text:
- Research article
- Open Access
Deep and comparative analysis of the mycelium and appressorium transcriptomes of Magnaporthe grisea using MPSS, RL-SAGE, and oligoarray methods
- Malali Gowda†1,
- RC Venu†1,
- Mohan B Raghupathy1,
- Kan Nobuta2,
- Huameng Li1,
- Rod Wing3,
- Eric Stahlberg4,
- Sean Couglan5,
- Christian D Haudenschild6,
- Ralph Dean7,
- Baek-Hie Nahm8,
- Blake C Meyers2Email author and
- Guo-Liang Wang1Email author
© Gowda et al; licensee BioMed Central Ltd. 2006
- Received: 14 June 2006
- Accepted: 08 December 2006
- Published: 08 December 2006
Rice blast, caused by the fungal pathogen Magnaporthe grisea, is a devastating disease causing tremendous yield loss in rice production. The public availability of the complete genome sequence of M. grisea provides ample opportunities to understand the molecular mechanism of its pathogenesis on rice plants at the transcriptome level. To identify all the expressed genes encoded in the fungal genome, we have analyzed the mycelium and appressorium transcriptomes using massively parallel signature sequencing (MPSS), robust-long serial analysis of gene expression (RL-SAGE) and oligoarray methods.
The MPSS analyses identified 12,531 and 12,927 distinct significant tags from mycelia and appressoria, respectively, while the RL-SAGE analysis identified 16,580 distinct significant tags from the mycelial library. When matching these 12,531 mycelial and 12,927 appressorial significant tags to the annotated CDS, 500 bp upstream and 500 bp downstream of CDS, 6,735 unique genes in mycelia and 7,686 unique genes in appressoria were identified. A total of 7,135 mycelium-specific and 7,531 appressorium-specific significant MPSS tags were identified, which correspond to 2,088 and 1,784 annotated genes, respectively, when matching to the same set of reference sequences. Nearly 85% of the significant MPSS tags from mycelia and appressoria and 65% of the significant tags from the RL-SAGE mycelium library matched to the M. grisea genome. MPSS and RL-SAGE methods supported the expression of more than 9,000 genes, representing over 80% of the predicted genes in M. grisea. About 40% of the MPSS tags and 55% of the RL-SAGE tags represent novel transcripts since they had no matches in the existing M. grisea EST collections. Over 19% of the annotated genes were found to produce both sense and antisense tags in the protein-coding region. The oligoarray analysis identified the expression of 3,793 mycelium-specific and 4,652 appressorium-specific genes. A total of 2,430 mycelial genes and 1,886 appressorial genes were identified by both MPSS and oligoarray.
The comprehensive and deep transcriptome analysis by MPSS and RL-SAGE methods identified many novel sense and antisense transcripts in the M. grisea genome at two important growth stages. The differentially expressed transcripts that were identified, especially those specifically expressed in appressoria, represent a genomic resource useful for gaining a better understanding of the molecular basis of M. grisea pathogenicity. Further analysis of the novel antisense transcripts will provide new insights into the regulation and function of these genes in fungal growth, development and pathogenesis in the host plants.
- Annotate Gene
- Antisense Transcript
- Alternative Transcript
- Massively Parallel Signature Sequencing
- Appressorium Formation
One of the most daunting challenges in the post-genomic era is to identify all transcribed regions in a sequenced genome. Although computational programs have played an important role in genome annotation, highly accurate prediction of the coding regions of many genes is still challenging [1]. Therefore, the experimental approaches such as ESTs (expressed sequenced tags) [2], full-length cDNA sequencing [3], SAGE (serial analysis of gene expression) [4, 5], MPSS (massively parallel signature sequencing) [6, 7], RATE (robust analysis of transcript ends [8] and microarrays [9, 10] are essential tools to validate the annotated putative transcriptional units (TUs) in the sequenced genomes.
Although EST and full-length cDNA sequencing are effective approaches for gene discovery and expression profiling, neither is cost-effective nor comprehensive enough to isolate the majority of rare transcripts. Since the fabrication of most types of microarrays relies on sequences that are derived from ESTs, full-length cDNAs or computer program-predicted coding regions, many rare transcripts may be absent from these arrays. SAGE and MPSS are two powerful methods for genome-wide expression profiling and novel gene identification [4, 5, 7]. Both methods determine the sequence of short tags derived from a defined position at the 3' regions of expressed mRNAs in a cell. SAGE uses traditional sequencing technologies, while MPSS uses a novel bead-based hybridization procedure [6]. The main advantage of MPSS over SAGE is that over one million signatures can be obtained from a library in comparison to 100,000 to 200,000 tags in a SAGE library. However, SAGE method is technically simple and can be performed in most molecular laboratories, whereas MPSS involves many complex cloning steps and library construction and sequencing is only performed at Solexa, Inc. The newly developed 5'-RATE method simplifies tag cloning and sequencing procedures and should have a broad application for transcriptome analysis of complex genomes [8].
Magnaporthe grisea, the causal agent of rice blast disease, causes severe yield losses to cereals, including the economically important crops, such as rice, barley, wheat, and millets [11]. This fungus completes its life cycle by conidia landing on the leaf surface, conidia germination, appressorium formation, penetration, establishment and new conidia formation within seven to ten days after infection [11, 12]. In addition to its economic importance, the recent release of the complete genome draft sequence [13] and characterization of several important avirulence/pathogenecity-related genes [14], makes M. grisea a model plant pathogen for studying host-pathogen interactions. Although 12,841 genes (Annotation release 5, [15]) are predicted in the genome of M. grisea, many of these genes have no experimental support. Identification and characterization of all expressed genes from the infectious (appressoria) and non-infectious (vegetative mycelia) structures are indispensable for understanding the mechanism of fungal pathogenesis and designing novel strategies to combat the disease.
The main objective of this study was to identify all expressed transcripts encoded in the M. grisea genome using MPSS, RL-SAGE and oligoarrays, and to compare the qualitative and quantitative measurements produced by these three methods. Using these three technologies, the M. grisea transcriptome in the vegetative mycelial stage and in the infectious appressorial stage was analyzed in depth, in which many novel sense and antisense transcripts and alternative splicing variants were discovered. We also compared the correlation coefficients of the gene expression patterns revealed by MPSS, RL-SAGE and oligoarray. Our results provide the most comprehensive analysis of the sense, antisense and alternative transcripts of a fungal genome published to date and represent a useful genomic resource for further detailed functional analysis of the expressed transcripts involved in M. grisea growth and pathogenesis.
Characterization of mycelium and appressorium transcriptomes of M. grisea by MPSS and RL-SAGE methods
MPSS and RL-SAGE libraries from mycelial and appressorial stages of 70-15 strain of M. grisea
Growth stage
24-hr after germination
96-hr liquid culture
96-hr liquid culture
Anchoring enzyme and tag size
GATC (Dpn II) + 13 bases
GATC (Dpn II) + 13 bases
CATG (Nla III) + 17 bases
No. of genomic virtual tags 1
No. of total tags per library
No. of unique tags per library
No. of copies per tag
2 to 28,845
1 to 30,910
1 to 7,383
No. of significant tags 2
No. of non-significant tags 3
No. of significant antisense tags 4
653 (5.1%)
1,825 (14.6%)
605 (3.6%)
An RL-SAGE library was constructed using the same RNA sample that was used for the construction of the mycelium MPSS library (Table 1). The average insert size of the RL-SAGE clones was approximately 1.1 kb. A total of 245,873 individual tags and 51,925 distinct tags were obtained from 7,292 sequence reads. Out of the total distinct tags, 16,580 (31.9%) were significant (= 2 copies) and 35,345 (68.1%) were non-significant (single copy) transcript tags.
Matching analysis of the MPSS and RL-SAGE tags to M. grisea genomic and expressed sequences
We compared the MPSS and RL-SAGE data to both genomic and expressed sequences of M. grisea to identify the genes from which these tags were obtained. The whole genome sequence from the Broad Institute (Release 5.0 on January 2006 at [15]) and the ESTs from TIGR (Release 5.0 on April 28, 2004 [16]) and the COGEME database [17]) were used to match MPSS and RL-SAGE tags. The matching results are summarized below.
1) MPSS tags
Matching analysis of MPSS and RL-SAGE tags to M. grisea EST and genomic sequences.
Significant tags (%)
Non Significant tags (%)
Significant tags (%)
Non Significant tags (%)
Significant tags (%)
Non Significant tags (%)
Genomic DNA
Both sense + antisense
10,957 (84.8)
2,335 (66.7)
10,649 (85.0)
5,912 (76.1)
10,772 (65.0)
8,950 (25.3)
5,597 (43.3)
753 (21.5)
5,557 (44.3%)
1,980 (25.5)
3,684 (22.2)
2,218 (6.3)
1,185 (9.2)
415 (11.8)
2,374 (18.9)
1,912 (24.6)
1,572 (9.5%)
1,419 (4.0)
Both sense + antisense
1,149 (32.8)
7,750 (61.8)
5,067 (30.6)
3,567 (10.0)
Coding region (CDS)
4,574 (35.4)
690 (19.7)
4,761 (38.0)
1,935 (24.9)
2,058 (12.4)
1,751 (5.0)
653 (5.1)
329 (9.4)
1,825 (14.6)
1,837 (23.6)
605 (3.6)
890 (2.5)
Both sense + antisense
5,205 (40.3)
1,016 (29.0)
6,560 (52.4)
3,759 (48.4)
2,648 (16.0)
2,638 (7.5)
500 bp upstream sequences2
1,730 (13.4)
367 (10.5)
1,352 (10.8)
715 (9.2)
2,548 (7.2)
1,399 (4.0)
699 (5.4)
284 (8.1)
906 (7.2)
655 (8.4)
2,495 (7.0)
1,204 (3.4)
Both sense + antisense
905 (7.0)
301 (8.6)
877 (7.0)
630 (8.1)
964 (5.8)
962 (2.7)
500 bp downstream sequences3
4,075 (31.5)
618 (17.7)
2,986 (23.8)
1,097 (14.1)
3,117 (6.0)
4,842 (29.2)
3,687 (28.5)
581 (16.6)
3,044 (24.3)
1,037 (13.4)
2,251 (4.3)
3,997 (24.1)
Both sense + antisense
600 (17.1)
3,065 (24.5)
1,005 (12.9)
5,131 (30.9)
2,799 (7.9)
Number of genes transcribed4
Novel transcript tags-TIGR ESTs5
4,478 (34.64)
1,220 (34.85)
3,297 (26.3)
2,281 (29.4)
5,887 (35.5)
5,499 (15.55)
Novel transcript tags-COGEME ESTs6
7,573 (58.6)
1,876 (53.6)
6,737 (53.8)
4,607 (59.3)
7,366 (44.4)
7,167 (20.3)
Putative intergenic tags7
1,618 (12.5)
571 (16.3)
1,211 (9.7)
977 (12.6)
2,716 (16.4)
3,004 (8.5)
Putative intergenic tags match ESTs8
394 (3.0)
70 (2.0)
395 (3.1)
189 (2.4)
596 (3.6)
437 (1.2)
2) RL-SAGE tags
In comparison with the MPSS libraries, more significant tags were identified in the mycelial RL-SAGE library. About 65% of significant RL-SAGE tags matched the M. grisea genomic sequences (Table 2). Unlike the MPSS tags, only 31% (5,067) of the significant tags from the RL-SAGE library matched to the M. grisea EST sequences (TIGR) and a small portion (16%) of the tags matched to the CDS of the annotated genes (Table 2). We also observed that about 31% of the mycelial RL-SAGE significant tags matched to the 500 bp downstream of the 3' region of the annotated genes and about 6% of the significant tags matched within 500 bp upstream of the 5' region of the annotated genes (Table 2). The reason for the high percentage of the RL-SAGE tags located within the 500 bp downstream region might be due to more NlaIII sites (RL-SAGE) than DpnII sites (MPSS) in the 3' UTR region. Combining the tags hit to CDS, 500 bp upstream and 500 bp downstream regions, we identified 6,028 genes in mycelia by the RL-SAGE method.
3) Identification of novel transcript tags in the MPSS and RL-SAGE libraries
A detailed analysis of the novel transcripts that did not match to any sequences in the ESTs and annotated genes was performed (Table 2). The percentage of the novel tags in the appressorium MPSS, mycelium MPSS, and mycelium RL-SAGE libraries was about 35%, 26%, and 35%, respectively [see Additional File 2]. A total of 3,339 genes in the RL-SAGE library identified by CDS and their 500 bp upstream and down stream regions did not match the ESTs either in TIGR or COGEME database. Similarly, 3,186 and 2,298 genes identified in the MPSS appressorial and mycelial library, respectively, also did not show any matches in TIGR or COGEME M. grisea ESTs. In the two MPSS libraries, about 67% and 76% of the non-significant appressorial and mycelial tags matched to the genome sequence, respectively (Table 2). Even among the tags that did not match the genome sequence, many of them were significant tags, which might be derived from the un-sequenced or intron-exon junctions in the genome. Some of them may be true transcripts encoded in the genome but could not be matched due to sequencing errors in the genome or in the EST, MPSS or RL-SAGE tag sequencing. To validate the MPSS and RL-SAGE results, two genes (MGG_04847.5 and MGG_0490.5) without any EST matches in the public databases were amplified and cloned (data not shown), demonstrating that the majority of the identified novel tags might be true transcripts.
Antisense transcripts for the annotated genes of M. grisea
1) Antisense transcript tags from the MPSS libraries
2) Antisense transcript tags from the RL-SAGE library
Although the total number of significant antisense transcript tags (3,558) corresponding to 3,100 genes identified from the mycelial RL-SAGE library was similar to that of the mycelial MPSS library, the tag distribution in the three regions of the annotated genes was different (Figure 1). The proportion of the RL-SAGE antisense tags located in the 500 bp downstream regions was twice (71.2%) that of the MPSS library (38.7%) at the mycelial stage. Similar with the sense tags, this difference might be due to the use of different anchoring enzymes in MPSS (Dpn II) and RL-SAGE (Nla III) library construction. Among the identified antisense tags, 364 were present in both RL-SAGE and MPSS libraries, 1,730 were specific to the MPSS libraries, and 431 were specific to the RL-SAGE library. The antisense tags and their frequency are listed in Additional File 4.
3) Annotated CDS regions with sense and antisense MPSS tags
Alternative sense and antisense ESTs, MPSS tags and RL-SAGE tags for M. grisea genes.
Annotated M. grisea gene
Gene ID
Putataive function
Sense tag*
Antisense tag*
Sense tag: CATG+17 bases (No. of tags in mycelia)
Antisense tag: CATG+17 bases (No. of tags in mycelia)
Sense tag: GATC+17 bases (No. of tags in appressoria:mycelia)
Antisense tag: GATC+17 bases (No. of tags in appressoria:mycelia)
Ribosomal protein, S9
Ribosomal protein, L34
DEAD box protein
MST12 – transcription factor
ATP synthase alpha chain-like protein
4) Annotated CDS regions with sense and antisense RL-SAGE tags
We identified that nearly 10% of the annotated genes in the M. grisea genome had bothsense and antisense tags from the mycelial RL-SAGE library(data not shown). The significant lower number of tag pairs at the protein coding region in the RL-SAGE library is likely because that the majority of the RL-SAGE tags were located in the 500 bp downstream region of the annotated genes (Figure 1). Some of the sense and antisense RL-SAGE tag pairs are also present in the MPSS libraries and public EST collections (Table 3).
Identification of alternative transcript tags in mycelia and appressoria
Since MPSS and RL-SAGE tags are derived from the 3' end of each transcript, the presence of more than one tag in the CDS of a gene suggests an alternative termination of the gene. These termination differences could be derived from either alternative polyadenylation or alternative splicing at the 3' end. All the transcripts from both cases are called alternative transcripts in this study. To determine the extent of transcriptional diversity in M. grisea, we assessed the proportion of the genes with alternative terminations in both libraries.
1) Alternative sense and antisense MPSS tags in mycelia and appressoria
Frequency of the alternatively transcribed sense and antisense tags for the coding regions of M. grisea.
Sense tags
Antisense tags
No. of alternative tags
No of antisense tags/gene
≥ 5
≥ 5
No of genes with ≥ 2 tags (%)
937 (20.8)
1,631 (35.0)
955 (27.5)
No of genes with ≥ 2 tags (%)
232 (19.9)
847 (31.8)
438 (24.0)
No of genes with single tag (%)
3,566 (79.2)
3,028 (65.0)
2,518 (72.5)
No of genes with single tag (%)
933 (80.1)
1,809 (68.2)
1,389 (76.0)
Similarly, many annotated genes had more than one antisense tags in the MPSS libraries (Table 4). Twenty percent of the annotated genes in appressoria and 32% in mycelia had more than one antisense tag. As with the sense transcript tags in mycelia, the same gene (MGG_13239.5) had thirteen alternative antisense tags (data not shown).
2) Alternative sense and antisense RL-SAGE tags in mycelia
About one-fourth (27.5%) of the annotated genes in mycelia were found to produce at least two alternative sense tags in the RL-SAGE library, which is less than that observed in the MPSS mycelial library (Table 4). In the RL-SAGE mycelial library, a hypothetical protein similar to reverse transcriptase (MGG_13890.5) was found to encode sixteen alternative transcript tags (data not shown). Many genes with known functions were found to encode alternative transcript tags [see Additional File 7]. A total of 10,629 alternative tags were commonly present in both RL-SAGE and MPSS [see Additional File 8].
Among the antisense RL-SAGE tags in the mycelium library, nearly a quarter (24%) had at least two alternative antisense transcript tags per gene (Table 4). A hypothetical gene (MGG_00329.5) was found to generate seven alternative antisense transcript tags (data not shown). Several genes with multiple sense and antisense alternative tags were also identified. For example, four sense and one antisense tags were obtained for the HSP70 gene (data not shown).
Characterization of the appressorium and mycelium transcriptomes by oligoarray hybridizations
To compare the transcriptional profiles generated from MPSS and RL-SAGE with that from oligoarray analysis, the same RNA samples used in MPSS and RL-SAGE library construction were hybridized to the M. grisea/O. sativa oligoarray [18]. Using a stringent cut off at false discovery rate (FDR) = 0.05 that corresponds to a p- value of 0.001, 9,138 genes (43.9%) were identified to be statistically significant expressed in mycelium and appressorium tissues. Among them, 8,569 probes are from M. grisea genes and 569 probes from rice genes. The hybridizations with the rice genes were likely due to sequence similarity between housekeeping genes in both organisms. Of the 8,569 M. grisea genes, 4,652 (54%) and 3,917 (46%) were differentially (2 fold) up-regulated in appressoria, and down-regulated in mycelia, respectively. We identified 846 M. grisea genes that were ≥ 3.0 fold significantly up-regulated in appressoria, and 792 genes that were ≤ 3.0 fold significantly down-regulated in mycelia. The top 20 highly and specifically up- and down-regulated genes in appressoria and mycelia are shown in [see Additional File 9].
Qualitative analysis of the M. grisea transcriptome using MPSS, RL-SAGE, and oligoarray platforms
Quantitative analysis of the mycelium and appressorium transcriptomes using MPSS, RL-SAGE, and oligoarray platforms
Comparison of M. grisea transcriptomes measured by MPSS, RL-SAGE and oligoarray approaches.
Technology Comparison
Correlation coefficient using all tags
Correlation coefficient using only significant tags
Correlation coefficient using tags ≥ 10 copies
Correlation coefficient using tags ≥ 25 copies
Appressoria: MPSS vs microarray 1
Appressoria: MPSS vs microarray 2
Mycelia: MPSS vs microarray 1
Mycelia: MPSS vs microarray 3
Mycelia: RL-SAGE vs microarray 1
Mycelia: RL-SAGE vs microarray 4
Mycelia: MPSS vs RL-SAGE 1
Mycelia: MPSS vs RL-SAGE 5
Conversely, a moderate correlation coefficiency (0.59) was obtained between MPSS and the oligoarray appressorial data without data filtering (Table 5). After the removal of the four outlying genes, the correlation coefficiency was increased from 0.65 to 0.74 for the MPSS tags with copy number = 10 (Table 5). In mycelia, the correlation was only about 0.5 for the genes with 25 or more copies, even after the removal of ten outlying genes. Comparing mycelial MPSS and RL-SAGE tags data, a low correlation was observed without data filtering. After the removal of the 18 outliers from the dataset, the correlation coefficiency was increased from 0.068 to 0.4 between mycelial MPSS and RL-SAGE tags (Table 5).
In summary, we found a low to moderate correlation among the expression data from the three platforms, especially those data between MPSS and oligoarray. In general, a better expression correlation was observed for high copy number tags in the MPSS and RL-SAGE libraries with their corresponding genes on the oligoarray.
Recent technological innovations have advanced genomics in an unprecedented way. Several complex genomes have been sequenced in recent years providing an excellent starting point to fully understand the genetic blueprint of an organism. However, identification of all the expressed portions of a sequenced genome is a challenging task, yet critical to the understanding of gene regulation and metabolic networks. The public availability of the whole genome sequence of M. grisea has established a solid foundation to further understand the pathogenicity mechanism of this notorious fungal plant pathogen which causes severe yield losses in rice growing countries [13]. Elucidating the transcriptome of M. grisea may ultimately lead to the development of novel approaches for combating rice blast disease. In the last few years, many researchers have adopted various gene expression profiling techniques to characterize the M. grisea transcriptome under various conditions or in different cell types including EST sequencing [20], microarrays [13], and SAGE [21]. However, these methods have only provided partial information about the M. grisea transcriptome due to technical limitations and the depth of the surveys performed in these studies. In this study, we employed three global and quantitative expression tools, namely MPSS, RL-SAGE and oligoarrays, to profile the M. grisea transcriptome at two developmental stages. In these experiments, the same RNA samples isolated from mycelia and appressoria were used so that the results from three different platforms could be readily compared. A total of 12,531 and 16,580 significant tags in mycelia have been identified by MPSS and RL-SAGE, respectively. In appressoria, 12,927 significant MPSS tags were identified. Many identified transcripts were not present in the existing EST or cDNA collections of M. grisea and many of them matched unannotated regions of the genome.
Both RL-SAGE and MPSS are tag-based approaches for transcriptome analysis and genome annotation. They are different from the conventional approach that focuses on the large clone collections following the principle of collecting a "representative clone" for each gene. Although conventional approaches are useful to catalog the expressed genes in certain tissues, especially moderately or highly expressed genes, many weakly expressed genes might have been missed in these collections. More importantly, it is not possible for the conventional approaches to address the questions regarding the dynamics of transcriptional regulation and regulatory principles like alternative promoter usage and splicing [22]. In contrast, RL-SAGE and MPSS methods isolate and sequence short tags (17–21 bp) from the 3' regions of most transcripts. At least 100,000 RL-SAGE tags or a million MPSS tags can be easily obtained from these libraries.
In this study, we obtained approximately 1.3 and 1.4 million tags from the mycelial and appressorial MPSS libraries, respectively. The matching rate of the significant tags from each library to the M. grisea draft genome sequence was about 85%, suggesting that the MPSS data have a very deep coverage of the transcriptome. In contrast, only about 50 to 60% of the significant tags matched to the existing EST collections in the public databases. A similar result was also obtained from the RL-SAGE library, suggesting that current M. grisea EST collections are incomplete. One possible explanation is that most M. grisea ESTs were sequenced from the 5' region of the transcripts [23], whereas most of the MPSS and RL-SAGE tags were derived from the 3' region of expressed genes. Due to sequencing cost limitations, we only sequenced only 7,000 clones and obtained a quarter million tags from the mycelium RL-SAGE library. The transcripts recovered from the MPSS and RL-SAGE methods were overlapping but not identical due to the use of different anchoring enzymes in the library construction. Interestingly, we found that the genome matching rate of RL-SAGE tags is lower than that of the MPSS tags. These are two possible reasons. First, sequencing errors might generate unmatched tags, especially for singleton tags. Second, the significant MPSS tags used for matching have = 4 copies whereas the significant RL-SAGE tags have = 2 copies, suggesting the MPSS tags selected for matching may be more reliable. The last reason is that most of the RL-SAGE tags matched putative 3' UTR region, which may frequently targeted for RNA variation as reported in mammalian system [24]. Nevertheless, our results demonstrated that MPSS and RL-SAGE methods are powerful techniques for deep transcriptome analysis and novel gene discovery. The two methods are complementary and different types of transcripts could be identified from each of these methods.
One of the advantages of tag-based techniques is the detection of alternatively terminated transcripts in the RNA population. From both MPSS and RL-SAGE libraries, we found many annotated genes have alternative transcript tags. Some of them have corresponding EST transcripts. The percentage of genes with evidence of alternative termination ranged from 27% in the RL-SAGE mycelium library to 35% in the MPSS mycelium library. The higher rate of alternative transcript tags in the MPSS library may be due to the fact that more MPSS tags (66%) matched to the coding regions of the annotated genes than that of the RL-SAGE tags (37%). It has been previously shown that a high rate of alternative transcripts was found in the protein coding regions (74%), and a low rate (4%) of alternative transcripts was found in the 3' UTR [25]. The reason for the lower rate of alternative transcripts in appressoria than in mycelia is not clear. In addition, cloning and sequencing confirmation of some alternative transcripts without ETS support is required. Regulation of the alternative transcripts and functions of these sense and antisense alternative transcripts in M. grisea warrant more detailed analyses.
Naturally occurring antisense transcripts were first observed in prokaryotes and viruses and later found in eukaryotes. There is evidence for the involvement of antisense transcripts in alternative transcription [26, 27], RNA editing [28, 29], DNA methylation [30, 31], genomic imprinting [32, 33], and X-chromosome inactivation [34]. In this study, many antisense tags were identified in mycelium or appressorium libraries, corresponding to more than 25% of the annotated genes. It is not clear at this point that why M. grisea genome encode so many antisense transcripts. Like the sense tags, almost two-thirds of antisense transcripts detected in the RL-SAGE library were located in the 500 bp downstream regions, whereas the majority of antisense tags from the MPSS library were located within the protein coding regions. Surprisingly, we found that almost one-fifth of the annotated genes encode both sense and antisense transcripts. In these genes, antisense transcripts could form double stranded RNA (dsRNA) with their sense transcripts. If a dsRNA is formed, it could be degraded to form small interfering RNAs that could decrease sense RNA abundance [35]. Alternatively, interference by RNA polymerase II transcription activity on the antisense strand could restrict sense-strand transcription [36].
Recently, few studies demonstrated the function of antisense transcripts in fungal growth and development. For example, the circadian clock gene in the fungus Neurospora, a close relative of M. grisea, is regulated by the presence of natural antisense transcripts [37, 38]. Casas-Flores et al [39] expressed an antisense version of the pkr-1 gene of Trichoderma atroviride, encoding the regulatory subunit of protein kinase A (PKA), resulted in a non-sporulating phenotype.
Through data mining for EST, MPSS and RL-SAGE antisense tags, we have identified longer antisense transcripts in M. grisea for transcription factors MST12 (MGG_12958.5) and DEAD box-containing protein (MGG_12894.5), ribosomal protein, S9 (MGG_12892.5), ribosomal protein, L34 (MGG_05296.5) and ATP synthase alpha chain (MGG_07752.5). MST12 (a yeast homologs of STE12) is essential for host penetration and invasive growth, but was not required for appressorium formation [40, 41]. MST12 is regulated by MAP kinase at the downstream of signal transduction cascade during pathogenesis [40, 41]. It will be interesting to know if the MST12-mediated signal transduction cascade is regulated by antisense mechanism or not. A detailed characterization of MST12 antisense transcript may reveal its novel role in pathogenesis.
MPSS, SAGE, and oligoarrays are three widely used methods for transcriptome profiling. We performed qualitative and quantitative comparative analysis of the mycelial and appressorial transcriptomes revealed by the three methods. More than 40% of the annotated genes were detected by both MPSS and RL-SAGE methods. There was a good correlation in gene expression levels between the appressorium MPSS expression data and appressorium oligoarray data (0.67) and a moderate correlation between the mycelium MPSS and the mycelium oligoarray data (0.49) after removing several outlying genes in the datasets. However, the correlation between RL-SAGE and MPSS or oligoarray data was not significant. The low correlation between RL-SAGE and oligoarray might be because that oligoarray probes are designed from the protein coding regions of the annotated genes and a large number of RL-SAGE tags are located in the 500 bp downstream regions (putative 3' UTRs). We speculate that a low correlation between MPSS and RL-SAGE is due to the following two reasons. First, the use of different anchoring enzymes would change the location of the MPSS and RL-SAGE tags within a given transcript. We found that the majority of the MPSS tags matched the protein coding regions and the majority of RL-SAGE tags matched within 500 bp downstream (putative 3' UTRs). In some cases, because the M. grisea annotation is incomplete, the MPSS and RL-SAGE tags derived from the same transcript may be mapped to two different predicted genes. Second, MPSS and RL-SAGE use two completely different library construction and sequencing procedures. Any bias in the PCR amplification of synthesized cDNAs could lead to generation of different tag populations. Nonetheless, these data provide the first detailed analysis of transcriptional activity in an important fungal pathogen of plants, and constitutes a starting point for large-scale functional analysis of many novel fungal genes identified in the study.
We sequenced one RL-SAGE library of mycelia and two MPSS libraries of appressoria and mycelia of M. grisea. Using the same RNA samples of appressoria and mycelia, oligoarray hybridization was performed to check if these three approaches can detect similar sets of expressed genes in M. grisea. The distinct transcripts detected by MPSS and RL-SAGE in appressoria and mycelia ranged from 12,000 to 16,000, which correspond to about 9,000 genes, representing 80% of the predicted genes in M. grisea [13]. A low to moderate correlation among the expression data from the three platforms was observed. MPSS and RL-SAGE methods identified many novel sense and antisense transcripts, which are differentially expressed at the two important growth stages of M. grisea. The identified novel transcripts, especially those specifically expressed in appressoria, are valuable genomic resource for a better understanding of the molecular basis of M. grisea pathogenicity. The established MPSS and RL-SAGE websites provide useful genomics resource for the public. The microarray (GSM126989) and RL-SAGE (GSM127012) data were deposited at the NCBI-GEO website and also the MPSS data can be downloaded at the M. grisea MPSS website [42].
Fungal strains, growth conditions and RNA isolation
The M. grisea strain 70–15 was chosen for the transcriptome profiling because of the availability of its whole genome sequence [13]. The mycelia of 70–15 was cultured on a liquid medium [0.2% (w/v) yeast extract and 1% (w/v) sucrose] for 72 h (28°C at 200 rpm). The harvested mycelia were filtered and grinded for RNA isolation using the TRIzol method (Invitrogen, CA). For isolation of total RNA from the germinating appressoria, the mycelia of 70–15 were grown for two weeks on oatmeal agar plates and then the conidia were induced under white fluorescence light for five days. About 2 ml of conidia suspension (5 × 105 spores ml-1) was sprayed on Falcon Petri plates (150 mm × 15 mm) (Falcon, NJ) and the lids were covered with moist filter papers. These plates were incubated at 28°C and appressorium formation was monitored under a microscope at 6 h intervals. Fungal tissue was harvested 24 h after incubation since over 90% of the conidia extended a germ tube from the basal and/or the apical cell. Using a sterile blade, appressoria were scrapped and transferred quickly to the TRIzol solution, and then the suspension was centrifuged at 12,000 × g at 5°C for 5 min. About 2 g of appressorium pellet was collected and subjected for total RNA isolation. The poly (A+) mRNA was isolated from the total RNA using the Oligotex mRNA midi kit (Qiagen, CA).
MPSS library construction and sequence analysis
MPSS library construction was carried out at Solexa, Inc. (Hayward, CA) as described by Brenner et al [6] and Meyers et al [7]. About 500 μg of total RNA isolated from mycelium and appressorium tissues (described above) were used in the MPSS library construction. The entire data set is available at [42]. All tags were normalized to tags per million (TPM) as described by Meyers et al [7].
RL-SAGE library construction and sequence analysis
About 50 ng of mRNA isolated from mycelium tissue was used for RL-SAGE library construction as described by Gowda et al [5]. A total of 7,292 sequence reads from the RL-SAGE library were sequenced at Arizona Genomics Institute. The ditags and distinct tags were extracted from these sequences using SAGEspy program developed at the Ohio Supercomputer Center [43]. All RL-SAGE tags from mycelium library are available from the MGOS database [44].
Annotation of MPSS and RL-SAGE tags
The distinct MPSS and RL-SAGE tag sequences were matched to the M. grisea reference sequences including the genomic DNA, annotated genes (CDS), and 500 bp upstream (putative 5'UTR) and downstream (putative 3'UTR) regions that are available from the Broad Institute (version 5.0, release in January, 2006 [15]). The EST dataset of M. grisea from the TIGR database was used for matching MPSS and RL-SAGE tags release 5.0 on April 28, 2004 [16]). We also used a number of tools developed at the Ohio Supercomputer Center [43], the Magnaporthe grisea Oryza sativa (MGOS) database [44] and the University of Delaware MPSS database [42]for data analyses. We identified the antisense transcripts from MPSS and RL-SAGE tags by converting all of the tags to antisense orientation using a reverse-complementation procedure before matching to the various sequences of M. grisea.
The M. grisea oligoarray and the hybridization procedures
The M. grisea oliogoarray chip containing 60-mer oligos representing a total of 21,885 probes was obtained from Agilent (G4137A; Wilmington, Delaware). Of the 21,885 probes, 13,666 are from the annotated genes of M. grisea and 7,144 probes are from the rice ESTs [18]. The remaining 1,075 probes include quality controls, positive controls and negative controls. The total RNA was isolated from mycelia and appressoria using the TRIzol reagent (Invitrogen) following the manufacturers suggested protocol. Prior to hybridizations, quality and quantity of the total RNA sample was confirmed by running an agarose gel electrophoresis and by using a spectrophotometer. In the oligoarray hybridization experiments, we included six technical replicates of one RNA sample from mycelial and appressorial tissue, of which three were dye-reversal. About 500 ng of total RNA was used as template for cRNA production, and Cyanine dyes were incorporated using the Agilent low RNA input linear amp kit (5198-3523; Agilent). Normal yields from 500 ng total RNA input using an in vitro transcription were 15 μg cRNA (15 pmole cyanine dye incorporated/ug cRNA). One μg of labeled cRNA (Cy3 and Cy5 labeled sample) was diluted to 175 μl and defragmented at 60°C for 30 min following the Agilent hybridization protocols (5184-3568; Agilent). Defragmented samples were diluted to 500 μl (30% formamide final concentration) and hybridized for 20 h at 40°C. Arrays were washed, dried and scanned on an Agilent G2565BA microarray scanner described by [9]. The raw TIFF images were analyzed using the Agilent Feature Extraction software v 8.1 using the recommended default settings.
Microarray data and KOG analysis
To minimize the variation in probe labeling and detection, intensities of Cy3- and Cy5-labeled probes were normalized using subgrid LOWESS normalization. Spots with lower signal intensity than the negative controls or with intensities less than twice the average background for the channel were manually blocked (flagged) from further analysis. This corrected and normalized dye bias data eliminated larger component of the variance, thus giving a greater confidence to the evaluated (treatment) and reference (control) data for testing the statistical significance. The genes with valid signals in all six replicates were exported to Partek Pro v.6.0 software (Partek Inc., Missouri). The normalized values were used to calculate the ratio of channel intensities (Cy5/Cy3), which were then log2 transformed. The transformed ratio was plotted in a histogram with ± two standard deviations away from the mean. A ± 1.7-fold increase or decrease in signal intensity or ± 0.77 on the Log2 scale from the histogram was considered to indicate genes that are differentially expressed. The normalized data were then subjected to ANOVA model using Boenferoni method [45, 46]. FDR (≤ 0.05) [47, 48] was calculated based on the p-value (≤ 0.001) from ANOVA. This stringent criterion limits the ability of the oligoarray experiment to detect small but biologically important changes between the appressorium and mycelium at approximately a 95% confidence interval. The genes that are significantly and differentially expressed in the appressorial and mycelial oligoarray data were used to compare the appressorial MPSS data with mycelial MPSS and RL SAGE data. Appressorium and mycelium-differentially expressed genes from the microarray analysis were functionally categorized using the euKaryotic Orthologous Groups (KOGs) database [19]. The gene sequences were blasted against the KOG database with E-value of 1.0e-20.
This work is supported by the Plant Genome Research Program of the National Science Foundation #0321437 and #0115642). We thank Dr. Jin-Rong Xu for his advice on the harvesting germinating appressoria and RNA isolation and Tae-Ho Lee for functional classification analysis using KOG programs.
Authors' Affiliations
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Setting up your email on the BlackBerry® Torch™ 9800 smartphone couldn't be any easier thanks to the Email Accounts setup wizard. This utility will allow you to integrate your personal and/or work email accounts with your BlackBerry® smartphone. Plus, if your email account is used for an instant messaging service such as Windows Live Messenger™ or Yahoo® Messenger, it will also help you setup those accounts.
Want to learn more about the Email Accounts setup wizard in BlackBerry® 6? Then just watch the above video!
Have you used the Email Accounts setup wizard in BlackBerry 6? Leave a comment and let me know!

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Street Fighter IV is coming this Winter
Although SF IV has already started its "career" at the Arcades, the release date for the PS3 version still remains unknown.
Even Yoshi Ono failed to give an answer to when his creation is going to visit us. But he did let us know that the development is almost finished, with Capcom now meddling with other sectors, like online features.
Ono didn't forget to mention that the console versions will come packed with new features, while – under pressure – he admitted that the title is to be released this Winter.
With this in mind, we easily come to the conclusion that SF IV will be finally released between December and February; no news yet about a worldwide release.
In his small but informative interview, Yoshi Ono also made some statements about the PSN title Super Street Fighter II HD Remix. Once again he didn't mention anything about the release date. Furthermore, he thanked all the beta testers for their support and made sure that the title will be available for download before the end of the year.


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Chuck Norris Sayings (beta)
Chuck Norris Facts and JokesBecome a fan of Tjshome.com on Facebook
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Chuck Norris died 20 years ago. Death just hasn't built up the courage to tell him yet.
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When Chuck Norris pokes you on Facebook you can feel it
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Chuck Norris runs android on his I-Phone
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Chuck Norris will whoop your @ss till your blood bleeds!
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The boogie man checks his closet every night for Chuck Norris
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"Oh c'mon guys. Chuck Norris is just a dumb actor. If he were some sort of superbeing, he'd come to my house and smash my face into my keyboa4egwsdvc8 ohuiblj4g evda zcg 9ob, jlmevda z"
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"Please.....Chuck Norris is no superman"". He is not even a good actor!"""
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Please No More Chuck Norris wanna be jokes...holly Crap...is this the only American Icon you yanks have?...
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Chuck Norris' sperm count is so high that b!tches have to chew before they can swallow his load
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Sarah Tun Words and Voices: Identity, News, and Connect
"I have come that they may have life, and that they may have it more abundantly." (John 10:10)
Sarah Tun offers two aspects of service to those seeking to fulfil God's Call on their lives. WORDS and VOICES are available to enable you to dig deep within yourself to discover all God has created you to be. Some messages are overtly Christian while others simply open the heart of readers and listeners, unlocking their deepest needs. SARAH TUN WORDS and VOICES is a platform which ministers to those desiring greater abundance for their lives and the lives of their loved ones.
"2017: the year of fulfilment. It is imperative that we "run the race" God has given each one of us (Hebrews 12:1). I sense a deep compulsion in my Spirit to press in at this time, like no other time before in my life. I urge you to do the same." ST
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Cricket365 – Cricket Live Streaming – Scores – Servers
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Monday, October 24, 2016
Ten of Water – Happiness is a verb
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Her turtle reminds me of a statue of Kuan Yin sitting on a turtle:ReplyDelete
Which makes me think that compassion includes self-compassion! :)
A good post for me to read this morning Ellen. Thank you. The Turtle is a common symbolic animal in many Indigenous cultures. Great Spirit created the earth on the Turtle's back and represents the earth.ReplyDelete
This water woman is looking compassionately at the Turtle. When I think about this in relation to your post, gratitude for me is directly connected to self-care and our happiness is proportional to our daily maintenance of much gratitude our daily lives.Replies
Self care is so important. I just had a long and luxurious shower which was so relaxing. I love turtles. This animal has been coming up quite a few times this last week. When I was about 16 years old I had red-eared turtle which I adoredDelete
Ellen I have a water problem right now as we've been experiencing drought in Nova Scotia. So you can be very grateful for your luxurious shower!Delete
My father gave me a florescent green and pink gorilla when I was a little. Didn't quite do it for me! ;) No turtles, but my late husband Bill, who was Metis, owned two beautiful turtles that I now mine, a brass one and the other made with Abalone shell from Mexico. I treasure them.
I can imagine how these two turtle have become very precious keepsakes for youDelete
So good feeling on this. Thank you for posting! I'm having a day of rest and still, even I've been not-living by the calendar for a few years, still I feel sometimes that weekends are for resting and by Monday you're up and ready to go again. Reading this, after a nap time, got me a new fresh start for the week remembering the gratitude for the situation that I am capable (also responsible) to rest whenever I need it. I don't have to schedule it. :) Love, AnniinaReplyDelete
My favorite absolutely free thing to do is smile at people coming toward me. Until I turned my negative thoughts to positive I was always one of the 'don't meet their eyes' walkers. I have the 5 of cups today and am still working out in my mind what that means to me.ReplyDelete
Self care is one of the most important things we can do for ourselves. Take it easy and take care of your injuries. It may be just the thing that needed to happen to you to keep you from being or doing something that could have had more painful injuries.ReplyDelete
It is amazing how powerful this sort of awareness and action can be! I'm sorry you are still having pain. Have you tried an herbal tea or tonic to help a bit, like skullcap? Sending you many well-wishes!! Hugs.ReplyDelete
It's interesting, I don't see her being by herself. For one thing, there's the turtle, for another the three part moon, suggestive of different life phases. And the waves of the ocean, of which we are each just a drop. I see her as the Goddess, connecting us all through her cycles, and through our emotions. Which of course leads me to that same place of making life a happier place by how we interact with others.ReplyDelete
It's funny, as I gave someone almost exactly those same examples last week, to describe my understanding of the angelic - messages from spirit that uplift us: a ray of sunshine, a smile from another, and a good card reading :)


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Canon PowerShot SX260 HS Review
Design & Features
The PowerShot SX260 HS is a more streamlined version of the SX230 that came before it. The camera is made almost entirely of metal, with everything feeling pretty solid. It's compact and easy to hold, though there's not a lot of room on the back of the camera for your thumb, with mine usually resting on the movie record button. I also don't like how the buttons on the back of the camera are flush with the body, so you can't tell what you're pressing without looking first.
Here's a look at how last year's PowerShot SX230 compares to the new SX260:
|The PowerShot SX230 and SX260, side-by-side (close to scale)
Images courtesy of Canon USA
The front view shows the SX260's new "flat top" design, since the GPS receiver has been pushed back into the body. On the back you can see that the SX260 loses the widescreen LCD of its predecessor (I view this as a good thing), and the buttons have been flattened to the flush design I mentioned earlier. The power button has moved from the back of the SX230 to the top of the SX260.
Images courtesy of Canon USA
The PowerShot SX260 comes in three colors: red, black, and green. My review unit was the green one, though I'd say it's closer to turquoise.
Now it's time to see how the PowerShot SX260 HS compares to other travel zooms in terms of size and weight. As you can see above, it's pretty compact for a camera with a 20X zoom lens!
As you can see, the SX260 is one of the smallest and lightest travel zooms out there.
Alright, let's begin our tour of the SX260 HS now, using our tabbed interface:
The biggest new feature on the SX260 is its new 20X lens. This F3.5-6.8 lens isn't exactly "fast" -- especially at the telephoto end -- so don't expect miracles in low light. The focal length is 4.5 - 90.0 mm , which is equivalent to 25 - 500 mm. The lens is not threaded, so conversion lenses and filters are not an option.
You need an image stabilization system with an ultra zoom camera, and the SX260 has one, of the lens-shift variety. This will reduce the risk of blur for still shots, and will smooth out your movies, as well. It has a "dynamic mode" for shooting videos which has extra shake reduction, as well as an Intelligent IS feature, which selects the right IS mode for the situation.
To the upper-left of the lens is the camera's pop-up flash, which is raised electronically (depending on the flash setting). The working range of the flash is 0.5 - 3.5 m at wide-angle and 1.0 - 2.0 m at telephoto (both at Auto ISO). If you want more flash power, consider picking up the slave flash I mentioned earlier, which has a range of up to 30 feet.
The last item of note on the front of the camera is the AF-assist lamp, which also serves as a visual countdown for the self-timer.
The first thing to point out on the back of the camera is the SX260's 3-inch LCD display. The LCD has gone back to a more traditional 4:3 aspect ratio, rather than the widescreen one on the SX230. This is good news for still shooters, as composing photos on a 16:9 display is awkward. The LCD retains the same 461,000 pixel resolution of its predecessor, and it offers good outdoor visibility. In low light situations the screen brightens up nicely, allowing you to see what you're trying to take a photo of.
Now let's talk dials and buttons. At the top-right of the photo is the mode dial, which is chock full of options. I'll tell you more about those options right after this tour.
Underneath that we have four buttons, plus the four-way controller / scroll wheel combo. The function of the four buttons is pretty obvious, and you can see that the four-way controller does a lot more than just navigate menus, as well. The scroll dial around the four-way controller can be used for adjusting manual settings, navigating the menu system, and replaying photos you've taken.
Up on top of the SX260 you can see the flash (closed here), speaker, and stereo microphones. The GPS has been integrated into the body, so the SX260 lacks the "hump" of its predecessor.
Continuing to the right we see the shutter release button, which has the zoom controller wrapped around it. The controller moves the lens from wide-angle to telephoto in just 1.9 seconds. I counted a little over twenty steps in the SX260's 20X zoom range, which doesn't allow for a lot of precision.
At the far right of the photo is the SX260's power button.
There's nothing to see on this side of the camera, aside from the popped-up flash. The lens is at the full wide-angle position here.
On the right side of the SX260 are its I/O ports, where are protected by a rubber cover. The ports here include USB + A/V and mini-HDMI. The optional AC adapter feeds through a "hole" in the battery compartment door.
That big ol' 20X lens as at full telephoto here.
On the bottom of the PowerShot SX260 you'll find a metal tripod mount (not visible in this photo) as well as the battery/memory card compartment. The door over this compartment is of average quality. Keep in mind that you won't be able to access its contents while the camera is on a tripod.
The include NB-6L lithium-ion battery can be seen at right.
There's no live histogram on the SX260
The first thing I want to talk about are the items on the SX260's fully loaded mode dial. They include:
Live View Control mode makes adjusting settings easy
There at least four point-and-shoot modes on the SX260, and that doesn't include the Scene or Creative Filter modes. For general use, there's Smart Auto mode, which selects one of 58 scene modes for you (plus the proper image stabilization mode). The SX260 even knows if you're using a tripod, and reacts accordingly. If you want a way to adjust exposure compensation, color saturation, and white balance without having to know any of those technical terms, then try the new Live View Control mode. If you want to capture a short video before each still photo, then try the Movie Digest mode. Finally, there's an Easy mode which lets you turn the flash on and off -- and that's it.
As you've seen, the SX260 is loaded with both scene modes and special effects. I want to mention a couple of the scene modes that need further explanation:
- Smart Shutter: choose from smile detection, or cool wink and face self-timers; smile detection waits until someone in your photo smiles, and then it'll start taking photos; the wink self-timer takes a photo two seconds after someone in the frame winks at the camera; face self-timer takes a photo 2 seconds after a new person (presumably the photographer) enters the frame
- High-speed Burst HQ: the camera takes eight photos in a row at 10.3 frames/second (Canon's numbers -- see mine later); do note that the LCD goes black while shooting is in-progress
- Handheld Night Scene: the camera takes several exposures and combines them into a single photo, which is hopefully sharp
- Stitch Assist: helps you line up photos side-by-side for later stitching into a single panorama (using the bundled software)
The SX260 has a limited set of manual controls. You can adjust the shutter speed and/or aperture to your liking, and manually adjust the focus or white balance. Unfortunately, there's no bracketing feature, white balance fine-tuning (except for underwater), or RAW format support available. Something else that bothered me is that the ISO is locked at 100 when the shutter speed drops below 1 second. While this reduces noise, it's also an unneeded restriction. You'll see how this affected my night test scenes on the next page of this review.
Function menu
I want to mention a few interesting options that are controlled by the four-way controller, which includes the Function menu (activated by pressing the Func/Set button). These options include:
- Self-timer: in addition to 2 and 10 second options, a custom mode lets you choose the number of shots and the amount of time before the first shot is taken
- My Colors: enhance colors or skin tones, take B&W or sepia photos, or manually adjust contrast/sharpness/saturation
- Underwater white balance compensation: for fine-tuning white balance when shooting underwater
- Still image aspect ratio: choose from 4:3, 16:9, 3:2, or 1:1
Not a whole lot of interesting options, as you can see. Unfortunately, the SX260 lacks the useful DR Correction and HDR features found on some of Canon's other higher-end cameras. It does have the i-Contrast feature (described below), but it's not as robust as the other two.
Shooting menu, with help info at bottom
The rest of the shooting-related options that I want to talk about can be found in the PowerShot SX260 HS's main menu. The menus are attractive, easy-to-navigate, and feature "hints & tips" that describe each option. The notable features here are:
- AF frame: choose from Face Detect, Tracking, and center AF; for center AF you can select from small or normal size focus points
- Digital zoom: normally I tell people to turn this off, but if you're willing to lower the resolution, you can use it without a reduction in image quality; for example, dropping down to 6 Megapixel gives you a whopping 50X of total zoom power
- Servo AF: the camera keeps focusing with the shutter release halfway-pressed, which is helpful for moving subjects
- Continuous AF: the camera is always focusing, even before you press the shutter release button; reduces focus times at the expense of battery life
- Flash exposure compensation/output: if you set the flash to manual control, you can adjust it in the same way that you do exposure; when the camera is in "M" mode, you can choose from 1/3, 2/3, or Full flash output
- Redeye correction: buried in the flash settings submenu, this option will digitally remove redeye from photos, as they are taken; look for the redeye test later in the review
- i-Contrast: improves overall image contrast; see below for example
- Blink detection: the camera will warn you if someone in the frame had their eyes closed
- IS settings: choose from continuous or "shoot only" stabilization, or turn it off entirely; a Powered IS mode is for shooting at full telephoto, and should be turned off when panning or walking
- Face ID settings: new to the SX260 is a face recognition feature; you can add a face along with a name and birthday; registered faces will get focus priority when they appear in the frame; you can add multiple angles to improve accuracy
- GPS Auto Time: sets the clock using the GPS system
- GPS settings: turn the GPS on and off, and activate a logging function which tracks your movements (at the expense of battery life)
While the SX260 lacks the more robust HDR and DR Correction tools of its more expensive siblings, its i-Contrast feature still reduces highlights and brightens shadows. The only real catch is that the camera may need to boost the ISO to 200 in order to make the magic happen. Here's an example of i-Contrast in action:
|i-Contrast off (default)
View Full Size Image
|i-Contrast on
View Full Size Image
While i-Contrast doesn't cure highlight clipping by any means, it definitely reduces it, which you can see on the left side of the tile floor. As I mentioned, the ISO went up to 200 with this feature on, which increases noise slightly. This isn't a great scene for seeing how i-Contrast brightens shadows, but from my own experiences, it'll do that too -- just not dramatically.
|i-Contrast examples added on 3/26/12
There's not a whole lot to tell you about the SX260's GPS feature. It's a bare bones system that logs your location and nothing more (sorry, landmark database fans). If you want to track your route, just turn on the logging function, though note that it'll put a heavy strain on your battery. Satellite acquisition times aren't wondrous. In a wide open area, it took the camera about a minute to figure out where I was. In the city, things are much more difficult, as is usually the case. Unless you're really lucky, don't expect the camera to find your location beneath the skyscrapers.
The PowerShot SX260's movie mode is essentially the same as the one on the SX230 that came before it. You can record Full HD video (1920 x 1080) at 24 frames/second with stereo sound, until the file size reaches 4GB (which takes about 14.5 minutes). While the 24p frame rate is popular with film makers, the average person may find it to be a bit choppy. The SX260 can also record at 1280 x 720 and 640 x 480 -- all at 30 frames/second. You can also use the Apple-developed iFrame codec, which is supposed to be easier to edit -- not that working with H.264 is that hard.
As you'd expect from this hybrid camera, you can use the optical zoom (complete with continuous AF) and image stabilizer while you're recording a movie. There aren't any manual controls, though, unless you count the wind filter.
Movies can also be recorded using the miniature effect (at 720p) and at high frame rates. These "super slow motion movies" are recorded at 120 or 240 fps, at resolutions of 640 x 480 and 320 x 240, respectively. When played back at normal speed, the videos taken at the high frame rates appear to move in slow motion, as their name implies. Many of the other Creative Filters can be used in movie mode, if regular color just isn't good enough for you.
There's no way to take a still photo while you're recording a movie.
Here's a brief sample movie for you, taken at the highest quality setting. Pardon the zoom action at the beginning of the clip.
Click to play movie (1920 x 1080, 24 fps, 35.1 MB, QuickTime/H.264 format)
Aside from the choppiness, the video quality is pretty good!
The PowerShot SX260 HS has a pretty nice playback mode. Some of the notable features here include:
- Movie Digest playback: I told you about this feature earlier; here's how you view the day's events
- Smart Shuffle: a bizarre feature which shows four photos similar to the one you're viewing
- Image Search: move through photos by date, category, file type, recognized face, or whether it's tagged as a favorite
- My Category: assign a category to a photo, which is then transferred over to the "Browser" software; in some cases, the camera has done this automatically
- Photobook: you can select photos to put into a photobook, which essentially just puts images into a separate folder, for easy uploading to photo printing sites
- i-Contrast: brightens dark areas of your photo
- Redeye correction: digitally remove this annoyance from a photo
- Erase range of photos: I normally don't mention image deletion features, but the ability to select a range of photos without having to click your way through thumbnails is very handy
Photo editing functions include the ability to rotate, resize, and crop. Movies can have unwanted footage trimmed off of the beginning or end of a clip.
The PowerShot SX260 HS shows just basic information about your photo by default. Pressing the Display button reveals more, and if you press "up" on the four-way controller, you'll see where the photo was taken.
The SX260 moves through photos without delay, even with the fancy transitions between each image. If you want to really go fast, just spin the dial on the back of the camera. Using the dial is another way to jump to photos taken on a certain date.


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Diaper-Free Before 3: The Healthier Way to Toilet Train and Help Your Child Out of Diapers Sooner
Potter/Ten Speed/Harmony/Rodale, Feb 10, 2010 - Family & Relationships - 224 pages
Conventional wisdom tells parents that they should delay potty training to toddler age, and only after seeing signs of readiness. But is that really the best way?
In Diaper-Free Before 3, Dr. Jill Lekovic presents the new case that early training--beginning as early as nine months olds--is most natural, healthy, and beneficial for your child, based on medical evidence. By incoporating the potty into your child's routine early on, toilet training becomes far less stressful for both parent and child. Dr. Lekovic's method, which she has used successfully with her own kids and recommends to patients, helps children become better aware of their body's signals, boosts confidence, and decreases the risk of urinary health problems.
The guide includes informative chapters on bedwetting, accidents, and adapting the method for day care, special-needs children, and older toddlers. Offering a technique that really works and turns toilet training into a positive experience, Diaper-Free Before 3 is sure to become a new parenting classic.
What people are saying - Write a review
LibraryThing ReviewUser Review - librarybrandy - LibraryThing
Why can't more parenting books be this short and skimmable? I mean, the authors MUST know their intended audience has young children? Anyway: this is blissfully short, and the first almost-half of the ... Read full review
Selected pages
how we got here and why it matters
healthy and happy toilet training
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Diaper-Free Before 3: The Healthier Way to Toilet Train and Help Your Child ...
Jill Lekovic, M.D.
No preview available - 2010
Diaper-Free Before 3: The Healthier Way to Toilet Train and Help Your Child ...
Jill Lekovic
No preview available - 2006
Common terms and phrases
adults autism avoid aware baby baby's bacteria bathroom bed-wetting behavior Berry Brazelton bladder control bottle bowel move bowel movement boys Brazelton caregivers cause chil child childcare circumcision clean cloth diapers completely constipation cotton training pants delayed toilet training diet disposable diapers doctor dren drink early empty encopresis enuresis fact feel frequent accidents girls go potty guidelines habits idea important infants introduce the potty involved Journal of Pediatrics juice kids let training look Maria Montessori ment milk months of age mother normal older parents pediatrician percent potty training practice preschool problems psychoanalysts readiness rectum routine schedule skills sleep started stool suggest teach tell things three-year-old tion toddler trips two-year-old underwear urinary system urinary tract infections urine Urology voiding waterproof cover


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Lee and Marina Oswald Timeline - June 1962 thru November 1963
June 14, 1962: The Oswalds arrive in Fort Worth, where they move in with Robert.
July 14, 1962: The Oswalds move in with Marguerite at 1501 W. 7th Street in Fort Worth.
August 10, 1962: The Oswalds move to 2703 Mercedes St. in Fort Worth. Marina claims abuse at this point.
Early October: Marina and June move in with Gary and Alexandra Taylor at 3519 Fairmont (Apt #12), Dallas, for 4 days. Alexandra is George De Mohrenschildt's daughter.
October 7, 1962: Marina and June move in with Mrs. Elena Hall at 4760 Trail Lake Drive, Ft. Worth, while LHO looks for work in Dallas.
October 15, 1962: LHO moves into the YMCA. (Lee's whereabouts unknown from 10/9 thru 10/14)
November 4, 1962: The Oswalds are reunited at 604 Elsbeth St. in Dallas, where LHO had found an apartment.
November 5, 1962: The Oswald's have a violent argument, and Marina and June move in with their friends, Teofil and Anna Meller. They live at 5930 LaVista Drive, Dallas.
November 10, 1962: Marina and June move to the apartment of Declan and Katya Ford, at 14057 Brookcrest, Dallas
November 17, 1962: Marina and June spend the day at the home of Mrs. Frank (Valentina) Ray. LHO calls and asks to visit Marina, who agrees to return to him. They return to the Elsbeth St. address that night.
March 2, 1963: The Oswalds move to an apartment on 214 West Neely Street, Dallas
April 24, 1963: LHO arrives in New Orleans and stays with his aunt Lillian Murret.
April 29, 1963: Marina and June move in with Ruth Paine in Paine's residence at 2515 W. 5th St, Irving, TX
May 11, 1963: LHO starts work (R. Coffee) and he, Marina and June move into their apartment at 4905 Magazine St., New Orleans.
September 23, 1963: Ruth with Marina and June leave for Irving and arrive on the 24th.
September 26, 1963 begins LHO's trip to Mexico City.
October 4, 1963: LHO arrives in Dallas and stays with Marina and June at the Paine's residence in Irving.
October 14, 1963: Ruth drives LHO to Dallas, where he later registers as O.H. Lee at a rooming house on 1026 North Beckley.


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Population and GNI data source
Guatemalan journalist and radio reporter Álvaro Alfredo Aceituno López was assassinated in Coatepeque.
Carlos Ponce, director for Latin America programs, calls on authorities to provide protection against attacks on journalists in Guatemala.
Freedom House calls for an unbiased investigation into the murder of Guatemalan environmentalist, Walter Méndez Barrios.
Freedom House issued the following statement after gunmen killed two well-known Guatemalan journalists, and wounded a third.


text: New Mexico Day Trips: Albuquerque to Las Cruces New Mexico is perfect for road trips! These 5 stops will make your Albuquerque to Las Cruces trip complete. Read More Albuquerque, birds, burger, college, cranes, day trips, getaway, green chile, green chile cheese burger, Hotel Encanto de Las Cruces, Las Cruces, Outdoor Recreation, pool, resort, Restaurant, Restaurants, trips, weekend getaway Albuquerque, Day Trips, Las Cruces Leave a Comment


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Line of Inquiry: The qualities that make me special
Visual Arts Line of Inquiry: Self Portraits
The kindergarten children made four different styles of self portrait for their Who We Are unit. One was abstract, one was realistic, one was a simple line drawing in their own personal style (they used bleeding tissue paper to make the background and loved it!). The kids are very proud of their work.
I found the poem template here.


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The Breakfast Club Interviews T.I.
May 18, 2012 · Posted in interview, Videos · 4 Comments
T.I. continued his NYC promo run with a stop at Power 105. The Breakfast Club asked Tip about his reality show, his family, working with B.o.B on a joint project, Grand Hustle, acting, and Trouble Man.
Follow @QuezKHH
4 Responses to "The Breakfast Club Interviews T.I."
breezy on May 18th, 2012 6:48 pm
he is still the king of the south
The Royal One on May 18th, 2012 10:45 pm
Best morning radio show in the country. Download iheartradio to listen to them, it's free. Power 105.1 in New York City. Weekdays from 6-10am. If your in the west coast like me it's 3-7am.
Leave a Reply


text: Got Issues With a Medicaid Managed Care Organization? State Now Offers 'Complaint Portal' Posted on: 2/2/2017 The state's initiative to transition the majority of Illinois Medicaid patients to managed care organizations (MCOs) caused significant confusion and unease among health care professionals and patients. As a result, ISMS conducted the Medicaid Managed Care survey, which brought to light the frustrations and hassles experienced by health care professionals due to the transition. Our survey findings have been shared with the state, MCOs, the media and other stakeholders to make the case for improving the system. The Illinois Department of Healthcare and Family Services (HFS) has launched a secure electronic web-based tool – the complaint portal – to assist you in resolving issues with Illinois Medicaid Managed Care Organizations (MCOs). However, please note that HFS requires that you attempt to work out the issue with the MCO BEFORE submitting a complaint via the new portal. When you access the portal to fill out the complaint form, read the top of the web page first for important instructions. Also be sure to have the following information handy: a description of the issue, the name of your contact at the MCO, and the patient ID number. Access the complaint portal. Issues that impact immediate access to care will be expedited. While this is a step in the right direction, keep in mind that the complaint portal may still not resolve all issues and concerns. The ISMS Advocacy team is interested in hearing about any problems you experience with the MCO initiative. Please email us with a brief description of your problem to help us track and monitor physician complaints.


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cartography, humour, maps, sea, stormchasing, walking, weather
I blame the Environment Agency. The moment you start telling people to stay away from the coast and issuing 'danger of death' warnings about the weather is the moment that certain people are going to start scanning OS map number 20 for the place where the steepest terrain meets the most torrid and treacherous bit of sea.
Once Bounder had identified a likely location where big seas and maximum peril could be experienced, we set off for Prawle Point. I have to admit to being more than usually game for this excursion, as I quite fancied filming and filing an Exceptional Weather Report. The previous evening, the local news had been exclusively about weather. In twenty-five minutes of weather coverage there must be, I reasoned, an opening for a fresh intrepid reporter and her trusty sidekick. I would stand, attractively windswept, (possibly in a long black, cashmere coat?) and wax lyrical about the mighty power of nature. I would then turn to Bounder, (dullard in a bobble hat) for informative stats and weathery terms like 'leeward' and 'isobar'. I felt sure Spotlight Southwest would be unable to resist.
Our journey to the coast was uneventful; the usual white knuckle ride through the lanes, with me losing all feeling in my hands because I'm gripping the seat so hard and Bounder telling me to stop screaming. Though storm debris littered the roads and massive puddles splashed up over the windscreen, we didn't actually crash into anything or sink.
Your Exceptional Weather Reporters finally screeched to a halt in a deserted car park. Even though the raging sea was probably half a mile away, being engulfed and swept away didn't seem fantastical. As we marched, zig-zagging towards the cliff path, so merciless was the wind that I put my hood up.
I was surprised to see the sheep that dotted the cliffside field, merely grazing in the face of such elemental furore rather than clumped in a terrified huddle or blown skyward like a fluffy apocalypse. Maybe they were just trying to look cool.
We soon reached the top of the hill and the pointiest bit of Prawle Point, where the Coastguard's lookout station is perched. As we moved from the east to the west side of the craggy protuberance, the wind increased from really windy to wind that makes your face go all G-forcey. I clung to the railing surrounding the lookout station, shouting "This is madness! We must turn back!". I couldn't hear Bounder's response, but his face made the shape of an "Eh?". I edged my way back round the Point, ensuring that, at no time did my hands lose contact with the railing. I raised terrified eyes to where the Coastguard was probably about to mount my rescue, but was disappointed to see him merely watching my progress around the railing with a look on his face similar to one Bounder sometimes gives me.*
On the less windy side I explained to Bounder that no weather report was worth dying for, and we should head back to the safety of the car**. Bounder, with a look in his eyes I'll never forget, replied "Ok, I think I'll carry on, see you in half an hourish."
To be continued…
*No, it wasn't one of lust.
**Relatively safe when stationary.


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Maps Collection
Category: E-Book
Maps (Collection)
English | PDF | 6 Books | 195.66 Mb
Antique Maps - 3rd ed. - 0714829544
Cybercartography - Theory and Practice - 0444516298
Map Projections - 3540367012
Maps That Made History - 1550025627
National Atlas USA States, Federal Lands and Indian Reservations
World Map
Antique Maps - 3rd ed. - 0714829544.pdf
Beautiful, evocative and historically significant, old maps are endlessly intriguing and keenly collected. Antique Maps is widely recognised as a standard reference work for novice map collectors and experts alike. It provides a historical background to the subject and advice on starting a collection. The invaluable biographical section lists major map makers between 1450 and 1850, with key dates and works.
Cybercartography - Theory and Practice - 0444516298.pdf
Cybercartography: Theory and Practice examines the major elements of cybercartography and emphasizes the importance of interaction between theory and practice in developing a paradigm which moves beyond the concept of Geographic Information Systems and Geographical Information Science. It argues for the centrality of the map as part of an integrated information, communication, and analytical package.
This volume is a result of a multidisciplinary team effort and has benefited from the input of partners from government, industry and other organizations. The international team reports on major original cybercartographic research and practice from a variety of disciplinary perspectives, including the humanities, social sciences including human factors psychology, cybernetics, English literature, cultural mediation, cartography, and geography. This new synthesis has intrinsic value for industries, the general public, and the relationships between mapping and the development of user-centered multimedia interfaces.
Map Projections - 3540367012.pdf
From the reviews: "This technical reference book covers Map Projections for the sphere, ellipsoid and rotational surfaces, and geodetic datum transformations. It is intended primarily for graduate students and lectures in GIS, photogrammetry, computer vision and remote sensing. ?It is also suitable for students and academics working in mapping sciences and related geographical fields. ?The book is recommended strongly to all who are practicing or who are interested in multimedia cartography or related cartographic mapping and visualization fields." (Eva Siekierska, Geomatica, Vol. 61 (2), 2007)
Maps That Made History - 1550025627.pdf
Few people can resist the appeal of old maps and plans. Even in these days of disposable mapping - from satellite navigation to customized road directions at the click of a mouse - the historical map continues to present a unique perspective on our changing world. Here we explore 25 glorious examples. The emphasis is on the story behind the map: what it reveals about its creator and users, from the first explorers to the railway builders. This thought-provoking collection features maps that chart societies as well as land, sea, and skies; maps that have influenced and inspired; and perhaps most revealing of all, maps that misrepresent.
National Atlas USA States, Federal Lands and Indian Reservations.pdf
World Map.pdf
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They're the only reason I'm able to walk around and not bump into anything (yup, I'm blind).
Giveaway closes on July 23, 2014. The vouchers are valid until July 30, 2014.
I've recently moved back to Langley and have been lazing my way around, devouring any and all food in sight. Really missed my grandma's cooking. I have never had to donate so many clothes at one time; it was a relief to have less but sad to see some stuff go. I'll be heading out to the Pemberton Music Festival in a week so I'm pumped up about that! I've also quit Topshop and was hired at Brandy Melville, so more on that later. Brandy's gonna be chill and I just can't wait to meet the entire team (such a small staff, so different from Topshop, I love it already).
Stay tuned for more giveaways and more of my ramblings.


text:
WASHINGTON — I'm still not sure what to make of the story of Eugene Lang and Public School 121, except to say that I'm awfully glad they met each other.
Lang, a 66-year-old millionaire industrialist, was the 1980 graduation speaker for the Harlem school he had attended 50 years earlier. For some reason (according to The New York Times, where I read the account) he abandoned the work-hard-and-you-too-can-be-a-success speech he had planned and instead said something that stood his low-income, dropout-prone audience on its collective ear.


text:
Today I want to talk a little about men; what we are called to do and acting upon our duties as men.
I went with our family to our church to see the movie Courageous and wow what a great movie. If you haven't seen it I highly recommend that you do. I have the link to the web site below.
This movie will challenge every man who is a Dad and Husband. I left our church deeply challenged as a Father and Husband. I don't want to be mediocre.
It calls us as men to lead our families, to teach our kids about God, to protect our families, to be a great husband. Sadly all over America we as men have and are neglecting these duties. We are missing the mark.
We need to make time for our Wives and kids. It should be our priority.
If we don't lead our kids and be a Dad to them then the streets and gangs will. So I am encouraging you today to make time for your family; spend quality time with them. Do it because you get to and not because you have to. Lead your family in doing family devotions, pray with them and for them.
Let's honor God by honoring our families.