Single-Cell RNA Sequencing Unravels Heterogeneity of the Stromal Niche in Cutaneous Melanoma Heterogeneous Spheroids.

Heterogeneous spheroids have recently acquired a prominent position in melanoma research because they incorporate microenvironmental cues relevant for melanoma. In this study, we focused on the analysis of microenvironmental factors introduced in melanoma heterogeneous spheroids by different dermal fibroblasts. We aimed to map the fibroblast diversity resulting from previously acquired damage caused by exposure to extrinsic and intrinsic stimuli. To construct heterogeneous melanoma spheroids, we used normal dermal fibroblasts from the sun-protected skin of a juvenile donor. We compared them to the fibroblasts from the sun-exposed photodamaged skin of an adult donor. Further, we analysed the spheroids by single-cell RNA sequencing. To validate transcriptional data, we also compared the immunohistochemical analysis of heterogeneous spheroids to melanoma biopsies. We have distinguished three functional clusters in primary human fibroblasts from melanoma spheroids. These clusters differed in the expression of (a) extracellular matrix-related genes, (b) pro-inflammatory factors, and (c) TGFß signalling superfamily. We observed a broader deregulation of gene transcription in previously photodamaged cells. We have confirmed that pro-inflammatory cytokine IL-6 significantly enhances melanoma invasion to the extracellular matrix in our model. This supports the opinion that the aspects of ageing are essential for reliable melanoma 3D modelling in vitro.

Analysis of HPV-Positive and HPV-Negative Head and Neck Squamous Cell Carcinomas and Paired Normal Mucosae Reveals Cyclin D1 Deregulation and Compensatory Effect of Cyclin D2.

Aberrant regulation of the cell cycle is a typical feature of all forms of cancer. In head and neck squamous cell carcinoma (HNSCC), it is often associated with the overexpression of cyclin D1 (CCND1). However, it remains unclear how CCND1 expression changes between tumor and normal tissues and whether human papillomavirus (HPV) affects differential CCND1 expression. Here, we evaluated the expression of D-type cyclins in a cohort of 94 HNSCC patients of which 82 were subjected to whole genome expression profiling of primary tumors and paired normal mucosa. Comparative analysis of paired samples showed that CCND1 was upregulated in 18% of HNSCC tumors. Counterintuitively, CCND1 was downregulated in 23% of carcinomas, more frequently in HPV-positive samples. There was no correlation between the change in D-type cyclin expression and patient survival. Intriguingly, among the tumors with downregulated CCND1, one-third showed an increase in cyclin D2 (CCND2) expression. On the other hand, one-third of tumors with upregulated CCND1 showed a decrease in CCND2. Collectively, we have shown that CCND1 was frequently downregulated in HNSCC tumors. Furthermore, regardless of the HPV status, our data suggested that a change in CCND1 expression was alleviated by a compensatory change in CCND2 expression.

Genome Sequence of Rhodococcus erythropolis Strain CCM2595, a Phenol Derivative-Degrading Bacterium.

We announce the completion of the genome sequence of a phenol derivative-degrading bacterium, Rhodococcus erythropolis strain CCM2595. This bacterium is interesting in the context of bioremediation for its capability to degrade phenol, catechol, resorcinol, hydroxybenzoate, hydroquinone, p-chlorophenol, p-nitrophenol, pyrimidines, and sterols.

Characterization of pbt genes conferring increased Pb2+ and Cd2+ tolerance upon Achromobacter xylosoxidans A8.

The cluster of pbtTFYRABC genes is carried by plasmid pA81. Its elimination from Achromobacter xylosoxidans A8 resulted in increased sensitivity towards Pb(2+) and Cd(2+). Predicted pbtTRABC products share strong similarities with Pb(2+) uptake transporter PbrT, transcriptional regulator PbrR, metal efflux P1-ATPases PbrA and CadA, undecaprenyl pyrophosphatase PbrB and its signal peptidase PbrC from Cupriavidus metallidurans CH34. Expression of pbtABC or pbtA in a metal-sensitive Escherichia coli GG48 rendered the strain Pb(2+)-, Cd(2+)- and Zn(2+)-tolerant and caused decreased accumulation of the metal ions. Accumulation of Pb(2+), but not of Cd(2+) or Zn(2+), was promoted in E. coli expressing pbtT. Additional genes of the pbt cluster are pbtF and pbtY, which encode the cation diffusion facilitator (CDF)-like transporter and a putative fatty acid hydroxylase of unknown function, respectively. Expression of pbtF did not confer increased metal tolerance upon E. coli GG48, although the protein showed measurable Pb(2+)-efflux activity. Unlike the pbtT promoter, promoters of pbtABC, pbtF and pbtY contain features characteristic of promoters controlled by metal-responsive transcriptional regulators of the MerR family. Upregulation of pbtABC, pbtF and pbtY upon Pb(2+), Cd(2+) and Zn(2+) exposure was confirmed in wild-type Achromobacter xylosoxidans A8. Gel shift assays proved binding of purified PbtR to the respective promoters.

New insight into transcription of human endogenous retroviral elements.

It is generally assumed that human endogenous retroviral elements (HERVs) belong to the class of genomic repetitive nucleotide sequences often called 'junk DNA'. These elements were categorized to families, and members of some of these families (e.g. HERV-H, HERV-W and HERV-K) were shown to be transcribed. These transcriptions were associated with several severe diseases such as mental disorders, AIDS, autoimmune diseases and cancer. In this review we discuss several bioinformatics strategies for genome-wide scan of HERVs transcription using high-throughput RNA sequencing on several platforms. We show that many more HERVs than previously described are transcribed to various levels and we discuss possible implications of these transcriptions.

Diversity, phylogeny and expression patterns of Pou and Six homeodomain transcription factors in hydrozoan jellyfish Craspedacusta sowerbyi.

Formation of all metazoan bodies is controlled by a group of selector genes including homeobox genes, highly conserved across the entire animal kingdom. The homeobox genes from Pou and Six classes are key members of the regulation cascades determining development of sensory organs, nervous system, gonads and muscles. Besides using common bilaterian models, more attention has recently been targeted at the identification and characterization of these genes within the basal metazoan phyla. Cnidaria as a diploblastic sister group to bilateria with simple and yet specialized organs are suitable models for studies on the sensory organ origin and the associated role of homeobox genes. In this work, Pou and Six homeobox genes, together with a broad range of other sensory-specific transcription factors, were identified in the transcriptome of hydrozoan jellyfish Craspedacusta sowerbyi. Phylogenetic analyses of Pou and Six proteins revealed cnidarian-specific sequence motifs and contributed to the classification of individual factors. The majority of the Craspedacusta sowerbyi Pou and Six homeobox genes are predominantly expressed in statocysts, manubrium and nerve ring, the tissues with sensory and nervous activities. The described diversity and expression patterns of Pou and Six factors in hydrozoan jellyfish highlight their evolutionarily conserved functions. This study extends the knowledge of the cnidarian genome complexity and shows that the transcriptome of hydrozoan jellyfish is generally rich in homeodomain transcription factors employed in the regulation of sensory and nervous functions.

Complete genome sequence of the haloaromatic acid-degrading bacterium Achromobacter xylosoxidans A8.

Achromobacter xylosoxidans strain A8 was isolated from soil contaminated with polychlorinated biphenyls. It can use 2-chlorobenzoate and 2,5-dichlorobenzoate as sole sources of carbon and energy. This property makes it a good starting microorganism for further development toward a bioremediation tool. The genome of A. xylosoxidans consists of a 7-Mb chromosome and two large plasmids (98 kb and 248 kb). Besides genes for the utilization of xenobiotic organic substrates, it contains genes associated with pathogenesis, toxin production, and resistance. Here, we report the complete genome sequence.

Complete genome sequence of the photosynthetic purple nonsulfur bacterium Rhodobacter capsulatus SB 1003.

Rhodobacter capsulatus SB 1003 belongs to the group of purple nonsulfur bacteria. Its genome consists of a 3.7-Mb chromosome and a 133-kb plasmid. The genome encodes genes for photosynthesis, nitrogen fixation, utilization of xenobiotic organic substrates, and synthesis of polyhydroxyalkanoates. These features made it a favorite research tool for studying these processes. Here we report its complete genome sequence.

Loss of the response regulator CtrA causes pleiotropic effects on gene expression but does not affect growth phase regulation in Rhodobacter capsulatus.

The purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus has been extensively studied for its metabolic versatility as well as for production of a gene transfer agent called RcGTA. Production of RcGTA is highest in the stationary phase of growth and requires the response regulator protein CtrA. The CtrA protein in Caulobacter crescentus has been thoroughly studied for its role as an essential, master regulator of the cell cycle. Although the CtrA protein in R. capsulatus shares a high degree of sequence similarity with the C. crescentus protein, it is nonessential and clearly plays a different role in this bacterium. We have used transcriptomic and proteomic analyses of wild-type and ctrA mutant cultures to identify the genes dysregulated by the loss of CtrA in R. capsulatus. We have also characterized gene expression differences between the logarithmic and stationary phases of growth. Loss of CtrA has pleiotropic effects, with dysregulation of expression of approximately 6% of genes in the R. capsulatus genome. This includes all flagellar motility genes and a number of other putative regulatory proteins but does not appear to include any genes involved in the cell cycle. Quantitative proteomic data supported 88% of the CtrA transcriptome results. Phylogenetic analysis of CtrA sequences supports the hypothesis of an ancestral ctrA gene within the alphaproteobacteria, with subsequent diversification of function in the major alphaproteobacterial lineages.

Biphenyl-metabolizing bacteria in the rhizosphere of horseradish and bulk soil contaminated by polychlorinated biphenyls as revealed by stable isotope probing.

DNA-based stable isotope probing in combination with terminal restriction fragment length polymorphism was used in order to identify members of the microbial community that metabolize biphenyl in the rhizosphere of horseradish (Armoracia rusticana) cultivated in soil contaminated with polychlorinated biphenyls (PCBs) compared to members of the microbial community in initial, uncultivated bulk soil. On the basis of early and recurrent detection of their 16S rRNA genes in clone libraries constructed from [(13)C]DNA, Hydrogenophaga spp. appeared to dominate biphenyl catabolism in the horseradish rhizosphere soil, whereas Paenibacillus spp. were the predominant biphenyl-utilizing bacteria in the initial bulk soil. Other bacteria found to derive carbon from biphenyl in this nutrient-amended microcosm-based study belonged mostly to the class Betaproteobacteria and were identified as Achromobacter spp., Variovorax spp., Methylovorus spp., or Methylophilus spp. Some bacteria that were unclassified at the genus level were also detected, and these bacteria may be members of undescribed genera. The deduced amino acid sequences of the biphenyl dioxygenase alpha subunits (BphA) from bacteria that incorporated [(13)C]into DNA in 3-day incubations of the soils with [(13)C]biphenyl are almost identical to that of Pseudomonas alcaligenes B-357. This suggests that the spectrum of the PCB congeners that can be degraded by these enzymes may be similar to that of strain B-357. These results demonstrate that altering the soil environment can result in the participation of different bacteria in the metabolism of biphenyl.

Assembly of the cnidarian camera-type eye from vertebrate-like components.

Animal eyes are morphologically diverse. Their assembly, however, always relies on the same basic principle, i.e., photoreceptors located in the vicinity of dark shielding pigment. Cnidaria as the likely sister group to the Bilateria are the earliest branching phylum with a well developed visual system. Here, we show that camera-type eyes of the cubozoan jellyfish, Tripedalia cystophora, use genetic building blocks typical of vertebrate eyes, namely, a ciliary phototransduction cascade and melanogenic pathway. Our findings indicative of parallelism provide an insight into eye evolution. Combined, the available data favor the possibility that vertebrate and cubozoan eyes arose by independent recruitment of orthologous genes during evolution.

Sulfoacetate released during the assimilation of taurine-nitrogen by Neptuniibacter caesariensis: purification of sulfoacetaldehyde dehydrogenase.

Taurine (2-aminoethanesulfonate) is a widespread natural product whose nitrogen moiety was recently shown to be assimilated by bacteria, usually with excretion of an organosulfonate via undefined novel pathways; other data involve transcriptional regulator TauR in taurine metabolism. A screen of genome sequences for TauR with the BLAST algorithm allowed the hypothesis that the marine gammaproteobacterium Neptuniibacter caesariensis MED92 would inducibly assimilate taurine-nitrogen and excrete sulfoacetate. The pathway involved an ABC transporter (TauABC), taurine:pyruvate aminotransferase (Tpa), a novel sulfoacetaldehyde dehydrogenase (SafD) and exporter(s) of sulfoacetate (SafE) (DUF81). Ten candidate genes in two clusters involved three sets of paralogues (for TauR, Tpa and SafE). Inducible Tpa and SafD were detected in cell extracts. SafD was purified 600-fold to homogeneity in two steps. The monomer had a molecular mass of 50 kDa (SDS-PAGE); data from gel filtration chromatography indicated a tetrameric native protein. SafD was specific for sulfoacetaldehyde with a K (m)-value of 0.12 mM. The N-terminal amino acid sequence of SafD confirmed the identity of the safD gene. The eight pathway genes were transcribed inducibly, which indicated expression of the whole hypothetical pathway. We presume that this pathway is one source of sulfoacetate in nature, where this compound is dissimilated by many bacteria.

Nucleotide sequence, organization and characterization of the (halo)aromatic acid catabolic plasmid pA81 from Achromobacter xylosoxidans A8.

The complete 98,192bp nucleotide sequence was determined for plasmid pA81, which is harbored by the haloaromatic acid-degrading bacterium Achromobacter xylosoxidans A8. The majority of the 103 open reading frames identified on pA81 could be categorized as either "backbone" genes, genes encoding (halo)aromatic compound degradation, or heavy metal resistance determinants. The backbone genes controlled conjugative transfer, replication and plasmid stability, and were well conserved with other IncP1-beta plasmids. Genes encoding (halo)aromatic degradation were clustered within a type I transposon, TnAxI, and included two ring-hydroxylating oxygenases (ortho-halobenzoate oxygenase, salicylate 5-hydroxylase) and a modified ortho-cleavage pathway for chlorocatechol degradation. The cluster of heavy metal resistance determinants was contained within a Type II transposon TnAxII, and included a predicted P-type ATPase and cation diffusion facilitator system. Genes identical to those carried by TnAxI and TnAxII were identified on other biodegradative/resistance plasmids and genomic islands, indicating an evolutionary relationship between these elements. Collectively, these insights further our understanding of how mobile elements, and interactions between mobile elements affect the fate of organic and inorganic toxicants in the environment.

Cubozoan crystallins: evidence for convergent evolution of pax regulatory sequences.

Cnidaria is the earliest-branching metazoan phylum containing a well-developed, lens-containing visual system located on specialized sensory structures called rhopalia. Each rhopalium in a cubozoan jellyfish Tripedalia cystophora has a large and a small complex, camera-type eye with a cellular lens containing distinct families of crystallins. Here, we have characterized J2-crystallin and its gene in T. cystophora. The J2-crystallin gene is composed of a single exon and encodes a 157-amino acid cytoplasmic protein with no apparent homology to known proteins from other species. The non-lens expression of J2-crystallin suggests nonoptical as well as crystallin functions consistent with the gene-sharing strategy that has been used during evolution of lens crystallins in other invertebrates and vertebrates. Although nonfunctional in transfected mammalian lens cells, the J2-crystallin promoter is activated by the jellyfish paired domain transcription factor PaxB in co-transfection tests via binding to three paired domain sites. PaxB paired domain-binding sites were also identified in the PaxB-regulated promoters of the J1A- and J1B-crystallin genes, which are not homologous to the J2-crystallin gene. Taken together with previous studies on the regulation of the diverse crystallin genes, the present report strongly supports the idea that crystallin recruitment of multifunctional proteins was driven by convergent changes involving Pax (as well as other transcription factors) in the promoters of nonhomologous genes within and between species as well as within gene families.

Retroposition of processed pseudogenes: the impact of RNA stability and translational control.

Human processed pseudogenes are copies of cellular RNAs reverse transcribed and inserted into the nuclear genome by the enzymatic machinery of L1 (LINE1) non-LTR retrotransposons. Although it is generally accepted that germline expression is crucial for the heritable retroposition of cellular mRNAs, little is known about the influences of RNA stability, mRNA quality control and compartmentalization of translation on the retroposition of processed pseudogenes. We found that frequently retroposed human mRNAs are derived from stable transcripts with translation-competent functional reading frames that are resistant to nonsense-mediated RNA decay. They are preferentially translated on free cytoplasmic ribosomes and encode soluble proteins. Our results indicate that interactions between mRNAs and L1 proteins seem to occur at free cytoplasmic ribosomes.

An approach towards experimental cDNA sequence determination of predicted genes: an example from Arabidopsis U3-55k homologues.

Experimental cDNA sequence determinations lag behind in silico gene structure predictions in some recently sequenced genomes. This may be due in part to low transcript abundance and/or the severely spatio-temporarily restricted expression pattern of some genes. Here we characterize the predicted repressed gene of Arabidopsis thaliana (At4g21130) that encodes a homologue of the Arabidopsis U3-55K-like protein (At4g05410) and of the U3-55K (RNU3IP2, Rrp9p) proteins from other eukaryotes. In man and yeast, U3-55K is involved in the processing of the pre-ribosomal RNA. Here we show that treatment with inhibitors of histone deacetylases (trichostatin A, sodium butyrate) or DNA methyltransferases (5-aza-2'-deoxycytidine) induces a low but distinct level of mRNA from the repressed Arabidopsis At4g21130 locus, which can be detected by RT-PCR amplification. Direct sequencing of PCR products reveals the open reading frame that differs, in part, from the hypothetical one and encodes a seven-WD-repeat protein highly conserved when compared to U3-55K proteins from various eukaryotic species. This suggests the conservation of its function. The described approach may help to determine the nucleotide sequences of transcripts from predicted genes with a low level of expression.

Global transcriptome analysis of the C57BL/6J mouse testis by SAGE: evidence for nonrandom gene order.

BACKGROUND: We generated the gene expression profile of the total testis from the adult C57BL/6J male mice using serial analysis of gene expression (SAGE). Two high-quality SAGE libraries containing a total of 76 854 tags were constructed. An extensive bioinformatic analysis and comparison of SAGE transcriptomes of the total testis, testicular somatic cells and other mouse tissues was performed and the theory of male-biased gene accumulation on the X chromosome was tested. RESULTS: We sorted out 829 genes predominantly expressed from the germinal part and 944 genes from the somatic part of the testis. The genes preferentially and specifically expressed in total testis and testicular somatic cells were identified by comparing the testis SAGE transcriptomes to the available transcriptomes of seven non-testis tissues. We uncovered chromosomal clusters of adjacent genes with preferential expression in total testis and testicular somatic cells by a genome-wide search and found that the clusters encompassed a significantly higher number of genes than expected by chance. We observed a significant 3.2-fold enrichment of the proportion of X-linked genes specific for testicular somatic cells, while the proportions of X-linked genes specific for total testis and for other tissues were comparable. In contrast to the tissue-specific genes, an under-representation of X-linked genes in the total testis transcriptome but not in the transcriptomes of testicular somatic cells and other tissues was detected. CONCLUSION: Our results provide new evidence in favor of the theory of male-biased genes accumulation on the X chromosome in testicular somatic cells and indicate the opposite action of the meiotic X-inactivation in testicular germ cells.

TrnL- trnF intergenic spacer and trnL intron define major clades within Luzula and Juncus (Juncaceae): importance of structural mutations.

Seven hundred fifty-two to one thousand ninety-seven base pairs of the trnL intron and trnL- trnF intergenic spacer of the chloroplast DNA of 55 Juncaceae taxa (Juncus, Luzula, Rostkovia, and Oxychloë) was sequenced. Seventeen structural mutations (13 indels marked A to M, 3 parts of the trnF pseudogene, and insertion "o" within a pseudogene) within the chloroplast trnL- trnF region were examined as possible indicators for phylogenetic relationships in Juncaceae. Juncus trifidus (section Steirochloa) was clearly separated from the other taxa by two large (>80 bp) indels. The "Southern Hemisphere clade" was strongly supported by a unique insertion (334 bp) in the trnL intron. The monophyly of Luzula was supported by three small (<10 bp) indels in the trnL-F spacer. They were found in all 22 examined members that represent the taxonomic and geographical diversity of the genus Luzula. A tandemly duplicated tRNA pseudogene was found in the Juncus subgenus Juncus species and is supported by four small unique indels too. The acceptor stem and D-domain-encoding regions are separated by a unique 8-bp insertion. The T-domain and acceptor stem-encoding regions were not found in the pseudogene repeats. Only the Juncus sections Ozophyllum and Iridifolii contain the 5' acceptor stem, D-domain, and anticodon domain of the tRNAF encoding DNA. The structural mutations in the trnL intron and the trnL- trnF intergenic spacer are useful for phylogenetic reconstruction in the Juncaceae.

WAViS server for handling, visualization and presentation of multiple alignments of nucleotide or amino acids sequences.

Web Alignment Visualization Server contains a set of web-tools designed for quick generation of publication-quality color figures of multiple alignments of nucleotide or amino acids sequences. It can be used for identification of conserved regions and gaps within many sequences using only common web browsers. The server is accessible at http://wavis.img.cas.cz.