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Oral cancer is one of the most common cancers worldwide and ranks fourth for the mortality rate of cancers in males in Taiwan. The oral microbiota is the microbial community in the oral cavity, which is essential for maintaining oral health, but the relationship between oral tumorigenesis and the oral microbiota remains to be clarified. This study evaluated the effect of microbiome dysbiosis on oral carcinogenesis in mice, and the impact of the microbiome and its metabolic pathways on regulating oral carcinogenesis. We found that antibiotics treatment decreases carcinogen-induced oral epithelial malignant transformation. Microbiome analysis based on 16S rRNA gene sequencing revealed that the species richness of fecal specimens was significantly reduced in antibiotic-treated mice, while that in the salivary specimens was not decreased accordingly. Differences in bacterial composition, including

Both greenness and air pollution have widely been linked with asthma. However, the potential mechanism has rarely been investigated. This study aimed to identify the association between residential greenness and air pollution (fine particulate matter [PM

The Drosophila transcription factor (TF) Zfh1 has distinct roles compared to the cell lineage-determining TFs in almost all mesoderm-derived tissues. Here, we link Zfh1 to the well-characterized mesodermal transcriptional network. We identify five enhancers integrating upstream regulatory inputs from mesodermal TFs and directing zfh1 expression in mesoderm. Most downstream Zfh1-target genes are co-bound by mesodermal TFs, suggesting that Zfh1 and mesodermal TFs act on the same sets of co-regulated genes during the development of certain mesodermal tissues. Furthermore, we demonstrate that Zfh1 is critical for the expression of a hemocyte marker gene peroxidasin and helps restrict the activity of a hemocyte-specific enhancer of serpent to hemocyte-deriving head mesoderm, suggesting a potential role of Zfh1 in hemocyte development.

The current study investigated the potential functions of amoeboid cell formation and migration in a thraustochytrid strain, Aurantiochytrium limacinum BL10. Our results showed that: (1) When the surface of an agar plate was inoculated with BL10, amoeboid cells mainly emerged on the periphery of isolated colonies. The amoeboid cells then migrated outwards to form small vegetative cell clusters, which favored rapid colony expansion. In addition, amoeboid cells were capable of self-recognition (i.e. they were able to distinguish BL10 from other thraustochytrid species), and could choose whether to evade (self colonies) or approach (non-self colonies). These observations indicated that amoeboid cells were employed by BL10 to help colonize empty territories and to outcompete other thraustochytrid species in previously colonized territories. (2) When the agar medium was soft, amoeboid cells were able to penetrate the surface and migrate throughout, thereby allowing BL10 to colonize the interior of the solid matrix. This finding suggested that amoeboid cell formation and migration may help Aurantiochytrium colonize the interior of solid matrices to obtain additional nutrients and spatial resources. The mechanisms underlying the regulation of amoeboid cell formation and migration as well as the extraordinary microbial social behaviors of BL10 are also discussed in this article.

Cancer-associated fibroblasts (CAFs) play an essential role in supporting cancer progression. However, the details and consequent effects in response to the communication between CAFs and angiogenesis remain largely uninvestigated, especially in anticancer drug treatments. We found that cisplatin and 5-fluorouracil could induce fibroblast differentiation toward myofibroblasts via CCAAT/enhancer-binding protein delta (CEBPD) and consequently promote proliferation, migration, and in vitro tube formation of vascular endothelial cells and angiogenesis in vivo. Stromal-cell-derived factor 4 (SDF4) is responsive to anticancer drugs via CEBPD activation in CAFs and contributes to create a permissive environment for tumor cell angiogenesis and promotion of distant metastasis. Importantly, we demonstrated that SDF4 interacts with CXCR4 to trigger VEGFD expression through the activation of the ERK1/2 and p38 pathways in endothelial cells. Taken together, our novel findings support that SDF4 can be a therapeutic target in inhibition of angiogenesis for chemotherapy drug-administrated cancer patients.

Chronic hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC). The pre-S2 mutant large HBV surface antigen (LHBS) is highly associated with HCC. This study analyzed the expression of the large form of surface protein in tumors and evaluated the LHBS with mutations within the pre-S2 region as a high-risk recurrence marker in HCC patients after curative hepatic resection. By analyses using immunohistochemical staining (n = 12) and western blotting (n = 22), the HBV surface protein, which is mainly comprised of the major form of HBV surface antigen, was greatly diminished in the tumors. However, LHBS was not significantly decreased in tumorous regions, suggesting that LHBS maintains its expression in cancer development. A cohort of 175 patients with HBV-related HCC who underwent curative hepatic resection was analyzed for pre-S gene mutations using Pre-S Gene Chip. Results of the multivariate regression analysis showed that the serum pre-S2 mutant level and the American Joint Committee on Cancer stage were the two main independent high-risk factors for recurrence. A Cox proportional hazards analysis also revealed a prediction model, which indicated the recurrence-free survival rate along with the time after surgery; this was developed and further validated in an independent HCC cohort. Receiver operating characteristic curve analysis revealed that the model showed close sensitivities in the main and validation cohorts (area under the curve values, 0.741 and 0.704, respectively). Conclusion: Unlike the major HBV surface antigen, LHBS is mostly expressed in the tumorous regions of HBV-induced HCC, indicating that it plays a unique role in tumor progression; the relative level of pre-S2 mutant in serum is, independently of tumor stage, an important high-risk marker for HCC recurrence after primary hepatic resection. (Hepatology 2018).

Background An attenuated mutant (designated NY303) of Vibrio vulnificus, which causes serious wound infection and septicemia in humans, was isolated fortuitously from a clinical strain YJ016. This mutant was defective in cytotoxicity, migration on soft agar and virulence in the mouse. The purpose of this study was to map the mutation in this attenuated mutant and further explore how the gene thus identified is involved in virulence. Methods The whole genome sequence of mutant NY303 determined by next-generation sequencing was compared with that of strain YJ016 to map the mutations. By isolating and characterizing the specific gene-knockout mutants, the gene associated with the phenotype of mutant NY303 was identified. This gene encodes a global regulator, Lrp. A mutant, YH01, deficient in Lrp was isolated and examined in vitro, in vivo and ex vivo to find the affected virulence mechanisms. The target genes of Lrp were further identified by comparing the transcriptomes, which were determined by RNA-seq, of strain YJ016 and mutant YH01. The promoters bound by Lrp were identified by genome footprinting-sequencing, and those related with virulence were further examined by electrophoretic mobility shift assay. Results A mutation in lrp was shown to be associated with the reduced cytotoxicity, chemotaxis and virulence of mutant NY303. Mutant YH01 exhibited a phenotype resembling that of mutant NY303, and was defective in colonization in the mouse and growth in mouse serum, but not the antiphagocytosis ability. 596 and 95 genes were down- and up-regulated, respectively, in mutant YH01. Many of the genes involved in secretion of the MARTX cytotoxin, chemotaxis and iron-acquisition were down-regulated in mutant YH01. The lrp gene, which was shown to be negatively autoregulated, and 7 down-regulated virulence-associated genes were bound by Lrp in their promoters. A 14-bp consensus sequence, mkCrTTkwAyTsTG, putatively recognized by Lrp was identified in the promoters of these genes. Conclusions Lrp is a global regulator involved in regulation of cytotoxicity, chemotaxis and iron-acquisition in V. vulnificus. Down-regulation of many of the genes associated with these properties may be responsible, at least partly, for loss of virulence in mutant NY303. Electronic supplementary material The online version of this article (doi:10.1186/s12929-017-0361-9) contains supplementary material, which is available to authorized users.

Homeostasis in the nervous system requires intricate regulation and is largely accomplished by the blood–brain barrier (BBB). The major gate keeper of the vertebrate BBB is vascular endothelial cells, which form tight junctions (TJs). To gain insight into the development of the BBB, we studied the carpet glia, a subperineurial glial cell type with vertebrate TJ-equivalent septate junctions, in the developing Drosophila eye. The large and flat, sheet-like carpet glia, which extends along the developing eye following neuronal differentiation, serves as an easily accessible experimental system to understand the cell types that exhibit barrier function. We profiled transcribed genes in the carpet glia using targeted DNA adenine methyl-transferase identification, followed by next-generation sequencing (targeted DamID-seq) and found that the majority of genes expressed in the carpet glia function in cellular activities were related to its dynamic morphological changes in the developing eye. To unravel the morphology regulators, we silenced genes selected from the carpet glia transcriptome using RNA interference. The Rho1 gene encoding a GTPase was previously reported as a key regulator of the actin cytoskeleton. The expression of the pathetic (path) gene, encoding a solute carrier transporter in the developing eye, is specific to the carpet glia. The reduced expression of Rho1 severely disrupted the formation of intact carpet glia, and the silencing path impaired the connection between the two carpet glial cells, indicating the pan-cellular and local effects of Rho1 and Path on carpet glial cell morphology, respectively. Our study molecularly characterized a particular subperineurial cell type providing a resource for a further understanding of the cell types comprising the BBB.

Chronic hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC). The pre-S

The Fanconi anemia pathway in coordination with homologous recombination is essential to repair interstrand crosslinks (ICLs) caused by cisplatin. TIP60 belongs to the MYST family of acetyltransferases and is involved in DNA repair and regulation of gene transcription. Although the physical interaction between the TIP60 and FANCD2 proteins has been identified that is critical for ICL repair, it is still elusive whether TIP60 regulates the expression of FA and HR genes. In this study, we found that the chemoresistant nasopharyngeal carcinoma cells, derived from chronic treatment of cisplatin, show elevated expression of TIP60. Furthermore, TIP60 binds to the promoters of FANCD2 and BRCA1 by using the chromatin immunoprecipitation experiments and promote the expression of FANCD2 and BRCA1. Importantly, the depletion of TIP60 significantly reduces sister chromatid exchange, a measurement of HR efficiency. The similar results were also shown in the FNACD2-, and BRCA1-deficient cells. Additionally, these TIP60-deficient cells encounter more frequent stalled forks, as well as more DNA double-strand breaks resulting from the collapse of stalled forks. Taken together, our results suggest that TIP60 promotes the expression of FA and HR genes that are important for ICL repair and the chemoresistant phenotype under chronic treatment with cisplatin.

Sulphur is an essential element for life and is ubiquitous in living systems. Yet how the sulphur atom is incorporated into many sulphur-containing secondary metabolites is poorly understood. For bond formation between carbon and sulphur in primary metabolites, the major ionic sulphur sources are the persulphide and thiocarboxylate groups on sulphur-carrier (donor) proteins. Each group is post-translationally generated through the action of a specific activating enzyme. In all reported bacterial cases, the gene encoding the enzyme that catalyses the carbon-sulphur bond formation reaction and that encoding the cognate sulphur-carrier protein exist in the same gene cluster. To study the production of the 2-thiosugar moiety in BE-7585A, an antibiotic from Amycolatopsis orientalis, we identified a putative 2-thioglucose synthase, BexX, whose protein sequence and mode of action seem similar to those of ThiG, the enzyme that catalyses thiazole formation in thiamine biosynthesis. However, no gene encoding a sulphur-carrier protein could be located in the BE-7585A cluster. Subsequent genome sequencing uncovered a few genes encoding sulphur-carrier proteins that are probably involved in the biosynthesis of primary metabolites but only one activating enzyme gene in the A. orientalis genome. Further experiments showed that this activating enzyme can adenylate each of these sulphur-carrier proteins and probably also catalyses the subsequent thiolation, through its rhodanese domain. A proper combination of these sulphur-delivery systems is effective for BexX-catalysed 2-thioglucose production. The ability of BexX to selectively distinguish sulphur-carrier proteins is given a structural basis using X-ray crystallography. This study is, to our knowledge, the first complete characterization of thiosugar formation in nature and also demonstrates the receptor promiscuity of the A. orientalis sulphur-delivery system. Our results also show that co-opting the sulphur-delivery machinery of primary metabolism for the biosynthesis of sulphur-containing natural products is probably a general strategy found in nature.

Gastric cancer is one of the most common causes of cancer death worldwide. Aberrant activation of JAK/STAT3 signaling is frequently observed in gastric cancer and associated with cancer progression. Our previous studies demonstrated that several STAT3 targets and tumor suppressors such as NR4A3 and GATA6 are found to be epigenetically silenced by DNA methylation in gastric cancer. However, the role of aberrant JAK/STAT3 signaling in the epigenetic changes of downstream targets is not fully understood. In this study, by using Methylation EPIC array, we compared the global methylation changes in gastric cancer patient samples with different STAT3 activation level. In silico analysis was also performed to identify STAT3 binding sites in those differentially methylated loci. Hypermethylated loci which are functionally relate carcinogenesis and mediated by STAT3 expression were examined. Interestingly, SMARCAL1, which was involved in chromosome stability and alternative lengthening of telomeres was identified. Unexpectedly, differentially methylated region (DMR) was not occurred in the promoter, but enhancer region located in the CpG island shore. Additionally, we also found a transcription factor binding site, YY1, which was a methylation-sensitive enhancer-promoter regulator, in this DMR region. By using a statistic shuffling model, we found that STAT3 related methylation changes were significantly associated with H3K4me1 and H3K27me3 at that region. TCGA dataset also indicated that this DMR with dramatic changes was occurred in chromosome instability (CIN) subtype of gastric cancer, whereas the expression of SMARCAL1 was decreased in precancerous lesion in previous published data. Taken together, STAT3-related methylation changes may regulate the activity of enhancer and affect the expression of SMARCAL1, during gastric cancer progression. Citation Format: Yu Ming Chuang, Sheng-Jou Hung, Jiang Liang Chou, Wan-Hong Huang, Pearlly S. Yan, Tsunglin Liu, Michael W.Y. Chan. Epigenetic control of chromosome instability by STAT3 in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4326.

MicroRNAs have been known to regulate almost all physiological and pathological processes by suppressing their target genes. In humans, more than 1000 microRNAs have been identified, each of which targets dozens or even hundreds of genes. Facing this huge repertoire of microRNA targeting, it is important to identify which microRNAs are active, i.e., down-regulating their targets, in specific physiological or pathological conditions. Predicting active microRNAs is different from predicting microRNA targets because the authentic target genes of a microRNA are often not directly and solely regulated by that microRNA, leading to inconsistent expression changes between the microRNA and its true targets. Several computational programs have been proposed to predict the activity of a microRNA from the expressions of its target genes. These programs performed well when being applied on the expression data obtained from distinct tissue types or from experiments that transfect a microRNA into cells (i.e., non-physiological). But the performance of microRNA activity prediction is not clear on the expression data from the same tissue type in two physiological conditions, e.g., liver tissues from cancer patients and healthy people. In this work, we evaluate the performance of two microRNA activity prediction programs using seven expression data sets, all of which compare samples in two physiological conditions, as well as propose a new approach that predicts microRNA activity with an accuracy of over 80%. Unlike current methods, which predict active microRNAs by comparing two groups of samples, e.g., tumor versus normal, our new approach compares each diseased sample with all the samples in the control group. In other words, it can predict the microRNA activity of a person. In this work, this new application is named to predict “personalized microRNA activity”.

Here, we announce the complete genome sequence of Klebsiella pneumoniae KP36, a strain isolated from a patient with a severe community-acquired urinary tract infection. This genome provides insights into the pathogenesis of a pandemic K. pneumoniae strain from a community-acquired urinary tract infection.

Background T cells and B cells are essential in the adaptive immunity via expressing T cell receptors and immunoglogulins respectively for recognizing antigens. To recognize a wide variety of antigens, a highly diverse repertoire of receptors is generated via complex recombination of the receptor genes. Reasonably, frequencies of the recombination events have been shown to predict immune diseases and provide insights into the development of immunity. The field is further boosted by high-throughput sequencing and several computational tools have been released to analyze the recombined sequences. However, all current tools assume regular recombination of the receptor genes, which is not always valid in data prepared using a RACE approach. Compared to the traditional multiplex PCR approach, RACE is free of primer bias, therefore can provide accurate estimation of recombination frequencies. To handle the non-regular recombination events, a new computational program is needed. Results We propose TRIg to handle non-regular T cell receptor and immunoglobulin sequences. Unlike all current programs, TRIg does alignments to the whole receptor gene instead of only to the coding regions. This brings new computational challenges, e.g., ambiguous alignments due to multiple hits to repetitive regions. To reduce ambiguity, TRIg applies a heuristic strategy and incorporates gene annotation to identify authentic alignments. On our own and public RACE datasets, TRIg correctly identified non-regularly recombined sequences, which could not be achieved by current programs. TRIg also works well for regularly recombined sequences. Conclusions TRIg takes into account non-regular recombination of T cell receptor and immunoglobulin genes, therefore is suitable for analyzing RACE data. Such analysis will provide accurate estimation of recombination events, which will benefit various immune studies directly. In addition, TRIg is suitable for studying aberrant recombination in immune diseases. TRIg is freely available at https://github.com/TLlab/trig. Electronic supplementary material The online version of this article (doi:10.1186/s12859-016-1304-2) contains supplementary material, which is available to authorized users.

To date, genome sequences (complete or in draft form) from only six baeocytous cyanobacteria in four genera have been reported: Xenococcus, Chroococcidiopsis, Pleurocapsa, and Stanieria. To expand our knowledge on the diversity of baeocytous cyanobacteria, this study sequenced the genome of GI1, which is a Myxosarcina-like baeocytous cyanobacterium. GI1 is of interest not only because of its phylogenetic niche, but also because it is a cyanobiont isolated from the marine cyanobacteriosponge Terpios hoshinota, which has been shown to cause the death of corals. The ~7 Mb draft GI1 genome contains 6,891 protein-coding genes and 62 RNA genes. A comparison of genomes among the sequenced baeocytous cyanobacterial strains revealed the existence or absence of numerous discrete genes involved in nitrogen metabolism. It will be interesting to determine whether these genes are important for cyanobacterial adaptations and interactions between cyanobionts and their marine sponge hosts.

Although dissimilatory arsenic reduction (DAsR) has been recognized as an important process for groundwater arsenic (As) enrichment, its characterization and association with in situ microbial activities and diversity in As-rich groundwater is barely studied. In this work, we collected As-rich groundwater at depths of 23, 300, and 313 m, respectively, from Yenshui-3, Budai-Shinwen, and Budai-4 of Chianan plain, southwestern Taiwan, and conducted incubation experiments using different electron donors, acceptors, and sulfate-reducing bacterial inhibitor (tungstate) to characterize DAsR. Moreover, bacterial diversity was evaluated using 454-pyrosequencing targeting bacterial 16S rRNAs. MPN technique was used to enumerate microorganisms with different in situ metabolic functions. The results revealed that DAsR in groundwater of Chianan plain was a biotic phenomenon (as DAsR was totally inhibited by filter sterilization), enhanced by the type of electron donor (in this case, lactate enhanced DAsR but acetate and succinate did not), and limited by the availability of arsenate. In addition to oxidative recycling of As(III), dissolution of As(V)-saturated manganese and iron minerals by indigenous dissimilatory Mn(IV)- and Fe(III)-reducing bacteria, and abiotic oxidation of As(III) with Mn(IV) regenerated As(V) in the groundwater. Sulfate-respiring bacteria contributed 7.4 and 28.2 % to the observed DAsR in groundwater of Yinshui-3 and Budai-Shinwen, respectively, whereas their contribution was negligible in groundwater of Budai-4. A noticeable variation in dominant genera Acinetobacter and Bacillus was observed within the groundwater. Firmicutes dominated in highly As-rich groundwater of Yenshui-3, whereas Proteobacteria dominated in comparatively less As-rich groundwater of Budai-Shinwen and Budai 4.

Background Genome sequencing and assembly are essential for revealing the secrets of life hidden in genomes. Because of repeats in most genomes, current programs collate sequencing data into a set of assembled sequences, called contigs, instead of a complete genome. Toward completing a genome, optical mapping is powerful in rendering the relative order of contigs on the genome, which is called scaffolding. However, connecting the neighboring contigs with nucleotide sequences requires further efforts. Nagarajian et al. have recently proposed a software module, FINISH, to close the gaps between contigs with other contig sequences after scaffolding contigs using an optical map. The results, however, are not yet satisfying. Results To increase the accuracy of contig connections, we develop OMACC, which carefully takes into account length information in optical maps. Specifically, it rescales optical map and applies length constraint for selecting the correct contig sequences for gap closure. In addition, it uses an advanced graph search algorithm to facilitate estimating the number of repeat copies within gaps between contigs. On both simulated and real datasets, OMACC achieves a

Reactive astrogliosis is a cellular manifestation of neuroinflammation and occurs in response to all forms and severities of the central nervous system (CNS)’s injury and disease. Both astroglial proliferation and antiapoptotic processes are aspects of astrogliosis. However, the underlying mechanism of this response remains poorly understood. In addition, little is known about why activated astrocytes are more resistant to stress and inflammation. CCAAT/enhancer binding protein delta (CEBPD) is a transcription factor found in activated astrocytes that surround β-amyloid plaques. In this study, we found that astrocytes activation was attenuated in the cortex and hippocampus of APPswe/PS1 E9 (AppTg)/Cebpd−/−mice. Furthermore, an increase in apoptotic astrocytes was observed in AppTg/Cebpd−/−mice, suggesting that CEBPD plays a functional role in enhancing the antiapoptotic ability of astrocytes. We found that Zinc Finger Protein 179 (ZNF179) was a CEBPD-regulated gene that played an antiapoptotic, but not proliferative, role in astrocytes. The transcriptions of the proapoptotic genes, insulin-like growth factor binding protein 3 (IGFBP3) and BCL2-interacting killer (BIK), were suppressed by ZNF179 via its interaction with the promyelocytic leukemia zinc finger (PLZF) protein in astrocytes. This study provides the first evidence that ZNF179, PLZF, IGFBP3, and BIK contributed to the novel CEBPD-induced antiapoptotic feature of astrocytes. Electronic supplementary material The online version of this article (doi:10.1007/s12035-014-8714-9) contains supplementary material, which is available to authorized users.

Assembling a large genome using next generation sequencing reads requires large computer memory and a long execution time. To reduce these requirements, we propose an extension-based assembler, called JR-Assembler, where J and R stand for "jumping" extension and read "remapping." First, it uses the read count to select good quality reads as seeds. Second, it extends each seed by a whole-read extension process, which expedites the extension process and can jump over short repeats. Third, it uses a dynamic back trimming process to avoid extension termination due to sequencing errors. Fourth, it remaps reads to each assembled sequence, and if an assembly error occurs by the presence of a repeat, it breaks the contig at the repeat boundaries. Fifth, it applies a less stringent extension criterion to connect low-coverage regions. Finally, it merges contigs by unused reads. An extensive comparison of JR-Assembler with current assemblers using datasets from small, medium, and large genomes shows that JR-Assembler achieves a better or comparable overall assembly quality and requires lower memory use and less central processing unit time, especially for large genomes. Finally, a simulation study shows that JR-Assembler achieves a superior performance on memory use and central processing unit time than most current assemblers when the read length is 150 bp or longer, indicating that the advantages of JR-Assembler over current assemblers will increase as the read length increases with advances in next generation sequencing technology.

Next-Generation-Sequencing is advantageous because of its much higher data throughput and much lower cost compared with the traditional Sanger method. However, NGS reads are shorter than Sanger reads, making de novo genome assembly very challenging. Because genome assembly is essential for all downstream biological studies, great efforts have been made to enhance the completeness of genome assembly, which requires the presence of long reads or long distance information. To improve de novo genome assembly, we develop a computational program, ARF-PE, to increase the length of Illumina reads. ARF-PE takes as input Illumina paired-end (PE) reads and recovers the original DNA fragments from which two ends the paired reads are obtained. On the PE data of four bacteria, ARF-PE recovered >87% of the DNA fragments and achieved >98% of perfect DNA fragment recovery. Using Velvet, SOAPdenovo, Newbler, and CABOG, we evaluated the benefits of recovered DNA fragments to genome assembly. For all four bacteria, the recovered DNA fragments increased the assembly contiguity. For example, the N50 lengths of the P. brasiliensis contigs assembled by SOAPdenovo and Newbler increased from 80,524 bp to 166,573 bp and from 80,655 bp to 193,388 bp, respectively. ARF-PE also increased assembly accuracy in many cases. On the PE data of two fungi and a human chromosome, ARF-PE doubled and tripled the N50 length. However, the assembly accuracies dropped, but still remained >91%. In general, ARF-PE can increase both assembly contiguity and accuracy for bacterial genomes. For complex eukaryotic genomes, ARF-PE is promising because it raises assembly contiguity. But future error correction is needed for ARF-PE to also increase the assembly accuracy. ARF-PE is freely available at http://140.116.235.124/~tliu/arf-pe/.

Next-generation-sequencing (NGS) has revolutionized the field of genome assembly because of its much higher data throughput and much lower cost compared with traditional Sanger sequencing. However, NGS poses new computational challenges to de novo genome assembly. Among the challenges, GC bias in NGS data is known to aggravate genome assembly. However, it is not clear to what extent GC bias affects genome assembly in general. In this work, we conduct a systematic analysis on the effects of GC bias on genome assembly. Our analyses reveal that GC bias only lowers assembly completeness when the degree of GC bias is above a threshold. At a strong GC bias, the assembly fragmentation due to GC bias can be explained by the low coverage of reads in the GC-poor or GC-rich regions of a genome. This effect is observed for all the assemblers under study. Increasing the total amount of NGS data thus rescues the assembly fragmentation because of GC bias. However, the amount of data needed for a full rescue depends on the distribution of GC contents. Both low and high coverage depths due to GC bias lower the accuracy of assembly. These pieces of information provide guidance toward a better de novo genome assembly in the presence of GC bias.

De novo genome assembly is always fragmented. Assembly fragmentation is more serious using the popular next generation sequencing (NGS) data because NGS sequences are shorter than the traditional Sanger sequences. As the data throughput of NGS is high, the fragmentations in assemblies are usually not the result of missing data. On the contrary, the assembled sequences, called contigs, are often connected to more than one other contigs in a complicated manner, leading to the fragmentations. False connections in such complicated connections between contigs, named a contig graph, are inevitable because of repeats and sequencing/assembly errors. Simplifying a contig graph by removing false connections directly improves genome assembly. In this work, we have developed a tool, SIMGraph, to resolve ambiguous connections between contigs using NGS data. Applying SIMGraph to the assembly of a fungus and a fish genome, we resolved 27.6% and 60.3% ambiguous contig connections, respectively. These results can reduce the experimental efforts in resolving contig connections., {"references":["O. M. Margulies, et al., \"Genome sequencing in microfabricated\nhigh-density picolitre reactors,\" Nature, vol. 437, pp. 376-80, Sep 15\n2005.","D. R. Bentley, \"Whole-genome re-sequencing,\" Curr Opin Genet Dev,\nvol. 16, pp. 545-52, Dec 2006.","A. Valouev, et al., \"A high-resolution, nucleosome position map of C.\nelegans reveals a lack of universal sequence-dictated positioning,\"\nGenome Res, vol. 18, pp. 1051-63, Jul 2008.","M. A. Batzer and P. L. Deininger, \"Alu repeats and human genomic\ndiversity,\" Nat Rev Genet, vol. 3, pp. 370-9, May 2002.","N. Nagarajan, et al., \"Finishing genomes with limited resources: lessons\nfrom an ensemble of microbial genomes,\" BMC Genomics, vol. 11, p.\n242, 2010.","D. B. Jaffe, et al., \"Whole-genome sequence assembly for mammalian\ngenomes: Arachne 2,\" Genome Res, vol. 13, pp. 91-6, Jan 2003.","F. C. Jones, et al., \"The genomic basis of adaptive evolution in threespine\nsticklebacks,\" Nature, vol. in press, 2012.","P. Flicek, et al., \"Ensembl 2011,\" Nucleic Acids Res, vol. 39, pp. D800-6,\nJan 2011.","E. W. Sayers, et al., \"Database resources of the National Center for\nBiotechnology Information,\" Nucleic Acids Res, Dec 2 2011.\n[10] R. Li, et al., \"SOAP2: an improved ultrafast tool for short read\nalignment,\" Bioinformatics, vol. 25, pp. 1966-7, Aug 1 2009.\n[11] J. R. Miller, et al., \"Aggressive assembly of pyrosequencing reads with\nmates,\" Bioinformatics, vol. 24, pp. 2818-24, Dec 15 2008.\n[12] E. W. Myers, et al., \"A whole-genome assembly of Drosophila,\" Science,\nvol. 287, pp. 2196-204, Mar 24 2000.\n[13] M. Boetzer, et al., \"Scaffolding pre-assembled contigs using SSPACE,\"\nBioinformatics, vol. 27, pp. 578-9, Feb 15 2011.\n[14] W. J. Kent, \"BLAT--the BLAST-like alignment tool,\" Genome Res, vol.\n12, pp. 656-64, Apr 2002."]}

Profiling has contributed to the rapid growth of knowledge concerning microRNAs (miRNAs). The ability to measure all the known miRNAs by hybridization or by quantitative PCR has yielded insights into oncogenesis, stem differentiation, development, and disease. The steps toward discovery have often begun with profiling, followed by target prediction for miRNAs that are differentially expressed and then experimental validation of the targets. Here we present some methodological detail on the profiling procedures used in the lab and their application.

Neurogenetics (2009) 10:169–170 DOI 10.1007/s10048-009-0180-6 LETTER TO THE EDITORS Reply to the “Letter to the Editors” by Steven Buyske K. Abu-Elneel & T. Liu & F. S. Gazzaniga & Y. Nishimura & D. P. Wall & D. H. Geschwind & K. Lao & K. S. Kosik Received: 26 January 2009 / Accepted: 26 January 2009 / Published online: 24 February 2009 # The Author(s) 2009. This article is published with open access at Springerlink.com Dear Editors: We appreciate the opportunity to clarify the statistical treatment of our data. During the preparation of the manuscript, we did consider the use of the t-test as proposed by Dr. Buyske. Indeed, the z-test requires that the standard deviation be known, while we estimated the standard deviation from the sample. However, the t-test is not ideal either. A t-test compares two groups of values and the statistics rely on the standard deviation of both groups. It is unconventional to use the t-test to compare a single value, i.e., that of the miRNA in each case of autism against a group of values. By definition, the single values do not K. Abu-Elneel : T. Liu : F. S. Gazzaniga : K. S. Kosik (*) Neuroscience Research Institute, Department of Molecular Cellular and Developmental Biology, University of California Santa Barbara, Santa Barbara, CA 93106, USA e-mail: kosik@lifesci.ucsb.edu Y. Nishimura : D. H. Geschwind Department of Neurology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095-1769, USA D. P. Wall The Center for Biomedical Informatics & Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA K. Lao Applied Biosystems, 850 Lincoln Centre Dr., Foster City, CA 94404, USA Present Address: F. S. Gazzaniga Biomedical Sciences, University of California San Francisco, San Francisco, CA 94143, USA have a standard deviation. For this reason, we chose the z- test as a first-level less stringent screen to assess differential expression between one autistic case and 13 normal cases for each miRNA. We had also addressed the issue raised by Dr. Buyske with a nonparametric approach—the Wilcoxon rank test. However, the statistical power of the rank order test is rather weak and, more importantly, we lose the information in the differences in magnitude of the miRNAs. A strength of the method we used to profile the miRNAs—real-time PCR—is its ability to detect a broad dynamic range of miRNA expression. Our method was preferable to capture this quantitative parameter. However, the miRNAs with p- values that rank among the top using the parametric test remain among the top with the nonparametric test. The statistical testing as suggested by Dr. Buyske does demonstrate some differentially expressed miRNAs. With a one-sided t-test, our top five miRNAs remained statistically significant. The main difference between the z-test and t- test in a sense is the p-value cutoff. The z-test is indeed less stringent than the t-test and the two-sided t-test further increases the stringency. On the other hand, the Bonferroni correction for multiple hypotheses testing, applied in the manuscript, is often considered overly stringent. If we control for multiple hypotheses with a 5% false discovery rate (FDR) rather than the Bonferroni correction on t- statistics, the number of dysregulated miRNAs goes from five to 13. One could further argue that even the FDR is overly stringent because many miRNAs are co-regulated, and therefore treating each as an independent query may be unnecessarily conservative. Whichever the test and correction, we did provide all p- values signifying the difference between each autistic case and the normal cases for each miRNA, and these p-values are not affected by the significance cutoff. These p-values

Motivation: Insertional RNA editing renders gene prediction very difficult compared to organisms without such RNA editing. A case in point is the mitochondrial genome of Physarum polycephalum in which only about one-third of the number of genes that are to be expected given its length are annotated. Thus, gene prediction methods that explicitly take into account insertional editing are needed for successful annotation of such genomes. Results: We annotate the mitochondrial genome of P.polycephalum using several different approaches for gene prediction in organisms with insertional RNA editing. We computationally validate our annotations by comparing the results from different methods against each other and as proof of concept experimentally validate two of the newly predicted genes. We more than double the number of annotated putative genes in this organism and find several intriguing candidate genes that are not expected in a mitochondrial genome. Availability: The C source code of the programs described here are available upon request from the corresponding author. Contact: bundschuh@mps.ohio-state.edu

microRNAs (miRNAs) are approximately 21 nt transcripts capable of regulating the expression of many mRNAs and are abundant in the brain. miRNAs have a role in several complex diseases including cancer as well as some neurological diseases such as Tourette's syndrome and Fragile x syndrome. As a genetically complex disease, dysregulation of miRNA expression might be a feature of autism spectrum disorders (ASDs). Using multiplex quantitative polymerase chain reaction (PCR), we compared the expression of 466 human miRNAs from postmortem cerebellar cortex tissue of individuals with ASD (n = 13) and a control set of non-autistic cerebellar samples (n = 13). While most miRNAs levels showed little variation across all samples suggesting that autism does not induce global dysfunction of miRNA expression, some miRNAs among the autistic samples were expressed at significantly different levels compared to the mean control value. Twenty-eight miRNAs were expressed at significantly different levels compared to the non-autism control set in at least one of the autism samples. To validate the finding, we reversed the analysis and compared each non-autism control to a single mean value for each miRNA across all autism cases. In this analysis, the number of dysregulated miRNAs fell from 28 to 9 miRNAs. Among the predicted targets of dysregulated miRNAs are genes that are known genetic causes of autism such Neurexin and SHANK3. This study finds that altered miRNA expression levels are observed in postmortem cerebellar cortex from autism patients, a finding which suggests that dysregulation of miRNAs may contribute to autism spectrum phenotype.

Background. microRNAs (miRNAs) are approximately 21 nucleotide non-coding transcripts capable of regulating gene expression. The most widely studied mechanism of regulation involves binding of a miRNA to the target mRNA. As a result, translation of the target mRNA is inhibited and the mRNA may be destabilized. The inhibitory effects of miRNAs have been linked to diverse cellular processes including malignant proliferation, apoptosis, development, differentiation, and metabolic processes. We asked whether endogenous fluctuations in a set of mRNA and miRNA profiles contain correlated changes that are statistically distinguishable from the many other fluctuations in the data set. Methodology/Principal Findings. RNA was extracted from 12 human primary brain tumor biopsies. These samples were used to determine genome-wide mRNA expression levels by microarray analysis and a miRNA profile by real-time reverse transcription PCR. Correlation coefficients were determined for all possible mRNA-miRNA pairs and the distribution of these correlations compared to the random distribution. An excess of high positive and negative correlation pairs were observed at the tails of these distributions. Most of these highest correlation pairs do not contain sufficiently complementary sequences to predict a target relationship; nor do they lie in physical proximity to each other. However, by examining pairs in which the significance of the correlation coefficients is modestly relaxed, negative correlations do tend to predict targets and positive correlations tend to predict physically proximate pairs. A subset of high correlation pairs were experimentally validated by over-expressing or suppressing a miRNA and measuring the correlated mRNAs. Conclusions/Significance. Sufficient information exists within a set of tumor samples to detect endogenous correlations between miRNA and mRNA levels. Based on the validations the causal arrow for these correlations is likely to be directed from the miRNAs to the mRNAs. From these data sets, we inferred and validated a tumor suppression pathway linked to miR-181c.

The catalog of RNAs present in dendrites represents the complete repertoire of local translation that contributes to synaptic plasticity. Most views hold that a pool of dendritic mRNAs is selectively transported to a dendritic destination. This view requires that some mRNAs in the dendrite are locally enriched relative to the cell body; however, quantitative comparisons that would support this assumption do not currently exist. These issues related to somatodendritic distribution of mRNAs also apply to the microRNAs, ∼21 nucleotide noncoding transcripts that bind to target mRNAs and either inhibit their translation or destabilize them. We combined laser capture with multiplex real-time RT (reverse transcription) PCR to quantify microRNAs in the neuritic and somatic compartments separately. The samples were standardized by RT-PCR measurements of a set of mRNAs, including known dendritic mRNAs, in these two compartments. Most neuronal miRNAs were detected in dendrites. With a few notable exceptions, most miRNAs were distributed through the somatodendritic compartment across a nearly constant gradient. Thus for lower-abundance miRNAs, the total neuronal concentration of the miRNA can remain readily detectable in the cell body but vanish from the dendrite. A very small number of miRNAs deviate from the distribution gradient across the miRNA population as relatively enriched or depleted in the dendrite.

RNA editing can be crucial for the expression of genetic information via inserting, deleting, or substituting a few nucleotides at specific positions in an RNA sequence. Within coding regions in an RNA sequence, editing usually occurs with a certain bias in choosing the positions of the editing sites. In the mitochondrial genes of {\it Physarum polycephalum}, many more editing events have been observed at the third codon position than at the first and second, while in some plant mitochondria the second codon position dominates. Here we propose an evolutionary model that explains this bias as the basis of selection at the protein level. The model predicts a distribution of the three positions rather close to the experimental observation in {\it Physarum}. This suggests that the codon position bias in {\it Physarum} is mainly a consequence of selection at the protein level., 4 pages, 1 figure, submitted to Phys. Rev. Lett

The analytical study of disordered system is usually difficult due to the necessity to perform a quenched average over the disorder. Thus, one may resort to the easier annealed ensemble as an approximation to the quenched system. In the study of RNA secondary structures, we explicitly quantify the deviation of this approximation from the quenched ensemble by looking at the correlations between neighboring bases. This quantified deviation then allows us to propose a constrained annealed ensemble which predicts physical quantities much closer to the results of the quenched ensemble without becoming technically intractable., 9 pages, 14 figures, submitted to Phys. Rev. E

The ensemble of RNA secondary structures of uniform sequences is studied analytically. We calculate the partition function for very long sequences and discuss how the cross-over length, beyond which asymptotic scaling laws apply, depends on thermodynamic parameters. For realistic choices of parameters this length can be much longer than natural RNA molecules. This has to be taken into account when applying asymptotic theory to interpret experiments or numerical results., Comment: 10 pages, 13 figures, published in Phys. Rev. E