Background:Hydroxylated biphenyls are currently recognized as secondary pollutants that are hazardous to animals and humans. Bacterial degradation is the most effective method for the degradation of hydroxylated biphenyls. Several strains capable of degrading polychlorinated biphenyls have been described, which also degrade hydroxylated biphenyls. Objectives:1) To study the biodegradative properties of the Rhodococcus opacus strain KT112-7 towards mono-hydroxylated biphenyls. 2) To analyze the genome of the Rhodococcus opacus strain KT112-7. 3) To identify the genetic basis for the unique biodegradative potential of the Rhodococcus opacus strain KT112-7. Methods:A genome analysis of the strain KT112-7 was conducted based on whole-genome sequencing using various programs and databases (Velvet, CONTIGuator, RAST, KEGG) for annotation and identification of protein-coding sequences. The strain KT112-7 was cultivated in a K1 mineral medium supplemented with mono-hydroxy biphenyls or mono-hydroxybenzoic acids as the carbon source. For the growth test mono-hydroxybiphenyls or mono-hydroxybenzoic acids were dosed at concentrations of 0.5 g/L and 1.0 g/L correspondently, and the bacterial growth was monitored by the optical density. For the biodegradative activity test, mono-hydroxybiphenyls were dosed at a concentration of 0.1 g/L in vials, inoculated with late exponential phase bacteria previously acclimated on biphenyl. Compound analysis was performed using GC-MS, HPLC, and spectrophotometry. Results:It was found that the genome of strain KT112-7 consists of a chromosome and 2 plasmids. Biphenyl degradation genes (bph genes) were identified on plasmid PRHWK1 and the chromosome, as well as hydroxybenzoic acid degradation genes on the chromosome. The strain KT112-7 was shown to degrade mono-hydroxylated biphenyls to basal metabolic compounds of the cell, with the highest destructive activity observed towards 3- and 4-hydroxylated biphenyls (98%). Discussion:Analysis of the translated sequences of the bph genes from strain KT112-7 revealed that the amino acid sequences of the bph operon, located on plasmid pRHWK1, exhibit high similarity to homologous enzymes of the "upper" pathway for biphenyl degradation in Rhodococcus jostii RHA1, whose bph genes are also plasmid-borne. The deduced amino acid sequences encoded by the bphA1A2B genes, located on the chromosome of strain KT112-7, show a high degree of similarity to enzymes from Rhodococcus strains that mediate naphthalene degradation. The genes of the "lower" biphenyl pathway in strain KT112-7 are located on the chromosome and share a high level of similarity with the bph genes of Acidovorax sp. KKS102. Analysis of the deduced sequences of the pcaGHBCDF, pcaIJfadA, and catABC genes, along with the metabolites identified during the cultivation of strain KT112-7 on hydroxylated biphenyls, suggests the presence of both classical and unique metabolic pathways for hydroxylated benzoic acids in strain KT112-7. Conclusion:The Rhodococcus opacus strain KT112-7 is characterized by genetic systems that contribute to its high biodegradative potential towards mono-hydroxylated biphenyls and their metabolites. Thus, the strain KT112-7 is promising for use in hydroxybiphenyl degradation technologies.

Molecular biomarkers, like gene transcripts or enzyme activities, are potentially powerful tools for early warning assessment of pollution. However, a thorough understanding of response and baseline variation is required to distinguish actual effects from pollution. Here, we assess the freshwater mussel Anodonta anatina as a biomarker model species for freshwater ecosystems, by testing responses of six transcriptional (cat, gst, hsp70, hsp90, mt, and sod) and two biochemical (AChE and GST) biomarkers to environmentally relevant Cu water concentrations. Mussels (n = 20), collected from a stream free from point source pollution, were exposed in the laboratory, for 96 h, to Cu treatments (< 0.2 μg/L, 0.77 ± 0.87 μg/L, and 6.3 ± 5.4 μg/L). Gills and digestive glands were extracted and analyzed for transcriptional and biochemical responses. Biological and statistical effect sizes from Cu treatments were in general small (mean log2 fold-change ≤ 0.80 and Cohen’s f ≤ 0.69, respectively), and no significant treatment effects were observed. In contrast, four out of eight biomarkers (cat, gst, hsp70, and GST) showed a significant sex:tissue interaction, and additionally one (sod) showed significant overall effects from sex. Specifically, three markers in gills (cat, mt, GST) and one in digestive gland (AChE) displayed significant sex differences, independent of treatment. Results suggest that sex or tissue effects might obscure low-magnitude biomarker responses and potential early warnings. Thus, variation in biomarker baselines and response patterns needs to be further addressed for the future use of A. anatina as a biomarker model species.

MOTIVATION:Medulloblastoma (MB) is a brain cancer predominantly arising in children. Roughly 70% of patients are cured today, but survivors often suffer from severe sequelae. MB has been extensively studied by molecular profiling, but often in small and scattered cohorts. To improve cure rates and reduce treatment side effects, accurate integration of such data to increase analytical power will be important, if not essential. RESULTS:We have integrated 23 transcription datasets, spanning 1350 MB and 291 normal brain samples. To remove batch effects, we combined the Removal of Unwanted Variation (RUV) method with a novel pipeline for determining empirical negative control genes and a panel of metrics to evaluate normalization performance. The documented approach enabled the removal of a majority of batch effects, producing a large-scale, integrative dataset of MB and cerebellar expression data. The proposed strategy will be broadly applicable for accurate integration of data and incorporation of normal reference samples for studies of various diseases. We hope that the integrated dataset will improve current research in the field of MB by allowing more large-scale gene expression analyses. AVAILABILITY AND IMPLEMENTATION:The RUV-normalized expression data is available through the Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) and can be accessed via the GSE series number GSE124814. SUPPLEMENTARY INFORMATION:Supplementary data are available at Bioinformatics online.

Objective We report 3 siblings with the characteristic features of ataxia-telangiectasia-like disorder associated with a homozygous MRE11 synonymous variant causing nonsense-mediated mRNA decay (NMD) and MRE11A deficiency. Methods Clinical assessments, next-generation sequencing, transcript and immunohistochemistry analyses were performed. Results The patients presented with poor balance, developmental delay during the first year of age, and suffered from intellectual disability from early childhood. They showed oculomotor apraxia, slurred and explosive speech, limb and gait ataxia, exaggerated deep tendon reflex, dystonic posture, and mirror movement in their hands. They developed mild cognitive abilities. Brain MRI in the index case revealed cerebellar atrophy. Next-generation sequencing revealed a homozygous synonymous variant in MRE11 (c.657C>T, p.Asn219=) that we show affects splicing. A complete absence of MRE11 transcripts in the index case suggested NMD and immunohistochemistry confirmed the absence of a stable protein. Conclusions Despite the critical role of MRE11A in double-strand break repair and its contribution to the Mre11/Rad50/Nbs1 complex, the absence of MRE11A is compatible with life.

The previously described chromium resistant bacterium, Enterobacter cloacae B2-DHA, was isolated from leather manufacturing tannery landfill in Bangladesh. Here we report the entire genome sequence of this bacterium containing chromium and other heavy metal resistance genes. The genome size and the number of genes, determined by massive parallel sequencing and comparative analysis with other known Enterobacter genomes, are predicted to be 4.22 Mb and 3958, respectively. Nearly 160 of these genes were found to be involved in binding, transport, and catabolism of ions as well as efflux of inorganic and organic compounds. Specifically, the presence of two chromium resistance genes, chrR and chrA was verified by polymerase chain reaction. The outcome of this research highlights the significance of this bacterium in bioremediation of chromium and other toxic metals from the contaminated sources.

Halomonads are moderately halophilic bacteria that are studied as models of prokaryotic osmoadaptation and sources of enzymes and chemicals for biotechnological applications. Despite the progress in understanding the diversity of these organisms, our ability to explain ecological, metabolic, and biochemical traits of halomonads at the genomic sequence level remains limited. This study addresses this gap by presenting draft genomes of Salinicola socius SMB35 T , Salinicola sp. MH3R3–1 and Chromohalobacter sp. SMB17, which were isolated from potash mine tailings in the Verkhnekamsk salt deposit area of Russia. The analysis of these genomes confirmed the importance of ectoines and quaternary amines to the capacity of halomonads to tolerate osmotic stress and adapt to hypersaline environments. The study also revealed that Chromohalobacter and Salinicola share 75–90% of the predicted proteome, but also harbor a set of genus-specific genes, which in Salinicola amounted to approximately 0.5 Mbp. These genus-specific genome segments may contribute to the phenotypic diversity of the Halomonadaceae and the ability of these organisms to adapt to changing environmental conditions and colonize new ecological niches.

Cancer is a disease characterized by abnormal proliferation of cells in the body and ranks as the second leading cause of death worldwide. In order to improve cancer patient care, a major focus of cancer research is to discover biomarkers. A biomarker is a biological molecule found in tissues or body fluids and can be used to predict or assess disease states. The aim of this thesis is to develop bioinformatics tools for discovery and evaluation of novel biomarkers from high-throughput datasets.MicroRNAs (miRNAs) are short non-coding RNAs that function as negative regulators of gene expression. Dysregulation of miRNAs in cancer is frequently reported, making them interesting as biomarkercandidates. GenoScan was developed for genome-wide discovery of miRNA-coding genes, as a first step in the identification of novel mi-RNA biomarkers.High-throughput technologies such as microarrays allow researchers to measure the expression of thousands of genes or miRNAs simultaneously. The Decision Trunk Classifier (DTC) algorithm has been developed to screen datasets from these experiments for biomarker candidates. When applied to a miRNA expression dataset for endometrial cancer (EC) samples vs. controls, a two-marker model with 98 % accuracy was generated. These miRNAs (hsa-miR-183-5p and hsamiRPlus-C1070) are promising as biomarkers for EC screening.The miREC database was developed to store gene and miRNA data from curated expression profiling studies of EC, as well as gene-miRNA regulatory connections. Using gene-miRNA interaction networks from miREC, the roles of miRNAs in cancer hallmark acquisition can be clarified. To further support exploratory analysis of expression data, DTC was extended with partial least squares regression models. The resulting PLS-DTC algorithm can be used to gain deeper insights into the perturbation of biological processes and pathways.

ABSTRACT Here, we report the genomic sequence and genetic composition of an arsenic-resistant bacterium, Lysinibacillus sphaericus B1-CDA. Assembly of the sequencing reads revealed that the genome size is ~4.5 Mb, encompassing ~80% of the chromosomal DNA.

Human pluripotent stem cells- (hPSCs-) derived hepatocytes have the potential to replace many hepatic models in drug discovery and provide a cell source for regenerative medicine applications. However, the generation of fully functional hPSC-derived hepatocytes is still a challenge. Towards gaining better understanding of the differentiation and maturation process, we employed a standardized protocol to differentiate six hPSC lines into hepatocytes and investigated the synchronicity of the hPSC lines by applying RT-qPCR to assess the expression of lineage-specific genes (OCT4, NANOG, T, SOX17, CXCR4, CER1, HHEX, TBX3, PROX1, HNF6, AFP, HNF4a, KRT18, ALB, AAT, and CYP3A4) which serve as markers for different stages during liver development. The data was evaluated using correlation and clustering analysis, demonstrating that the expression of these markers is highly synchronized and correlated well across all cell lines. The analysis also revealed a distribution of the markers in groups reflecting the developmental stages of hepatocytes. Functional analysis of the differentiated cells further confirmed their hepatic phenotype. Taken together, these results demonstrate, on the molecular level, the highly synchronized differentiation pattern across multiple hPSC lines. Moreover, this study provides additional understanding for future efforts to improve the functionality of hPSC-derived hepatocytes and thereby increase the value of related models.

ABSTRACT Previously, we reported a chromium-resistant bacterium, Enterobacter cloacae B2-DHA, isolated from the landfills of tannery industries in Bangladesh. Here, we investigated its genetic composition using massively parallel sequencing and comparative analysis with other known Enterobacter genomes. Assembly of the sequencing reads revealed a genome of ~4.21 Mb in size.

Myosin-1C (MYO1C) is a tumor suppressor candidate located in a region of recurrent losses distal to TP53. Myo1c can tightly and specifically bind to PIP2, the substrate of Phosphoinositide 3-kinase (PI3K), and to Rictor, suggesting a role for MYO1C in the PI3K pathway. This study was designed to examine MYO1C expression status in a panel of well-stratified endometrial carcinomas as well as to assess the biological significance of MYO1C as a tumor suppressor in vitro. We found a significant correlation between the tumor stage and lowered expression of MYO1C in endometrial carcinoma samples. In cell transfection experiments, we found a negative correlation between MYO1C expression and cell proliferation, and MYO1C silencing resulted in diminished cell migration and adhesion. Cells expressing excess of MYO1C had low basal level of phosphorylated protein kinase B (PKB, a.k.a. AKT) and cells with knocked down MYO1C expression showed a quicker phosphorylated AKT (pAKT) response in reaction to serum stimulation. Taken together the present study gives further evidence for tumor suppressor activity of MYO1C and suggests MYO1C mediates its tumor suppressor function through inhibition of PI3K pathway and its involvement in loss of contact inhibition.

Background: MicroRNAs are small non-coding RNAs that have been implicated in tumor initiation and progression. In a previous study we identified 138 miRNAs as differentially expressed in endometrial adenocarcinoma compared to normal tissues. One of these miRNAs was miRNA-34a, which regulates several genes involved in the Notch pathway, which is frequently altered in endometrial cancer. The aims of this study were to verify the differential expression of a subset of miRNAs and to scrutinize the regulatory role of mir-34a on the target genes NOTCH1 and DLL1.Methods: Twenty-five miRNAs that were previously identified as differentially expressed were subjected to further analysis using qPCR. To investigate the regulation of NOTCH1 and DLL1 by mir-34a, we designed gain- and loss-of-function experiments in Ishikawa and HEK293 cell lines by transfection with a synthetic mir-34a mimic and a mir-34a inhibitor.Results: Of the 25 validated miRNAs, seven were down-regulated and 18 were up-regulated compared to normal endometrium, which was fully consistent with our previous findings. In addition, the up-regulation of mir-34a led to a significant decrease in mRNA levels of NOTCH1 and DLL1, while down-regulation led to a significant increase in mRNA levels of these two genes.Conclusions: We verified both up-regulated and down-regulated miRNAs in the tumor samples, indicating various roles of microRNAs during tumor development. Mir-34a functions as a regulator by decreasing the expression of NOTCH1 and DLL1. Our study is the first to identify a correlation between mir-34a and its target genes NOTCH1 and DLL1 in endometrial adenocarcinoma.

Genome analysis of Enterobacter cloacae B2-DHA : A bacterium resistant to chromium and/or other heavy metals

BACKGROUND:In humans, muscle-specific nicotinergic acetylcholine receptor (AChR) is a transmembrane protein with five different subunits, coded by CHRNA1, CHRNB, CHRND and CHRNG/CHRNE. The gamma subunit of AChR encoded by CHRNG is expressed during early foetal development, whereas in the adult, the γ subunit is replaced by a ε subunit. Mutations in the CHRNG encoding the embryonal acetylcholine receptor may cause the non-lethal Escobar variant (EVMPS) and lethal form (LMPS) of multiple pterygium syndrome. The MPS is a condition characterised by prenatal growth failure with pterygium and akinesia leading to muscle weakness and severe congenital contractures, as well as scoliosis.RESULTS:Our whole exome sequencing studies have identified one novel and two previously reported homozygous mutations in CHRNG in three families affected by non-lethal EVMPS. The mutations consist of deletion of two nucleotides, cause a frameshift predicted to result in premature termination of the foetally expressed gamma subunit of the AChR.CONCLUSIONS:Our data suggest that severity of the phenotype varies significantly both within and between families with MPS and that there is no apparent correlation between mutation position and clinical phenotype. Although individuals with CHRNG mutations can survive, there is an increased frequency of abortions and stillbirth in their families. Furthermore, genetic background and environmental modifiers might be of significance for decisiveness of the lethal spectrum, rather than the state of the mutation per se. Detailed clinical examination of our patients further indicates the changing phenotype from infancy to childhood.

ABSTRACT We report the 5,728,255-bp draft genome sequence of Rhodococcus ruber P25, isolated from a soil polluted with halogenated aromatic compounds in the city of Perm, Russia. The strain degrades polychlorinated biphenyls and a broad range of aromatic compounds. It possesses genes that mediate the degradation of biphenyls/polychlorinated biphenyls, naphthalene, and monoaromatic compounds.

In silico prediction of novel miRNAs from genomic sequences remains a challenging problem. This study presents a genome-wide miRNA discovery software package called GenoScan and evaluates two hairpin classification methods. These methods, one ensemble-based and one using logistic regression were benchmarked along with 15 published methods. In addition, the sequence-folding step is addressed by investigating the impact of secondary structure prediction methods and the choice of input sequence length on prediction performance. Both the accuracy of secondary structure predictions and the miRNA prediction are evaluated. In the benchmark of hairpin classification methods, the regression model achieved highest classification accuracy. Of the structure prediction methods evaluated, ContextFold achieved the highest agreement between predicted and experimentally determined structures. However, both the choice of secondary structure prediction method and input sequence length had limited impact on hairpin classification performance.

BACKGROUND:Endometrial cancer (EC) is the most frequently diagnosed gynecological malignancy and the fourth most common cancer diagnosis overall among women. As with many other forms of cancer, it has been shown that certain miRNAs are differentially expressed in EC and these miRNAs are believed to play important roles as regulators of processes involved in the development of the disease. With the rapidly growing number of studies of miRNA expression in EC, there is a need to organize the data, combine the findings from experimental studies of EC with information from various miRNA databases, and make the integrated information easily accessible for the EC research community.FINDINGS:The miREC database is an organized collection of data and information about miRNAs shown to be differentially expressed in EC. The database can be used to map connections between miRNAs and their target genes in order to identify specific miRNAs that are potentially important for the development of EC. The aim of the miREC database is to integrate all available information about miRNAs and target genes involved in the development of endometrial cancer, and to provide a comprehensive, up-to-date, and easily accessible source of knowledge regarding the role of miRNAs in the development of EC. Database URL: http://www.mirecdb.org .CONCLUSIONS:Several databases have been published that store information about all miRNA targets that have been predicted or experimentally verified to date. It would be a time-consuming task to navigate between these different data sources and literature to gather information about a specific disease, such as endometrial cancer. The miREC database is a specialized data repository that, in addition to miRNA target information, keeps track of the differential expression of genes and miRNAs potentially involved in endometrial cancer development. By providing flexible search functions it becomes easy to search for EC-associated genes and miRNAs from different starting points, such as differential expression and genomic loci (based on genomic aberrations).

Bull spermatozoa are rich in active miRNAs, and it has been shown that specific spermborne miRNAs can be linked to fertility. Thus, expression profiling of spermatozoa could be helpful for understanding male fertility and the ability of spermatozoa to initiate and sustain zygotic, embryonic and foetal development. Herein we hypothesized that bulls with moderate to high fertility can be identified by differences in amounts of certain miRNAs between their ejaculates. RNA samples from spermatozoa of eight brother pairs (one bull with high and one with moderate NRR in each pair) of the Holstein breed were prepared. miRNA was isolated, and the expression of 178 miRNAs was determined by RT-qPCR. Important findings were that highly expressed miRNAs, not linked to NRR status, were identified in the bull sperm samples, which indicate that these miRNAs have an important role in early embryogenesis. A large fraction of the targets genes were phosphoproteins and genes involved in the regulation of transcription. Seven miRNAs (mir-502-5p, mir-1249, mir-320a, mir-34c-3p, mir-19b-3p, mir-27a-5p and mir-148b-3p) were differentially expressed between bulls with moderate and high NRR with a strong tendency towards a higher expression of miRNAs in bulls with moderate fertility. Thus, bulls with a moderate NRR negatively regulate the expression of protein-coding genes, which leads to problems during the pregnancy.