extfieldsofstudy:
Computer Science
Biology
Medicine

text:
The UCSC Genome Browser database: 2017 update

Since its 2001 debut, the University of California, Santa Cruz (UCSC) Genome Browser (http://genome.ucsc.edu/) team has provided continuous support to the international genomics and biomedical communities through a web-based, open source platform designed for the fast, scalable display of sequence alignments and annotations landscaped against a vast collection of quality reference genome assemblies. The browser's publicly accessible databases are the backbone of a rich, integrated bioinformatics tool suite that includes a graphical interface for data queries and downloads, alignment programs, command-line utilities and more. This year's highlights include newly designed home and gateway pages; a new ‘multi-region’ track display configuration for exon-only, gene-only and custom regions visualization; new genome browsers for three species (brown kiwi, crab-eating macaque and Malayan flying lemur); eight updated genome assemblies; extended support for new data types such as CRAM, RNA-seq expression data and long-range chromatin interaction pairs; and the unveiling of a new supported mirror site in Japan.

INTRODUCTION
With the revolutionary rate at which vast amounts of biological data are now being generated, new technologies and resources that can accommodate computational challenges are constantly in demand. The UCSC Genome Browser (1) continually strives to meet the needs of biological researchers in the face of exponential growth. This update gives a brief overview of the Genome Browser tools and data, and describes what has been added, changed, and improved since the previous 2016 update (2).

Genome assemblies
From its original focus on the early drafts of the human genome, the Genome Browser database now offers genomic data for nearly 100 organisms, many with multiple assemblies. Available as genome browsers and as downloadable data, this year there are three new species assemblies available (brown kiwi, crab-eating macaque, Malayan flying lemur) and new assembly versions for the following eight species: C. elegans, cat, chicken, gray mouse lemur, platypus, rhesus macaque monkey, western clawed frog, and western gorilla (3)(4)(5)(6)(7)(8). For a detailed list of new and updated UCSChosted genomes, see Table 1.

Genome browser annotation data tracks
Assemblies for all species contain a set of basic annotations ('tracks'), some of which are automatically generated by UCSC (e.g., assembly and gaps, the percentage of guanine and cytosine bases, repeat regions). Popular research species such as human and mouse are much more richly annotated, with tracks showcasing data from international sources.
The UCSC Genome Browser team regularly updates data tracks and adds new annotations. Although some annota- tions are mapped from the previous assembly (particularly in recent human assemblies), most data sets are obtained from the original data providers or generated from original sources. All assemblies for which GenBank provides cDNA sequences receive weekly automatic updates for relevant tracks (e.g., RefSeq Genes, ESTs, mRNAs). Other new and updated data for most assemblies include genome alignment data (chain and net tracks), predicted Augustus Genes (11,12) for all new assembly databases and Ensembl Genes (13).
The following descriptions are a sampling of new and updated data tracks in the Genome Browser database, grouped by track category. For a complete list of new and updated tracks released in this last year, see Supplementary Table S1.
Mapping and sequencing. Tracks in this group annotate the foundational structure of the assembly and constructed contigs. Beyond automatically updated tracks, most of the prominent new tracks in the Mapping and Sequencing category this year were added to the human GRCh38/hg38 assembly. These include a new fluorescent in situ hybridization (FISH) clones track (14) lifted from NCBI36/hg18, and a new track that annotates STS markers (15,16). A Clone Ends track was added showing mapped clone end libraries for mouse (GRCm38/mm10) and rat (RGSC 6.0/rn6) from NCBI's Clone DB (17).
Genes and gene predictions. This group includes computationally predicted gene sets (e.g., Ensembl (13), Augustus), high-quality manually curated and evidence-based automated gene predictions for the human and mouse genomes from the GENCODE project (18), and subsets from NCBI such as manually curated sets from the Reference Sequence collection (RefSeq) (19). This year there were several major gene set additions and updates, primarily to the latest human and mouse assemblies. Existing Ensembl gene annotations were updated to version 81, and new Ensembl tracks were added to several assemblies: Drosophila (dm6), rat (rn6) and zebrafish (danRer10). Two major gene sets (GENCODE Genes, UCSC Genes) were updated, as described below. GENCODE genes. The GENCODE Genes set is now the default gene set for the latest GRCh38/hg38 human assembly, replacing the previous default set, UCSC Genes. This transition was made to reduce the number of competing gene transcript sets used by the bioinformatics community, which have caused confusion in downstream analyses due to subtle differences among the sets. As of September 2016, the browser displays the v24 version, released by GENCODE in August 2015. The v24 gene set has increased by 2604 new transcripts to a total of 197 782 total transcripts. Only the Basic subset is displayed in the Genome Browser by default; the underlying database tables contain the Comprehensive D628 Nucleic Acids Research, 2017, Vol. 45, Database issue set, which can be viewed by clicking the 'show comprehensive set' checkbox on the track controls page. The database schema retains most of the same table names (starting with 'known' or 'kg'). Every transcript has a UCSC identifier (e.g., uc001abz.5) in addition to the GENCODE identifier (e.g., ENST00000327044.6).

UCSC genes.
The UCSC Genes set (20) has been updated for the mm10 mouse assembly. The new release increased by 515 new transcripts to a total of 63 759 total transcripts, 95% of which remained the same between versions. The total number of canonical genes has increased by 121 to a total of 33 079.
Phenotype and literature. This group contains tracks linking to phenotype references and literature (e.g., published articles) in which referenced sequences or identifiers are mapped to the assembly. The GRCh37/hg19 human assembly is currently the most comprehensively annotated with 21 major tracks, displaying data related to genetic phenotypes such as Online Mendelian Inheritance in Man (OMIM) allelic variants and positions of OMIM genes (21) as well as variants and copy number variants from the National Institutes of Health (NIH) funded ClinVar (22) and ClinGen (23) datasets. One highlight in this category is the addition of a super-track showing the genomic mapping of biomedical sequences submitted as part of patent application documents worldwide, obtained from the Cambia Lens Pat-Seq database (24) and aligned to the human (hg19), mouse (mm10) and Ebola virus (eboVir3) genome assemblies (25).
Other new track additions for the recent human assemblies include new copy number variant tracks for CNV Developmental Delay and ClinGen Benign Aggregate, both for hg38 and hg19. In hg38's new non-coding RNA track (lifted from hg19), four different types of RNA data are positioned in three different sub-tracks: sno/miRNAs, tRNAs (also added as a new track in hg19), and lincRNAs (26)(27)(28)(29).
As of 2016, proprietary OMIM data can now be accessed on a per-chromosome basis from the Table Browser and the Data Integrator; whole genome downloads are not available, per agreement with OMIM.
Expression. This category contains tracks related to the synthesis of functional gene products. A new gene expression track based on data from the NIH Genotype-Tissue Expression (GTEx) project (30,31) was released for the most recent human assemblies, hg38 and hg19. The GTEx track displays tissue-specific gene expression based on RNA-seq in 53 tissues from 570 donors obtained from the GTEx midpoint data release (V6, October 2015). This browser track uses a new bar graph display with a graph shown for each gene, and a colored bar indicating each tissue assayed ( Figure 1). The 'squish' view colors the gene by most highly expressed tissue. A box plot showing the range of expression levels across samples is displayed on the details page for each gene in the GTEx track, and also on the details page for that gene on the browser's default gene set.
Additional GTEx expression data are available in two UCSC-hosted track hubs. The first is the GTEx RNA-Seq Signal hub, hosting ∼7000 tracks, one for each tissue sample in the midpoint data release. The second is the GTEx Analysis hub, which currently hosts summary tracks of allele-specific expression (per-tissue and cross-tissue). GTEx gene expression has also been incorporated in the Gene Sorter tool, replacing GNF Atlas2 as the default gene expression set.
Regulation. This group contains tracks related to regulation of gene expression, including a wide range of mechanisms used to increase or decrease the production of gene products. The most notable tracks in this category are found primarily on the hg19 assembly, representing data from the ENCyclopedia Of DNA Elements (ENCODE) project (32). The UCSC Genome Browser offers ENCODE data through 2012 (33). The entire collection of ENCODE results from 2007 until present day are available from the EN-CODE Project Portal (http://encodeproject.org) (34) hosted by Stanford University. The major integrated ENCODE tracks have been mapped to hg38; however, the bulk of annotations remain on hg19.
Comparative genomics. Conservation tracks combine multi-species genome-wide alignments and estimated evolutionary conservation and acceleration scores to enable visualization and analysis of evolutionary processes. In September 2015, a new 100-vertebrate conservation track was released on the hg38 human assembly that compares pairwise alignments for 100 species from a variety of clades. Another addition is the C. elegans (ce11) 26-species conservation track, which shows the multiple alignments and measurements of evolutionary conservation for 26 nematode species.
Variation. Major variant annotation tracks include small variations from NCBI's dbSNP database (35), including tracks for older dbSNP releases in human assemblies, along with variant calls and high-confidence regions from the 1000 Genomes Consortium (36).

New dbSNP Build 146 and Build 147.
Data from NCBI's dbSNP Builds 146 and 147 have been added to the two most recent human assemblies (hg38, hg19), and Build 146 annotations were also added to the mm10 mouse assembly. Both builds 146 and 147 contain >150 million variants for human.
New phase 3 data of the 1000 Genomes Project. Data from Phase 3 of the 1000 Genomes Project (representing 2504 individuals from 26 populations worldwide) are now available for the hg19 human assembly. These data include nearly 90 million single nucleotide variants (SNVs), insertions/deletions (indels), and structural variants (SVs). For each variant, a detail page incorporates links to the variant in the dbSNP database, quality scores and allele frequency information for various populations. In addition to variant calls, the 'Paired-end Accessible Regions' tracks encompass genomic regions estimated to be accessible to next generation sequencing technologies using short paired-end reads, based on depth and quality of mapped reads. Tissues are identified on mouseover and the full legend is shown on the track description and details pages. The GTEx transcript model used to quantify expression level is displayed below the graph, colored by gene class using GENCODE conventions. The GTEx RNA-seq tracks show per sample read density; in this image, eight tissues from a single GTEx donor. The GTEx Analysis Hub is also connected and therefore is visible in the track menu, but no tracks are selected for display. Tissue colors in all GTEx tracks are assigned using GTEx project conventions.

Home and gateway pages
While the home page of the UCSC Genome Browser is the front door to the online browser, the gateway page is the entrance to visualizing all genomes hosted by the browser, providing opportunities to access 96 unique species (all eukaryotic, except for one virus), whose assembly versions combine to a total of 175.
Both the home page and the gateway page have been redesigned this year to be more interactive and intuitive. The new home page (Supplementary Figure S1) includes more graphics, less text, and better access to Genome Browser tools and content, such as the ability to access popular organisms from the site-wide menu. The newly designed gateway page (Supplementary Figure S2) features an autocomplete search for both UCSC-hosted and public assembly hubs, an interactive phylogenetic tree menu for selecting species, quick access shortcuts to popular browsers, and a new style and color scheme. From both the gateway page and the genome browsers, the ability to search genomes by submitting a chromosomal position, gene symbol, or search term remains a much-used feature. For the human genome, the hg38 assembly is now the default; the previous default assembly, hg19, can be quickly selected from the Genomes menu in the site-wide top menu bar.

Visualizing genomes: genome browser tracks display
The Genomes Browser tracks display can be seen as the 'heart' of the browser. The selected reference genome assembly provides navigational structure for browsing sequence alignments and annotations. The fast, responsive interface shows details from the nucleotide base level out to wholechromosome perspectives. Major features of the browser display include a powerful search for all data tracks; track configuration and reordering; reverse strand view; and a variety of navigation options to zoom, pan, or quickly jump between features.
New multi-region display feature. The multi-region display configuration was developed to omit introns or intergenic D630 Nucleic Acids Research, 2017, Vol. 45, Database issue regions from the display, resulting in a Genome Browser display with exon-only or gene-only views (Figure 2), much like a handheld paper fan where portions of the design are hidden behind creased folds. This custom viewing mode can be launched from the browser multi-region button, from the 'View' menu on the navigation bar, or (in the case of the exon-only view) by using the keyboard shortcut 'e v' while on the graphic browser window.
From the multi-region configuration window, clicking the checkbox to turn on alternating region highlights will help differentiate alternating regions; every other region will appear with a tinted hue, thus creating a series of vertical stripes in the graphic browser window as the genome is 'sliced' to display only the defined regions.
The multi-region configuration window offers several display choices: exon-only, gene-only, alternate haplotype view, or custom BED regions. For the exon-only and geneonly modes, the browser automatically chooses which gene track to use as the source for the region coordinates, and the user may configure the padding between features. Available on human assemblies hg17 and newer is the option to display an alternate haplotype within the context of the reference genome (Supplementary Figure S3). A particularly powerful feature of the multi-region configuration is the option to specify the URL of a remotely hosted BED (Browser Extensible Data) file containing coordinates that define custom genomic regions, even those that span chromosomes.
New density graph feature. Another new visualization option for tracks is a density graph track display, useful for viewing tracks with many items in a large region (e.g., shortread sequence alignments from a BAM file). Selecting this option from the track settings will replace the visualization of dense data tracks with bar charts that show data coverage. This graph can be seen in tracks such as gene prediction tracks (e.g., RefSeq Genes and UCSC Genes), alignment tracks (e.g., GenBank mRNAs), and cross-species chained alignment tracks. The density coverage graph can also be configured for visibility in custom tracks, track hubs and assembly hubs with supported formats.
New keyboard shortcuts. From the Genome Browser data tracks webpage, pressing the question mark '?' key will open a menu of keyboard shortcuts, which has been expanded to include several new options (e.g., toggling zoom views, navigating upstream or downstream in a chromosome, hiding all tracks and launching the 'view DNA sequence in external tools' feature).
New browser menu options. There have been three major changes to the browser's site-wide top menu bar. First, the 'Genomes' menu option now allows quick access to the latest two human and mouse assemblies. Secondly, the 'View > In External Tools' menu provides an option to export the current browser region to external tools (e.g., Ensembl, NCBI MapView (37), CRISPOR (38)). Lastly, the 'Genome Browser' menu now features a 'Configure' link to set visibilities for all tracks on the currently browsed assembly. The 'Reset All User Settings' option in the same menu will immediately restore the UCSC Genome Browser on the current Internet browser to a default state by clearing connec-tions to external hubs, removing loaded custom tracks, and reverting to site-wide default configurations (such as default track views for the hg38 assembly).

New features in gene description page.
In the Genome Browser tracks display, clicking on a gene from a browser's default gene set will display a gene description page containing links to other tools and databases. From this description page, a chart showing RNA-Seq Expression Data from GTEx (53 Tissues, 570 Donors) is now displayed on the two latest human assemblies. Other changes for the gene description page include updated data and links to the MalaCards human disease database (39) and updated Wikipedia links.

New sessions gallery and public sessions
One widely used feature of the browser is the 'Sessions' tool, which allows the user to save the configuration settings of the current browser view for later viewing and sharing (40). Sessions are now arranged in a table with filtering and ordering options based on session name, primary assembly, and creation date. From the session list, clicking on the session name displays that session in the browser. Session expirations have been eliminated to assist with long-term data sharing.
A new Session Gallery has been added, featuring sample sessions created by the UCSC Genome Browser team, which highlight topics of interest to the genomics community. To date, sessions in the gallery include the display of coding and wobble bases, alt-splicing, evolution, variation and disease, topics derived from commonly asked questions on the browser user support mailing list, and sessions used in browser training workshops.
A new 'Public Sessions' page has been released, displaying Genome Browser sessions that users have opted to share to this publicly accessed list. In the Session tool's listing of saved sessions for a user's account, a checkbox can be marked to enable public viewing permissions, thus adding the session to the public list. From the Public Sessions page, sessions can be filtered by assembly, name, popularity, creation date, or a phrase from the description. Public Sessions can be reached from the site-wide menu (My Data > Public Sessions).

Table-based browsing and querying
There are several major interactive web-based tools purposed for browsing and querying underlying tables in the UCSC Genome Browser, including most track data.
First, and most widely used, the web-based Table Browser tool (41) can be accessed directly from the Genome Browser menu, pre-loading current browser configurations for the querying, intersecting, filtering and downloading of genomic data. Another tool is the web-based Data Integrator (42), specializing in combining and exporting data from multiple tracks simultaneously. The Variant Annotation Integrator (VAI) (42) annotates user-provided variant calls with predicted functional effects and data from other tracks. database to store track data. For many years, the Table  Browser has allowed related tables to be joined with the main track table so that their fields can be included in the output. For example, protein product accessions and descriptions associated with RefSeq Genes can be joined by selecting the protAcc and product fields from the refLink table. The Data Integrator now offers the same functionality via the 'Choose fields. . . ' button and dialog box.
Updated dbNSFP data in hgVai. For hg38, the VAI now offers selected annotations from version 3.1a of the Database of Non-Synonymous Functional Predictions (db-NSFP), which provides pre-computed scores and predictions of functional significance from a variety of tools.

Custom tracks, track hubs, assembly hubs and public hubs
The UCSC Genome Browser offers three primary options for viewing custom genomic data in the form of custom tracks, track hubs and assembly hubs. These tools provide methods for publicly sharing or privately viewing custom genome assemblies, large-scale genomic annotations, and data tracks.
The collection of data types supported by the custom track, track hub, and assembly hub tools continues to expand. New supported data types include big-Psl, bigChain and bigMaf (which store alignments, pairwise alignments, and multiple alignments, respectively), and CRAM. CRAM is similar to BAM, but denser, using an external reference sequence index. Another new UCSC-specific file type, longTabix, is now supported as a custom track or track hub. This format stores chromatin interactions of paired chromosomal regions. Regional pairs are visualized within the browser with an 'arc' representation, similar to chromatin interactions displayed in the WashU EpiGenome Browser (43). For a complete listing of data types supported by track hubs and custom tracks, see Supplementary Table S2.
Custom tracks. The simplest way to view custom data against a UCSC-hosted genome assembly is by uploading a file to the browser as a custom track in one of many supported file formats, some of which are new as of this last year (Supplementary Table S2). For most big* compressed formats (e.g., bigWig), the URL to the hosted files alone can be pasted into the custom tracks data-loading dialog box, without additional track information needed.
As with other major tools such as the Table Browser, custom tracks are now integrated with the Data Integrator tool. The new option to view dense data as a bar graph is available for supported custom track data types.
Track hubs. Track hubs (44) are web-accessible directories of genomic data that can be viewed as a data portal through the UCSC Browser. The new option to view dense data as a bar graph is available for supported data types.
Public UCSC-reviewed track and assembly hubs. The UCSC Genome Browser team continues to promote the use of public track and assembly hubs to display large data sets The UniProt Consortium from consortia and external labs. As of September 2016, there are over 45 public hubs linked for display in the UCSC Genome Browser. Of these, thirteen were added this last year. For a complete listing of recently added public hubs, see Table 2.

Alternative UCSC genome browsers: GBiB, mirrors
The UCSC Genome Browser is available as a stand-alone virtual machine, the Genome Browser in a Box (GBiB) (45), that can be used on a personal computer. GBiB provides a complete solution for browsing confidential data, while requiring a small memory footprint. Alternatively, the Genome Browser can be fully or partially mirrored at a third-party site, thus allowing customizations of any browser component or data. The 'My Data' menu now includes a new GBiB Shared Data Folder, providing navigation to locally shared files that can be entered as URLs in the custom tracks or track hubs tools. The GBiB custom track tool provides local file-loading capabilities by pointing to the '/folders' directory with the bigDataUrl parameter.
A new supported Genome Browser mirror (genomeasia.ucsc.edu) has been released to provide an access point of faster functionality for those users who are geographically closer to Asia than to the United States. This UCSCadministered mirror site is physically located at the RIKEN Yokohama Campus in Japan. Technical support continues to be provided for full and partial mirrors of the Genome Browser offered by academic and noncommercial institu-tions. Mirror sites can now specify the location of a Galaxy (46) instance to the Table Browser.

Public MySQL server access
As an alternative to the graphical Table Browser tool, the UCSC-hosted MySQL database is publicly accessible and synchronized on a weekly basis from the main databases on the UCSC Genome Browser website. Many of the command-line utilities from the source tree can be used with the public MySQL databases. These data are stored on a central file server and MySQL server, totaling approximately 20 terabytes of disk space, and growing daily.
This past year, additional security was implemented in the form of SSL (Secure Sockets Layer) connectivity between the browser hosts and MySQL servers. Also, the location of related tables that support the GenBank and Ref-Seq tracks (e.g., RefSeq, Other RefSeq, mRNA, EST) have moved from individual assembly databases to one shared metadata database, hgFixed.

Utilities and source code
The UCSC Genome Browser team provides ∼250 application binaries built for standalone command-line use on Linux and Mac OSX platforms. There are several new additions to the command-line utility collection, including file conversion tools (pslToBigPsl, bigPslToPsl, chain-ToPslBasic) and utilities for loading specific file types into a database (hgLoadChain, hgLoadMaf, hgLoadNet, hgLoadMafSummary).

Nucleic Acids Research, 2017, Vol. 45, Database issue D633
The entire source code tree, which is written primarily in C, HTML, and Javascript, is publicly available. To install the package, consult the detailed README files in the source tree (http://genome-source.cse.ucsc.edu/gitweb/).
Both the binaries and source code are available for download. Licenses are required for commercial use (https:// genome-store.ucsc.edu/), with several exceptions (see http: //genome.ucsc.edu/license/).

Support, training and documentation
This last year, in-person trainings, workshops and presentations were conducted in the United States and international locations, including Germany, Brazil, South Korea, Japan, Sweden, Spain and Denmark. More information regarding workshop requests can be found online (http://bit. ly/ucscTraining).
Multi-media learning materials continue to be developed, such as user guides (e.g., the new multi-region display), online training videos (e.g., how to view exons only and export exon coordinates), announcements of major new data releases, and new blog posts.

FUTURE PLANS
The UCSC Genome Browser team will continue to release new genome assemblies for species not yet hosted at UCSC, updates for existing genomes, new and updated annotations, new software and overall site improvements. In the coming year, the new Genome Browser in the Cloud (GBiC) will be released. The GBiC is a cloud-based solution that is similar to the stand-alone virtual machine, GBiB.
The Genome Browser team has been working to provide an NCBI RefSeq Gene composite track that will provide alignments and annotations directly from NCBI in addition to providing UCSC's BLAT (47) alignments of RefSeq sequences and resulting transcript models.
Other planned software changes include new features for the multi-region display, improved track sorting, improved track hub searching, extensions to the GTEx track display, a publications track for the human assembly (hg38), and a CRISPR/cas9 track.

CONTACT US
All UCSC Genome Browser contact information can be found on the website at http://genome.ucsc.edu/contacts. html.

SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.

ACKNOWLEDGEMENTS
Sincere gratitude is extended to the worldwide users of the UCSC Genome Browser, from college students who are just beginning their journey to experienced researchers. Special appreciation is extended to funders, collaborators, and contributors who give value beyond measure to the research possibilities within the browser. Much thanks is given to the exceptional group of system administrators, the management team, and all staff who support the UCSC Genome Browser.

National
Human


extfieldsofstudy: Biology Medicine

text:
Microbial metabolite butyrate facilitates M2 macrophage polarization and function

Metabolites from intestinal microbes modulate the mucosal immune system by regulating the polarization and expansion of T cells. Whether the microbial metabolites influence macrophage polarization, however, is poorly understood. Here, we show that the large bowel microbial fermentation product, butyrate, facilitates M2 macrophage polarization, in vitro and in vivo. The supernatant from butyrate-treated M2 macrophage increased the migration and enhanced the wound closure rate of MLE-12 cells. Butyrate attenuated intestinal inflammation in mice with dextran sulfate sodium (DSS)-induced colitis, with a significant increase in colonic expression of the M2 macrophage-associated protein, Arg1. M2 macrophage treated with butyrate, had increased activation of the H3K9/STAT6 signaling pathway, suggesting a mechanism for butyrate facilitated M2 macrophage polarization. Collectively, our study indicated that commensal microbe-derived butyrate is a novel activator of STAT6-mediated transcription through H3K9 acetylation driving M2 macrophage polarization, and delineated new insights into the immune interplay underlying inflammatory bowel disease.

Results
Butyrate facilitates polarization of M2-BMDMs. The cells were morphologically typical of bone marrow-derived macrophages (BMDMs) (Supplementary Fig. 1a) and 99.9% were F4/80 + as determined by flow cytometry (Supplementary Fig. 1b,c). Flow cytometry and Cell Counting Kit-8 assays showed no changes in BMDMs' viability following butyrate treatment ( Supplementary Fig. 2). To investigate the effect of butyrate on polarization of M2 macrophage, we first examined the hallmarks of M2 macrophage expression in BMDMs directly treated with butyrate. The results showed that treatment of M0 macrophages with butyrate did not stimulate the upregulation of M2 markers (Arg1, Fizz1, Ym1 and MR) ( Supplementary Fig. 3). The data suggested that butyrate did not itself drive polarization of M0 macrophages towards the M2 phenotype. To determine whether butyrate facilitated IL-4-driven M2 polarization, M0 macrophages were activated by IL-4 in the presence of various concentrations (1 to 200 μg/ml) of butyrate. The relative expression of genes encoding Arg1, Fizz1, and Ym1, but not MR, was considerably higher in M2-BMDMs exposed to 50 μg/ml butyrate for 24 h (Fig. 1a). Meanwhile, stimulation with butyrate did not alter the surface expression of costimulatory molecules, CD80 or CD86, on M2-BMDMs ( Supplementary Fig. 4). Butyrate clearly increased the expression of M2 macrophage marker genes in M2-BMDMs and 50 μg/mL was then used to determine its influence the progress of polarization, again triggered by IL-4. Compared to its absence, exposure of M2-BMDMs to butyrate significantly enhanced expression of Arg1, Fizz1, and Ym1 after 12 h treatment with IL-4 and the effect on Arg1 was most striking (Fig. 1b). Western blot analysis of Arg1 protein content at 24 h was consistent with the enhanced gene expression (Fig. 1c). Collectively, these data indicated that butyrate enhanced the IL-4-induced expression of M2-associated markers.
Butyrate enhances migration and wound-healing properties in M2-BMDMs. M2 macrophage promotes the resolution of wound healing by facillating cell migration to damaged tissues 14,15 . To analyze if butyrate influences M2 macrophage function, wound-healing assay was performed using MLE-12 bronchial cells in an in vitro scrape model. As shown in Fig. 2a, supernatants from M2-BMDMs increased wound closure compared with those from the M0-BMDMs (p < 0.05) and this effect on re-epithelialization was improved further when M2-BMDMs had been treated with butyrate. As shown in Fig. 2b, medium conditioned by M2-BMDMs for 24 h promoted the migration of MLE-12 cells in transwell filter assays. The number of MLE-12 cells in the permeating septum after 12 h more than doubled (p < 0.05) in the presence of medium conditioned by M2-BMDMs (a) M0-BMDMs were directly exposed in series concentrations of butyrate for 24 h. Expression of Arg1, Fizz1, Ym1, and MR was measured by quantitative RT-PCR. (b) The relative quantity of Arg1, Fizz1, Ym1, and MR mRNA transcribed by nonpolarized (M0-BMDMs, gray bars) or M2-BMDM after 3, 6, 12, and 24 h with IL-4 in the absence (white bars) or presence (black bars) of 50 μg/mL butyrate. (c) Arg1 protein levels in the absence or presence of 50 μg/mL butyrate in M2-BMDMs after 24 h treatment. Data are mean ± SD for at least three independent experiments. Comparisons between means used t tests (*P < 0.05, **P < 0.01, ***P < 0.001).
rather than M0 and doubled again (p < 0.01) when conditioning had occurred in the presence of butyrate. Together, these data demonstrated that butyrate significantly enhanced the functional ability of M2-BMDMs promote cell migration and wound-healing. Consistent with this, the expression of relevant C-C cytokine genes, CCL2, CCL17, and CCL22 was consistently higher in butyrate-treated M2-BMDMs (Fig. 2c).
Butyrate ameliorates dextran sulfate sodium (DSS)-induced colitis in mice. M2 macrophage plays an important role in colonic infection with parasitic nematodes 16 . Furthermore, an increase in intestinal M2 macrophage has been associated with reducing the severity of colitis in mice 17 . We first evaluated the therapeutic effect of butyrate on DSS-induced colitis as a model of inflammatory bowel disease (IBD). Butyrate-treated mice showed much milder symptoms than the untreated colitis control mice, as evidenced by a slower rate of weight loss (Fig. 3a) and a lower disease activity index (DAI; Fig. 3b). Meanwhile, butyrate could promote the growth in mice and the results are consistent with the butyrate can improve the growth performance of animals [18][19][20] . Histological examination of the colons showed less mucosal damage, demonstrating the protective effect of butyrate against colitis in the DSS-induced colitis model (Fig. 3c,d). Following butyrate treatment, reduced levels of proinflammatory cytokines, namely IL-6, TNF-α , and IL-1β , were detected in the serum compared with control mice (Fig. 3e). Moreover, we found that butyrate-treated mice had more Arg1 protein expression in colonic tissues (Fig. 3f). Taken together, these data showed that butyrate-treated mice have reduced clinical disease and histological damage compared with control mice, and this protection during DSS-induced colitis may correlate with increased in Arg1 protein expression in the colon during inflammation.

M2 macrophage confers protection to mice during DSS-induced colitis.
To further elucidate the importance of M2-BMDMs in IBD, we adoptively transferred M0-and M2-BMDMs into colitis-bearing recipient mice. There was no significant difference between the mice treated with M0-BMDMs and the colitis-only control group. In contrast, mice receiving M2-BMDMs showed significantly less weight loss compared with the colitis-only control group, with lowered DAI (Fig. 4a,b) and improved histological condition of the colon (Fig. 3c,d). Moreover, the M2-BMDM recipient mice possessed less IL-6, TNF-α , and IL-1β in the serum than the colitis-only control mice (Fig. 4e). Furthermore, increased Arg1 protein expression was detected in the colonic tissues of M2-BMDM recipient mice than in control tissues (Fig. 4f). These data demonstrated that M2 macrophage can protect against DSS-induced colitis in mice. Migrated cells at the lower surface of the transwell filter were stained with DAPI and counted. (c) Total RNA was isolated from M0-BMDMs, and M2-BMDMs, untreated or treated with butyrate, and analyzed by RT-PCR for the expression of representative M2 macrophage chemokine genes. Data are shown as mean ± SD for three independent experiments, Comparisons between means used t tests (*P < 0.05, **P < 0.01, ***P < 0.001).
Butyrate reduces histone H3K9 deacetylase in M2-BMDMs. Butyrate, as a histone deacetylase (HDAC) inhibitor, is known to regulate gene expression epigenetically 22,23 . Whether butyrate alters the histone acetylation of BMDMs to epigenetically regulate the transcription of genes responsible for M2-BMDM development or function is still unknown. We hypothesized that butyrate enhanced the polarization of M2 macrophage involved the HDACs inhibition. To test this hypothesis, M2-BMDMs were stimulated with butyrate for 6 h and the expression of HDAC genes was analyzed. The expression of HDAC1, HDAC6, HDAC7, and HDAC9 was reduced in M2-BMDMs, while no differences were observed in HDAC2 and HDAC3 gene expression. Butyrate-treated M2-BMDMs had lower HDAC1 gene expression than M2-BMDMs (Fig. 6a). Western blot analysis demonstrated that butyrate treatment of M2-BMDMs enhanced histone 3 lysine 9 acetylation (H3K9) (Fig. 6b). These results suggested that butyrate enhanced M2-BMDMs polarization partly through HDAC1 inhibition and histone H3K9 acetylation.

Discussion
In this study, we have identified that a metabolite derived from commensal microbes, butyrate, effectively facilitates M2 macrophage polarization. Although butyrate did not directly affect the polarization of M0-BMDMs to M2-BMDMs, it could promote a greater degree of polarization in IL-4-induced M2 macrophage. Additionally, butyrate enhanced the critical functions of M2 macrophage by improving re-epithelialization and the migration of MLE-12 cells. Furthermore, we demonstrated that butyrate attenuates intestinal inflammation in mice DSS-induced colitis model partly via M2 polarized macrophage.
The DSS-induced colitis model is suitable to study the contribution of innate immune cells to colitis, particularly to epithelial repair mechanisms 24,25 . After DSS-induced gut injury, macrophages are recruited to the colon and play a central role in disease pathogenesis 26,27 . Previous studies showed that a defect in M2 macrophage polarization led to increased severity in DSS-induced colitis, whereas adoptive transfer of in vitro-derived M2 macrophage could attenuate colonic inflammation in mice 17,28,29 . Recent evidence showed that another SCFA, acetate, in the drinking water markedly attenuated disease indices, with increased colon length, decreased DAI, and reduced TNF-α levels in germ-free mice treated with DSS 30 . Here, we employed the DSS-induced colitis  model and demonstrated that M2 macrophage were indeed responsible for the therapeutic effects of butyrate on IBD. These findings supported the role of M2 macrophage polarization in the development of inflammatory diseases, and they showed that commensal microbe-derived butyrate, as a new effector molecule, could ameliorate disease severity in DSS-induced colitis by enhancing the polarization of M2 macrophage. It has been suggested that Arg1, expressed by M2 macrophage, is important for tissue repair in diverse models and tissues 14,15,[31][32][33] . Our results show increased Arg1 protein in butyrate-treated mice, associated with less mucosal damage and lower levels of proinflammatory cytokines.
It is well known that the classical pathway for IL-4-induced M2 macrophage polarization involves the STAT6 signaling pathway. The polarization of M2 macrophage is mediated by IL-4-dependent activation of STAT6, which leads to the expression of Fizz1, Ym-1, and Arg1 in macrophages 34 . These results provided a molecular mechanism for the butyrate-mediated protection against DSS-induced colitis, possibly by enhancing STAT6 signaling in intestinal tissues. Another possible mechanism of butyrate-enhanced M2 polarization may be by inhibiting the activity of HDACs. Previous studies have demonstrated that butyrate inhibited the activity of HDACs in many different cell types 22 . H3K9 acetylation is considered a hallmark of transcriptional activation. Another SCFA propionate treatment of Ffar2 +/+ mice reduced Treg HDAC6 and HDAC9 expression and enhanced H3K9 acetylation 6 . Treatment of BMDMs with butyrate led to a downregulation of LPS-induced proinflammatory mediators by inhibition of H3K9 acetylation 12 . To determine whether butyrate behaved as an HDAC inhibitor involved in the polarization of M2-BMDM, we checked the levels of HDACs in M2-BMDMs without butyrate. Compared with M0-BMDMs, the expression of HDAC1, HDAC6, and HDAC9 were decreased. Western blot analysis also demonstrated that H3K9 acetylation was enhanced in M2-BMDMs. Furthermore, HDAC1 expression was further reduced and the H3K9 acetylation was increased in butyrate-treated M2-BMDMs. The level of histone acetylation is tightly regulated by the opposing activities of HAT and HDACs 35 . C646 is capable of attenuating HDACs, such as in butyrate, trichostatin, and cAMP-induced H3K9 acetylation 36,37 . To further determine whether acetylation of H3K9 mediated the polarization of M2 macrophage, we treated M2-BMDMs with C646. We showed that the expression of Arg1 and H3K9 acetylation was decreased in C646-treated M2-BMDMs. Moreover, C646 efficiently inhibited the effect of butyrate on facilitating M2-BMDMs polarization. These results suggest that butyrate may facilitate M2 macrophage partly through HDAC1 inhibition.
We next investigated how butyrate modulates STAT6 phosphorylation, which is required for IL-4 induced M2-BMDMs polarization. We hypothesized that H3K9 may play an important role in regulating the level of phosphorylated STAT6 and controlling M2 macrophage polarization. Our results showed that C646 could effectively reduce the level of phosphorylated STAT6 in both M2-BMDMs and butyrate-treated-M2-BMDMs. These data demonstrated that butyrate enhanced the STAT6 phosphorylation partly via inhibiting HDAC1 gene expression and increasing H3K9 acetylation. The STAT6 signaling pathway requires various co-activators or co-repressors to promote and regulate transcription of endogenous genes. Consistent with this concept, The polypyrimidine tract binding protein (PTB)-associated splicing factor (PSF) recruits HDAC1 to the STAT6 complex, which reduces H3 acetylation at the promoter regions of the Igε gene and inhibits STAT6 signing in lymphomas 38 . IL-4 induced expression of the reticulocyte-type 15-lipoxygenase-1 (15-LOX-1) gene requires the enhancement of H3 acetylation and the activation of the STAT6 signaling pathway in A549 lung epithelial cells 39 . Our findings also support the concept that histone acetylation correlates with their post-translational modifications and coordinately regulate STAT6-mediated gene transcription. Butyrate-mediated activation of H3K9 acetylation and the STAT6 signaling pathway, and its influence on the polarization of M2 macrophage are still unclear. SCFAs bind the G-protein-coupled receptor 43 (GPR43; also known as FFAR2), which is expressed on immune cells, mediating resolution of inflammatory responses 6,30,40 . GPR43 is expressed on the colonic epithelium and butyrate provides a major source of energy for colonocytes 41 . Gpr43 −/− mice showed aggravated or unresolving inflammation in DSS-induced colitis. In a T-cell-dependent model of colitis, Gpr43 −/− mice also had more severe disease with increased colon histological scores 30 . SCFAs mediate the size and function of the colonic Treg pool and protect against colitis in a Gpr43-dependent manner in mice 5,6 . Therefore, using Gpr43 −/− mice might enable the dissection of a precise mechanism of action for butyrate on H3K9 acetylation/STAT6 signaling in the polarization of M2-BMDMs. Moreover, the inflammatory process is highly complicated, and considerably more information is required for an improved understanding about macrophages in vivo. Because macrophages are able to perform mixed M1/M2 phenotypes in pathological conditions, further studies may focus not only on M2 polarization but also on the changes in M1/M2 polarization treated with butyrate during the development of inflammatory bowel disease. In addition, the polarization of either M2 macrophage or Treg could be facilitated or induced by butyrate. Future studies may delineate a dynamic view of this inflammatory process, take M2 macrophage and Treg in systemic and local milieu into account, and define the kinetics, plasticity, reversibility, and memory of their responses in order to full elucidate the mechanism that butyrate ameliorates inflammatory bowel disease in mice.
In sum, our studies indicated that commensal microbe-derived butyrate enhanced the IL-4-mediated STAT6 transcription, achieved through H3K9 acetylation to enhance M2-BMDMs polarization, and delineated new insights into the immune interplay underlying inflammatory bowel disease. Co., Ltd (SLAC; Shanghai, China). All mice were maintained in the Experimental Animal Center of Zhejiang University. Experiments were conducted in accordance with the Chinese guidelines for animal welfare, approved by the Animal Welfare Committee of Animal Science College, Zhejiang University. M2 macrophage culture. Bone marrow-derived macrophages (BMDMs) were generated by isolating bone marrow from femurs and culturing in high glucose Dulbecco's modified Eagle's medium (DMEM, Hyclone, Logan, UT, USA) containing 10% (vol/vol) fetal bovine serum (Gibco) with macrophage colony-stimulating factor (M-CSF; 15 ng/mL; Peprotech) at 37 °C with 5% CO 2 .Half of the medium was replaced with fresh medium containing M-CSF every 3 days. After 6 days of culture, IL-4 (15 ng/mL) was added to generate full alternatively activated M2-BMDMs for 24 h. Untreated BMDMs were retained as nonpolarized macrophages (M0-BMDMs). The BMDMs were subsequently cultured for 1.5-24 h in the presence of sodium butyrate (Sigma-Aldrich) or histone acetyltransferase (HAT) inhibitor C646 (Selleck).
Cell viability assays. Cells (5, 000 cells/well) were seeded into a 96-well plate and treated with variable concentrations of butyrate for 24 h. Subsequently, the cells were labeled using a Cell Counting Kit-8 (CKK8) (Dojindo, Tabaru, Japan) for 2 h. Absorbance at 450 nm was measured with a plate reader and cell viability was expressed as the percentage of the absorbance of treated cells versus untreated cells.
Western blotting. Cell lysates containing extracted proteins were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked at room temperature for 1 h in 5% fat-free milk, incubated overnight at 4 °C with a primary antibody, followed by incubation with the appropriate HRP-conjugated reporter antibody for 1 h at room temperature. The enhanced chemiluminescence Fluorchem E system (Cell Biosciences, Santa Clara, CA) was used for developing. Wound-healing assay. The wound-healing assay was performed as previously described in vitro 42 .
Transformed mouse lung epithelial cells (MLE-12) were grown to confluency in a 24-well plate. Confluent cell monolayers were mechanically wounded by scraping a sterile conventional pipette tip across the monolayer. Wounded MLE-12 monolayers were washed three times with PBS (pH 7.4) to remove loose cells and debris, and then were incubated for 12 h at 37 °C with 5% CO 2 in the presence of supernatants collected from M2-BMDMs untreated or stimulated with butyrate for 24 h. A magnification of 10× was used to allow a major surface area to be viewed. Photographs were taken and analyzed using Image J software to measure the re-epithelialization area.
Migration assays. A migration assay of MLE-12 cells was performed using matrix-coated polycarbonate filters (8-μm pore size, 24 well, Transwell; Becton Dickinson). MLE-12 cells were resuspended in 500 μL DMEM without fetal bovine serum and seeded to the upper chamber. Supernatant (500 μL) from M2-BMDMs cultured in the presence or absence of butyrate was added to the lower chamber. After 24 h, cells that had migrated into the lower wells were collected, stained with DAPI (Roche Diagnostics), and counted under a microscope.
Cytokine assay. Cytokine levels (IL-1β , IL-6, and TNF-α ) in sera of mice were measured by cytometric bead array immunoassay (CBA; BD Biosciences) according to the manufacturer's instructions.
Histology and immunohistochemistry. Colon tissue samples were fixed in PBS containing 10% neutral-buffered formalin. Paraffin-embedded sections (5 μm) were stained with hematoxylin and eosin. The degree of histological damage and inflammation was graded by a blinded veterinary pathologist. The following variables were scored: (i) amount of inflammation (0, none;1, mild; 2, moderate; 3, severe; 4, accumulation of inflammatory cells in the gut lumen), (ii) distribution of lesions (0, none; 1, focal; 2, multifocal; 3, nearly diffuse; 4, diffuse), and (iii) depth of inflammation and layers involved (0, none; 1, mucosa only; 2, mucosa and submucosa; 3, limited transmural involvement; 4, transmural). The overall histological score was the sum of the three variables (maximum score 12). Consecutive cryostat colon sections were used for immunohistochemistry to detect deposited Arg1 protein. The mean density was shown as an average integrated optical density/area using the Image-Pro Plus software (version 6) (Media Cybernetics, USA).

Statistical analysis.
All data are expressed as means ± SD. Comparisons between two groups was performed using the Student's t test, whereas differences between > 2 groups were performed using one-way ANOVA, (with post hoc comparisons using Dunnett tests) with GraphPad Prism version 4.0 statistical software. Differences were considered significant if P < 0.05.


text:
Candida albicans Dbf4-dependent Cdc7 kinase plays a novel role in the inhibition of hyphal development

Candida albicans is an opportunistic human fungal pathogen. The ability to switch among multiple cellular forms is key to its pathogenesis. The Dbf4-dependent protein kinase gene CDC7 is conserved due to its role in initiating DNA replication. Because a C. albicans Cdc7 (Cacdc7) homozygous null was not viable, we generated a C. albicans strain with a deleted C. albicans CDC7 (CaCDC7) allele and an expression-repressible allele. Surprisingly, cells of the strain grew as hyphae under the repressed conditions. The in vitro kinase assays confirmed that CaCdc7 (K232) and CaCdc7 (T437) are critical for catalytic and phosphoacceptor of activation activity, respectively. C. albicans cells formed hyphae when expressing either the catalytically inactive CaCdc7 (K232R) or the phosphoacceptor-deficient CaCdc7 (T437A). While CaCdc7 interacted with CaDbf4, cells of the strain in which CaCDC7 was repressed were not rescued by constitutively expressing C. albicans DBF4 or vice versa. We conclude that CaDBF4-dependent CaCDC7 is an essential gene suppressing the hyphal development.

remains incompletely understood. Moreover, the importance or requirement of some of the key factors, such as Swe1, for DNA replication stress-induced filamentous development does not seem to be conserved between the two yeasts [24][25][26][27] . Until a recent global analysis of C. albicans morphology 28 , no reports pointed to a direct involvement of DDK in filamentation.
To verify the role of DDK in C. albicans, we have characterized CaDBF4 29 and C. albicans CDC7 (CaCDC7). We generated a C. albicans strain capable of repressing the expression of CaCDC7 and examined the cellular morphology when CaCDC7 is depleted. Strains constitutively expressing the catalytically inactive CaCdc7 or the phosphoacceptor-deficient CaCdc7 were generated to verify the requirement of kinase activity for cellular morphology. Additionally, the functional dependency of CaCdc7 and CaDbf4 was tested by the yeast two-hybrid assay and by constitutively expressing CaCdc7 in a CaDBF-deletion strain and vice versa.

Results and Discussion
C. albicans CDC7 is a structural homolog of S. cerevisiae CDC7. By using Blast to compare the Candida Genome Database with the entire sequence of the S. cerevisiae Cdc7 protein, a single CaCDC7 located on chromosome 2 was identified that belongs to Contig19-10183 [70303..72273] orf19.3561, Assembly 19, which has one reading frame of 1971 bp and potentially encodes a 72 kD protein of 657 amino acid residues. To analyze the structure of the protein encoded by CaCDC7, the protein sequence derived from the 1971 bp ORF was aligned and compared to other Cdc7 protein sequences across the evolutionary spectrum by ClustalW 30,31 . As shown in Supplementary Fig. S1, all twelve conserved serine/threonine protein kinase subdomains as defined by Hanks, S.K., and Quinn, A.M. 32 can clearly be identified in the protein sequence of CaCdc7. Moreover, the invariant amino acid residues in the subdomains, which are found throughout protein serine/threonine kinases, are also conserved in CaCdc7. Based on Supplementary Fig. S1, Fig. 1 was generated as a schematic diagram comparing C. albicans Cdc7 (CaCdc7), S. cerevisiae Cdc7 (ScCdc7), and human Cdc7 (HsCdc7). As shown in Fig. 1, in domain II of the ATP-binding region, CaCdc7 contains a lysine at residue 232 (K232), the same as lysine 76 (K76) 33,34 in the budding yeast ScCdc7 and lysine 90 (K90) in human Cdc7 35 , which is the site required for catalytic activity. In subdomain VIII of the phosphoacceptor region, CaCdc7 also contains a threonine at residue 437 (T437), equivalent to threonine 281 (T281) in ScCdc7 and threonine 376 (T376) in humans, which is the phosphorylation site for kinase activation 33,34 . Additionally, the spacing between subdomains VII and VIII is worth noting, because the same region in ScCdc7 is needed for its mitotic function 36 and is unique among all known kinases 32 . Notably, CaCdc7 possesses Cdc7 characteristic insertions of KI-0, KI-1, KI-2, and KI-3, and distinct regions located at the N-and C-terminus. Importantly, the C-terminal tail of Cdc7 is known to be essential for interacting with Dbf4 37 . Together with KI-2 and KI-3, such an interaction becomes efficient 38 . Nonetheless, the functional significance of the less-conserved C-terminal tail of CaCdc7 is unclear. Unique features are also visible in CaCdc7. The most striking is an extended stretch of approximately 140 amino acid residues, which are quite hydrophobic, at the amino-terminus of CaCdc7. The region of insertion KI-2 of CaCdc7 between 355 and 393 is rich in threonine and serine ( Supplementary Fig. S1). Nevertheless, the functional significance of these differences is unknown. Despite the differences between CaCdc7 and its counterparts, particularly ScCdc7, the overall organization and sequence of CaCdc7 is very similar to Cdc7. We conclude that CaCdc7 encodes a serine/threonine kinase with homology both in sequence and organization to Cdc7 across the evolutionary spectrum.
Construction of the CDC7 expression-repressible C. albicans strain. To establish the function of CaCDC7 in C. albicans, we sought to construct a Cacdc7 deletion mutant. However, as CaCDC7 encodes a protein with the structural homologue of known Cdc7 proteins whose function is the initiation of DNA replication, we predicted that the cdc7 homozygous null mutant is lethal. We addressed this issue by generating a strain with one CaCDC7 allele deleted and the other under the control of MET3 promoter (MET3p). To delete Figure 1. Schematic diagram of the comparison of C. albicans Cdc7 (CaCdc7), S. cerevisiae Cdc7 (ScCdc7), and human Cdc7 (HsCdc7). The indicated conserved subdomains of the serine/threonine kinases are shown, each of which is specified by a roman numeral (from I to XI). The subdomain VI is further separated into the subdomains VIa and VIb. The kinase insert with conserved residues (KI-0) is shown. The kinase inserts of KI-1, KI-2, and KI-3 are indicated. The positions of the essential lysine residue for catalytic activity of the three Cdc7s, CaCdc7 (K232), ScCdc7 (K76), and HsCdc7 (K90), are shown. The positions of the phosphoacceptor threonine residue for kinase activation of the three Cdc7s, CaCdc7 (T437), ScCdc7 (T281), and HsCdc7 (T376), are shown.
The size of the XbaI fragment containing CaCDC7 shifted from 8392 bp to 1924 bp and from 8392 bp to 6698 bp, respectively, demonstrating that the CaCDC7 alleles were either integrated with the mini-Ura-blaster cassette or MET3p-driven CaCDC7 ( Fig. 2A). The size of the HindIII fragment containing CaCDC7 shifted from 7578 bp to 6223 bp, demonstrating that the CaCDC7 alleles integrated with the mini-Ura-blaster cassette had lost URA3 (Fig. 2B). These results indicate that the strain CaCDC7 M3/− has the expected genome organization of C. albicans.
CDC7 is an essential gene and depletion of CDC7 leads to hyphal growth in C. albicans. To test the repressibility of CaCDC7 in the strain CaCDC7 M3/− , we grew strains CaCDC7 M3/− , BWP17 (CaCDC7+ /+ ), and CaCDC7+ /− cells in SD medium with or without 2.5 mM methionine/cysteine (Met/Cys) and extracted RNA for RT-PCR analysis. The expression of CaCDC7 in the strain CaCDC7 M3/− was significantly reduced under the repressed conditions compared to the de-repressed conditions (Fig. 3A), suggesting that the expression of CaCDC7 in the strain CaCDC7 M3/− was solely controlled by MET3p and the other allele was deleted.
We next established the requirement for CaCDC7 in C. albicans by growing strains on selective media in the presence or absence of Met/Cys. Strain CaCDC7 M3/− formed colonies in the presence of Met/Cys with wrinkled surfaces (unpublished data). The results suggested that CaCDC7 suppresses filamentous development and may not be essential. This result is consistent with our finding that repression of CaDBF4 expression leads to filamentous growth in C. albicans 29 . To confirm the necessity of CaCDC7 in C. albicans, we established a Cacdc7 homozygous null mutant. However, whereas the Cacdc7 heterozygous null mutants were generated with ease, no Cacdc7 homozygous null mutants were obtained. Our result is in agreement with a recent report where the functional copy of the essential CaCDC7 gene in the heterozygous strain was governed by a tetracycline-repressible promoter to allow functional study 28 . This result is also consistent with the CaDBF4 data, in which Cadbf4 homozygous null mutants were unable to survive 29 . These results suggest that C. albicans CDC7 and its regulator encoded CaDBF4 gene, like their counterparts across evolutionary spectrum, possesses a conserved role in the initiation of DNA replication. In the MET3 promoter-driven system, the difference in expression can reach 85-fold between repressed and de-repressed conditions 40 . We reasoned that under the repressed condition, CaCDC7 was depleted while a limited amount of CaCdc7 was able to function, although to a lesser extent. While Cdc7 depletion substantially inhibits proliferation in cancer cells 41,42 , the depletion of CaCDC7 appeared to reduce proliferation, as the growth rate of CaCDC7 M3/− was lower than that of its parental BWP17 when cells were grown in medium with Met/Cys ( Supplementary Fig. S2). Taken together, these results suggest that the function of CaCDC7 is tightly associated to that of CaDBF4 in C. albicans and that the CaDbf4-dependent CaCdc7 kinase is essential, as is its counterpart in S. cerevisiae 9,10 .
To determine the phenotypic consequences, in particular the cellular morphology, of CaCDC7 M3/− under the repressed condition, we grew CaCDC7 M3/− cells in medium with or without 2.5 mM Met/Cys and examined the phenotypic consequences microscopically. The cells formed germ tubes after 4 h of repression and continued to grow as hyphae from 8 h to 24 h under the repressed condition (Fig. 3B). These observations were comparable to those of cells repressing CaDBF4 expression 29 , suggesting that CaCDC7 and CaDBF4 function as a DDK for the YTH transition and that CaCDC7 may have an additional role in morphogenesis. To definitely determine that CaCDC7 is the gene for suppression of the yeast-to-hypha transition, we performed a rescue assay where a constitutive ACT1 promoter (ACT1p)-driven CaCDC7 was introduced into the CaCDC7 M3/− strain. These cells grew in the yeast form even in the presence of Met/Cys ( Supplementary Fig. S3), confirming that CaCDC7 is responsible for the inhibition of hyphal growth in C. albicans. Together with the fact that repression of CaDBF4 expression led to filamentous growth in C. albicans 29 , these data suggest that the CaCDC7 and CaDBF4 encoded proteins likely act together to perform their function. The hyphal growth during the CaCDC7 or CaDBF4 depleted condition may be the consequence of constrained function that exerts as a stress condition. In S. cerevisiae, genotoxic stress conditions that reduce DNA synthesis can induce filamentous differentiation through Mec1-Rad53-Swe1-Cdc28-Clb2 17 . However, in C. albicans, DNA damage-induced cell cycle delay leading to polarized growth is only partially dependent on Swe1 24 , suggesting that genotoxic stress-induced filamentous growth is not or is only partially mediated by Swe1 in C. albicans. However, no evidence indicates that CDC7 interacts with the above components in C. albicans. Hence, whether genotoxic stress-induced filamentous growth is mediated by CaCDC7 in C. albicans remains to be verified.
The conserved residues are essential for the catalytic activity of CaCdc7 and the phosphoacceptor of activation. To verify if the CaCDC7-encoded protein product has kinase activity and is capable of being phosphorylated, we created plasmids p6HF-ACT1p-CaCDC7, p6HF-ACT1p-CaCDC7 (K232R) and p6HF-ACT1p-CaCDC7 (T437A) capable of constitutively expressing either the wild-type CaCDC7, the catalytically inactive CaCdc7 (K232R), or the phosphoacceptor-deficient CaCdc7 (T437A) (Fig. 4A), each of which was (A) The alignment of subdomains II and VII among the homologues of Cdc7, revealing lysine 232 as a conserved residue for catalytic activity and threonine 437 as a conserved phosphoacceptor residue. These were used to generate the catalytically inactive CaCdc7 (K232R) or the phosphoacceptor-deficient CaCdc7 (T437A). (B) An in vitro kinase assay was used to verify that CaCdc7 (K232) and CaCdc7 (T437) are required for the catalytic activity and activation by phosphorylation. Cells of strain CaCDC7 M3/− were transformed with p6HF-ACT1p-CaCDC7, which is capable of constitutively expressing wild-type CaCdc7 (CaCDC7), catalytically inactive CaCdc7 (K232R) or the phosphoacceptor-deficient CaCdc7 (T437A). Cells of each strain were grown in SD medium with the required supplements in the presence (+ Met/Cys) of each of 2.5 mM methionine and cysteine for 12 h prior to purification of CaCdc7 for the in vitro kinase assay, followed by western blot analysis with specific antibodies to FLAG (CaCdc7-6xHisFLAG tagged), histone H1, and phosphorylated histone H1. The purified CaCdc7 was also subjected to FSBA treatment as described in the Materials and Methods. (C) Phosphorylation of CaCdc7 is activated by hydroxyurea (HU). The cells carrying p6HF-ACT1p-CaCDC7 were grown in same the conditions and media as in (B) treated with or without 0.1 mM HU for 1 h. The purified CaCdc7 from non-HU treated cells was exposed to CIP phosphatase for the indicated times as described in the Materials and Methods.
Scientific RepoRts | 6:33716 | DOI: 10.1038/srep33716 introduced into cells of the strain CaCDC7 M3/− . We grew cells of these strains exponentially in the medium with Met/Cys. After harvesting and lysing, each of the wild-type and mutant CaCdc7s was purified by Ni 2+ -NTA agarose and subjected to an in vitro kinase assay. While the wild-type CaCdc7 was able to phosphorylate histone H1, this phosphorylation was significantly inhibited by FSBA, which blocks the catalytic activity of a kinase (Fig. 4B). This result was consistent with the inability of catalytically inactive CaCdc7 (K232R) to phosphorylate histone H1 (Fig. 4B). We noted that the FSBA appeared to block autophosphorylation of CaCdc7, which was indicated by the decrease in the upper band of the CaCdc7 doublet (Fig. 4B). These results confirmed that CaCdc7 possesses kinase activity and that the structurally predicted K232 ( Fig. 1) 33,34 is indeed the site essential for catalytic activity. Additionally, the phosphoacceptor-deficient CaCdc7 (T437A) almost lost the ability to phosphorylate histone H1 (Fig. 4B), indicating that the site is the conserved phosphorylation site ( Fig. 1) 33,34 that is required for activating kinase activity. The reduction of the upper band of the CaCdc7 doublet from the purified wild-type CaCdc7 in a time-dependent manner when treated with phosphatase confirmed that the upper band of the CaCdc7 doublet is the phosphorylated form of CaCdc7 (Fig. 4C). A reduction in the phosphorylated form of CaCdc7 was observed when cells were treated with HU for 1 h before western blot analysis (Fig. 4C). This result is in agreement with the downregulation of S. cerevisiae DDK activity mediated by the phosphorylation of Dbf4 through Rad53 in S. cerevisiae 12,15,43,44 and in Xenopus egg extracts 45 under DNA stress, which likely resulted from inhibiting the phosphorylation site of kinase activation.
The catalytic activity of CaCdc7 and the phosphorylated activation of CaCdc7 are required for suppression of hyphal growth in C. albicans. To investigate whether the ability of cells to suppress the YTH transition requires kinase activity and phosphorylation of DDK, CaCDC7 M3/− cells containing plasmids p6HF-ACT1p-CaCDC7, p6HF-ACT1p-CaCDC7 (K232R), p6HF-ACT1p-CaCDC7 (T437A) or the empty plasmid p6HF-ACT1p were grown in medium with or without Met/Cys. Regardless of the expression of endogenous CaCDC7 under the control of MET3p being repressed or de-repressed, the expression levels of different versions of the CaCdc7 protein driven under ACT1p control were similar (Fig. 5A). It was apparent that cells with repressed MET3p-controlled endogenous CaCDC7 expression but constitutive expression of the ACT1p-driven mutants CaCdc7 (K232R) or CaCdc7 (T437A) grew as the hyphal form (Fig. 5B), which is in contrast to those expressing ACT1p-driven wild type CaCdc7, which remained as yeast cells. These results suggest that the catalytic activity of CaCdc7 and the phosphorylation of CaCdc7 are required for the function of CaCdc7 on the suppression of the YTH transition.
By examining cells of the strains repressing the expression of CaCDC7 in a longer time, we confirmed that the mutant strains were able to proliferate, though in a reduced rate as verified by the growth curve ( Supplementary  Fig. S2). The constitutive expression of CaCDC7 but none of the two CaCDC7 mutants allows recovery of this growth defect (Supplementary Fig. S2). These data indicate that cells depleted with CaCDC7 or expressing the defective CaCdc7 mutants delay the cell cycle progression. We note that cdc7 conditional mutants of S. cerevisiae show a dumbbell shape 46 , unlike polarized growth of C. albicans expressing the CDC7 mutations. It is known that pseudohyphae and true hyphae emerge in response to cell-cycle arrest in C. albicans 47 . Hence, the C. albicans expressing the mutant CDC7 may delay the cell cycle at the S phase. However, the cells were still able to grow, and the extension of hyphal development was more prominent at a later time point of 32 h (Supplementary Fig. S4). Moreover, because the cell cycle stage of cells repressing with CaCDC7 was different, the budded daughter cells, like the mother cells, switch to the hyphal mode of growth can grow as extended hyphal form (Fig. 5). These results suggest that cells depleted with CaCDC7 or expressing the defective CaCDC7 mutants delay in the cell cycle, which accompanies the reduced rate of hyphal extension. To assess further if CaCDC7 is required for YTH, cells of the strains, together with BWP17 were subjected to grow in 37 °C, a condition known to induce hyphal growth. As shown in the Supplementary Fig. S5A, however, the hyphal formation appeared in the CaCDC7-repressede but not the CaCDC7-derepressed condition at 37 °C, suggesting that the initiation and continuation of filamentation are dependent on CaCdc7 in C. albicans. Importantly, unlike the wild-type strain SC5314, BWP17was unable to induce hyphal development but pseudohyphae-like type when cultured in the SD minimum medium at 37 °C ( Supplementary Fig. S5B).
We are not aware of any Cdc7 homologs across the evolutionary spectrum that are involved in morphogenesis until the latest report. Based on the GRACE strain collection 48,49 , the report identified over a hundred strains as negative regulators of filamentation. The expression of CaCDC7 of the GRACE strain was controlled by the tet-operator; hence, cells formed filaments under the repressed condition 28 . However, the kinase activity of CaCdc7 and its activation by phosphorylation has not been determined.

CDC7 and DBF4 are interdependent for the function of morphological control in C. albicans.
The protein products encoded by CDC7 and DBF4 function together as a DDK for the initiation of DNA replication 10 . Although our study suggests that DDK in C. albicans plays a significant role in morphogenesis, we wondered whether the presence of C. albicans DDK functions in hypha-suppression in C. albicans. The definitive determination of how CaCdc7 and CaDbf4 constitute DDK required verification of their direct interaction. To do so, we adopted the yeast two-hybrid assay. The diploid S. cerevisiae cells were able to grow on media lacking leucine and tryptophan, indicating the presence of both plasmids based on the Gal4 DNA binding domain with the TRP1 selection marker and the Gal4 activation domain with the LEU2 selection marker (Fig. 6A). However, only those cells expressing CaCdc7 fused with the Gal4 DNA binding domain and CaDbf4 fused with the Gal4 activation domain concurrently were able to grow on the plate without histidine (Fig. 6B). These results suggest that CaCdc7 and CaDbf4 can associate physically. Moreover, diploid S. cerevisiae cells capable of simultaneously expressing CaCdc7 fused with the Gal4 DNA binding domain and CaDbf4 fused with the Gal4 activation domain were able to grow on the plate without histidine, even in the presence of 20 mM 3-amino-1,2,4-triazole (3-AT), the inhibitor of imidazole glycerol-phosphate dehydratase encoded by the reporter gene HIS3. These results indicate that the interaction between CaCdc7 and CaDbf4 is relatively stable and thus likely to be genuine (Fig. 6C).
To assess the functional dependency between CaCDC7 and CaDBF4, we generated plasmids p6HF-ACT1p-CaCDC7 and p6HF-ACT1p-CaDBF4 that are capable of constitutively expressing either CaCDC7 or CaDBF4, and introduced them into CaDBF4 M3/− /− 29 and CaCDC7 M3/− , respectively. The ability of a DDK gene that can be constitutively expressed to suppress the loss of the other DDK gene that is repressed in the presence of Met/Cys was assessed. Constitutive expression of CaCDC7 (Fig. 7A) did not suppress the loss of CaDBF4 (Fig. 7B), suggesting that the function of CaDBF4 requires the presence of CaCDC7. Similarly, constitutive expression of CaDBF4 (Fig. 7C) was unable to suppress the loss of CaCDC7 (Fig. 7D). The epistasis analyses and yeast two-hybrid assay results confirm that CaCDC7 and CaDBF4 encode proteins that form functional DDK and are functionally interdependent. Additionally, we introduced a Tet-on expression system 50 into BWP17 where CaCDC7 or CaDBF4 were massively overproduced in the presence of doxycycline under serum-induced hyphal growth conditions. Cells under such conditions grew as hyphae ( Supplemmentary Fig. S6), suggesting that either DDK control of the yeast-to-hypha transition is not via the serum-induced signaling pathway, but instead through other pathways, such as DNA replication stress, or that the blockage of serum-induced filamentous growth could be bypassed by other factors. It is equally possible that DDK control of the yeast-to-hypha transition constitutes a novel pathway.

Conclusion
In this paper, we describe the characterization of the CaCDC7 gene. To our surprise, we found that C. albicans CDC7 plays a role as a negative regulator of the YTH transition. Additionally, kinase activity is required for the function of CaCdc7 and the presence of other kinases for the function of CaCdc7. Moreover, the function of CaCdc7 is dependent on CaDbf4 for suppression of the hyphal mode of growth. To the best of our knowledge, no known role for Dbf4-dependent Cdc7 homologs across the evolutionary spectrum relevant to morphogenesis has been identified. The uncovering of CaCdc7 function in morphogenesis may be of evolutionary significance in that essential elements in the cell cycle have evolved in the direction of morphogenesis for adapting the host-pathogen interaction. It would be crucial to elucidate the regulation of DDK in the network controlling morphogenesis. Elucidating the regulation of DDK in the system that controls morphogenesis should lend insight into understanding why DDK is involved in morphogenesis.

Methods
General manipulation, media and growth conditions. The E. coli strain DH5α was used for routine manipulation of the plasmids, and all C. albicans strains (Table 1) were derived from the auxotrophic strain BWP17 (arg4/arg4 his1/his1 ura3/ura3) 51 . The wild-type SC5314 strain 52 was also used. The media usage and routine growth conditions of the strains of E. coli and C. albicans were as previously described 53 . E. coli strain DH5α was transformed with plasmid DNA by CaCl 2 as described 54 or by electroporation 55 . Candida albicans strains were transformed by the LiAc/PEG/ssDNA method 56 or electroporation 57 .
Strain construction. The nucleotide sequence of C. albicans was obtained from the Candida Genome Database (http://www.candidagenome.org/). The CaCDC7 gene including regions flanking the protein coding sequence was PCR-generated with the primers CaCDC7-xba-F and CaCDC7-xba-R (Table 2) and genomic DNA extracted from C. albicans strain BWP17. The CaCDC7 gene was cloned into the plasmid vector pBluescript II (+ ) to obtain the plasmid pBII-CaCDC7. CaCDC7 was deleted in the C. albicans auxotrophic strain BWP17 with the mini-Ura-blaster cassette dpl200-URA3-dpl200 derived from pDDB57 39 . Briefly, the mini-Ura-blaster cassette flanked by 60 bp of upstream and downstream homology to the CaCDC7 open reading frame was amplified by PCR with the template pBII-CaCDC7 and a pair of primers, CaCDC7-Spe-F and CaCDC7-Spe-R ( Table 2). The mini-Ura-blaster cassette flanked by the short homology regions of CaCDC7 was then transformed into BWP17 cells and the uridine prototrophic strains (Ura + ) CaCDC7+ /U3− (Table 1) containing the heterozygous deletion of CaCDC7 were selected for. To allow CaCDC7 expression under the control of MET3p, a partial CaCDC7 Figure 6. Specific interaction between CaCDC7 and CaDBF4 verified by a yeast two-hybrid assay.
(A) Verification of diploid S. cerevisiae cells carrying the plasmids required for the test. The plasmids pACT2 and pACT2-CaDBF4 (capable of expressing Gal4 activation domain fused with CaDbf4) were transformed into AH109, from which AD and AD CaDBF4 were obtained, respectively. The plasmids pGBKT7 and pGBKT7-CaCDC7 (capable of expressing Gal4 DNA biding domain fused with CaCdc7) were transformed into Y187, from which BD and BD CaCDC7 were obtained, respectively. AH109 and Y187 derivatives were mated to obtain diploid S. cerevisiae cells of AD/BD, AD CaDBF4/BD, AD/BD CaCDC7, and AD CaDBF4/BD CaCDC7. Ten-fold serially diluted S. cerevisiae diploid cells (10 4 ∼ 10 1 ) were grown on semi-solid agar plates with selective minimum media lacking leucine and tryptophan (SD− LW). (B) Verification of the interaction between CaCdc7 and CaDbf4. Ten-fold serially diluted S. cerevisiae diploid cells (10 4 ∼ 10 1 ), the same as in (A) were grown on semi-solid agar plates with selective minimum media lacking leucine, tryptophan, and histidine, supplemented with 2.5 mM 3-AT, the antagonist of the HIS3 gene product (SD− LWH+ 2.5 mM 3-AT). (C) Strong interaction occurs between CaCdc7 and CaDbf4. Serially diluted S. cerevisiae diploid cells of AD CaDBF4/BD CaCDC7 were grown on semi-solid agar plates with selective media lacking leucine, tryptophan, and histidine but with the indicated concentration of 3-amino-triazole (3-AT). The cells were able to proliferate normally at the concentration of 20 mM due to a relatively strong interaction between CaCdc7 and CaDbf4.
Scientific RepoRts | 6:33716 | DOI: 10.1038/srep33716 two pairs of oligonucleotides, CaDBF4-XmaI-F and CaDBF4-XhoI-R (Table 2) and CaCDC7-NcoI(hybrid)-F and CaCDC7-PstI(hybrid)-R (Table 2), respectively. The plasmids pGBKT7-CaCDC7 and pACT2-CaDBF4 were introduced into the haploid S. cerevisiae strain Y187 59 and AH109, a derivative of strain PJ69-2A 60 resulting from the introduction of the lacZ reporter gene into PJ69-2A (Table 1), respectively, with opposite mating types and with the reporter systems HIS3, ADE2, and LacZ. These strains were used to determine the activation of the system and the interaction between CaCdc7 and CaDbf4 upon fusion to become diploid.
Isolation of genomic DNA and Southern blotting. Genomic DNA from the C. albicans strains was isolated by the MasterPure TM Yeast DNA Purification Kit (EPICENTRE). Southern blotting was performed according to standard protocols with the aid of the Rapid Downward Transfer System (TURBOBLOTTER TM ) using 10 μ g of the restriction enzyme-digested genomic DNA. The probe was generated by the PCR DIG probe synthesis kit (Roche) with a genomic DNA template extracted from BWP17 and a pair of primers, CaCDC7-Probe-F and CaCDC7-Probe-R ( Table 2). The DNA on the blot was hybridized with the probe using DIG Easy Hyb (Roche).
To reveal the gene deletion, the DIG Luminescent Detection Kit (Roche) was used after hybridization, and the blot was exposed to X-ray film for up to 24 h as appropriate.
RT-PCR analysis. Cells were grown to mid-log phase and total RNA was extracted using the MasterPure TM Yeast RNA Purification Kit (EPICENTRE) following the manufacturer's instructions. Then, 5 μ g of total RNA was used to generate cDNA by using the SuperScript III Reverse Transcriptase Kit (Invitrogen) following the manufacturer's instructions. The cDNA was then subjected to PCR with a pair of CaCDC7-specific primers, CaCDC7-internal-F-2 and CaCDC7-BspEI-R (Table 2), targeted downstream of the coding sequence. A 366 bp product was generated.
Immunoblot analysis. The total protein was extracted from cultured cells as previously described 61 . The protein was partially purified from cells bearing the p6HF-ACT1p plasmid with the open reading frame of the gene integrated at RP10 capable of generating a tagged (6 × His and FLAG) protein using Ni 2+ -NTA-agarose beads (Qiagen Inc.) essentially as previously described 61 . Precipitated proteins were resolved by 10% SDS-PAGE and transferred electrophoretically to PVDF membranes (PerkinElmer, Boston, USA) and probed with a polyclonal antibody to FLAG (Sigma) in a 1:2000 dilution and visualized using the SuperSignal West Pico Chemiluminescent Substrate Kit (PIERCE). The detected proteins were recorded with the Luminescent Image Analyzer (FUJIFILM LAS-1000) and analyzed by ImageGauge 3.46 and L Process v 1.96 (FUJIFILM).
In vitro kinase assay. The in vitro kinase assays using CaCdc7-6xHisFlAG and purified bovine histone H1 (Upstate) as substrates were performed using a non-radioactive assay essentially as previously described 62 . Briefly, the Ni 2+ -NTA-agarose bead (Qiagen Inc.)-purified CaCdc7 was washed three times with kinase reaction buffer [25 mM Tris pH 7.5, 5 mM β -glycerophosphate, 2 mM DTT, 0.1 mM Na 3 VO 4 , 10 mM MgCl 2 and a serine/threonine phosphatase inhibitor cocktail (Calbiochem)] and used in the kinase reaction with 5 μ g histone H1 as substrate in the presence of 200 μ M ATP in a 20 μ l kinase assay buffer. The kinase reaction was incubated at 30 °C for 45 minutes, and the end products were resolved by 12% SDS-PAGE and detected by immunoblotting using anti-Histone H1 (H11-4, Sigma; 1:500) and anti-phospho-Histone H1 (12D11, Millipore; 1:1000 monoclonal antibodies) according to the manufacturer's instructions.
Chemical treatment. Phosphatase treatment was performed with calf intestinal alkaline phosphatase (CIP) (NEB). Briefly, the Ni 2+ -NTA-agarose bead (Qiagen Inc.)-purified CaCdc7 was washed three times with CIP buffer (1XNE Buffer 3) and resuspended in 20 μ l CIP buffer before the addition of 20 units of CIP and incubation at 37 °C for the required period of time. The CIP was inactivated by heating to 75 °C for 10 minutes in the presence of 5 mM EDTA. Irreversible protein kinase inhibitor treatment was performed with 5′ -fluorosulfonylbenzoyl-5′ -adenosine (FSBA), essentially as previously described 63 . Briefly, the Ni 2+ -NTA-agarose bead-purified CaCdc7 was subjected to three successive treatments by 1 mM FSBA (Sigma) at 30 °C for 15 min in kinase buffer without DTT. The product was then washed three times with kinase buffer to eliminate the FSBA before performing the in vitro kinase assay. Hydroxyurea (HU) was used to block DNA replication, which is known to attenuate Cdc7 kinase activity 15 . Cells in culture were treated with 0.1 M HU before the Ni 2+ -NTA-agarose bead purification of CaCdc7.

Cellular image observation and recording. Cells in liquid culture were visualized and recorded with a
Nikon 50i microscope at 400x magnification. Colonies were photographed with a MEIJI stereoscopic microscope EMZ5 at 40x magnification.