Prevalence and Intra-Family Phylogenetic Divergence of Burkholderiaceae-Related Endobacteria Associated with Species of Mortierella.

Endofungal bacteria are widespread within the phylum Mucoromycota, and these include Burkholderiaceae-related endobacteria (BRE). However. the prevalence of BRE in Mortierellomycotinan fungi and their phylogenetic divergence remain unclear. Therefore, we examined the prevalence of BRE in diverse species of Mortierella. We surveyed 238 isolates of Mortierella spp. mainly obtained in Japan that were phylogenetically classified into 59 species. BRE were found in 53 isolates consisting of 22 species of Mortierella. Among them, 20 species of Mortierella were newly reported as the fungal hosts of BRE. BRE in a Glomeribacter-illycoavidus Glade in the family Burkholderiaceae were separated phylogenetically into three groups. These groups consisted of a group containing Mycoavidus cysteinexigens, which is known to be associated with M. elongata, and two other newly distinguishable groups. Our results demonstrated that BRE were harbored by many species of Mortierella and those that associated with isolates of Mortierella spp. were more phylogenetically divergent than previously reported

Comparison of lung cancer cell lines representing four histopathological subtypes with gene expression profiling using quantitative real-time PCR

<p>Abstract</p> <p>Background</p> <p>Lung cancers are the most common type of human malignancy and are intractable. Lung cancers are generally classified into four histopathological subtypes: adenocarcinoma (AD), squamous cell carcinoma (SQ), large cell carcinoma (LC), and small cell carcinoma (SC). Molecular biological characterization of these subtypes has been performed mainly using DNA microarrays. In this study, we compared the gene expression profiles of these four subtypes using twelve human lung cancer cell lines and the more reliable quantitative real-time PCR (qPCR).</p> <p>Results</p> <p>We selected 100 genes from public DNA microarray data and examined them by DNA microarray analysis in eight test cell lines (A549, ABC-1, EBC-1, LK-2, LU65, LU99, STC 1, RERF-LC-MA) and a normal control lung cell line (MRC-9). From this, we extracted 19 candidate genes. We quantified the expression of the 19 genes and a housekeeping gene, <it>GAPDH</it>, with qPCR, using the same eight cell lines plus four additional validation lung cancer cell lines (RERF-LC-MS, LC-1/sq, 86-2, and MS-1-L). Finally, we characterized the four subtypes of lung cancer cell lines using principal component analysis (PCA) of gene expression profiling for 12 of the 19 genes (<it>AMY2A</it>, <it>CDH1</it>, <it>FOXG1</it>, <it>IGSF3</it>, <it>ISL1</it>, <it>MALL</it>, <it>PLAU</it>, <it>RAB25</it>, <it>S100P</it>, <it>SLCO4A1</it>, <it>STMN1</it>, and <it>TGM2</it>). The combined PCA and gene pathway analyses suggested that these genes were related to cell adhesion, growth, and invasion. <it>S100P </it>in AD cells and <it>CDH1 </it>in AD and SQ cells were identified as candidate markers of these lung cancer subtypes based on their upregulation and the results of PCA analysis. Immunohistochemistry for S100P and RAB25 was closely correlated to gene expression.</p> <p>Conclusions</p> <p>These results show that the four subtypes, represented by 12 lung cancer cell lines, were well characterized using qPCR and PCA for the 12 genes examined. Certain genes, in particular <it>S100P </it>and <it>CDH1</it>, may be especially important for distinguishing the different subtypes. Our results confirm that qPCR and PCA analysis provide a useful tool for characterizing cancer cell subtypes, and we discuss the possible clinical applications of this approach.</p

Detection and isolation of a new member of Burkholderiaceae-related endofungal bacteria from Saksenaea boninensis sp. nov., a new thermotolerant fungus in Mucorales

Abstract Thermotolerance in Mucorales (Mucoromycotina) is one of the factors to be opportunistic pathogens, causing mucormycosis. Among thermotolerant mucoralean fungi, Burkholderiaceae-related endobacteria (BRE) are rarely found and the known range of hosts is limited to Rhizopus spp. The phylogenetic divergence of BRE has recently expanded in other fungal groups such as Mortierellaceae spp. (Mortierellomycotina); however, it remains unexplored in Mucorales. Here, we found a thermotolerant mucoralean fungus obtained from a litter sample collected from Haha-jima Island in the Ogasawara (Bonin) Islands, Japan. The fungus was morphologically, phylogenetically, and physiologically characterized and proposed as a new species, Saksenaea boninensis sp. nov. Besides the fungal taxonomy, we also found the presence of BRE in isolates of this species by diagnostic PCR amplification of the 16S rRNA gene from mycelia, fluorescence microscopic observations, and isolation of the bacterium in pure culture. Phylogenetic analysis of the 16S rRNA gene of BRE revealed that it is distinct from all known BRE. The discovery of a culturable BRE lineage in the genus Saksenaea will add new insight into the evolutional origin of mucoralean fungus-BRE associations and emphasize the need to pay more attention to endofungal bacteria potentially associated with isolates of thermotolerant mucoralean fungi causing mucormycosis

Multiple sequence alignments: Detection and isolation of a new member of Burkholderiaceae‑related endofungal bacteria from Saksenaea boninensis sp. nov., a new thermotolerant fungus in Mucorales

&lt;p&gt;&lt;strong&gt;Methods:&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;Nucleotide sequences were aligned independently for each region using MAFFT v7.212 (Katoh and Standley, 2013). The obtained alignment blocks were subject to Gblocks 0.91b (Castresana, 2000) to remove poorly aligned positions with the relaxed selection setting described in Talavera &amp; Castresana (2007) using the following parameters (-t = d -b2 = 9 -b3 = 10 -b4 = 5 -b5 = h). After automatically removing gaps, the alignment blocks were viewed using MEGA 6.06 software (Tamura et al., 2013) and poorly aligned positions at either end of the alignments were removed manually. Pairwise distances of the nucleotide sequences (ITS2, ITS1-5.8S-ITS2, LSU, and tef1) of the ex-type strains of seven &lt;i&gt;Saksenaea&lt;/i&gt; spp. and the representative isolate &lt;i&gt;S. boninensis&lt;/i&gt; Sak4 were calculated by MEGA 6.06 software (Tamura et al. 2013). Multiple sequence alignment of 16S rRNA gene of the family &lt;i&gt;Burkholderiaceae&lt;/i&gt; was prepared for the phylogeny of a bacterial endosymbiont. Multiple sequence alignments of &nbsp;ITS, LSU, and tef1 genes of &lt;i&gt;Saksenaea&lt;/i&gt; spp. (Mucorales) were separately prepared for the phylogeny of a fungal host. Concatenated dataset of these genes were also prepared. All nucleotide sequences were retrieved from GenBank (See "Sequence_ID.csv" and taxon names of each alignment). &nbsp;&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Description of files:&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;A. Phylogeny of the family &lt;/strong&gt;&lt;i&gt;&lt;strong&gt;Burkholderiaceae&lt;/strong&gt;&lt;/i&gt;&lt;strong&gt; (Bacterial endosymbiont):&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;1. Burkholderiaceae_16S_RAW.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of 16S rRNA gene of the family &lt;i&gt;Burkholderiaceae&lt;/i&gt;.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;2. Burkholderiaceae_16S_aligned.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of 16S rRNA gene of the family &lt;i&gt;Burkholderiaceae&lt;/i&gt;.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;B. Phylogenies of &lt;/strong&gt;&lt;i&gt;&lt;strong&gt;Saksenaea&lt;/strong&gt;&lt;/i&gt;&lt;strong&gt; spp. (Fungal host):&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;1. Sequence_ID_v2.csv&lt;/p&gt;&lt;p&gt;Taxon names, accession numbers, and sequence ID for the concatenated multiple sequence alignment are listed.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;2. Saksenaea_ITS_RAW_v2.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of ITS1-5.8S-ITS2 region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. &nbsp;&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;3. Saksenaea_ITS_aligned_v2.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of ITS1-5.8S-ITS2 region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. Only used for ITS1-5.8S-ITS2 phylogeny.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;4. Saksenaea_LSU_RAW_v2.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of LSU gene region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. &nbsp;&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;5. Saksenaea_LSU_aligned_v2.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of LSU gene region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. Only used for LSU phylogeny.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;6. Saksenaea_tef1_RAW_v2.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of tef1 gene region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. &nbsp;&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;7. Saksenaea_tef1_aligned_v2.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of tef1 gene region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. Only used for tef1 phylogeny.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;&lt;Concatenated dataset 1 (ITS2, LSU, tef1)&gt;&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;8. Saksenaea_ITS2_for_concatenated_RAW_v2.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of ITS2 region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. used for preparation of a concatenated dataset 1.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;9. Saksenaea_ITS2_for_concatenated_aligned_v2.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of ITS2 region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. used for preparation of a concatenated dataset 1.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;10. Saksenaea_LSU_for_concatenated_RAW_v2.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of LSU gene region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. used for preparation of concatenated datasets 1 and 2.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;11. Saksenaea_LSU_for_concatenated_aligned_v2.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of LSU gene region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. used for preparation of concatenated datasets 1 and 2.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;12. Saksenaea_tef1_for_concatenated_RAW_v2.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of tef1 gene region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. used for preparation of concatenated datasets 1 and 2.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;13. Saksenaea_tef1_for_concatenated_aligned_v2.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of tef1 gene region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. used for preparation of concatenated datasets 1 and 2.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;14. Saksenaea_ITS2_LSU_tef1_concatenated_dataset1.fasta&lt;/p&gt;&lt;p&gt;Concatenated dataset of three multiple sequence alignments (9, 11, and 13). This concatenated dataset was used for the main phylogeny of &lt;i&gt;Saksenaea&lt;/i&gt; spp.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;&lt;Concatenated dataset 2 (ITS1-5.8S-ITS2, LSU, tef1)&gt;&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;15. Saksenaea_ITS_for_concatenated_RAW_v2.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of ITS1-5.8S-ITS2 region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. used for preparation of a concatenated dataset 2.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;16. Saksenaea_ITS_for_concatenated_aligned_v2.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of ITS1-5.8S-ITS2 region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. used for preparation of a concatenated dataset 2.&lt;/p&gt;&lt;p&gt;Blank sequences were inserted for five isolates of &lt;i&gt;Saksenaea longicolla&lt;/i&gt; after the alignment.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;17.Saksenaea_ITS_LSU_tef1_concatenated_dataset2.fasta&lt;/p&gt;&lt;p&gt;Concatenated dataset of three multiple sequence alignments (15, 11, and 13). This concatenated dataset was used for the main phylogeny of &lt;i&gt;Saksenaea&lt;/i&gt; spp.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;C. Pairwise distances of the ex-type strains of &lt;/strong&gt;&lt;i&gt;&lt;strong&gt;Saksenaea&lt;/strong&gt;&lt;/i&gt;&lt;strong&gt; spp.&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;1. Saksenaea_ITS_type_RAW.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of ITS1-5.8S-ITS2 region of the ex-type strains of &lt;i&gt;Saksenaea&lt;/i&gt; spp.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;2. Saksenaea_ITS2_type_aligned.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of ITS2 region of the ex-type strains of &lt;i&gt;Saksenaea&lt;/i&gt; spp.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;3.Saksenaea_ITS_type_aligned.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of ITS1-5.8S-ITS2 region of the ex-type strains of &lt;i&gt;Saksenaea&lt;/i&gt; spp. without &lt;i&gt;Saksenaea longicolla&lt;/i&gt;.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;4. Saksenaea_LSU_type_RAW.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of LSU gene region of the ex-type strains of &lt;i&gt;Saksenaea &lt;/i&gt;spp.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;5. Saksenaea_LSU_type_aligned.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of LSU gene region of the ex-type strains of &lt;i&gt;Saksenaea&lt;/i&gt; spp.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;6. Saksenaea_tef1_type_RAW.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of tef1 gene region of the ex-type strains of &lt;i&gt;Saksenaea&lt;/i&gt; spp.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;7. Saksenaea_tef1_type_aligned.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of tef1 gene region of the ex-type strains of &lt;i&gt;Saksenaea&lt;/i&gt; spp.&lt;/p&gt