNA transcription [48] and alternative splicing and/or transcriptional initiation [49,50]. This may be achieved by designing exon-specific array features, as well as antisense oligonucleotides. The oligonucleotide collection could also be expanded by sequences corresponding to intergenic genomic regions. Inclusion of such elements was found to be extremely useful for identifying protein-binding DNA regions by chromatin-immunoprecipitation as well as genes not detected by automated gene-prediction algorithms [51].
Within both the trophozoite and schizont categories, large numbers of genes belong to functionally related processes. These include genes encoding ribosomal subunits, multiple factors for transcription and translation, enzymes of biosynthetic and catabolic pathways, or merozoite adherence and invasion machinery. These results are consistent with predictions that a large number of plasmodial genes undergo strict stage-specific transcriptional regulation, and that such (co-)regulation is shared among functionally related genes [15,52]. Naturally, a 'fine-resolution' global gene-expression profile including the different steps of the plasmodial life cycle for multiple divergent strains will be necessary to characterize fully the intraerythrocytic life of the parasite. At present, our laboratory is analyzing a global gene-expression profile of the 48-hour erythrocytic life cycle with 1-hour resolution for three strains of P. falciparum.
In a number of model organisms, high-resolution gene-expression maps have served as extremely powerful tools for discovery and characterization of novel genes as well as exploration of multiple cellular functions [9,11]. The gene-expression maps typically comprise genome-wide expression profiles at a number of different stages of cellular development, profiles of multiple strains and genetic variants, and global expression responses to number of growth perturbations and growth-inhibitory drugs. Following a similar approach in P. falciparum is most likely to provide substantial information about the many ORFs that lack functional annotation. Further understanding of cellular physiology of this parasite including basic metabolic functions and the intricate interactions between the parasite cell and human host immune system will be a key step in uncovering new targets for antimalarial drug discoveries and vaccine development.
Materials and methods
Microarray fabrication
The 70-bp oligonucleotides were synthesized (Operon Technologies, CA), resuspended in 3 × SSC to a final concentration of 60 pmol/μl, and spotted onto poly-L-lysine-coated microscopic slides, as previously described [53]. All oligo sequences are available at [23].
Cell cultures
P. falciparum parasite cells (W2 strain) were cultured as described [54] with slight modifications: 2% suspension of purified human red blood cells in RPMI1640 media supplemented with 0.25% AlbumaxI (GIBCO/Invitrogen, San Diego, CA), 2 g/l sodium bicarbonate, 0.1 mM hypoxanthine, 25 mM HEPES pH 7.4, and 50 μg/I gentamycin. Cells were synchronized by two consecutive sorbitol treatments on two consecutive cell cycles (a total of four treatments) and harvested at the subsequent trophozoite stage (18-24 h post-invasion) and schizont stage (36-42 h post-invasion). For the trophozoite stage collection, visual inspection of the Giemsa stains show a nearly pure trophozoite population with less than 1% schizonts. For the schizont stage collection, we estimate the amount of ring contamination to be around 3%. The cells were harvested in prewarmed PBS at 37°C, and spun at 1,500 g for 5 min. Cell pellets were rapidly frozen in liquid nitrogen and stored at -80°C.
RNA preparation and microarray hybridization
Total RNA was prepared directly from the frozen pellets of parasitized erythrocytes, where approximately 1 ml of cell pellet was lysed in 7.5 ml Trizol (GIBCO) and RNA was extracted according to the manufacturer's instructions. mRNA was isolated from total RNA preparations using the Oligotex mRNA Mini Kit (Qiagen, Valencia, CA). For the hybridization experiments, 12 μg total RNA was used for first-strand cDNA synthesis as follows: RNA was mixed with a mixture of random hexamer (pdN6) oligonucelotides and oligo-(dT20) at final concentration 125 μg/μl for each oligonucleotide. The mixture was heated to 70°C for 10 min and then incubated on ice for 10 min. Reverse transcription was started by adding dNTPs to a final concentration of 1 mM dATP and 500 μM each: dCTP, dGTP, dTTP and 5-(3-aminoallyl)-2'-deoxyuridine-5'-triophosphate, (aa-dUTP) (Sigma), with 150 units of StrataScript (Stratagene, La Jolla, CA). The reaction was carried out at 42°C for 120 min and the residual RNA was hydrolyzed with 0.1 mM EDTA and 0.2 M NaOH at 65°C for 15 min. The resulting aa-dUTP-containing cDNA was coupled to CyScribe Cy3 or Cy5 (Amersham, Piscataway, NJ) monofunctional dye in the presence of 0.1 M NaHCO3 pH 9.0. Coupling reactions were incubated for a minimum of 1 h at room temperature. The labeled product was purified using QIAquick PCR purification system (Qiagen). Hybridizations and final washing procedures were carried out as described [9] with slight modifications. Briefly, the hybridization medium contained 3 × SSC, 1.5 μg/μl poly(A) DNA (Pharmacia Biotech, Uppsala), and 0.5% SDS. Hybridizations were incubated at 65°C for 8-16 h. Arrays were washed in 2 × SSC/0.2% SDS and then 0.1 × SSC at room temperature. The microarrays were scanned with a GenePix 4000B scanner and the images analyzed using GenePix Pro 3.0 software (Axon Instruments, Union City, CA). Subsequently, the data were normalized using the AMAD microarray database and subjected to the cluster analysis using the CLUSTER and TREEVIEW software, as described [53]. For the CLUSTER analysis, low-quality features and features with a signal level less than fivefold the background were filtered from the initial raw data set, yielding 4,737 elements. Subsequently, features with an arbitrary twofold fluorescence signal difference in at least four experiments were considered. All programs and microarray-related protocols are available online [55].
Probe preparation and northern blot analysis
The northern blot probes were generated by PCR using the following oligonucleotide sequences:
FWD-M11919_1: 5'-TAGAAAACAGAGCTAGCTACAGAG; REV-M11919_1: 5'-AGTTGGTTTTCCTTTGGCTGTGTG; FWD-M1282_7: 5'-CTGTAGGTGGTATCCCTTTACAAG; REV-M12812_7: 5'-GACAAATAATAATGCCATACCAGG; FWD-I12861_2: 5'-AAATGCAGTTGTTACTGTCCCTG; REV-I12861_2: 5'-GCTCTTTTGTCAGTTCTTAAATCG; FWD-F5910_2: 5'-ACAACCAGTTTGCTCTGCTTATC; REV-F5910_2: 5'-GGCCGACATTAATTGCTTATATGC; FWD-M38757_7: 5'-TAGAAGTATATCATTCCGAAGGTG; REV-M38757_7: 5'-GTAGAAGCTTCAATATCAAGCTC; FWD-M1282_7: 5'-CTGTAGGTGGTATCCCTTTACAAG; REV-M12812_7: 5'-GCTAATGCCTTCATTCTCTTAGTT; FWD-Ks44_1: 5'-GGCAAGCTATAACAAATCCTGAGA; REV-Ks44_1: 5'-GCTAAAGCGGCAGCAGTTGGTTCA.
Total RNA (10 μg) or poly(A)+ RNA (0.4 μg) was resolved on a denaturing 1% agarose gel, transferred to nitrocellulose membrane and hybridized with a radiolabeled probe as described [56]. The blots were analyzed using ImageQuant v1.2 (Molecular Dynamics, Sunnyvale, CA).
Additional data files
The predicted ORFs and GenePix results (GPR) files containing raw data for Figure 5 and from 10 additional hybridizations are available as additional data files with the online version of this paper and from [23]. Data for Figure 5: three hybridizations (1, 2,3) with trophozoite RNA labeled with Cy3 and schizont RNA labeled with Cy5; Three hybridizations (4,5,6) with trophozoite RNA labeled with Cy5 and schizont RNA labeled with Cy3. Additional hybridizations: Six hybridizations (7,8,9,10,11,12) with trophozoite RNA labeled with Cy3 and schizont RNA labeled with Cy5; four hybridizations (14,15,15,16) with trophozoite RNA labeled with Cy5 and schizont RNA labeled with Cy3.
ORF predictions of August 2000 were predicted from contig sequences available in August 2000, using GlimmerM software. These predictions were used to design the first set of 70 mer oligonucleotides and includes genes from the plastid genome. ORF predictions of October 2000 were predicted from contig sequences available in October 2000, using GlimmerM software. These predictions also include genes from the plastid and mitochondrial genomes.
We thank Pradip Rathod and his lab for ongoing discussion and assistance. We also thank the Howard Hughes Medical Institute, Anita Sil, Phil Rosenthal, David Roos and Patrick O. Brown. Z.B., M.P.J., and J.L.D. are supported by an award from the Searle Foundation and the Burroughs Wellcome Fund.
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text:
Now we come to that most wonderful and sacred time of worship when we open God's Word and listen to Him speak to us. Second Timothy is our chapter, is our book, chapter 4 our hapter, 2 Timothy chapter 4, we're looking at verses 1 to 5. We have spent two weeks in these five verses, we shall spend this week and yet one more week. The richness of this truth has captured my heart and I trust it's captured your heart as well.
As you know at Grace Community Church this month is commitment month and it's fitting that we would spend it studying this portion of Scripture, for in these five verses Paul is calling young Timothy to complete commitment to the ministry given him by his Lord. And here in chapter 4 for one final time in this last chapter of Paul's last letter, he compels Timothy to the highest level of self-sacrificing duty in the cause of Jesus Christ. This is the last call to excellence in ministry. In a sense, these five verses sum up everything that Paul has already demanded of Timothy.
As I looked back and reread 1 and 2 Timothy this week and chronicled in my mind everything that Paul expected out of Timothy, I was quite amazed at the breadth of responsibility he was given. Let me just rehearse for you briefly the standard of ministry that Paul set for Timothy in these two epistles. Paul instructed Timothy that he must correct those teaching false doctrine and call them to a pure heart, a good conscience and a sincere faith. He instructed Timothy that he must fight for divine truth and for God's purposes keeping his own faith and a good conscience. He instructed Timothy that he must pray for the lost and lead the men of the church to do the same, that he must call the women to fulfill their God-given role of submission and to raise up godly children setting an example of faith, love and sanctity with self-restraint.
He instructed Timothy that he must carefully select spiritual leaders for the church on the basis of their godliness and virtue. That he must point out error and those who taught it. That he must be constantly being nourished up himself on the words of the Scripture and its sound teaching, avoiding all myths and false doctrines. He must discipline himself for the purpose of godliness. He must boldly command as he teaches the truth of God's Word. He must be a model and an example of spiritual virtue that all can follow. He must faithfully read, explain and apply the Scriptures publicly. He must be progressing toward Christ-likeness in his own life. He must be gracious and gentle in confronting the sin of his people. He is to give special consideration and care to those who are widows. He must honor faithful pastors who work hard and never receive an accusation against any of them unless it is substantiated by two or three witnesses. He must choose church leaders with great care, seeing to it that they are both mature and proven. He must take care of his physical condition so he is strong to serve. He must flee the love of money and pursue righteousness, godliness and faith, love, perseverance and gentleness.
He must fight for the faith against all enemies and all attacks. He must keep all the Lord's commandments. He must guard the Word of God as a sacred trust and a treasure. He must keep the gift of God in him fresh and useful. He must not be timid but powerful. He must never be ashamed of Christ or anyone who serves Christ. He must hold tightly to the truth and guard it. He must be strong in character. He must reproduce himself in faithful men. He must suffer difficulty and persecution for the sake of Christ willingly. He must keep his eyes on Christ at all times.
He must lead with authority. He must interpret and apply the Scripture accurately. He is to avoid useless conversation that leads only to ungodliness. He is to be an instrument of honor, set apart from sin and useful to the Lord. He is to flee youthful lusts and pursue righteousness, faith and love. He is to refuse to be drawn into philosophical and theological wrangling. He must not be an arguer but kind, teachable, gentle, and patient even when he is wronged. And he must face dangerous times with a deep knowledge of the Word of God.
Quite a list, isn't it? That is the kind of charge that ought to be given to every young man who pursues the ministry. And I'll tell you, that takes a total commitment of life to fulfill. But what a privilege it is to have been given such a high calling. James Mason Brown was right when he said years ago, "The only true happiness comes from squandering ourselves for a great purpose...the only true happiness comes from squandering ourselves for a great purpose." And honestly, folks, the best of life belongs to those who give themselves up totally for a purpose. And the supreme purpose is the glory of God and the gospel of His Son, the Lord Jesus Christ. So the best of life belongs to those who give themselves up for the glory of God and the gospel of Christ.
Approaching that, one needs a perspective. And it struck me that in all of this which Paul has said to Timothy the emphasis is on character...the emphasis is on commitment, not on success. Nothing is said about how many people are to be in his church. Nothing is said about how large his church is or how much money it takes in or how influential it is. Nothing is said about how the world is supposed to revere, esteem or accept him. In fact, there is no mention of success. That's never the issue. The issue here is not success, the issue is excellence. And if I might, I'd like to speak to the distinction between those two things, if I may, for a moment.
None of Paul's instruction to Timothy has anything to do with success. It all has to do with a commitment to personal excellence. It all has to do with Timothy being all that God wants him to be based upon his God-given potential. Frankly, some men seek success rather than excellence. At times all of us are tempted to seek success rather than excellence. In John Johnson's book entitled Christian Excellence, he suggests that success and excellence are competing ideals. In other words, he suggests that man's energies and man's creativity is devoted to the emphasis of one or the other of those two things. He is driven to be successful or he is driven to be excellent. Being successful does not necessarily mean you'll be excellent, and being excellent does not necessarily mean, in the world's eyes, you'll be successful.
Let me see if I can't help you to understand the difference. To define success we could say that success is attaining cultural goals which elevate one's importance in the culture. Did you get that? Success is attaining or achieving cultural goals which elevate one's importance in the culture. Status, and usually success brings four things...power, prestige, wealth and privilege. Power, you have a sphere of influence, of sphere of domination. Prestige, you are esteemed, you are honored, you are looked up to, you are admired, you are envied. Wealth, you make it. Privilege, you get to do what other people don't get to do. You have access, you are on the in. Success...success is attaining cultural goals which elevate one's importance in the culture.
Let me ask you a question. What does success have to do with character? Not much. I would venture to say the powerful prestigious wealth and privileged people in our culture are not necessarily people who have excellence of character. Success still though is the pursuit of most people.
Well what about excellence, what is it? Let me give you a definition. Excellence is the pursuit of quality in one's work and effort whether the culture recognizes it or not. Excellence is the pursuit of quality in one's work and effort whether the culture recognizes it or not. Success seeks satis...success seeks status, excellence seeks satisfaction, having done your best. Success is in relation to others in the culture. Excellence is in relation to your own potential. Success is being esteemed by people, excellence is being the best I can be. Success seeks to please men, excellence seeks to please God. Success offers a hoped-for goal, excellence offers a present standard. Success bases our worth on a comparison with others, excellence gauges our value by measuring us against our own potential. Success grants its rewards to a few, but is the dream of the multitudes. Excellence is available to all human beings but is accepted only by a few. Success focuses its attention on the external, becoming the taste maker for the insatiable appetites of the conspicuous consumer. But excellence beams the spotlight not on the external but on the internal spirit, becoming the quiet but pervasive conscience of the conscientious person who years for integrity. Success engenders fantasy and a compulsive groping for the pot of gold at the end of the rainbow. Excellence brings us down to reality and a deep gratitude for the promise of joy when we do our best.
Success encourages expedience and compromise which prompts us to treat people as means to our end. Excellence cultivates principles and consistency which ensures that we'll treat all persons as intrinsically valuable, the apex of God's heavenly creation. Beloved, success pales in comparison to excellence. Excellence deals with character. Excellence deals with integrity. Excellence deals with being the best you can be. Success has no relation to any of those things necessarily. Success may be cheap. It's often discounted. You can take short cuts to get there. You can lie and cheat and steal and rob, but you will pay the full price for excellence. It's never discounted, never. It's never reduced in price. It will always cost you everything, always...always. But it's the most lasting rewarding ideal.
Listen to the words of David Neff(?), "Success is the key they hand you when they like you. It doesn't matter why. They just give you the key that unlocks the upscale condo, triggers the powerful purr of your new Mercedes. And accesses the executive washroom. Success is what they give you. Excellence is different. It's what's within you. It's what you do that stretches mind and muscle. They'll hand you success when your ratings are up, your sales soar, or when the eager masses plunk down their grubby bucks to buy your stuff, and they'll snatch your success away at daybreak. Success loves its one-night stands at the Ritz, but never expects to say `I do.' Success is a day-tripper, success is a tease. Success will forget you...and then I love this line...excellence endures when the crowd moves on." Excellence endures when the crowd moves on.
What drives you spiritually? Success? Or excellence? Bernard, the old Medieval saint had a study desk and over the study desk he had these words, "Bernard, what are you here for?" He faced them every day. "Bernard, what are you here for?" Asking himself the deepest question, what is your raison d'etre, as the French say, what is your reason to be, are you pursuing success or excellence? In 1932 George Henderson of South Africa wrote, and I like this quote, "The desire to preach helpfully without making the necessary preparation for it is not true ambition, it is merely inflammation of the wishbone." Listen, our society is full of people who have inflammation of the wishbone. They wish for a lot of stuff but they don't understand what it takes to make the necessary preparation to produce excellence. Many times we seek success rather than excellence. One is pride, the other is humility. One says I am content to be the best that I can be, the other says I'm not content until I'm better than you. That's success.
You look at it from a ministry viewpoint as Henderson also said, "Some young fellows have such a high estimate of their powers that they chafe at the restrictions which hedge them round, they feel that they were made for the universe instead of for the little village in which their ministry is exercised. Scripture, however, reminds us that a man's gift makes room for him, Proverbs 18:16, and nature teaches us that when God intends a creature to fly, he provides it with wings. If therefore we aspire to positions which we are unable, acceptably to occupy, we do but manifest our incompetence." Boy, that's profound.
If you pursue success at a level beyond your ability, you will only magnify your incompetence for everyone to see. How much better that you just be all you can be at the level God designed you to be?
The great Doctor Joseph Parker had an interesting sermon called "Clocks and Watches," he preached years ago. Quoting from that he told this story. "A little watch so delicately made was dissatisfied with its little sphere in a lady's pocket. It envied Big Ben, the great tower clock of London. And one day as it passed with her ladyship over Westminster Bridge, the little watch said, `I wish I could be up there, I could then serve the multitude.' You shall have your opportunity, little watch. And thus dramatically the little watch was taken over to the tower and by a slender thread was pulled all the way to the top where it hung directly in front of Big Ben.
"When it reached the very top, its owner standing on the ground looked up and said, `Where are you, little watch? I can't see you.'"
And then pausing in his dramatic style, Joseph Parker said, "Its elevation had become its annihilation."
You want to be exactly what God made you to be, but you want to be the best, that's excellence. Success may be your annihilation.
What Paul is saying to Timothy here is something along this line. He is not calling for Timothy to be successful, he's calling for Timothy to be excellent. There are eight elements required here in this text for excellence on the part of the preacher. Let's go back over the ones we've looked at just by mention.
First of all, he has already noted that an excellent preacher understands the seriousness of his commission, verse 1, "I solemnly charge you in the presence of God, even Christ Jesus, who is to judge the living and the dead and by His appearing and His Kingdom." That is the seriousness of his commission. If you're going to be excellent in ministry, you have to realize you're going to be judged. You also have to realize that the one who is going to judge you is the one in whose presence you are now ministering. So your judge is present now in seeing everything you do. That's the seriousness of the commission. You are now functioning in your ministry in full view of the one who will pass judgment on you. So, there must be an understanding of the seriousness of the commission.
Excellence in ministry demands that you understand the content of the commission, verse 2, "Preach the Word." The content for a faithful minister is clearly defined as the Word of God. Our task is to proclaim divine revelation, this holy book. We are to proclaim it.
Thirdly, the scope of his commission. He is to realize how far-reaching the commission to preach is. As to time, he is to be ready in season and out of season. As one writer said, there are only two times when you're allowed to preach...in season and out of season. That means at all times, in all places.
And then he talks about not only the time of ministry scope but the tone of it. It's to be negative...reprove, rebuke. Positive...exhort with patience and teaching. And so any excellent minister will understand the seriousness of his commission, the content of his commission, the scope of his commission. He is to preach. He is to preach all the time. He is to preach as the old creed said, "No sacrifice but Calvary, no priest but Christ, no confessional but the throne of grace, and no authority but the Word of God." His preaching is to be listened to, therefore it ought to be interesting. It is to be understood, therefore it should be clear. It is to be useful, therefore it should be practical. It is to be obeyed, therefore it should be biblical. The faithful preacher will have excellence in his preaching because he understands the seriousness, the content and the scope of his ministry.
That brings us to the fourth point that we'll look at this morning, the urgency of his commission. And this is a dramatic part of the text. As Paul has been calling Timothy to excellence in preaching and teaching in the first two verses, he now speaks to him about the opposition. There is a certain urgency in ministry because of what it says in verses 3 and 4, let's look at them. "For the time will come..." Stop at that point. That's a prophecy, that introduces a prophecy...the time will come. It's very much like chapter 3 verse 1, "Realize this, that in the last days dangerous times will come." It's very much like 1 Timothy 4:1, the Spirit explicitly says, "In the latter times some will depart or fall away from the faith." This is in a sense then the third prophecy to Timothy. The first one said, "Timothy, the time is going to come when people will depart from the faith." The second one, "Dangerous times are coming in the church." The third one, here it comes, "The time will come when they will not endure sound doctrine, but wanting to have their ears tickled, they will accumulate for themselves teachers in accordance to their own desires and will turn away their ears from the truth and will be turned aside to myths."
Herein, dear friends, is the urgency of the commission. We have to do it now because the time will come when people don't listen. The times come through all of church history, there have been those times in different nations at different eras when they did not hear the Word of God. The word "time" by the way is kairos, not chronos, not chronological time, but epochal time, the season, the era. It's the same word used in chapter 3 verse 1 where it says, "Dangerous times," it means seasons, eras, epochs, intervals. There will come in the future, Paul says to Timothy, those times and seasons when men will not listen. They will not hear.
You go back to Matthew chapter 10 and verse 16 as our Lord was warning His disciples, He said, "I send you out as sheep in the midst of wolves, therefore be shrewd as serpents and innocent as doves, beware of men for they will deliver you up to the courts and scourge you in their synagogues and you shall even be brought before governors and kings for My sake as a testimony to them and the Gentiles. But when they deliver you up, do not become anxious about how or what you will speak, it will be given you in that hour what you are to speak, for it is not you who speak but it is the Spirit of your Father who speaks in you. And brother will deliver up brother to death, and a father his child and children will rise up against their parents and cause them to be put to death. And you will be hated by all on account of My name but it is the one who has endured to the end who will be saved, but whenever they persecute you in this city, flee to the next, for truly I say to you, you shall not finish going through the cities of Israel until the Son of Man comes." Perhaps we can gather from the general gist of that text the promise that they were going to have trouble...that troublesome times were coming, times when men not only would not listen but would persecute the messengers of God.
In John 15 and 16, it's the same thing. Jesus said if they hated Me, they'll hate you. The time will come when their hatred will turn to avid persecution. He begins the sixteenth chapter of John's gospel, "These things I have spoken to you that you may be kept from stumbling, they will make you outcasts from the synagogues, the hour is coming for everyone who kills you to think that he is offering service to God." So He warned His disciples, Paul warns his disciple Timothy, times and seasons will come when people will not listen to the truth. Therefore fearless preaching becomes all the more necessary in light of what is ahead of us. It's not going to be able to be done at all times, do it while you can. Preach while they listen. Preach while you have a responsive audience because the time will come when people will not tolerate the truth.
Times like this have come and you can chronicle them in church history, you can study them, times when the people turned their back on the truth of God. Notice what he says in verse 3, "The time will come when they will not endure sound teaching." The word "endure," anecho means to hear, to support, to hold to. The best way to say it in English is to tolerate, they will not tolerate sound teaching. They don't want any part of sound teaching. They're not interested in sound teaching. That word "sound" there is an interesting word in the Greek, hugiainoses(?) is the form here. It's the word from which we get the English word hygienic. They don't want to hear clean teaching. They don't want to hear healthy teaching. They're not interested in that. They'll turn a deaf ear to that. In fact, in 1 Timothy 1:10 he said there are immoral men, homosexuals, kidnappers, liars, perjurers, and whatever else is contrary to healthy teaching. And where you have a society pervaded by people whose lives are contrary to healthy teaching, then you're going to have resistance to healthy teaching. You have a society filled with homosexuals and liars and perjurers and murderers and those who would overturn God's order for the sexes and feminists and all the rest of that stuff, where you have a society pervaded by that, you have resistance to the truth. No question.
Notice the word "they," please, in verse 3. "When they will not endure." The question here interpretively is to whom does they refer? Does it refer to the world outside? Well surely it does in part, but does it refer to the church? It must because those referred to as "they" are the ones to whom Timothy will preach and that no doubt will embrace the church. There would come a time when professing Christians would be included in the "they" like the church right there at Ephesus where Timothy was laboring. And they too would not be interested in hearing healthy teaching, that is the truth from God's Word. There will always be in the church a narrow gate, a narrow way, few there be that find it. There are many kinds of soil and many kinds of false conversions but not much good soil. There are many hard hearts. When God called Isaiah in chapter 6 and Isaiah said, "Here am I, send me." He said, "I have to tell you one thing, their eyes are blind, their ears are deaf, their hearts are fat, they won't listen." There's only a small remnant, He said, a tenth that will hear. People will not tolerate truth, they will not tolerate healthy teaching.
I look back on our country. I think there was a time in the United States of America, this great country, when people honored God's Word, when they listened to sound teaching, when they listened to the truth of God, when they revered the minister of God. That's not true anymore. And it's not just the world that doesn't want to hear the truth, even the church doesn't want to hear the truth. Within the large framework of what is known as the church, Christendom is a small group of people who believe the truth and are willing to face the truth, hear the truth, the healthy teaching. And today the church is advocating rights for homosexuals, feminists doctrine overturning God's design for women and I was just reading this past week the excerpts from the Feminist Bible wherever it refers to God as Father, it's changed, it's changed into some kind of non-descript sexless term so that women aren't offended. It doesn't seem to bother anybody that you can't just change the Bible unless you strike a blow at God.
But they don't mind doing that. They don't want to hear the truth. They don't want to hear the truth. This society doesn't want to hear the truth of what God teaches about the role of a woman. This society doesn't want to hear the truth about what God teaches about homosexuality. And there are many in the church who don't want to hear it either. This society does not want to hear the truth about abortion and there are many in the church that don't want to hear it. Frankly, I'm convinced that most churches in this nation would not tolerate for two weeks strong biblical teaching that confronted their doctrinal error, refuted that error, confronted their sin, convicted them of sin and called them to obey the truth. I think they'd throw you out in two weeks...in most cases. They don't want to hear healthy teaching. Why? Because the society is so corrupt and perverse the church has become the world, it now owns that philosophy and, beloved, I am telling you, unless something dramatic changes in this country the pressure is going to come greater and greater and greater against anyone who speaks the truth...because not only does the society not want to hear it, even the church doesn't want to hear it and the church, much of the liberal church, the church that doesn't affirm biblical truth, is going to side with the world against what they see as the fringe of the church. I know in the last just few years, the attacks keep coming more rapidly against me and of course that's true of anyone who stands for healthy teaching in our society. And I get more than most because of the wide-profile of ministry through radio and tapes. But it wouldn't surprise me at all for me to be in hot water with all kinds of groups who don't want to hear sound teaching..which could lead to all kinds of interesting conflict. Believe me, people can be volatile about that.
Now what is they want to hear? Look at verse 3, "Well they will accumulate for themselves," that's a very interesting little thing, you can underline that..."for themselves." You see, they're in this for themselves. "They will accumulate or mass or pile up teachers in accordance to their own epithumia, lusts. Give us people who say what we want to hear." See, that's the issue.
Jeremiah directed himself at that in chapter 5. Let's digress a moment from 2 Timothy and note with me Jeremiah 5 and verse 31, actually verse 30. His language is graphic. "An appalling and horrible thing has happened in the land." He chooses some strong adjectives. "An appalling and horrible thing has happened in the land." What is it, Jeremiah, what is it? "The prophets prophesy falsely and the priests rule on their own authority...and here it is...and my people...what?...love it that way...they love it." They want somebody to say what they want to hear. They want the prophet to lie, to tell them what they want to hear. They want the priest to function on his own authority, not divine authority.
Ezekiel confronted it, graphic terms. Look at chapter 33 of Ezekiel. And chapter 33 verse 30 is such a poignant passage. "But as for you, son of man," God speaks to Ezekiel, "your fellow citizens who talk about you by the walls and in the doorways of the houses, speak to one another, each to his brother saying, Come now, and hear what the message is which comes forth from the Lord." They talk about you, Ezekiel. They stand by the wall and talk about you. They talk about you in the doorways of their houses. And they say to each other...let's go listen to what the Lord's saying through Ezekiel. Verse 31, "And they come to you as a people come and they sit before you as my people and hear your words...here it is...but they do not do them for they do the lustful desire expressed by their mouth and their heart goes after their gain and you, Ezekiel, verse 32, you're like a sensual song by one who has a beautiful voice and plays well on instrument for they hear your words but they do not practice them. So when it comes to pass, as surely it will, then they will know that a prophet has been in their midst." They'll wake up too late. They'd listen to you out of fascination. You're like a sensual song from someone who has a beautiful voice, you're very entertaining, he must have had some oratorical capability. They want to accumulate teachers, however, who tell them what their lusts already dictate.
Now you can go back to 2 Timothy. They will accumulate for themselves teachers in accordance to their own lusts. Boy, that's true today. Churches are filled with people saying exactly what the congregation wants to hear, right? What do they want to hear? They're like those in Acts 17, "Tell us some new thing." They want what feeds their ego. They want what makes them feel good about themselves. "Give us something sensational, give us something entertaining, give us something ego-fulfilling, makes us feel better about ourselves, build up our personal satisfaction." And preachers who blast their lusts and who offend their sin, they reject. So they accumulate a mass of teachers who feed their lusts, which by the way are an insatiable desire and catering to that insatiability is folly of the worst order because it's absolutely unable to be satisfied. But that's the kind of teachers and preachers they want. And the preacher, get this, the preacher they least like to hear brings the message they most need to hear, right? Truth seldom gets a hearing...seldom gets a hearing.
That Bible scholar Vincent who did a monumental work on New Testament Word Studies said this, "If people desire a calf to worship, a ministerial calf maker is readily found." They're always there...always there.
Now their appetite has a terrible end...verse 4. It says, "And will turn away their ears from the truth and will be turned aside to myths." First there's a deliberate refusal and then they become a victim of that refusal. The first verb is an active, the second is a passive. They themselves actively turn away their ears from the truth and then they are turned as victims toward myths. You can't live in a vacuum, that's the point. If you turn away from the truth, you become the victim of Satan who will turn you into errors. It's so common today, those who resent the truth. Beloved, do you understand how many people resent the truth and how many people even in the church resent the truth? If you preach the truth boldly and you preach the truth out of God's Word, they resent that, they don't want to hear that, they don't want anything to do with that. So they turn their ears from the truth and then by an outside force they become the victims of those who turn them to myths...those hypocritical lie speakers who are the agents of Satan himself. That verb "to be turned aside" is to twist or dislocate a limb, their minds are dislocated, out of joint, non-functioning. They have a dislocated mind and their believing delusions and muthos, myths, fables, not the truth.
That's what we have today. The trash, the myths, the fables, the theological error that's being pervasively preached and taught and advocated, people are flooding into that. Why? Because they don't want to hear the truth and so they become victims of that because you can't exist in a vacuum. As soon as you turn away from the truth, you become the pawn of Satan, he moves you.
What's behind it all? Go back to verse 3 and let's pick up the key phrase, here's the motive for the whole deal. Why will not they endure sound doctrine? Why do they accumulate teachers? Why do they turn away from the truth? Because down deep inside they simply want to have their ears...what?...tickled. They don't want to be confronted. They don't want to be convicted. They just want to be entertained. You see, it refers to speaking things which produce pleasant sensations, that's the idea. They just want to feel good. They want their ears scratched, no deeper penetration, just give me some nice stories...little positive thinking, a little psychology, a little ego feeding, a little self-congratulation. Might even talk about some of the tragedies of human kind so I feel sort of like there's a cause. Make me feel good about myself. Just tickle my ears, give me some nice sensations.
You want to know something about the gospel? The gospel does not tickle your ears, it boxes them. Not only boxes them, it burns them. It pounds your ears, it reproves, it rebukes, it convicts. This is what preachers face. Herein lies the urgency of our ministry. In my short life time there has been an escalating indifference to the truth, is that not true in yours? Even in the church. And we must preach while we can what we can before they stop hearing all together. Soon, I think, soon I think, tolerance for the truth is very very low.
I was reading last...two weeks ago...about a young girl who was the valedictorian of her high school graduating class. She was to give the speech at graduation. In her speech, she was a Christian, she used the word "Lord" once in a statement something like this, "As I look forward to life, I'm grateful that the most important person in my life is my Lord." Nothing more than that. The school demanded that she remove the word "Lord" from the speech. She refused to do that so they didn't let her give it. This is the United States of America. On our coins we have, "In God we trust," but you can't say "Lord" in a high school graduation, even if that's your belief. Frightening! Intolerance for the truth, even anything that comes close to the truth. A faithful preacher knows then that there's an urgency about what he does. I don't know how much time we'll have to keep declaring the truth before they rise up against us, but we keep doing it while we can.
So the one who serves with excellence is one who is compelled by the seriousness of his commission, the content, the scope, the urgency. Let me give you one more this morning, this is so good, just briefly. The attitude of his commission...the attitude. What is the pervasive attitude that we are to have? Look at verse 5, just the first..."But you, but you in contrast to those flaky people who won't listen to truth and turn away, you be sober in all things." What do you mean by that? Well what does he mean be sober, not drunk? Well, that certainly is part of it, but that's not what he means. Sober means self- contained, steady. Let me give you my favorite synonym, stable, unmoved, unwavering, steadfast. Says...Hort(?), the Greek scholar, "It describes a mental state in which every faculty is at full command, in which an individual is able to look at all facts and all consideration right in the face with deliberation."
This is a solid person, a stable person, who is like an athlete who has brought all his passions and all his appetites and all his nerves under complete control to perform at a maximum. To put it in the negative, a preacher is not to be flaky, not to be trendy, not to be a victim of whims, not to be inconsistent, to be stable. In the midst of a changing world, in the midst of a changing church, in the midst of a rocking and reeling society, in the midst of all of this animosity and antagonism toward the truth, you better be a steadfast, stable, firm rock or you'll compromise when the pressure's on. You be stable in all things. Don't you become a victim. Stability is the issue here. We've all had enough of flaky preachers, trendy preachers, whimsical preachers who flip-flop depending on the tide of the mob. What we need are those who live totally steadfast in an unstable world, who know their priorities. We need men of God whose heads are clear of deceit, whose heads are clear of false teaching, whose heads are clear of whimsy, who are unaffected by the people's desire to have their ears tickled. O how I pray that God would raise up powerful preachers. O how wearing it is to hear insipid innocuous pablum dribbled out of pulpits instead of the profound Word of God. I think this also implies an unruffled alertness, a watchfulness. There's something very balanced. There's something very consistent. There's something very solid. There's something very clear- minded about the noble preacher, he never picks up the ear tickling stuff that entertains the lusts of the wanton masses. He's just unmoved by it.
Timothy, would have to face that. He would have to face the fact that the heat would really come because they wouldn't want to hear what he was saying and he needed to be firm and say it anyway because he had to discharge his commission before God. Excellence, not success, excellence...not how do I stack up with others in my culture, but how do I stack up with God? Not am I better than someone else, but am I the best that I can be? That's the issue, always. Paul is calling Timothy to excellence and calling me to excellence and calling you to excellence. And so we need to look at our lives, don't we? All you can be is all you can be. And if you try to be more, you'll be a pocket watch hanging in front of Big Ben and nobody will know you're there and your elevation will your annihilation. And if you pursue success you'll compromise, but if you pursue excellence, you can't compromise...excellence doesn't have any room for compromise. You can go the cheap route to success but it will cost you everything to go the route of excellence. You can get your success here and now, or you can have excellence reward you forever and ever and ever. The choice is yours and it's yours every day, every hour of every day. It comes down to that. Let's pray together.
I think of the words in this same passage, Father, where Paul says, "I have fought the good fight, I have finished the course, I have kept the faith." That little statement, "I finished the course," is so thrilling, to be able to come to the end of your life and know you finished your course, you did it with excellence. Help us, Father, to take the lead of Paul and be all that we can be, not in our flesh but in Your Spirit, not in our strength, but in Your strength, doing all we can do and yet knowing as Paul said in Colossians that it's the Spirit which works in us mightily. May we be noble servants for You, knowing the urgency of a time when they will not listen, maintaining the attitude of steadfastness in the midst of all the change. Raise up a generation of people like this for Your glory in Christ's name. Amen.

subset: commoncrawl


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"Study requires calm, talent requires study. Without study there is no way to expand talent; without calm there is no way to accomplish study."
| Zhuge Liang's advice to his son |
Zhuge Liang (Kongming), born 181 lived in the turbulent times of the declining HAN Dynasty and during the times of the Three Kingdoms. From his death in 234 until now, memories, stories and legends have created a myth around Zhuge Liang ... by many regarded as the most intelligent and successful strategist of all times. However not all stories are true, so let's start looking at what we really know about Zhuge Liang: Chen Shou wrote Sanguozhi (Records of Three Kingdoms) in 274 - only 40 years after Kongming's death. This book is regarded as the closest we will get to the person of Zhuge Liang. So let's start our journey here:
by Chen Shou in Sanguozhi (Records of the Three Kingdoms)
As the Chancellor of the State, Zhuge Liang pacified the commoners, elucidating proper behaviour and the moral standard. He simplified the administration and established timely, just policies; speaking frankly and sincerely. In dealing with the loyal and useful, he would reward them even if he felt enmity against them. In dealing with the criminal and heinous, he would dispense punishment if they were friends or relatives. In dealing with those who confessed and truly repented, he would be merciful even if their crimes were serious. In dealing with those who were glib-tongued and deceitful, he would be punitive even if their crimes were trivial. A benevolence no matter how minute would be rewarded. A malevolence no matter how slight would be castigated. In all affairs his administration was precise and penetrating, organising from the fundamentals, serving needs according to the true situation. He detested most the practise of deception. In the finality, all within the state esteemed him. Even though the legal system was severe, none despised him because his justice was fair and open. It can be said that he was exceptional in administration and the like of Guan Zhong and Xiao He. Yet year after year he marshalled the army and never attained success, probably because flexibility of strategy was never his forte.
Read the full Zhuge Liang (Kongming) Biography from Chen Shou's Sanguozhi here: Zhuge Liang Biography.
In the winter of 208 Cao Cao led a large army on a southern campaign. The allied armies of Sun Quan and Liu Bei were massed on the south bank of the Yangtze River. They planned to attack the warships of Cao Cao with blazing barges. When all the preparations for the attack were completed, they called to mind that the wind in winter always blew from the northwest, and so would blow the fire barges back toward their own ships. So an east wind was needed for the attack. Zhuge Liang said that he could summon an east wind, and he did so... The result was that Cao Cao's ships were burnt to ashes.
Later, the phrase came to be used to describe a situation in which all is ready except what is crucial for the fulfillment of an undertaking.
This allusion is taken from The Romance of the Three Kingdoms.


text:
Complete video documentation of Los Angeles Bread Bakers' January 5 meeting on the background of AB1616, California's Cottage Food Law is available at: http://youtu.be/CAeW_ccTXTQA detailed Q&A session with L.A. County Health representatives at that meeting is also available at: http://youtu.be/dasVKuFZupo
The three short videos on this page are of Mark Stambler's comments on how AB1616 came to be. They are incorporated in the background video on YouTube, above. Mark, a bread baker and co-founder of Los Angeles Bread Bakers, is the recipient of the first Class B permit.
Part one.
Part two of Mark Stambler's remarks.
This is part three of Mark Stambler's remarks.


text: This is a high quality battery-operated Silver Bell LED String Light with 24 Warm White LEDs (Light Emitting Diode). With the environmental friendly and also the durability characteristics, it is specially designed for using in places that have no power supply. It gives festive lighting to your decorations for Christmas parties, 4th of July Celebrations and Daily Decor. Designed with steady ON and a variety of flashing modes, it is a great idea to attract any of your guests. Operated by 3 AA batteries, it is very convenient for indoor/ outdoor decoration without any household power supply, perfect choice to place on a fireplace mantel, front door wreath, BBQ picnic table, or centerpiece.


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You can capture video of all of the amazing things happening on your desktop with one of Linux's many screencasting applications. These programs are perfect for creating demonstrations for blogs and tutorials, and for illustrating projects with more than just still images.
Many different programs are available, and they all provide a different set of features, options, and output formats. When choosing, consider the degree of control you want to have over your video resolution and whether the video is intended to work on non-Linux operating systems. Common open source output formats, such as FLAC and Ogg Theora, work natively on Linux but require software and plugins on proprietary operating systems. User interfaces also vary greatly; some applications are nothing more than an icon in the system tray while others depend on large interfaces with many options.
I tested three popular screencasting applications to see which is best for everyday use. I evaluated the user interface, the quality and variety of output formats, and the ease of installation and obtaining the required dependencies. I tested the programs on a MacBook Intel Core 2 Duo with 4GB of RAM, dual-booting into Ubuntu 8.04 Hardy Heron.
Istanbul is a simple desktop recorder that lives in your system tray. Clicking on the icon starts the recording session, and clicking it again ends it. The video is encoded in the Ogg Theora format. You can capture the full screen, a selected window, or a portion of the desktop, with or without sound.
This application doesn't have a complicated user interface, making it easy to use, but there are some drawbacks. Although you do have some control over the resolution size, you cannot change the output format of the video file. This isn't much of a problem if you're going to upload the video to a Flash-based video site, but the Ogg Theora format doesn't work out of the box of the main proprietary operating systems without an application like the VLC media player. Therefore, you'll have to convert to a different format if your intended audience requires it.
Istanbul is easy to install, and it is included in most distribution repositories. In Ubuntu, I was able to install it with the Synaptic Package Manager. It requires PyGTK and GStreamer, which are common dependencies.
Wink is geared toward making video tutorials; it compiles a series of screenshots to a Flash format, and allows you to edit the video directly and add text boxes, navigation buttons, and still images. It includes many advanced features, such as the ability to create preloaders (elements that load portions of the video before it starts playing) and control bars for the Flash playback.
The interface is not as minimal as Istanbul's, but it is still relatively straightforward. The main drawback of Wink is the video output formats: Flash and Windows .exe programs are good for viewing the video when the file is local or when it is hosted on a personal Web site, but you can't upload Flash and .exe files to most social networking and video sharing sites. Wink is good for corporate entities who want to share a tutorial with staff, but it is less useful for home users who want to show off their desktop on YouTube.
Most distributions include Wink in their repositories, and you can install it with any package management application. Unfortunately, though, Wink doesn't work with Ubuntu 8.04 as of the writing of this article without some hacking. I tested it for this review with a previous version of Ubuntu.
With XVidCap, you can capture desktop video and take single-frame screenshots. You can record portions of the screen by dragging a red selection rectangle over the area you wish to capture. The program can handle a wide variety of output formats, including MPEG, AVI, Flash, and QuickTime.
The interface consists of a small toolbar with more options tucked away inside a Preferences panel. XVidCap uses the FFmpeg libraries to capture video or a series of images, and it can also embed audio. Some people claim that XVidCap can dramatically slow down your computer if you try to record a large area. I didn't have that problem on my machine, but I had 4GB of RAM.
Installation of XVidCap is easy because it comes in the repositories for most distributions. It requires common dependencies, such as libavcodec (the FFmpeg codec libraries for video), and Cairo, Glade Interface Designer, and GTK+ to render the graphical interface.
XVidCap is the most versatile of these three tools for recording your desktop and creating screencasts. Istanbul is a close second, but its lack of support for a variety of different output types is a drawback. Wink is nice, but it's limited in use to only a few tasks, and it's not for users who merely want to record their desktops. XVidCap can handle many popular video formats, allowing it to play nice with both Windows and Mac OS X. The red selection box is kind of clunky, and there is no easy way to record only a specific window without it, but XVidCap gets the job done in an efficient manner.


text: Barby007 12 Posted January 5, 2013 Hey everyone! I just started playing Netrunner and I LOVE IT!! I think it's the best game FFG has IMO. I have sided with being a runner. I really dislike playing Corporation. Not my play style at all. I have become quite attracted to the runner "Mac", the Shaper. I feel she is great based on just playing her using core set cards only. On the other hand, I would like to pick a good Runner because my goal is to eventually play at the tournament level. I love being able to play great players. Also, I would like to be good enough to be able to support my LGS and judge Netrunner leagues for them. I have the Core Set and the "What Lies Beneath" expansion pack. In your opinions, 1. Which runner do ya'll feel have the best chance of winning? 2. What is a good list for the runner of choice? 3. Why is that particular runner better than the other 3 (Expansion guy included)? Thanks, Barby Quote Share this post Link to post Share on other sites


text:
Neo-sexualism;Neo-sexualist: Persons who participate in constant behavior and or speech with innuendos or sexual overlays when unwanted or misplaced in certain situations and/or locations.
Person A recently found that their house was vandalized, person A turns to person B and says: "My back door was broken in!" Person B responds: "That's what she said." Person A responds: "You're such a Neo-sexualist."
Thus person B is participating in Neo-sexualism.
Thus person B is participating in Neo-sexualism.
by Satoshi Sama April 15, 2010
buy the domain for your cat vlog


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I was browsing hoopshype earlier and I noticed that there is the possibility that the management is shopping Baron Davis?! (Allen Iverson is mentioned too). Any possibility that management could be secreting brewing up a trade for BD? And for who? If possible what do you guys think we could get for him?
Btw, heres the link: http://www.oregonlive.com/canzano/index.ssf?/mtlogs/olive_JohnCanzano/archives/2006_07.html#166832
BD on the Block
Discuss anything related to Golden State Warriors basketball here
Moderators: Mr. Crackerz, JREED, Guybrush, 32
I've kinda backed away from following trades so much after the Harrington deal fell through on our part...but with the possibility of BD departing the Bay Area, this would be something interesting to debate about. I would hate to see him go like Murphy, since I feel he should be given one more shot to bring the team to a playoff status.
#32 wrote:A BD-for-Iverson swap would mean death for the Warrior franchise. Iverson would get us to the first-round... and then we'd commence to tank, like the Sixers currently are. I hope we give BD another year...
will the real BD please stand up?
****, I'd be disappointed to see BD leave, since we were all so psyched up when he came to town. I'd like to see him and J-Rich get us into that postseason hehe.
#32 wrote:I would, too. One full year together isn't enough for me.
a starting five with BD, JRich and Ike would constitute the most entertaining Ws team in at least a decade
coltraning wrote:#32 wrote:I would, too. One full year together isn't enough for me.
a starting five with BD, JRich and Ike would constitute the most entertaining Ws team in at least a decade
if they won that is! Otherwise that'd be entertainment for the fans of the opposition
We'll anyways, I hope that the possiblity of BD on the market is bs, having arguably the most entertaining backcourt in the nba and a booming Ike Diogu is something I'm dying to see. And the new schedule for the 06-07 year came out, seems like the team is playing 16 out of the first 23 games at home. Seems eerily similar to last year. They were 12-6 by the end of November and just tanked after 06 came around. What needs to change in order for them to consistently keep the wins up by the end of 06?
ChurroChou wrote:What needs to change in order for them to consistently keep the wins up by the end of 06?
Their ability to finish in the fourth quarter. The Warriors (in more than half the games they lost) were never completely left in the dust; they blew leads and/or made too many costly mistakes that allow the opposition to capitalize (free throws, melting defense, ect). If the Warriors hope to establish themselves as more than a league doormat, they need to prove they can hang in the fourth... and with Derek Fisher (arguably, our best 4th quarter player) gone, it seems even harder this year.