ALBERT

Description: Genes, Vol. 9, Pages 103: Microfluidic Devices for Drug Delivery Systems and Drug Screening Genes doi: 10.3390/genes9020103 Authors: Samar Damiati Uday B. Kompella Safa A. Damiati Rimantas Kodzius Microfluidic devices present unique advantages for the development of efficient drug carrier particles, cell-free protein synthesis systems, and rapid techniques for direct drug screening. Compared to bulk methods, by efficiently controlling the geometries of the fabricated chip and the flow rates of multiphase fluids, microfluidic technology enables the generation of highly stable, uniform, monodispersed particles with higher encapsulation efficiency. Since the existing preclinical models are inefficient drug screens for predicting clinical outcomes, microfluidic platforms might offer a more rapid and cost-effective alternative. Compared to 2D cell culture systems and in vivo animal models, microfluidic 3D platforms mimic the in vivo cell systems in a simple, inexpensive manner, which allows high throughput and multiplexed drug screening at the cell, organ, and whole-body levels. In this review, the generation of appropriate drug or gene carriers including different particle types using different configurations of microfluidic devices is highlighted. Additionally, this paper discusses the emergence of fabricated microfluidic cell-free protein synthesis systems for potential use at point of care as well as cell-, organ-, and human-on-a-chip models as smart, sensitive, and reproducible platforms, allowing the investigation of the effects of drugs under conditions imitating the biological system.

Description: Genes, Vol. 9, Pages 102: Dietary Fiber Treatment Corrects the Composition of Gut Microbiota, Promotes SCFA Production, and Suppresses Colon Carcinogenesis Genes doi: 10.3390/genes9020102 Authors: Faraz Bishehsari Phillip A. Engen Nailliw Z. Preite Yunus E. Tuncil Ankur Naqib Maliha Shaikh Marco Rossi Sherry Wilber Stefan J. Green Bruce R. Hamaker Khashayarsha Khazaie Robin M. Voigt Christopher B. Forsyth Ali Keshavarzian Epidemiological studies propose a protective role for dietary fiber in colon cancer (CRC). One possible mechanism of fiber is its fermentation property in the gut and ability to change microbiota composition and function. Here, we investigate the role of a dietary fiber mixture in polyposis and elucidate potential mechanisms using TS4Cre×cAPCl°x468 mice. Stool microbiota profiling was performed, while functional prediction was done using PICRUSt. Stool short-chain fatty acid (SCFA) metabolites were measured. Histone acetylation and expression of SCFA butyrate receptor were assessed. We found that SCFA-producing bacteria were lower in the polyposis mice, suggesting a decline in the fermentation product of dietary fibers with polyposis. Next, a high fiber diet was given to polyposis mice, which significantly increased SCFA-producing bacteria as well as SCFA levels. This was associated with an increase in SCFA butyrate receptor and a significant decrease in polyposis. In conclusion, we found polyposis to be associated with dysbiotic microbiota characterized by a decline in SCFA-producing bacteria, which was targetable by high fiber treatment, leading to an increase in SCFA levels and amelioration of polyposis. The prebiotic activity of fiber, promoting beneficial bacteria, could be the key mechanism for the protective effects of fiber on colon carcinogenesis. SCFA-promoting fermentable fibers are a promising dietary intervention to prevent CRC.

Description: Genes, Vol. 9, Pages 372: TERribly Difficult: Searching for Telomerase RNAs in Saccharomycetes Genes doi: 10.3390/genes9080372 Authors: Maria Waldl Bernhard C. Thiel Roman Ochsenreiter Alexander Holzenleiter João Victor de Araujo Oliveira Maria Emília M. T. Walter Michael T. Wolfinger Peter F. Stadler The telomerase RNA in yeasts is large, usually >1000 nt, and contains functional elements that have been extensively studied experimentally in several disparate species. Nevertheless, they are very difficult to detect by homology-based methods and so far have escaped annotation in the majority of the genomes of Saccharomycotina. This is a consequence of sequences that evolve rapidly at nucleotide level, are subject to large variations in size, and are highly plastic with respect to their secondary structures. Here, we report on a survey that was aimed at closing this gap in RNA annotation. Despite considerable efforts and the combination of a variety of different methods, it was only partially successful. While 27 new telomerase RNAs were identified, we had to restrict our efforts to the subgroup Saccharomycetacea because even this narrow subgroup was diverse enough to require different search models for different phylogenetic subgroups. More distant branches of the Saccharomycotina remain without annotated telomerase RNA.

Description: Genes, Vol. 9, Pages 373: Insights into Avian Incomplete Dosage Compensation: Sex-Biased Gene Expression Coevolves with Sex Chromosome Degeneration in the Common Whitethroat Genes doi: 10.3390/genes9080373 Authors: Hanna Sigeman Suvi Ponnikas Elin Videvall Hongkai Zhang Pallavi Chauhan Sara Naurin Bengt Hansson Non-recombining sex chromosomes (Y and W) accumulate deleterious mutations and degenerate. This poses a problem for the heterogametic sex (XY males; ZW females) because a single functional gene copy often implies less gene expression and a potential imbalance of crucial expression networks. Mammals counteract this by dosage compensation, resulting in equal sex chromosome expression in males and females, whereas birds show incomplete dosage compensation with significantly lower expression in females (ZW). Here, we study the evolution of Z and W sequence divergence and sex-specific gene expression in the common whitethroat (Sylvia communis), a species within the Sylvioidea clade where a neo-sex chromosome has been formed by a fusion between an autosome and the ancestral sex chromosome. In line with data from other birds, females had lower expression than males at the majority of sex-linked genes. Results from the neo-sex chromosome region showed that W gametologs have diverged functionally to a higher extent than their Z counterparts, and that the female-to-male expression ratio correlated negatively with the degree of functional divergence of these gametologs. We find it most likely that sex-linked genes are being suppressed in females as a response to W chromosome degradation, rather than that these genes experience relaxed selection, and thus diverge more, by having low female expression. Overall, our data of this unique avian neo-sex chromosome system suggest that incomplete dosage compensation evolves, at least partly, through gradual accumulation of deleterious mutations at the W chromosome and declining female gene expression.

Description: Genes, Vol. 9, Pages 379: Genomic and Biotechnological Characterization of the Heavy-Metal Resistant, Arsenic-Oxidizing Bacterium Ensifer sp. M14 Genes doi: 10.3390/genes9080379 Authors: George C diCenzo Klaudia Debiec Jan Krzysztoforski Witold Uhrynowski Alessio Mengoni Camilla Fagorzi Adrian Gorecki Lukasz Dziewit Tomasz Bajda Grzegorz Rzepa Lukasz Drewniak Ensifer (Sinorhizobium) sp. M14 is an efficient arsenic-oxidizing bacterium (AOB) that displays high resistance to numerous metals and various stressors. Here, we report the draft genome sequence and genome-guided characterization of Ensifer sp. M14, and we describe a pilot-scale installation applying the M14 strain for remediation of arsenic-contaminated waters. The M14 genome contains 6874 protein coding sequences, including hundreds not found in related strains. Nearly all unique genes that are associated with metal resistance and arsenic oxidation are localized within the pSinA and pSinB megaplasmids. Comparative genomics revealed that multiple copies of high-affinity phosphate transport systems are common in AOBs, possibly as an As-resistance mechanism. Genome and antibiotic sensitivity analyses further suggested that the use of Ensifer sp. M14 in biotechnology does not pose serious biosafety risks. Therefore, a novel two-stage installation for remediation of arsenic-contaminated waters was developed. It consists of a microbiological module, where M14 oxidizes As(III) to As(V) ion, followed by an adsorption module for As(V) removal using granulated bog iron ores. During a 40-day pilot-scale test in an abandoned gold mine in Zloty Stok (Poland), water leaving the microbiological module generally contained trace amounts of As(III), and dramatic decreases in total arsenic concentrations were observed after passage through the adsorption module. These results demonstrate the usefulness of Ensifer sp. M14 in arsenic removal performed in environmental settings.

Description: Genes, Vol. 9, Pages 382: Multimodal 3D DenseNet for IDH Genotype Prediction in Gliomas Genes doi: 10.3390/genes9080382 Authors: Sen Liang Rongguo Zhang Dayang Liang Tianci Song Tao Ai Chen Xia Liming Xia Yan Wang Non-invasive prediction of isocitrate dehydrogenase (IDH) genotype plays an important role in tumor glioma diagnosis and prognosis. Recently, research has shown that radiology images can be a potential tool for genotype prediction, and fusion of multi-modality data by deep learning methods can further provide complementary information to enhance prediction accuracy. However, it still does not have an effective deep learning architecture to predict IDH genotype with three-dimensional (3D) multimodal medical images. In this paper, we proposed a novel multimodal 3D DenseNet (M3D-DenseNet) model to predict IDH genotypes with multimodal magnetic resonance imaging (MRI) data. To evaluate its performance, we conducted experiments on the BRATS-2017 and The Cancer Genome Atlas breast invasive carcinoma (TCGA-BRCA) dataset to get image data as input and gene mutation information as the target, respectively. We achieved 84.6% accuracy (area under the curve (AUC) = 85.7%) on the validation dataset. To evaluate its generalizability, we applied transfer learning techniques to predict World Health Organization (WHO) grade status, which also achieved a high accuracy of 91.4% (AUC = 94.8%) on validation dataset. With the properties of automatic feature extraction, and effective and high generalizability, M3D-DenseNet can serve as a useful method for other multimodal radiogenomics problems and has the potential to be applied in clinical decision making.

Description: Genes, Vol. 9, Pages 386: Structural and Evolutionary Insights within the Polysaccharide Deacetylase Gene Family of Bacillus anthracis and Bacillus cereus Genes doi: 10.3390/genes9080386 Authors: Athena Andreou Petros Giastas Elias Christoforides Elias E. Eliopoulos Functional and folding constraints impose interdependence between interacting sites along the protein chain that are envisaged through protein sequence evolution. Studying the influence of structure in phylogenetic models requires detailed and reliable structural models. Polysaccharide deacetylases (PDAs), members of the carbohydrate esterase family 4, perform mainly metal-dependent deacetylation of O- or N-acetylated polysaccharides such as peptidoglycan, chitin and acetylxylan through a conserved catalytic core termed the NodB homology domain. Genomes of Bacillus anthracis and its relative Bacillus cereus contain multiple genes of putative or known PDAs. A comparison of the functional domains of the recently determined PDAs from B. anthracis and B. cereus and multiple amino acid and nucleotide sequence alignments and phylogenetic analysis performed on these closely related species showed that there were distinct differences in binding site formation, despite the high conservation on the protein sequence, the folding level and the active site assembly. This may indicate that, subject to biochemical verification, the binding site-forming sequence fragments are under functionally driven evolutionary pressure to accommodate and recognize distinct polysaccharide residues according to cell location, use, or environment. Finally, we discuss the suggestion of the paralogous nature of at least two genes of B. anthracis, ba0330 and ba0331, via specific differences in gene sequence, protein structure, selection pressure and available localization patterns. This study may contribute to understanding the mechanisms under which sequences evolve in their structures and how evolutionary processes enable structural variations.

Description: Genes, Vol. 9, Pages 385: Synteny-Based Development of CAPS Markers Linked to the Sweet kernel LOCUS, Controlling Amygdalin Accumulation in Almond (Prunus dulcis (Mill.) D.A.Webb) Genes doi: 10.3390/genes9080385 Authors: Francesca Ricciardi Jorge Del Cueto Nicoletta Bardaro Rosa Mazzeo Luigi Ricciardi Federico Dicenta Raquel Sánchez-Pérez Stefano Pavan Concetta Lotti The bitterness and toxicity of wild-type seeds of Prunoideae is due to the cyanogenic glucoside amygdalin. In cultivated almond (Prunus dulcis (Mill.) D.A. Webb), a dominant mutation at the Sk locus prevents amygdalin accumulation and thus results in edible sweet kernels. Here, we exploited sequence similarity and synteny between the genomes of almond and peach (Prunus persica (L.) Batsch) to identify cleaved amplified polymorphic sequence (CAPS) molecular markers linked to the Sk locus. A segregant F1 population was used to map these markers on the Sk genomic region, together with Sk-linked simple sequence repeat (SSR) markers previously described. Molecular fingerprinting of a cultivar collection indicated the possibility to use CAPS polymorphisms identified in this study in breeding programs arising from different parental combinations. Overall, we highlight a set of codominant markers useful for early selection of sweet kernel genotypes, an aspect of primary importance in almond breeding. In addition, by showing collinearity between the physical map of peach and the genetic map of almond with respect to the Sk genomic region, we provide valuable information for further marker development and Sk positional cloning.

Description: Genes, Vol. 9, Pages 393: A High-Quality, Long-Read De Novo Genome Assembly to Aid Conservation of Hawaii’s Last Remaining Crow Species Genes doi: 10.3390/genes9080393 Authors: Jolene T. Sutton Martin Helmkampf Cynthia C. Steiner M. Renee Bellinger Jonas Korlach Richard Hall Primo Baybayan Jill Muehling Jenny Gu Sarah Kingan Bryce M. Masuda Oliver A. Ryder Abstract: Genome-level data can provide researchers with unprecedented precision to examine the causes and genetic consequences of population declines, which can inform conservation management. Here, we present a high-quality, long-read, de novo genome assembly for one of the world’s most endangered bird species, the ʻAlalā (Corvus hawaiiensis; Hawaiian crow). As the only remaining native crow species in Hawaiʻi, the ʻAlalā survived solely in a captive-breeding program from 2002 until 2016, at which point a long-term reintroduction program was initiated. The high-quality genome assembly was generated to lay the foundation for both comparative genomics studies and the development of population-level genomic tools that will aid conservation and recovery efforts. We illustrate how the quality of this assembly places it amongst the very best avian genomes assembled to date, comparable to intensively studied model systems. We describe the genome architecture in terms of repetitive elements and runs of homozygosity, and we show that compared with more outbred species, the ʻAlalā genome is substantially more homozygous. We also provide annotations for a subset of immunity genes that are likely to be important in conservation management, and we discuss how this genome is currently being used as a roadmap for downstream conservation applications.

Description: Genes, Vol. 9, Pages 391: PRDM Histone Methyltransferase mRNA Levels Increase in Response to Curative Hormone Treatment for Cryptorchidism-Dependent Male Infertility Genes doi: 10.3390/genes9080391 Authors: Faruk Hadziselimovic Gieri Cathomas Gilvydas Verkauskas Darius Dasevicius Michael B. Stadler There is a correlation between cryptorchidism and an increased risk of testicular cancer and infertility. During orchidopexy, testicular biopsies are performed to confirm the presence of type A dark (Ad) spermatogonia, which are a marker for low infertility risk (LIR). The Ad spermatogonia are absent in high infertility risk (HIR) patients, who are treated with a gonadotropin-releasing hormone agonist (GnRHa) to significantly lower the risk of infertility. Despite its prevalence, little is known about the molecular events involved in cryptorchidism. Previously, we compared the transcriptomes of LIR versus HIR patients treated with and without hormones. Here, we interpreted data regarding members of the positive regulatory domain-containing (PRDM) family; some of which encoded histone methyltransferases that are important for reproduction. We found there were lower levels of PRDM1, PRDM6, PRDM9, PRDM13, and PRDM14 mRNA in the testes of HIR patients compared with LIR patients, and that PRDM7, PRDM9, PRDM12, and PRDM16 were significantly induced after GnRHa treatment. Furthermore, we observed PRDM9 protein staining in the cytoplasm of germ cells in the testes from LIR and HIR patients, indicating that the mRNA and protein levels corresponded. This result indicated that the curative hormonal therapy for cryptorchidism involved conserved chromatin modification enzymes.

Description: Genes, Vol. 9, Pages 396: Genomic Diversity of Listeria monocytogenes Isolated from Clinical and Non-Clinical Samples in Chile Genes doi: 10.3390/genes9080396 Authors: Viviana Toledo Henk C. den Bakker Juan Carlos Hormazábal Gerardo González-Rocha Helia Bello-Toledo Magaly Toro Andrea I. Moreno-Switt Listeria monocytogenes is the causative agent of listeriosis, which is an uncommon but severe infection associated with high mortality rates in humans especially in high-risk groups. This bacterium survives a variety of stress conditions (e.g., high osmolality, low pH), which allows it to colonize different niches especially niches found in food processing environments. Additionally, a considerable heterogeneity in pathogenic potential has been observed in different strains. In this study, 38 isolates of L. monocytogenes collected in Chile from clinical samples (n = 22) and non-clinical samples (n = 16) were analyzed using whole genome sequencing (WGS) to determine their genomic diversity. A core genome Single Nucleotide Polymorphism (SNP) tree using 55 additional L. monocytogenes accessions classified the Chilean isolates in lineages I (n = 25) and II (n = 13). In silico, Multi-locus sequence typing (MLST) differentiated the isolates into 13 sequence types (ST) in which the most common were ST1 (15 isolates) and ST9 (6 isolates) and represented 55% of the isolates. Genomic elements associated with virulence (i.e., LIPI-1, LIPI-3, inlA, inlB, inlC, inlG, inlH, inlD, inlE, inlK, inlF, and inlJ) and stress survival (i.e., stress survival islet 1 and stress survival islet 2) were unevenly distributed among clinical and non-clinical isolates. In addition, one novel inlA premature stop codon (PMSC) was detected. Comparative analysis of L. monocytogenes circulating in Chile revealed the presence of globally distributed sequence types along with differences among the isolates analyzed at a genomic level specifically associated with virulence and stress survival.

Description: Genes, Vol. 9, Pages 282: The Complete Mitochondrial Genome of Glyptothorax macromaculatus Provides a Well-Resolved Molecular Phylogeny of the Chinese Sisorid Catfishes Genes doi: 10.3390/genes9060282 Authors: Yunyun Lv Yanping Li Zhiqiang Ruan Chao Bian Xinxin You Junxing Yang Wansheng Jiang Qiong Shi Previous phylogenetic analyses of the Chinese sisorid catfishes have either been poorly resolved or have not included all the 12 sisorid genera. Here, we successfully assembled the first complete mitochondrial genome of the sisorid fish Glyptothorax macromaculatus. Based on this novel mitochondrial genome and previously published mitochondrial genomes in the Sisoridae, we generated maximum likelihood and Bayesian phylogenies. We dated our preferred topology using fossil calibration points. We also tested the protein-coding genes in the mitochondrial genomes of the glyptosternoid fishes for signals of natural selection by comparing the nucleotide substitution rate along the branch ancestral to the glyptosternoid fishes to other branches in our topology. The mitochondrial sequence structure of G. macromaculatus was similar to those known from other vertebrates, with some slight differences. Our sisorid phylogenies were well-resolved and well-supported, with exact congruence between the different phylogenetic methods. This robust phylogeny clarified the relationships among the Chinese sisorid genera and strongly supported the division of the family into three main clades. Interestingly, the glyptosternoid divergence time predicted by our molecular dating analysis coincided with the uplift of the Tibetan Plateau, suggesting that geology may have influenced speciation in the Sisoridae. Among the mitochondrial protein-coding genes, atp8 may have most rapidly evolved, and atp6 may have been subjected to positive selection pressure to adapt to high elevations. In summary, this study provided novel insights into the phylogeny, evolution and high-altitude adaptions of the Chinese sisorid fishes.

Description: Genes, Vol. 9, Pages 281: High-Throughput Incubation and Quantification of Agglutination Assays in a Microfluidic System Genes doi: 10.3390/genes9060281 Authors: David Castro David Conchouso Rimantas Kodzius Arpys Arevalo Ian G. Foulds In this paper, we present a two-phase microfluidic system capable of incubating and quantifying microbead-based agglutination assays. The microfluidic system is based on a simple fabrication solution, which requires only laboratory tubing filled with carrier oil, driven by negative pressure using a syringe pump. We provide a user-friendly interface, in which a pipette is used to insert single droplets of a 1.25-µL volume into a system that is continuously running and therefore works entirely on demand without the need for stopping, resetting or washing the system. These assays are incubated by highly efficient passive mixing with a sample-to-answer time of 2.5 min, a 5–10-fold improvement over traditional agglutination assays. We study system parameters such as channel length, incubation time and flow speed to select optimal assay conditions, using the streptavidin-biotin interaction as a model analyte quantified using optical image processing. We then investigate the effect of changing the concentration of both analyte and microbead concentrations, with a minimum detection limit of 100 ng/mL. The system can be both low- and high-throughput, depending on the rate at which assays are inserted. In our experiments, we were able to easily produce throughputs of 360 assays per hour by simple manual pipetting, which could be increased even further by automation and parallelization. Agglutination assays are a versatile tool, capable of detecting an ever-growing catalog of infectious diseases, proteins and metabolites. A system such as this one is a step towards being able to produce high-throughput microfluidic diagnostic solutions with widespread adoption. The development of analytical techniques in the microfluidic format, such as the one presented in this work, is an important step in being able to continuously monitor the performance and microfluidic outputs of organ-on-chip devices.

Description: Genes, Vol. 9, Pages 301: Decision Variants for the Automatic Determination of Optimal Feature Subset in RF-RFE Genes doi: 10.3390/genes9060301 Authors: Qi Chen Zhaopeng Meng Xinyi Liu Qianguo Jin Ran Su Feature selection, which identifies a set of most informative features from the original feature space, has been widely used to simplify the predictor. Recursive feature elimination (RFE), as one of the most popular feature selection approaches, is effective in data dimension reduction and efficiency increase. A ranking of features, as well as candidate subsets with the corresponding accuracy, is produced through RFE. The subset with highest accuracy (HA) or a preset number of features (PreNum) are often used as the final subset. However, this may lead to a large number of features being selected, or if there is no prior knowledge about this preset number, it is often ambiguous and subjective regarding final subset selection. A proper decision variant is in high demand to automatically determine the optimal subset. In this study, we conduct pioneering work to explore the decision variant after obtaining a list of candidate subsets from RFE. We provide a detailed analysis and comparison of several decision variants to automatically select the optimal feature subset. Random forest (RF)-recursive feature elimination (RF-RFE) algorithm and a voting strategy are introduced. We validated the variants on two totally different molecular biology datasets, one for a toxicogenomic study and the other one for protein sequence analysis. The study provides an automated way to determine the optimal feature subset when using RF-RFE.

Description: Genes, Vol. 9, Pages 312: The Case of X and Y Localization of Nucleolus Organizer Regions (NORs) in Tragulus javanicus (Cetartiodactyla, Mammalia) Genes doi: 10.3390/genes9060312 Authors: Anastasia A. Proskuryakova Anastasia I. Kulemzina Polina L. Perelman Natalia A. Serdukova Oliver A. Ryder Alexander S. Graphodatsky There are differences in number and localization of nucleolus organizer regions (NORs) in genomes. In mammalian genomes, NORs are located on autosomes, which are often situated on short arms of acrocentric chromosomes and more rarely in telomeric, pericentromeric, or interstitial regions. In this work, we report the unique case of active NORs located on gonоsomes of a eutherian mammal, the Javan mouse-deer (Tragulus javanicus). We have investigated the position of NORs by FISH experiments with ribosomal DNA (rDNA) sequences (18S, 5.8S, and 28S) and show the presence of a single NOR site on the X and Y chromosomes. The NOR is localized interstitially on the p-arm of the X chromosome in close proximity with prominent C-positive heterochromatin blocks and in the pericentromeric area of mostly heterochromatic Y. The NOR sites are active on both the X and Y chromosomes in the studied individual and surrounded by GC enriched heterochromatin. We hypothesize that the surrounding heterochromatin might have played a role in the transfer of NORs from autosomes to sex chromosomes during the karyotype evolution of the Javan mouse-deer.

Description: Genes, Vol. 9, Pages 311: Genome-Wide Identification and Functional Prediction of Novel Drought-Responsive lncRNAs in Pyrus betulifolia Genes doi: 10.3390/genes9060311 Authors: Jinxing Wang Jing Lin Jialiang Kan Hong Wang Xiaogang Li Qingsong Yang Hui Li Youhong Chang Increasing evidence shows that long noncoding RNAs (lncRNAs) play important roles in developmental regulation and many other biological processes in plants. However, identification of lncRNAs in Pyrus betulifolia is limited compared with studies of functional gene expression. Using high-throughput sequencing technology, the transcriptome of P. betulifolia under drought stress was analyzed to identify lncRNAs. A total of 14,478 lncRNAs were identified, of which 251 were found to be drought-responsive. The putative target genes of these differentially expressed lncRNAs were significantly enriched in metabolic processes, organic substance metabolic processes, macromolecule metabolic processes, and heterocyclic compound binding. Real-time quantitative polymerase chain reaction validation suggested that the results of the RNA sequencing data analysis were reliable. This study will provide genetic resources for pear breeding and provide reference to other pomological studies.

Description: Genes, Vol. 9, Pages 308: Construction of Red Fox Chromosomal Fragments from the Short-Read Genome Assembly Genes doi: 10.3390/genes9060308 Authors: Halie M. Rando Marta Farré Michael P. Robson Naomi B. Won Jennifer L. Johnson Ronak Buch Estelle R. Bastounes Xueyan Xiang Shaohong Feng Shiping Liu Zijun Xiong Jaebum Kim Guojie Zhang Lyudmila N. Trut Denis M. Larkin Anna V. Kukekova The genome of a red fox (Vulpes vulpes) was recently sequenced and assembled using next-generation sequencing (NGS). The assembly is of high quality, with 94X coverage and a scaffold N50 of 11.8 Mbp, but is split into 676,878 scaffolds, some of which are likely to contain assembly errors. Fragmentation and misassembly hinder accurate gene prediction and downstream analysis such as the identification of loci under selection. Therefore, assembly of the genome into chromosome-scale fragments was an important step towards developing this genomic model. Scaffolds from the assembly were aligned to the dog reference genome and compared to the alignment of an outgroup genome (cat) against the dog to identify syntenic sequences among species. The program Reference-Assisted Chromosome Assembly (RACA) then integrated the comparative alignment with the mapping of the raw sequencing reads generated during assembly against the fox scaffolds. The 128 sequence fragments RACA assembled were compared to the fox meiotic linkage map to guide the construction of 40 chromosomal fragments. This computational approach to assembly was facilitated by prior research in comparative mammalian genomics, and the continued improvement of the red fox genome can in turn offer insight into canid and carnivore chromosome evolution. This assembly is also necessary for advancing genetic research in foxes and other canids.

Description: Genes, Vol. 9, Pages 317: The Role of aDNA in Understanding the Coevolutionary Patterns of Human Sexually Transmitted Infections Genes doi: 10.3390/genes9070317 Authors: Ville N. Pimenoff Charlotte J. Houldcroft Riaan F. Rifkin Simon Underdown Analysis of pathogen genome data sequenced from clinical and historical samples has made it possible to perform phylogenetic analyses of sexually transmitted infections on a global scale, and to estimate the diversity, distribution, and coevolutionary host relationships of these pathogens, providing insights into pathogen emergence and disease prevention. Deep-sequenced pathogen genomes from clinical studies and ancient samples yield estimates of within-host and between-host evolutionary rates and provide data on changes in pathogen genomic stability and evolutionary responses. Here we examine three groups of pathogens transmitted mainly through sexual contact between modern humans to provide insight into ancient human behavior and history with their pathogens. Exploring ancient pathogen genomic divergence and the ancient viral-host parallel evolutionary histories will help us to reconstruct the origin of present-day geographical distribution and diversity of clinical pathogen infections, and will hopefully allow us to foresee possible environmentally induced pathogen evolutionary responses. Lastly, we emphasize that ancient pathogen DNA research should be combined with modern clinical pathogen data, and be equitable and provide advantages for all researchers worldwide, e.g., through shared data.

Description: Genes, Vol. 9, Pages 322: APC and MUTYH Analysis in FAP Patients: A Novel Mutation in APC Gene and Genotype-Phenotype Correlation Genes doi: 10.3390/genes9070322 Authors: Giovanna D’Elia Gemma Caliendo Amelia Casamassimi Michele Cioffi Anna Maria Molinari Maria Teresa Vietri APC and MUTYH genes are mutated in 70–90% and 10–30% of familial adenomatous polyposis cases (FAP) respectively. An association between mutation localization and FAP clinical phenotype is reported. The aims of this study were to determine APC and MUTYH mutational status in a small cohort of FAP patients and to evaluate the genotype-phenotype correlation in mutated patients. Here, we report the identification of a novel APC germline mutation, c.510_511insA. Overall, mutational analysis showed pathogenic mutations in 6/10 patients: 5/10 in APC and 1/10 in MUTYH. Additionally, we found three variants of unknown significance in MUTYH gene that showed no evidence of possible splicing defects by in silico analysis. Molecular analysis was also extended to family members of mutated patients. A genotype-phenotype correlation was observed for colonic signs whereas a variation of disease onset age was revealed for the same mutation. Moreover, we found an intrafamilial variability of FAP onset age. Regarding extracolonic manifestations, the development of desmoid tumors was related to surgery and not to mutation position, while a genotype-phenotype correspondence was observed for the onset of thyroid or gastric cancer. These findings can be useful in association to clinical data for early surveillance and suitable treatment of FAP patients.

Description: Genes, Vol. 9, Pages 327: Transcriptome-Wide Identification of an Aurone Glycosyltransferase with Glycosidase Activity from Ornithogalum saundersiae Genes doi: 10.3390/genes9070327 Authors: Shuai Yuan Ming Liu Yan Yang Jiu-Ming He Ya-Nan Wang Jian-Qiang Kong Aurone glycosides display a variety of biological activities. However, reports about glycosyltransferases (GTs) responsible for aurones glycosylation are limited. Here, the transcriptome-wide discovery and identification of an aurone glycosyltransferase with glycosidase activity is reported. Specifically, a complementary DNA (cDNA), designated as OsUGT1, was isolated from the plant Ornithogalum saundersiae based on transcriptome mining. Conserved domain (CD)-search speculated OsUGT1 as a flavonoid GT. Phylogenetically, OsUGT1 is clustered as the same phylogenetic group with a putative 5,6-dihydroxyindoline-2-carboxylic acid (cyclo-DOPA) 5-O-glucosyltransferase, suggesting OsUGT1 may be an aurone glycosyltransferase. The purified OsUGT1 was therefore used as a biocatalyst to incubate with the representative aurone sulfuretin. In vitro enzymatic analyses showed that OsUGT1 was able to catalyze sulfuretin to form corresponding monoglycosides, suggesting OsUGT1 was indeed an aurone glycosyltransferase. OsUGT1 was observed to be a flavonoid GT, specific for flavonoid substrates. Moreover, OsUGT1 was demonstrated to display transglucosylation activity, transferring glucosyl group to sulfuretin via o-Nitrophenyl-β-d-glucopyranoside (oNP-β-Glc)-dependent fashion. In addition, OsUGT1-catalyzed hydrolysis was observed. This multifunctionality of OcUGT1 will broaden the application of OcUGT1 in glycosylation of aurones and other flavonoids.

Description: Genes, Vol. 9, Pages 387: Olfactory Communication via Microbiota: What Is Known in Birds? Genes doi: 10.3390/genes9080387 Authors: Öncü Maraci Kathrin Engel Barbara A. Caspers Animal bodies harbour a complex and diverse community of microorganisms and accumulating evidence has revealed that microbes can influence the hosts’ behaviour, for example by altering body odours. Microbial communities produce odorant molecules as metabolic by-products and thereby modulate the biochemical signalling profiles of their animal hosts. As the diversity and the relative abundance of microbial species are influenced by several factors including host-specific factors, environmental factors and social interactions, there are substantial individual variations in the composition of microbial communities. In turn, the variations in microbial communities would consequently affect social and communicative behaviour by influencing recognition cues of the hosts. Therefore, microbiota studies have a great potential to expand our understanding of recognition of conspecifics, group members and kin. In this review, we aim to summarize existing knowledge of the factors influencing the microbial communities and the effect of microbiota on olfactory cue production and social and communicative behaviour. We concentrate on avian taxa, yet we also include recent research performed on non-avian species when necessary.

Description: Genes, Vol. 9, Pages 392: RNA Structure Elements Conserved between Mouse and 59 Other Vertebrates Genes doi: 10.3390/genes9080392 Authors: Bernhard Thiel Roman Ochsenreiter Veerendra Gadekar Andrea Tanzer Ivo L. Hofacker In this work, we present a computational screen conducted for functional RNA structures, resulting in over 100,000 conserved RNA structure elements found in alignments of mouse (mm10) against 59 other vertebrates. We explicitly included masked repeat regions to explore the potential of transposable elements and low-complexity regions to give rise to regulatory RNA elements. In our analysis pipeline, we implemented a four-step procedure: (i) we screened genome-wide alignments for potential structure elements using RNAz-2, (ii) realigned and refined candidate loci with LocARNA-P, (iii) scored candidates again with RNAz-2 in structure alignment mode, and (iv) searched for additional homologous loci in mouse genome that were not covered by genome alignments. The 3’-untranslated regions (3’-UTRs) of protein-coding genes and small noncoding RNAs are enriched for structures, while coding sequences are depleted. Repeat-associated loci make up about 95% of the homologous loci identified and are, as expected, predominantly found in intronic and intergenic regions. Nevertheless, we report the structure elements enriched in specific genome elements, such as 3’-UTRs and long noncoding RNAs (lncRNAs). We provide full access to our results via a custom UCSC genome browser trackhub freely available on our website (http://rna.tbi.univie.ac.at/trackhubs/#RNAz).

Description: Genes, Vol. 9, Pages 411: Pervasive Modulation of Obesity Risk by the Environment and Genomic Background Genes doi: 10.3390/genes9080411 Authors: Sini Nagpal Greg Gibson Urko M. Marigorta The prevalence of the so-called diseases of affluence, such as type 2 diabetes or hypertension, has increased dramatically in the last two generations. Although genome-wide association studies (GWAS) have discovered hundreds of genes involved in disease etiology, the sudden increase in disease incidence suggests a major role for environmental risk factors. Obesity constitutes a case example of a modern trait shaped by contemporary environment, although with considerable debates about the extent to which gene-by-environment (G×E) interactions accentuate obesity risk in individuals following obesogenic lifestyles. Although interaction effects have been robustly confirmed at the FTO locus, accumulating evidence at the genome-wide level implicates a role for polygenic risk-by-environment interactions. Through a variety of analyses using the UK Biobank, we confirm that the genomic background plays a major role in shaping the expressivity of alleles that increase body mass index (BMI).

Description: Genes, Vol. 9, Pages 417: Membrane Surface Features of Blastocystis Subtypes Genes doi: 10.3390/genes9080417 Authors: John Anthony Yason Kevin Shyong Wei Tan Blastocystis is a common intestinal protistan parasite with global distribution. Blastocystis is a species complex composed of several isolates with biological and morphological differences. The surface coats of Blastocystis from three different isolates representing three subtypes were analyzed using scanning electron microscopy. This structure contains carbohydrate components that are also present in surface glycoconjugates in other parasitic protozoa. Electron micrographs show variations in the surface coats from the three Blastocystis isolates. These differences could be associated with the differences in the pathogenic potential of Blastocystis subtypes. Apart from the surface coat, a plasma membrane-associated surface antigen has been described for Blastocystis ST7 and is associated with programmed cell death features of the parasite.

Description: Genes, Vol. 9, Pages 416: Maneuvers on PCNA Rings during DNA Replication and Repair Genes doi: 10.3390/genes9080416 Authors: Dea Slade DNA replication and repair are essential cellular processes that ensure genome duplication and safeguard the genome from deleterious mutations. Both processes utilize an abundance of enzymatic functions that need to be tightly regulated to ensure dynamic exchange of DNA replication and repair factors. Proliferating cell nuclear antigen (PCNA) is the major coordinator of faithful and processive replication and DNA repair at replication forks. Post-translational modifications of PCNA, ubiquitination and acetylation in particular, regulate the dynamics of PCNA-protein interactions. Proliferating cell nuclear antigen (PCNA) monoubiquitination elicits ‘polymerase switching’, whereby stalled replicative polymerase is replaced with a specialized polymerase, while PCNA acetylation may reduce the processivity of replicative polymerases to promote homologous recombination-dependent repair. While regulatory functions of PCNA ubiquitination and acetylation have been well established, the regulation of PCNA-binding proteins remains underexplored. Considering the vast number of PCNA-binding proteins, many of which have similar PCNA binding affinities, the question arises as to the regulation of the strength and sequence of their binding to PCNA. Here I provide an overview of post-translational modifications on both PCNA and PCNA-interacting proteins and discuss their relevance for the regulation of the dynamic processes of DNA replication and repair.

Description: Genes, Vol. 9, Pages 419: Comparative Genomics and Description of Putative Virulence Factors of Melissococcus plutonius, the Causative Agent of European Foulbrood Disease in Honey Bees Genes doi: 10.3390/genes9080419 Authors: Marvin Djukic Silvio Erler Andreas Leimbach Daniela Grossar Jean-Daniel Charrière Laurent Gauthier Denise Hartken Sascha Dietrich Heiko Nacke Rolf Daniel Anja Poehlein In Europe, approximately 84% of cultivated crop species depend on insect pollinators, mainly bees. Apis mellifera (the Western honey bee) is the most important commercial pollinator worldwide. The Gram-positive bacterium Melissococcus plutonius is the causative agent of European foulbrood (EFB), a global honey bee brood disease. In order to detect putative virulence factors, we sequenced and analyzed the genomes of 14 M. plutonius strains, including two reference isolates. The isolates do not show a high diversity in genome size or number of predicted protein-encoding genes, ranging from 2.021 to 2.101 Mbp and 1589 to 1686, respectively. Comparative genomics detected genes that might play a role in EFB pathogenesis and ultimately in the death of the honey bee larvae. These include bacteriocins, bacteria cell surface- and host cell adhesion-associated proteins, an enterococcal polysaccharide antigen, an epsilon toxin, proteolytic enzymes, and capsule-associated proteins. In vivo expression of three putative virulence factors (endo-alpha-N-acetylgalactosaminidase, enhancin and epsilon toxin) was verified using naturally infected larvae. With our strain collection, we show for the first time that genomic differences exist between non-virulent and virulent typical strains, as well as a highly virulent atypical strain, that may contribute to the virulence of M. plutonius. Finally, we also detected a high number of conserved pseudogenes (75 to 156) per genome, which indicates genomic reduction during evolutionary host adaptation.

Description: Genes, Vol. 9, Pages 427: Genetic Variants in pre-miR-146a, pre-miR-499, pre-miR-125a, pre-miR-605, and pri-miR-182 Are Associated with Breast Cancer Susceptibility in a South American Population Genes doi: 10.3390/genes9090427 Authors: Sebastián Morales Tomas De Mayo Felipe Andrés Gulppi Patricio Gonzalez-Hormazabal Valentina Carrasco José Miguel Reyes Fernando Gómez Enrique Waugh Lilian Jara Breast cancer (BC) is one of the most frequent tumors affecting women worldwide. microRNAs (miRNAs) single-nucleotide polymorphisms (SNPs) likely contribute to BC susceptibility. We evaluated the association of five SNPs with BC risk in non-carriers of the BRCA1/2-mutation from a South American population. The SNPs were genotyped in 440 Chilean BRCA1/2-negative BC cases and 1048 controls. Our data do not support an association between rs2910164:G>C or rs3746444:A>G and BC risk. The rs12975333:G>T is monomorphic in the Chilean population. The pre-miR-605 rs2043556-C allele was associated with a decreased risk of BC, both in patients with a strong family history of BC and in early-onset non-familial BC (Odds ratio (OR) = 0.5 [95% confidence interval (CI) 0.4–0.9] p = 0.006 and OR = 0.6 [95% CI 0.5–0.9] p = 0.02, respectively). The rs4541843-T allele is associated with increased risk of familial BC. This is the first association study on rs4541843 and BC risk. Previously, we showed that the TOX3-rs3803662:C>T was significantly associated with increased risk of familial BC. Given that TOX3 mRNA is a target of miR-182, and that both the TOX3 rs3803662-T and pri-miR-182 rs4541843-T alleles are associated with increased BC risk, we evaluated their combined effect. Risk of familial BC increased in a dose-dependent manner with the number of risk alleles (p-trend = 0.0005), indicating an additive effect.

Description: Genes, Vol. 9, Pages 426: RBPvsMIR: A Computational Pipeline to Identify Competing miRNAs and RNA-Binding Protein Pairs Regulating the Shared Transcripts Genes doi: 10.3390/genes9090426 Authors: Xing Zhao Danze Chen Yujie Cai Fan Zhang Jianzhen Xu Gene post-transcription regulation involves several critical regulators such as microRNAs (miRNAs) and RNA-binding proteins (RBPs). Accumulated experimental evidences have shown that miRNAs and RBPs can competitively regulate the shared targeting transcripts. Although this establishes a novel post-transcription regulation mechanism, there are currently no computational tools to scan for the possible competing miRNA and RBP pairs. Here, we developed a novel computational pipeline—RBPvsMIR—that enables us to statistically evaluate the competing relationship between miRNAs and RBPs. RBPvsMIR first combines with previously successful miRNAs and RBP motifs discovery applications to search for overlapping or adjacent binding sites along a given RNA sequence. Then a permutation test is performed to select the miRNA and RBP pairs with the significantly enriched binding sites. As an example, we used RBPvsMIR to identify 235 competing RBP-miRNA pairs for long non-coding RNA (lncRNA) MALAT1. Wet lab experiments verified that splicing factor SRSF2 competes with miR-383, miR-502 and miR-101 to regulate MALAT1 in esophageal squamous carcinoma cells. Our study also revealed the global mutual exclusive pattern for miRNAs and RBP to regulate human lncRNAs. In addition, we provided a convenient web server (http://bmc.med.stu.edu.cn/RBPvsMIR), which should accelerate the exploration of competing miRNAs and RBP pairs regulating the shared targeting transcripts.

Description: Genes, Vol. 9, Pages 432: Bioinformatics Tools and Benchmarks for Computational Docking and 3D Structure Prediction of RNA-Protein Complexes Genes doi: 10.3390/genes9090432 Authors: Chandran Nithin Pritha Ghosh Janusz M. Bujnicki RNA-protein (RNP) interactions play essential roles in many biological processes, such as regulation of co-transcriptional and post-transcriptional gene expression, RNA splicing, transport, storage and stabilization, as well as protein synthesis. An increasing number of RNP structures would aid in a better understanding of these processes. However, due to the technical difficulties associated with experimental determination of macromolecular structures by high-resolution methods, studies on RNP recognition and complex formation present significant challenges. As an alternative, computational prediction of RNP interactions can be carried out. Structural models obtained by theoretical predictive methods are, in general, less reliable compared to models based on experimental measurements but they can be sufficiently accurate to be used as a basis for to formulating functional hypotheses. In this article, we present an overview of computational methods for 3D structure prediction of RNP complexes. We discuss currently available methods for macromolecular docking and for scoring 3D structural models of RNP complexes in particular. Additionally, we also review benchmarks that have been developed to assess the accuracy of these methods.

Description: Genes, Vol. 9, Pages 431: Comparative Transcriptomics of Root Development in Wild and Cultivated Carrots Genes doi: 10.3390/genes9090431 Authors: Gabriela Machaj Hamed Bostan Alicja Macko-Podgórni Massimo Iorizzo Dariusz Grzebelus The carrot is the most popular root vegetable worldwide. The genetic makeup underlying the development of the edible storage root are fragmentary. Here, we report the first comparative transcriptome analysis between wild and cultivated carrot roots at multiple developmental stages. Overall, 3285, 4637, and 570 genes were differentially expressed in the cultivated carrot in comparisons made for young plants versus developing roots, young plants versus mature roots, and developing roots versus mature roots, respectively. Of those, 1916, 2645, and 475, respectively, were retained after filtering out genes showing similar profiles of expression in the wild carrot. They were assumed to be of special interest with respect to the development of the storage root. Among them, transcription factors and genes encoding proteins involved in post-translational modifications (signal transduction and ubiquitination) were mostly upregulated, while those involved in redox signaling were mostly downregulated. Also, genes encoding proteins regulating cell cycle, involved in cell divisions, development of vascular tissue, water transport, and sugar metabolism were enriched in the upregulated clusters. Genes encoding components of photosystem I and II, together with genes involved in carotenoid biosynthesis, were upregulated in the cultivated roots, as opposed to the wild roots; however, they were largely downregulated in the mature storage root, as compared with the young and developing root. The experiment produced robust resources for future investigations on the regulation of storage root formation in carrot and Apiaceae.

Description: Genes, Vol. 9, Pages 434: Functional Analyses of a Putative, Membrane-Bound, Peroxisomal Protein Import Mechanism from the Apicomplexan Protozoan Toxoplasma gondii Genes doi: 10.3390/genes9090434 Authors: Alison J. Mbekeani Will A. Stanley Vishal C. Kalel Noa Dahan Einat Zalckvar Lilach Sheiner Wolfgang Schliebs Ralf Erdmann Ehmke Pohl Paul W. Denny Peroxisomes are central to eukaryotic metabolism, including the oxidation of fatty acids—which subsequently provide an important source of metabolic energy—and in the biosynthesis of cholesterol and plasmalogens. However, the presence and nature of peroxisomes in the parasitic apicomplexan protozoa remains controversial. A survey of the available genomes revealed that genes encoding peroxisome biogenesis factors, so-called peroxins (Pex), are only present in a subset of these parasites, the coccidia. The basic principle of peroxisomal protein import is evolutionarily conserved, proteins harbouring a peroxisomal-targeting signal 1 (PTS1) interact in the cytosol with the shuttling receptor Pex5 and are then imported into the peroxisome via the membrane-bound protein complex formed by Pex13 and Pex14. Surprisingly, whilst Pex5 is clearly identifiable, Pex13 and, perhaps, Pex14 are apparently absent from the coccidian genomes. To investigate the functionality of the PTS1 import mechanism in these parasites, expression of Pex5 from the model coccidian Toxoplasma gondii was shown to rescue the import defect of Pex5-deleted Saccharomyces cerevisiae. In support of these data, green fluorescent protein (GFP) bearing the enhanced (e)PTS1 known to efficiently localise to peroxisomes in yeast, localised to peroxisome-like bodies when expressed in Toxoplasma. Furthermore, the PTS1-binding domain of Pex5 and a PTS1 ligand from the putatively peroxisome-localised Toxoplasma sterol carrier protein (SCP2) were shown to interact in vitro. Taken together, these data demonstrate that the Pex5–PTS1 interaction is functional in the coccidia and indicate that a nonconventional peroxisomal import mechanism may operate in the absence of Pex13 and Pex14.

Description: Genes, Vol. 9, Pages 435: FDHE-IW: A Fast Approach for Detecting High-Order Epistasis in Genome-Wide Case-Control Studies Genes doi: 10.3390/genes9090435 Authors: Shouheng Tuo Detecting high-order epistasis in genome-wide association studies (GWASs) is of importance when characterizing complex human diseases. However, the enormous numbers of possible single-nucleotide polymorphism (SNP) combinations and the diversity among diseases presents a significant computational challenge. Herein, a fast method for detecting high-order epistasis based on an interaction weight (FDHE-IW) method is evaluated in the detection of SNP combinations associated with disease. First, the symmetrical uncertainty (SU) value for each SNP is calculated. Then, the top-k SNPs are isolated as guiders to identify 2-way SNP combinations with significant interaction weight values. Next, a forward search is employed to detect high-order SNP combinations with significant interaction weight values as candidates. Finally, the findings were statistically evaluated using a G-test to isolate true positives. The developed algorithm was used to evaluate 12 simulated datasets and an age-related macular degeneration (AMD) dataset and was shown to perform robustly in the detection of some high-order disease-causing models.

Description: Genes, Vol. 9, Pages 433: Specific LTR-Retrotransposons Show Copy Number Variations between Wild and Cultivated Sunflowers Genes doi: 10.3390/genes9090433 Authors: Flavia Mascagni Alberto Vangelisti Tommaso Giordani Andrea Cavallini Lucia Natali The relationship between variation of the repetitive component of the genome and domestication in plant species is not fully understood. In previous work, variations in the abundance and proximity to genes of long terminal repeats (LTR)-retrotransposons of sunflower (Helianthus annuus L.) were investigated by Illumina DNA sequencingtocompare cultivars and wild accessions. In this study, we annotated and characterized 22 specific retrotransposon families whose abundance varies between domesticated and wild genotypes. These families mostly belonged to the Chromovirus lineage of the Gypsy superfamily and were distributed overall chromosomes. They were also analyzed in respect to their proximity to genes. Genes close to retrotransposon were classified according to biochemical pathways, and differences between domesticated and wild genotypes are shown. These data suggest that structural variations related to retrotransposons might have occurred to produce phenotypic variation between wild and domesticated genotypes, possibly by affecting the expression of genes that lie close to inserted or deleted retrotransposons and belong to specific biochemical pathways as those involved in plant stress responses.

Description: Genes, Vol. 9, Pages 437: Network-Based Approaches to Explore Complex Biological Systems towards Network Medicine Genes doi: 10.3390/genes9090437 Authors: Giulia Fiscon Federica Conte Lorenzo Farina Paola Paci Network medicine relies on different types of networks: from the molecular level of protein–protein interactions to gene regulatory network and correlation studies of gene expression. Among network approaches based on the analysis of the topological properties of protein–protein interaction (PPI) networks, we discuss the widespread DIAMOnD (disease module detection) algorithm. Starting from the assumption that PPI networks can be viewed as maps where diseases can be identified with localized perturbation within a specific neighborhood (i.e., disease modules), DIAMOnD performs a systematic analysis of the human PPI network to uncover new disease-associated genes by exploiting the connectivity significance instead of connection density. The past few years have witnessed the increasing interest in understanding the molecular mechanism of post-transcriptional regulation with a special emphasis on non-coding RNAs since they are emerging as key regulators of many cellular processes in both physiological and pathological states. Recent findings show that coding genes are not the only targets that microRNAs interact with. In fact, there is a pool of different RNAs—including long non-coding RNAs (lncRNAs) —competing with each other to attract microRNAs for interactions, thus acting as competing endogenous RNAs (ceRNAs). The framework of regulatory networks provides a powerful tool to gather new insights into ceRNA regulatory mechanisms. Here, we describe a data-driven model recently developed to explore the lncRNA-associated ceRNA activity in breast invasive carcinoma. On the other hand, a very promising example of the co-expression network is the one implemented by the software SWIM (switch miner), which combines topological properties of correlation networks with gene expression data in order to identify a small pool of genes—called switch genes—critically associated with drastic changes in cell phenotype. Here, we describe SWIM tool along with its applications to cancer research and compare its predictions with DIAMOnD disease genes.

Description: Genes, Vol. 9, Pages 439: Wnt Effector TCF4 Is Dispensable for Wnt Signaling in Human Cancer Cells Genes doi: 10.3390/genes9090439 Authors: Dusan Hrckulak Lucie Janeckova Lucie Lanikova Vitezslav Kriz Monika Horazna Olga Babosova Martina Vojtechova Katerina Galuskova Eva Sloncova Vladimir Korinek T-cell factor 4 (TCF4), together with β-catenin coactivator, functions as the major transcriptional mediator of the canonical wingless/integrated (Wnt) signaling pathway in the intestinal epithelium. The pathway activity is essential for both intestinal homeostasis and tumorigenesis. To date, several mouse models and cellular systems have been used to analyze TCF4 function. However, some findings were conflicting, especially those that were related to the defects observed in the mouse gastrointestinal tract after Tcf4 gene deletion, or to a potential tumor suppressive role of the gene in intestinal cancer cells or tumors. Here, we present the results obtained using a newly generated conditional Tcf4 allele that allows inactivation of all potential Tcf4 isoforms in the mouse tissue or small intestinal and colon organoids. We also employed the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to disrupt the TCF4 gene in human cells. We showed that in adult mice, epithelial expression of Tcf4 is indispensable for cell proliferation and tumor initiation. However, in human cells, the TCF4 role is redundant with the related T-cell factor 1 (TCF1) and lymphoid enhancer-binding factor 1 (LEF1) transcription factors.

Description: Genes, Vol. 9, Pages 441: The drnf1 Gene from the Drought-Adapted Cyanobacterium Nostoc flagelliforme Improved Salt Tolerance in Transgenic Synechocystis and Arabidopsis Plant Genes doi: 10.3390/genes9090441 Authors: Lijuan Cui Yinghui Liu Yiwen Yang Shuifeng Ye Hongyi Luo Baosheng Qiu Xiang Gao Environmental abiotic stresses are limiting factors for less tolerant organisms, including soil plants. Abiotic stress tolerance-associated genes from prokaryotic organisms are supposed to have a bright prospect for transgenic application. The drought-adapted cyanobacterium Nostoc flagelliforme is arising as a valuable prokaryotic biotic resource for gene excavation. In this study, we evaluated the salt-tolerant function and application potential of a candidate gene drnf1 from N. flagelliforme, which contains a P-loop NTPase (nucleoside-triphosphatase) domain, through heterologous expression in two model organisms Synechocystis sp. PCC 6803 and Arabidopsis thaliana. It was found that DRNF1 could confer significant salt tolerance in both transgenic organisms. In salt-stressed transgenic Synechocystis, DRNF1 could enhance the respiration rate; slow-down the accumulation of exopolysaccharides; up-regulate the expression of salt tolerance-related genes at a higher level, such as those related to glucosylglycerol synthesis, Na+/H+ antiport, and sugar metabolism; and maintain a better K+/Na+ homeostasis, as compared to the wild-type strain. These results imply that DRNF1 could facilitate salt tolerance by affecting the respiration metabolism and indirectly regulating the expression of important salt-tolerant genes. Arabidopsis was employed to evaluate the salt tolerance-conferring potential of DRNF1 in plants. The results show that it could enhance the seed germination and shoot growth of transgenic plants under saline conditions. In general, a novel prokaryotic salt-tolerant gene from N. flagelliforme was identified and characterized in this study, enriching the candidate gene pool for genetic engineering in plants.

Description: Genes, Vol. 9, Pages 446: Bioinformatics Analysis of SNPs in IL-6 Gene Promoter of Jinghai Yellow Chickens Genes doi: 10.3390/genes9090446 Authors: Shijie Xin Xiaohui Wang Guojun Dai Jingjing Zhang Tingting An Wenbin Zou Genxi Zhang Kaizhou Xie Jinyu Wang The proinflammatory cytokine, interleukin-6 (IL-6), plays a critical role in many chronic inflammatory diseases, particularly inflammatory bowel disease. To investigate the regulation of IL-6 gene expression at the molecular level, genomic DNA sequencing of Jinghai yellow chickens (Gallus gallus) was performed to detect single-nucleotide polymorphisms (SNPs) in the region −2200 base pairs (bp) upstream to 500 bp downstream of IL-6. Transcription factor binding sites and CpG islands in the IL-6 promoter region were predicted using bioinformatics software. Twenty-eight SNP sites were identified in IL-6. Four of these 28 SNPs, three [−357 (G > A), −447 (C > G), and −663 (A > G)] in the 5′ regulatory region and one in the 3′ non-coding region [3177 (C > T)] are not labelled in GenBank. Bioinformatics analysis revealed 11 SNPs within the promoter region that altered putative transcription factor binding sites. Furthermore, the C-939G mutation in the promoter region may change the number of CpG islands, and SNPs in the 5′ regulatory region may influence IL-6 gene expression by altering transcription factor binding or CpG methylation status. Genetic diversity analysis revealed that the newly discovered A-663G site significantly deviated from Hardy-Weinberg equilibrium. These results provide a basis for further exploration of the promoter function of the IL-6 gene and the relationships of these SNPs to intestinal inflammation resistance in chickens.

Description: Genes, Vol. 9, Pages 445: Metagenomic Composition Analysis of an Ancient Sequenced Polar Bear Jawbone from Svalbard Genes doi: 10.3390/genes9090445 Authors: Diogo Pratas Morteza Hosseini Gonçalo Grilo Armando J. Pinho Raquel M. Silva Tânia Caetano João Carneiro Filipe Pereira The sequencing of ancient DNA samples provides a novel way to find, characterize, and distinguish exogenous genomes of endogenous targets. After sequencing, computational composition analysis enables filtering of undesired sources in the focal organism, with the purpose of improving the quality of assemblies and subsequent data analysis. More importantly, such analysis allows extinct and extant species to be identified without requiring a specific or new sequencing run. However, the identification of exogenous organisms is a complex task, given the nature and degradation of the samples, and the evident necessity of using efficient computational tools, which rely on algorithms that are both fast and highly sensitive. In this work, we relied on a fast and highly sensitive tool, FALCON-meta, which measures similarity against whole-genome reference databases, to analyse the metagenomic composition of an ancient polar bear (Ursus maritimus) jawbone fossil. The fossil was collected in Svalbard, Norway, and has an estimated age of 110,000 to 130,000 years. The FASTQ samples contained 349 GB of nonamplified shotgun sequencing data. We identified and localized, relative to the FASTQ samples, the genomes with significant similarities to reference microbial genomes, including those of viruses, bacteria, and archaea, and to fungal, mitochondrial, and plastidial sequences. Among other striking features, we found significant similarities between modern-human, some bacterial and viral sequences (contamination) and the organelle sequences of wild carrot and tomato relative to the whole samples. For each exogenous candidate, we ran a damage pattern analysis, which in addition to revealing shallow levels of damage in the plant candidates, identified the source as contamination.

Description: Genes, Vol. 9, Pages 444: Can Genetic Factors Compromise the Success of Dental Implants? A Systematic Review and Meta-Analysis Genes doi: 10.3390/genes9090444 Authors: Joel Ferreira Santiago Junior Claudia Cristina Biguetti Mariza Akemi Matsumoto Guilherme Abu Halawa Kudo Raquel Barroso Parra da Silva Patrícia Pinto Saraiva Walid D. Fakhouri Dental implants provide a predictable treatment option for partial and complete edentulism via the placement of a fixed permanent artificial root to support prosthetic dental crowns. Despite the high survival rates, long-term dental implant failures are still reported, leading to implant removals and additional financial and health burdens. While extrinsic factors that improve the success rate of implants have been well explored, the impact of genetic factors on this matter is poorly understood. A systematic review and meta-analysis study was conducted to determine whether genetic factors contribute to an increased risk of dental implant failures. A comprehensive search for peer-reviewed articles on dental implants and genetic factors was performed using various literature database libraries. The study design was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines, and the obtained records were registered in the International Prospective Register of Systematic Reviews (PROSPERO) database. According to the exclusion/inclusion criteria, 13 studies were eligible for this study out of 809 articles. The meta-analysis of the combined association studies of DNA variations and dental implants did not indicate an increased risk for implant failure due to DNA variations in IL-1B, IL-10 and TNF-α. This study emphasizes the need for larger randomized controlled clinical trials to inform clinicians and patients about the role of genetic factors on dental implant survival and the success rate in healthy and compromised patients.

Description: Genes, Vol. 9, Pages 452: Recent Selection on a Class I ADH Locus Distinguishes Southwest Asian Populations Including Ashkenazi Jews Genes doi: 10.3390/genes9090452 Authors: Sheng Gu Hui Li Andrew Pakstis William Speed David Gurwitz Judith Kidd Kenneth Kidd The derived human alcohol dehydrogenase (ADH)1B*48His allele of the ADH1B Arg48His polymorphism (rs1229984) has been identified as one component of an East Asian specific core haplotype that underwent recent positive selection. Our study has been extended to Southwest Asia and additional markers in East Asia. Fst values (Sewall Wright’s fixation index) and long-range haplotype analyses identify a strong signature of selection not only in East Asian but also in Southwest Asian populations. However, except for the ADH2B*48His allele, different core haplotypes occur in Southwest Asia compared to East Asia and the extended haplotypes also differ. Thus, the ADH1B*48His allele, as part of a core haplotype of 10 kb, has undergone recent rapid increases in frequency independently in the two regions after divergence of the respective populations. Emergence of agriculture may be the common factor underlying the evident selection.

Description: Genes, Vol. 9, Pages 448: Epigenetic Control of Pancreatic Regeneration in Diabetes Genes doi: 10.3390/genes9090448 Authors: Shruti Balaji Tiziana Napolitano Serena Silvano Marika Elsa Friano Anna Garrido-Utrilla Josipa Atlija Patrick Collombat Both type 1 and type 2 diabetes are conditions that are associated with the loss of insulin-producing β-cells within the pancreas. An active research therefore aims at regenerating these β-cells with the hope that they could restore euglycemia. The approaches classically used consist in mimicking embryonic development, making use of diverse cell sources or converting pre-existing pancreatic cells. Despite impressive progresses and promising successes, it appears that we still need to gain further insight into the molecular mechanisms underlying β-cell development. This becomes even more obvious with the emergence of a relatively new field of research, epigenetics. The current review therefore focuses on the latest advances in this field in the context of β-cell (neo-)genesis research.

Description: Genes, Vol. 9, Pages 455: Correction of a Splicing Mutation Affecting an Unverricht-Lundborg Disease Patient by Antisense Therapy Genes doi: 10.3390/genes9090455 Authors: Liliana Matos Ana Joana Duarte Diogo Ribeiro João Chaves Olga Amaral Sandra Alves Unverricht-Lundborg disease (ULD) is a common form of progressive myoclonic epilepsy caused by mutations in the cystatin B gene (CSTB) that encodes an inhibitor of several lysosomal cathepsins. Presently, only pharmacological treatment and psychosocial support are available for ULD patients. To overcome the pathogenic effect of the ULD splicing mutation c.66G>A (exon 1), we investigated whether an antisense oligonucleotide therapeutic strategy could correct the defect in patient cells. A specific locked nucleic acid (LNA) antisense oligonucleotide was designed to block a cryptic 5′ss in intron 1. Overall, this approach allowed the restoration of the normal splicing pattern. Furthermore, the recovery was both sequence and dose-specific. In general, this work provides a proof of principle on the correction of a CSTB gene defect causing ULD through a mutation-specific antisense therapy. It adds evidence to the feasibility of this approach, joining the many studies that are paving the way for translating antisense technology into the clinical practice. The insights detailed herein make mutation-based therapy a clear candidate for personalized treatment of ULD patients, encouraging similar investigations into other genetic diseases.

Description: Genes, Vol. 9, Pages 457: Correction: Li, X.; et al. Identification and Characterization of the WOX Family Genes in Five Solanaceae Species Reveal Their Conserved Roles in Peptide Signaling. Genes 2018, 9, 260. Genes doi: 10.3390/genes9090457 Authors: Xiaoxu Li Madiha Hamyat Cheng Liu Salman Ahmad Xiaoming Gao Cun Guo Yuanying Wang Yongfeng Guo The authors wish to make the following change in their paper [...]

Description: Genes, Vol. 9, Pages 461: Evolutionary Emergence of Drug Resistance in Candida Opportunistic Pathogens Genes doi: 10.3390/genes9090461 Authors: Ewa Ksiezopolska Toni Gabaldón Fungal infections, such as candidiasis caused by Candida, pose a problem of growing medical concern. In developed countries, the incidence of Candida infections is increasing due to the higher survival of susceptible populations, such as immunocompromised patients or the elderly. Existing treatment options are limited to few antifungal drug families with efficacies that vary depending on the infecting species. In this context, the emergence and spread of resistant Candida isolates are being increasingly reported. Understanding how resistance can evolve within naturally susceptible species is key to developing novel, more effective treatment strategies. However, in contrast to the situation of antibiotic resistance in bacteria, few studies have focused on the evolutionary mechanisms leading to drug resistance in fungal species. In this review, we will survey and discuss current knowledge on the genetic bases of resistance to antifungal drugs in Candida opportunistic pathogens. We will do so from an evolutionary genomics perspective, focusing on the possible evolutionary paths that may lead to the emergence and selection of the resistant phenotype. Finally, we will discuss the potential of future studies enabled by current developments in sequencing technologies, in vitro evolution approaches, and the analysis of serial clinical isolates.

Description: Genes, Vol. 9, Pages 460: A Statistical Method for Observing Personal Diploid Methylomes and Transcriptomes with Single-Molecule Real-Time Sequencing Genes doi: 10.3390/genes9090460 Authors: Yuta Suzuki Yunhao Wang Kin Fai Au Shinichi Morishita We address the problem of observing personal diploid methylomes, CpG methylome pairs of homologous chromosomes that are distinguishable with respect to phased heterozygous variants (PHVs), which is challenging due to scarcity of PHVs in personal genomes. Single molecule real-time (SMRT) sequencing is promising as it outputs long reads with CpG methylation information, but a serious concern is whether reliable PHVs are available in erroneous SMRT reads with an error rate of ∼15%. To overcome the issue, we propose a statistical model that reduces the error rate of phasing CpG site to 1%, thereby calling CpG hypomethylation in each haplotype with >90% precision and sensitivity. Using our statistical model, we examined GNAS complex locus known for a combination of maternally, paternally, or biallelically expressed isoforms, and observed allele-specific methylation pattern almost perfectly reflecting their respective allele-specific expression status, demonstrating the merit of elucidating comprehensive personal diploid methylomes and transcriptomes.

Description: Genes, Vol. 9, Pages 466: Comprehensive Transcriptome Profiling and Identification of Potential Genes Responsible for Salt Tolerance in Tall Fescue Leaves under Salinity Stress Genes doi: 10.3390/genes9100466 Authors: Erick Amombo Xiaoning Li Guangyang Wang Shao An Wei Wang Jinmin Fu Soil salinity is a serious threat to plant growth and crop productivity. Tall fescue utilization in saline areas is limited by its inferior salt tolerance. Thus, a transcriptome study is a prerequisite for future research aimed at providing deeper insights into the molecular mechanisms of tall fescue salt tolerance as well as molecular breeding. Recent advances in sequencing technology offer a platform to achieve this. Here, Illumina RNA sequencing of tall fescue leaves generated a total of 144,339 raw reads. After de novo assembly, unigenes with a total length of 129,749,938 base pairs were obtained. For functional annotations, the unigenes were aligned to various databases. Further structural analyses revealed 79,352 coding DNA sequences and 13,003 microsatellites distributed across 11,277 unigenes as well as single nucleotide polymorphisms. In total, 1862 unigenes were predicted to encode for 2120 transcription factors among which most were key salt-responsive. We determined differential gene expression and distribution per sample and most genes related to salt tolerance and photosynthesis were upregulated in 48 h vs. 24 h salt treatment. Protein interaction analysis revealed a high interaction of chaperonins and Rubisco proteins in 48 h vs. 24 h salt treatment. The gene expressions were finally validated using quantitative polymerase chain reaction (qPCR), which was coherent with sequencing results.

Description: Genes, Vol. 9, Pages 465: Molecular Genotyping (SSR) and Agronomic Phenotyping for Utilization of Durum Wheat (Triticum durum Desf.) Ex Situ Collection from Southern Italy: A Combined Approach Including Pedigreed Varieties Genes doi: 10.3390/genes9100465 Authors: Stefania Marzario Giuseppina Logozzo Jacques L. David Pierluigi Spagnoletti Zeuli Tania Gioia In South Italy durum wheat (Triticum durum Desf.) has a long-time tradition of growing and breeding. Accessions collected and now preserved ex situ are a valuable genetic resource, but their effective use in agriculture and breeding programs remains very low. In this study, a small number (44) of simple sequence repeats (SSR) molecular markers were used to detect pattern of diversity for 136 accessions collected in South Italy over time, to identify the genepool of origin, and establish similarities with 28 Italian varieties with known pedigree grown in Italy over the same time-period. Phenotyping was conducted for 12 morphophysiological characters of agronomic interest. Based on discriminant analysis of principal components (DAPC) and STRUCTURE analysis six groups were identified, the assignment of varieties reflected the genetic basis and breeding strategies involved in their development. Some “old” varieties grown today are the result of evolution through natural hybridization and conservative pure line selection. A small number of molecular markers and little phenotyping coupled with powerful statistical analysis and comparison to pedigreed varieties can provide enough information on the genetic structure of durum wheat germplasm for a quick screening of the germplasm collection able to identify accessions for breeding or introduction in low input agriculture.

Description: Genes, Vol. 9, Pages 464: B Chromosomes of the Asian Seabass (Lates calcarifer) Contribute to Genome Variations at the Level of Individuals and Populations Genes doi: 10.3390/genes9100464 Authors: Aleksey Komissarov Shubha Vij Andrey Yurchenko Vladimir Trifonov Natascha Thevasagayam Jolly Saju Prakki Sai Rama Sridatta Kathiresan Purushothaman Alexander Graphodatsky László Orbán Inna Kuznetsova The Asian seabass (Lates calcarifer) is a bony fish from the Latidae family, which is widely distributed in the tropical Indo-West Pacific region. The karyotype of the Asian seabass contains 24 pairs of A chromosomes and a variable number of AT- and GC-rich B chromosomes (Bchrs or Bs). Dot-like shaped and nucleolus-associated AT-rich Bs were microdissected and sequenced earlier. Here we analyzed DNA fragments from Bs to determine their repeat and gene contents using the Asian seabass genome as a reference. Fragments of 75 genes, including an 18S rRNA gene, were found in the Bs; repeats represented 2% of the Bchr assembly. The 18S rDNA of the standard genome and Bs were similar and enriched with fragments of transposable elements. A higher nuclei DNA content in the male gonad and somatic tissue, compared to the female gonad, was demonstrated by flow cytometry. This variation in DNA content could be associated with the intra-individual variation in the number of Bs. A comparison between the copy number variation among the B-related fragments from whole genome resequencing data of Asian seabass individuals identified similar profiles between those from the South-East Asian/Philippines and Indian region but not the Australian ones. Our results suggest that Bs might cause variations in the genome among the individuals and populations of Asian seabass. A personalized copy number approach for segmental duplication detection offers a suitable tool for population-level analysis across specimens with low coverage genome sequencing.

Description: Genes, Vol. 9, Pages 463: Maintaining Genome Integrity during Seed Development in Phaseolus vulgaris L.: Evidence from a Transcriptomic Profiling Study Genes doi: 10.3390/genes9100463 Authors: José Ricardo Parreira Alma Balestrazzi Pedro Fevereiro Susana de Sousa Araújo The maintenance of genome integrity is crucial in seeds, due to the constant challenge of several endogenous and exogenous factors. The knowledge concerning DNA damage response and chromatin remodeling during seed development is still scarce, especially in Phaseolus vulgaris L. A transcriptomic profiling of the expression of genes related to DNA damage response/chromatin remodeling mechanisms was performed in P. vulgaris seeds at four distinct developmental stages, spanning from late embryogenesis to seed desiccation. Of the 14,001 expressed genes identified using massive analysis of cDNA ends, 301 belong to the DNA MapMan category. In late embryogenesis, a high expression of genes related to DNA damage sensing and repair suggests there is a tight control of DNA integrity. At the end of filling and the onset of seed dehydration, the upregulation of genes implicated in sensing of DNA double-strand breaks suggests that genome integrity is challenged. The expression of chromatin remodelers seems to imply a concomitant action of chromatin remodeling with DNA repair machinery, maintaining genome stability. The expression of genes related to nucleotide excision repair and chromatin structure is evidenced during the desiccation stage. An overview of the genes involved in DNA damage response and chromatin remodeling during P. vulgaris seed development is presented, providing insights into the mechanisms used by developing seeds to cope with DNA damage.

Description: Genes, Vol. 9, Pages 402: Speciation Theory of Carcinogenesis Explains Karyotypic Individuality and Long Latencies of Cancers Genes doi: 10.3390/genes9080402 Authors: Ankit Hirpara Mathew Bloomfield Peter Duesberg It has been known for over 100 years that cancers have individual karyotypes and arise only years to decades after initiating carcinogens. However, there is still no coherent theory to explain these definitive characteristics of cancer. The prevailing mutation theory holds that cancers are late because the primary cell must accumulate 3–8 causative mutations to become carcinogenic and that mutations, which induce chromosomal instability (CIN), generate the individual karyotypes of cancers. However, since there is still no proven set of mutations that transforms a normal to a cancer cell, we have recently advanced the theory that carcinogenesis is a form of speciation. This theory predicts carcinogens initiate cancer by inducing aneuploidy, which automatically unbalances thousands of genes and thus catalyzes chain-reactions of progressive aneuploidizations. Over time, these aneuploidizations have two endpoints, either non-viable karyotypes or very rarely karyotypes of new autonomous and immortal cancers. Cancer karyotypes are immortalized despite destabilizing congenital aneuploidy by clonal selections for autonomy—similar to those of conventional species. This theory predicts that the very low probability of converting the karyotype of a normal cell to that of a new autonomous cancer species by random aneuploidizations is the reason for the karyotypic individuality of new cancers and for the long latencies from carcinogens to cancers. In testing this theory, we observed: (1) Addition of mutagenic and non-mutagenic carcinogens to normal human and rat cells generated progressive aneuploidizations months before neoplastic transformation. (2) Sub-cloning of a neoplastic rat clone revealed heritable individual karyotypes, rather than the non-heritable karyotypes predicted by the CIN theory. (3) Analyses of neoplastic and preneoplastic karyotypes unexpectedly identified karyotypes with sets of 3–11 new marker chromosomes without detectable intermediates, consistent with single-step origins. We conclude that the speciation theory explains logically the long latencies from carcinogen exposure and the individuality of cancers. In addition, the theory supports the single-step origins of cancers, because karyotypic autonomy is all-or-nothing. Accordingly, we propose that preneoplastic aneuploidy and clonal neoplastic karyotypes provide more reliable therapeutic indications than current analyses of thousands of mutations.

Description: Genes, Vol. 9, Pages 405: Sequencing of Supernumerary Chromosomes of Red Fox and Raccoon Dog Confirms a Non-Random Gene Acquisition by B Chromosomes Genes doi: 10.3390/genes9080405 Authors: Alexey I. Makunin Svetlana A. Romanenko Violetta R. Beklemisheva Polina L. Perelman Anna S. Druzhkova Kristina O. Petrova Dmitry Yu. Prokopov Ekaterina N. Chernyaeva Jennifer L. Johnson Anna V. Kukekova Fengtang Yang Malcolm A. Ferguson-Smith Alexander S. Graphodatsky Vladimir A. Trifonov B chromosomes (Bs) represent a variable addition to the main karyotype in some lineages of animals and plants. Bs accumulate through non-Mendelian inheritance and become widespread in populations. Despite the presence of multiple genes, most Bs lack specific phenotypic effects, although their influence on host genome epigenetic status and gene expression are recorded. Previously, using sequencing of isolated Bs of ruminants and rodents, we demonstrated that Bs originate as segmental duplications of specific genomic regions, and subsequently experience pseudogenization and repeat accumulation. Here, we used a similar approach to characterize Bs of the red fox (Vulpes vulpes L.) and the Chinese raccoon dog (Nyctereutes procyonoides procyonoides Gray). We confirm the previous findings of the KIT gene on Bs of both species, but demostrate an independent origin of Bs in these species, with two reused regions. Comparison of gene ensembles in Bs of canids, ruminants, and rodents once again indicates enrichment with cell-cycle genes, development-related genes, and genes functioning in the neuron synapse. The presence of B-chromosomal copies of genes involved in cell-cycle regulation and tissue differentiation may indicate importance of these genes for B chromosome establishment.

Description: Genes, Vol. 9, Pages 403: Habitat Fragmentation Reduces Genetic Diversity and Connectivity of the Mexican Spotted Owl: A Simulation Study Using Empirical Resistance Models Genes doi: 10.3390/genes9080403 Authors: Ho Yi Wan Samuel A. Cushman Joseph L. Ganey We evaluated how differences between two empirical resistance models for the same geographic area affected predictions of gene flow processes and genetic diversity for the Mexican spotted owl (Strix occidentalis lucida). The two resistance models represented the landscape under low- and high-fragmentation parameters. Under low fragmentation, the landscape had larger but highly concentrated habitat patches, whereas under high fragmentation, the landscape had smaller habitat patches that scattered across a broader area. Overall habitat amount differed little between resistance models. We tested eight scenarios reflecting a factorial design of three factors: resistance model (low vs. high fragmentation), isolation hypothesis (isolation-by-distance, IBD, vs. isolation-by-resistance, IBR), and dispersal limit of species (200 km vs. 300 km). Higher dispersal limit generally had a positive but small influence on genetic diversity. Genetic distance increased with both geographic distance and landscape resistance, but landscape resistance displayed a stronger influence. Connectivity was positively related to genetic diversity under IBR but was less important under IBD. Fragmentation had a strong negative influence on the spatial patterns of genetic diversity and effective population size (Ns). Despite habitats being more concentrated and less widely distributed, the low-fragmentation landscape had greater genetic diversity than the high-fragmentation landscape, suggesting that highly concentrated but larger habitat patches may provide a genetic refuge for the Mexican spotted owl.

Description: Genes, Vol. 9, Pages 404: Membrane-Associated Proteins in Giardia lamblia Genes doi: 10.3390/genes9080404 Authors: María C. Touz Constanza Feliziani Andrea S. Rópolo The manner in which membrane-associated proteins interact with the membrane defines their subcellular fate and function. This interaction relies on the characteristics of the proteins, their journey after synthesis, and their interaction with other proteins or enzymes. Understanding these properties may help to define the function of a protein and also the role of an organelle. In the case of microorganisms like protozoa parasites, it may help to understand singular features that will eventually lead to the design of parasite-specific drugs. The protozoa parasite Giardia lamblia is an example of a widespread parasite that has been infecting humans and animals from ancestral times, adjusting itself to the changes of the environment inside and outside the host. Several membrane-associated proteins have been posted in the genome database GiardiaDB, although only a few of them have been characterized. This review discusses the data regarding membrane-associated proteins in relationship with lipids and specific organelles and their implication in the discovery of anti-giardial therapies.

Description: Genes, Vol. 9, Pages 406: Nitrogen Supply and Leaf Age Affect the Expression of TaGS1 or TaGS2 Driven by a Constitutive Promoter in Transgenic Tobacco Genes doi: 10.3390/genes9080406 Authors: Yihao Wei Aibo Shi Xiting Jia Zhiyong Zhang Xinming Ma Mingxin Gu Xiaodan Meng Xiaochun Wang Glutamine synthetase (GS) plays a key role in nitrogen metabolism. Here, two types of tobacco transformants, overexpressing Triticum aestivum GS1 (TaGS1) or GS2 (TaGS2), were analysed. Four independent transformed lines, GS1-TR1, GS1-TR2, GS2-TR1 and GS2-TR2, were used for the nitrogen treatment. Under nitrogen-sufficient conditions, the leaves of GS2-TR showed high accumulation of the TaGS2 transcript, while those of GS1-TR showed a low TaGS1 transcript levels. However, compared with nitrogen-sufficient conditions, the TaGS1 transcript level increased in the leaves under nitrogen starvation, but the TaGS2 transcript level decreased. In addition, the TaGS1 and TaGS2 transcript levels were highest in the middle leaves under nitrogen-sufficient and starvation conditions. These results show that nitrogen supply and leaf age regulate TaGS expression, even when they are driven by a super-promoter. Additionally, in regard to nitrogen metabolism level, the lower leaves of the GS1-TR exhibited lower NH4+ and higher amino acid contents, while the upper leaves exhibited higher amino acid, soluble protein and chlorophyll contents. The leaves of the GS2-TR exhibited lower NH4+ but higher amino acid, soluble protein and chlorophyll contents. Given the role that GS isoforms play in nitrogen metabolism, these data suggest that TaGS1 overexpression may improve nitrogen transport, and that TaGS2 overexpression may improve nitrogen assimilation under nitrogen stress.

Description: Genes, Vol. 9, Pages 407: Seasonal and Sexual Differences in the Microbiota of the Hoopoe Uropygial Secretion Genes doi: 10.3390/genes9080407 Authors: Sonia M. Rodríguez-Ruano Manuel Martín-Vivaldi Juan M. Peralta-Sánchez Ana B. García-Martín Ángela Martínez-García Juan J. Soler Eva Valdivia Manuel Martínez-Bueno The uropygial gland of hoopoe nestlings and nesting females hosts bacterial symbionts that cause changes in the characteristics of its secretion, including an increase of its antimicrobial activity. These changes occur only in nesting individuals during the breeding season, possibly associated with the high infection risk experienced during the stay in the hole-nests. However, the knowledge on hoopoes uropygial gland microbial community dynamics is quite limited and based so far on culture-dependent and molecular fingerprinting studies. In this work, we sampled wild and captive hoopoes of different sex, age, and reproductive status, and studied their microbiota using quantitative polymerase chain reaction (qPCR), fluorescence in situ hybridization (FISH) and pyrosequencing. Surprisingly, we found a complex bacterial community in all individuals (including non-nesting ones) during the breeding season. Nevertheless, dark secretions from nesting hoopoes harbored significantly higher bacterial density than white secretions from breeding males and both sexes in winter. We hypothesize that bacterial proliferation may be host-regulated in phases of high infection risk (i.e., nesting). We also highlight the importance of specific antimicrobial-producing bacteria present only in dark secretions that may be key in this defensive symbiosis. Finally, we discuss the possible role of environmental conditions in shaping the uropygial microbiota, based on differences found between wild and captive hoopoes.

Description: Genes, Vol. 9, Pages 408: Authenticity Testing and Detection of Eurycoma longifolia in Commercial Herbal Products Using Bar-High Resolution Melting Analysis Genes doi: 10.3390/genes9080408 Authors: Nur Fadhila Fadzil Alina Wagiran Faezah Mohd Salleh Shamsiah Abdullah Nur Hazwani Mohd Izham The present study demonstrated High Resolution Melting (HRM) analysis combined with DNA barcode (Bar-HRM) as a fast and highly sensitive technique for detecting adulterants in Eurycoma longifolia commercial herbal products. Targeting the DNA barcoding of the chloroplastic region-ribulose biphosphate carboxylase large chain (rbcL) and the nuclear ribosomal region- internal transcribed spacer 2 (ITS2), PCR amplification and HRM analysis using saturated Eva green dye as the source of fluorescence signals, was accomplished by employing a real-time cycler. The results were further validated by sequencing to identify unknown sequence from Genbank database and to generate phylogenetic tree using neighbour joint (NJ) analysis. Both of the DNA markers exhibited a distinguishable melting temperature and shape of the normalised curve between the reference and the adulterants. In the case of species identification, ITS2 was more successful in differentiating between species. Additionally, detection of admixture sample containing small traces of targeted E. longifolia DNA (w/v) can be detected as low as 5% for rbcL and less than 1% for ITS2, proving the sensitivity and versatility of the HRM analysis. In conclusion, the Bar-HRM analysis is a fast and reliable technique that can effectively detect adulterants in herbal products. Therefore, this will be beneficial for regulatory agencies in order to regulate food safety issues.

Description: Genes, Vol. 9, Pages 414: Genome-Wide Quantification of the Effect of Gene Overexpression on Escherichia coli Growth Genes doi: 10.3390/genes9080414 Authors: Hao Chen Sumana Venkat Jessica Wilson Paige McGuire Abigail L. Chang Qinglei Gan Chenguang Fan Recombinant protein production plays an essential role in both biological studies and pharmaceutical production. Escherichia coli is one of the most favorable hosts for this purpose. Although a number of strategies for optimizing protein production have been developed, the effect of gene overexpression on host cell growth has been much less studied. Here, we performed high-throughput tests on the E. coli a complete set of E. coli K-12 ORF archive (ASKA) collection to quantify the effects of overexpressing individual E. coli genes on its growth. The results indicated that overexpressing membrane-associated proteins or proteins with high abundances of branched-chain amino acids tended to impair cell growth, the latter of which could be remedied by amino acid supplementation. Through this study, we expect to provide an index for a fast pre-study estimate of host cell growth in order to choose proper rescuing approaches when working with different proteins.

Description: Genes, Vol. 9, Pages 415: Identification of Novel Candidate Markers of Type 2 Diabetes and Obesity in Russia by Exome Sequencing with a Limited Sample Size Genes doi: 10.3390/genes9080415 Authors: Yury A. Barbitoff Elena A. Serebryakova Yulia A. Nasykhova Alexander V. Predeus Dmitrii E. Polev Anna R. Shuvalova Evgenii V. Vasiliev Stanislav P. Urazov Andrey M. Sarana Sergey G. Scherbak Dmitrii V. Gladyshev Maria S. Pokrovskaya Oksana V. Sivakova Aleksey N. Meshkov Oxana M. Drapkina Oleg S. Glotov Andrey S. Glotov Type 2 diabetes (T2D) and obesity are common chronic disorders with multifactorial etiology. In our study, we performed an exome sequencing analysis of 110 patients of Russian ethnicity together with a multi-perspective approach based on biologically meaningful filtering criteria to detect novel candidate variants and loci for T2D and obesity. We have identified several known single nucleotide polymorphisms (SNPs) as markers for obesity (rs11960429), T2D (rs9379084, rs1126930), and body mass index (BMI) (rs11553746, rs1956549 and rs7195386) (p < 0.05). We show that a method based on scoring of case-specific variants together with selection of protein-altering variants can allow for the interrogation of novel and known candidate markers of T2D and obesity in small samples. Using this method, we identified rs328 in LPL (p = 0.023), rs11863726 in HBQ1 (p = 8 × 10−5), rs112984085 in VAV3 (p = 4.8 × 10−4) for T2D and obesity, rs6271 in DBH (p = 0.043), rs62618693 in QSER1 (p = 0.021), rs61758785 in RAD51B (p = 1.7 × 10−4), rs34042554 in PCDHA1 (p = 1 × 10−4), and rs144183813 in PLEKHA5 (p = 1.7 × 10−4) for obesity; and rs9379084 in RREB1 (p = 0.042), rs2233984 in C6orf15 (p = 0.030), rs61737764 in ITGB6 (p = 0.035), rs17801742 in COL2A1 (p = 8.5 × 10−5), and rs685523 in ADAMTS13 (p = 1 × 10−6) for T2D as important susceptibility loci in Russian population. Our results demonstrate the effectiveness of whole exome sequencing (WES) technologies for searching for novel markers of multifactorial diseases in cohorts of limited size in poorly studied populations.

Description: Genes, Vol. 9, Pages 430: New Insights into the Melanophilin (MLPH) Gene Affecting Coat Color Dilution in Rabbits Genes doi: 10.3390/genes9090430 Authors: Julie Demars Nathalie Iannuccelli Valerio Joe Utzeri Gerard Auvinet Juliette Riquet Luca Fontanesi Daniel Allain Coat color dilution corresponds to a specific pigmentation phenotype that leads to a dilution of wild type pigments. It affects both eumelanin and pheomelanin containing melanosomes. The mode of inheritance of the dilution phenotype is autosomal recessive. Candidate gene approaches focused on the melanophilin (MLPH) gene highlighted two variants associated with the dilution phenotype in rabbits: The c.111-5C>A variant that is located in an acceptor splice site or the c.585delG variant, a frameshift mutation. On the transcript level, the skipping of two exons has been reported as the molecular mechanism responsible for the coat color dilution. To clarify, which of the two variants represents the causal variant, (i) we analyzed their allelic segregation by genotyping Castor and Chinchilla populations, and (ii) we evaluated their functional effects on the stability of MLPH transcripts in skin samples of animals with diluted or wild type coat color. Firstly, we showed that the c.585delG variant showed perfect association with the dilution phenotype in contrast to the intronic c.111-5C>A variant. Secondly, we identified three different MLPH isoforms including the wild type isoform, the exon-skipping isoform and a retained intron isoform. Thirdly, we observed a drastic and significant decrease of MLPH transcript levels in rabbits with a coat color dilution (p-values ranging from 10−03 to 10−06). Together, our results bring new insights into the coat color dilution trait.

Description: Genes, Vol. 9, Pages 429: Profiling DNA Methylation Based on Next-Generation Sequencing Approaches: New Insights and Clinical Applications Genes doi: 10.3390/genes9090429 Authors: Daniela Barros-Silva C. Joana Marques Rui Henrique Carmen Jerónimo DNA methylation is an epigenetic modification that plays a pivotal role in regulating gene expression and, consequently, influences a wide variety of biological processes and diseases. The advances in next-generation sequencing technologies allow for genome-wide profiling of methyl marks both at a single-nucleotide and at a single-cell resolution. These profiling approaches vary in many aspects, such as DNA input, resolution, coverage, and bioinformatics analysis. Thus, the selection of the most feasible method according with the project’s purpose requires in-depth knowledge of those techniques. Currently, high-throughput sequencing techniques are intensively used in epigenomics profiling, which ultimately aims to find novel biomarkers for detection, diagnosis prognosis, and prediction of response to therapy, as well as to discover new targets for personalized treatments. Here, we present, in brief, a portrayal of next-generation sequencing methodologies’ evolution for profiling DNA methylation, highlighting its potential for translational medicine and presenting significant findings in several diseases.

Description: Genes, Vol. 9, Pages 428: Listeria monocytogenes Isolated from Illegally Imported Food Products into the European Union Harbor Different Virulence Factor Variants Genes doi: 10.3390/genes9090428 Authors: Kathrin Rychli Beatrix Stessl Kati Szakmary-Brändle Anja Strauß Martin Wagner Dagmar Schoder Unregulated international flow of foods poses a danger to human health, as it may be contaminated with pathogens. Recent studies have investigated neglected routes of pathogen transmission and reported the occurrence of Listeria monocytogenes in food illegally imported into the European Union (EU), either confiscated at four international airports or sold illegally on the Romanian black market. In this study we investigated the genotype diversity and the amino acid sequence variability of three main virulence factors of 57 L. monocytogenes isolates. These isolates were derived from 1474 food samples illegally imported into the EU and originated from 17 different countries. Multilocus sequence typing revealed 16 different sequence types (STs) indicating moderate genotype diversity. The most prevalent STs were ST2, ST9, and ST121. The pulsed-field gel electrophoresis (PFGE) analysis resulted in 34 unique pulsotypes. PFGE types assigned to the most prevalent STs (ST2, ST9, and ST121) were highly related in their genetic fingerprint. Internalin A (InlA) was present in 20 variants, including six truncated InlA variants, all harbored by isolates of ST9 and ST121. We detected eight ST-specific listeriolysin O (LLO) variants, and among them, one truncated form. The actin-assembly-inducing protein ActA was present in 15 different ST-specific variants, including four ActA variants with an internal truncation. In conclusion, this study shows that L. monocytogenes, isolated from illegally imported food, have moderate genotype diversity, but diverse virulence factors variants, mainly of InlA.

Description: Genes, Vol. 9, Pages 436: Assessing Metagenomic Signals Recovered from Lyuba, a 42,000-Year-Old Permafrost-Preserved Woolly Mammoth Calf Genes doi: 10.3390/genes9090436 Authors: Giada Ferrari Heidi E. L. Lischer Judith Neukamm Enrique Rayo Nicole Borel Andreas Pospischil Frank Rühli Abigail S. Bouwman Michael G. Campana The reconstruction of ancient metagenomes from archaeological material, and their implication in human health and evolution, is one of the most recent advances in paleomicrobiological studies. However, as for all ancient DNA (aDNA) studies, environmental and laboratory contamination need to be specifically addressed. Here we attempted to reconstruct the tissue-specific metagenomes of a 42,000-year-old, permafrost-preserved woolly mammoth calf through shotgun high-throughput sequencing. We analyzed the taxonomic composition of all tissue samples together with environmental and non-template experimental controls and compared them to metagenomes obtained from permafrost and elephant fecal samples. Preliminary results suggested the presence of tissue-specific metagenomic signals. We identified bacterial species that were present in only one experimental sample, absent from controls, and consistent with the nature of the samples. However, we failed to further authenticate any of these signals and conclude that, even when experimental samples are distinct from environmental and laboratory controls, this does not necessarily indicate endogenous presence of ancient host-associated microbiomic signals.

Description: Genes, Vol. 9, Pages 440: L Chromosome Behaviour and Chromosomal Imprinting in Sciara Coprophila Genes doi: 10.3390/genes9090440 Authors: Prim B. Singh Stepan N. Belyakin The retention of supernumerary chromosomes in the germ-line of Sciara coprophila is part of a highly-intricate pattern of chromosome behaviours that have fascinated cytogeneticists for over 80 years. Germ-line limited (termed L or “limited”) chromosomes are cytologically heterochromatic and late-replicating, with more recent studies confirming they possess epigenetic hallmarks characteristic of constitutive heterochromatin. Little is known about their genetic constitution although they have been found to undergo cycles of condensation and de-condensation at different stages of development. Unlike most supernumeraries, the L chromosomes in S. coprophila are thought to be indispensable, although in two closely related species Sciara ocellaris and Sciara reynoldsi the L chromosomes, have been lost during evolution. Here, we review what we know about L chromosomes in Sciara coprophila. We end by discussing how study of the L chromosome condensation cycle has provided insight into the site and timing of both the erasure of parental “imprints” and also the placement of a putative “imprint” that might be carried by the sperm into the egg.

Description: Genes, Vol. 9, Pages 451: Genome-Wide Identification of PIFs in Grapes (Vitis vinifera L.) and Their Transcriptional Analysis under Lighting/Shading Conditions Genes doi: 10.3390/genes9090451 Authors: Kekun Zhang Ting Zheng Xudong Zhu Songtao Jiu Zhongjie Liu Le Guan Haifeng Jia Jinggui Fang Phytochrome-interacting factors (PIFs), as the basic helix–loop–helix (bHLH) transcription factors, are the primary signaling partners for phytochromes (PHY) that play a key role in PHY-mediated light signal transduction. At present, there are few studies on PIFs in fruit trees. In order to clarify the status of PIFs in grapevines, we identified members of the grape PIFs family and conducted phylogenetic and expression analysis. We identified PIF1, PIF3, PIF4, and PIF7 in PIFs families of the grapevine (Vitis vinifera L.), which were distributed on four different chromosomes with similar gene structures. Except for the closer relationship with PIF1 of citrus, PIFs of grape were distant from the other fruit species such as apple, pear, peach, and strawberry. The VvPIFs (except VvPIF4) were located in the syntenic block with those from Arabidopsis thaliana, Solanum lycopersicum, or Citrus sinensis. In addition to PIF1, all PIFs in grapevines have conserved active PHYB binding (APB) sequences. VvPIF1 has a conserved PIF1-specific active PHYA binding (APA) sequence, while amino acid mutations occurred in the specific APA sequence in VvPIF3. Interestingly, two specific motifs were found in the PIF4 amino acid sequence. The photoreceptor-related elements in the VvPIFs promoter region were the most abundant. PIF1, LONG HYPOCOTYL 5 (HY5) and PIF3, PIF4, GIBBERELLIC ACID INSENSITIVE 1 (GAI1) may interact with each other and participate together in light signal transduction. The relative expression levels of the VvPIFs showed diverse patterns in the various organs at different developmental stages, of which PIF4 was most highly expressed. Prior to maturation, the expression of PIF4 and PIF7 in the skin of the different cultivars increased, while the expression of all PIFs in the flesh decreased. The transcription level of PIFs in grape leaves was sensitive to changes in lighting and shading. Shading treatment was beneficial for enhancing the transcription level of VvPIFs, but the effect on VvPIF3 and VvPIF4 was time-controlled. We concluded that PIFs in grapevines are both conservative and species-specific. The identification and analysis of grape PIFs could provide a theoretical foundation for the further construction of grape light regulation networks.

Description: Genes, Vol. 9, Pages 453: Molecular Characterization of Near Full-Length Genomes of Hepatitis B Virus Isolated from Predominantly HIV Infected Individuals in Botswana Genes doi: 10.3390/genes9090453 Authors: Motswedi Anderson Wonderful Tatenda Choga Sikhulile Moyo Trevor Graham Bell Tshepiso Mbangiwa Bonolo Bonita Phinius Lynnette Bhebhe Theresa Kibirige Sebunya Shahin Lockman Richard Marlink Anna Kramvis Max Essex Rosemary Mubanga Musonda Jason Tory Blackard Simani Gaseitsiwe The World Health Organization plans to eliminate hepatitis B and C Infections by 2030. Therefore, there is a need to study and understand hepatitis B virus (HBV) epidemiology and viral evolution further, including evaluating occult (HBsAg-negative) HBV infection (OBI), given that such infections are frequently undiagnosed and rarely treated. We aimed to molecularly characterize HBV genomes from 108 individuals co-infected with human immunodeficiency virus (HIV) and chronic hepatitis B (CHB) or OBI identified from previous HIV studies conducted in Botswana from 2009 to 2012. Full-length (3.2 kb) and nearly full-length (~3 kb) genomes were amplified by nested polymerase chain reaction (PCR). Sequences from OBI participants were compared to sequences from CHB participants and GenBank references to identify OBI-unique mutations. HBV genomes from 50 (25 CHB and 25 OBI) individuals were successfully genotyped. Among OBI participants, subgenotype A1 was identified in 12 (48%), D3 in 12 (48%), and E in 1 (4%). A similar genotype distribution was observed in CHB participants. Whole HBV genome sequences from Botswana, representing OBI and CHB, were compared for the first time. There were 43 OBI-unique mutations, of which 26 were novel. Future studies using larger sample sizes and functional analysis of OBI-unique mutations are warranted.

Description: Genes, Vol. 9, Pages 447: Role of Homologous Recombination Genes in Repair of Alkylation Base Damage by Candida albicans Genes doi: 10.3390/genes9090447 Authors: Toni Ciudad Alberto Bellido Encarnación Andaluz Belén Hermosa Germán Larriba Candida albicans mutants deficient in homologous recombination (HR) are extremely sensitive to the alkylating agent methyl-methane-sulfonate (MMS). Here, we have investigated the role of HR genes in the protection and repair of C. albicans chromosomes by taking advantage of the heat-labile property (55 °C) of MMS-induced base damage. Acute MMS treatments of cycling cells caused chromosome fragmentation in vitro (55 °C) due to the generation of heat-dependent breaks (HDBs), but not in vivo (30 °C). Following removal of MMS wild type, cells regained the chromosome ladder regardless of whether they were transferred to yeast extract/peptone/dextrose (YPD) or to phosphate buffer saline (PBS); however, repair of HDB/chromosome restitution was faster in YPD, suggesting that it was accelerated by metabolic energy and further fueled by the subsequent overgrowth of survivors. Compared to wild type CAI4, chromosome restitution in YPD was not altered in a Carad59 isogenic derivative, whereas it was significantly delayed in Carad51 and Carad52 counterparts. However, when post-MMS incubation took place in PBS, chromosome restitution in wild type and HR mutants occurred with similar kinetics, suggesting that the exquisite sensitivity of Carad51 and Carad52 mutants to MMS is due to defective fork restart. Overall, our results demonstrate that repair of HDBs by resting cells of C. albicans is rather independent of CaRad51, CaRad52, and CaRad59, suggesting that it occurs mainly by base excision repair (BER).

Description: Genes, Vol. 9, Pages 450: Phosphorylation-Dependent Inhibition of Akt1 Genes doi: 10.3390/genes9090450 Authors: Nileeka Balasuriya McShane McKenna Xuguang Liu Shawn S. C. Li Patrick O’Donoghue Protein kinase B (Akt1) is a proto-oncogene that is overactive in most cancers. Akt1 activation requires phosphorylation at Thr308; phosphorylation at Ser473 further enhances catalytic activity. Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal auto-inhibitory pleckstrin homology (PH) domain. As it was previously difficult to produce Akt1 in site-specific phosphorylated forms, the contribution of each activating phosphorylation site to auto-inhibition was unknown. Using a combination of genetic code expansion and in vivo enzymatic phosphorylation, we produced Akt1 variants containing programmed phosphorylation to probe the interplay between Akt1 phosphorylation status and the auto-inhibitory function of the PH domain. Deletion of the PH domain increased the enzyme activity for all three phosphorylated Akt1 variants. For the doubly phosphorylated enzyme, deletion of the PH domain relieved auto-inhibition by 295-fold. We next found that phosphorylation at Ser473 provided resistance to chemical inhibition by Akti-1/2 inhibitor VIII. The Akti-1/2 inhibitor was most effective against pAkt1T308 and showed four-fold decreased potency with Akt1 variants phosphorylated at Ser473. The data highlight the need to design more potent Akt1 inhibitors that are effective against the doubly phosphorylated and most pathogenic form of Akt1.

Description: Genes, Vol. 9, Pages 449: A Computational Method for Classifying Different Human Tissues with Quantitatively Tissue-Specific Expressed Genes Genes doi: 10.3390/genes9090449 Authors: JiaRui Li Lei Chen Yu-Hang Zhang XiangYin Kong Tao Huang Yu-Dong Cai Tissue-specific gene expression has long been recognized as a crucial key for understanding tissue development and function. Efforts have been made in the past decade to identify tissue-specific expression profiles, such as the Human Proteome Atlas and FANTOM5. However, these studies mainly focused on “qualitatively tissue-specific expressed genes” which are highly enriched in one or a group of tissues but paid less attention to “quantitatively tissue-specific expressed genes”, which are expressed in all or most tissues but with differential expression levels. In this study, we applied machine learning algorithms to build a computational method for identifying “quantitatively tissue-specific expressed genes” capable of distinguishing 25 human tissues from their expression patterns. Our results uncovered the expression of 432 genes as optimal features for tissue classification, which were obtained with a Matthews Correlation Coefficient (MCC) of more than 0.99 yielded by a support vector machine (SVM). This constructed model was superior to the SVM model using tissue enriched genes and yielded MCC of 0.985 on an independent test dataset, indicating its good generalization ability. These 432 genes were proven to be widely expressed in multiple tissues and a literature review of the top 23 genes found that most of them support their discriminating powers. As a complement to previous studies, our discovery of these quantitatively tissue-specific genes provides insights into the detailed understanding of tissue development and function.

Description: Genes, Vol. 9, Pages 454: Genetic Imbalances in Argentinean Patients with Congenital Conotruncal Heart Defects Genes doi: 10.3390/genes9090454 Authors: Marisol Delea Lucía D. Espeche Carlos D. Bruque María Paz Bidondo Lucía S. Massara Jaen Oliveri Paloma Brun Viviana R. Cosentino Celeste Martinoli Norma Tolaba Claudina Picon María Eugenia Ponce Zaldua Silvia Ávila Viviana Gutnisky Myriam Perez Lilian Furforo Noemí D. Buzzalino Rosa Liascovich Boris Groisman Mónica Rittler Sandra Rozental Pablo Barbero Liliana Dain Congenital conotruncal heart defects (CCHD) are a subset of serious congenital heart defects (CHD) of the cardiac outflow tracts or great arteries. Its frequency is estimated in 1/1000 live births, accounting for approximately 10–30% of all CHD cases. Chromosomal abnormalities and copy number variants (CNVs) contribute to the disease risk in patients with syndromic and/or non-syndromic forms. Although largely studied in several populations, their frequencies are barely reported for Latin American countries. The aim of this study was to analyze chromosomal abnormalities, 22q11 deletions, and other genomic imbalances in a group of Argentinean patients with CCHD of unknown etiology. A cohort of 219 patients with isolated CCHD or associated with other major anomalies were referred from different provinces of Argentina. Cytogenetic studies, Multiplex-Ligation-Probe-Amplification (MLPA) and fluorescent in situ hybridization (FISH) analysis were performed. No cytogenetic abnormalities were found. 22q11 deletion was found in 23.5% of the patients from our cohort, 66% only had CHD with no other major anomalies. None of the patients with transposition of the great vessels (TGV) carried the 22q11 deletion. Other 4 clinically relevant CNVs were also observed: a distal low copy repeat (LCR)D-E 22q11 duplication, and 17p13.3, 4q35 and TBX1 deletions. In summary, 25.8% of CCHD patients presented imbalances associated with the disease.

Description: Genes, Vol. 9, Pages 459: Long Non-Coding RNAs as Endogenous Target Mimics and Exploration of Their Role in Low Nutrient Stress Tolerance in Plants Genes doi: 10.3390/genes9090459 Authors: Priyanka Borah Antara Das Matthew J. Milner Arif Ali Alison R. Bentley Renu Pandey Long non-coding RNA (lncRNA) research in plants has recently gained momentum taking cues from studies in animals systems. The availability of next-generation sequencing has enabled genome-wide identification of lncRNA in several plant species. Some lncRNAs are inhibitors of microRNA expression and have a function known as target mimicry with the sequestered transcript known as an endogenous target mimic (eTM). The lncRNAs identified to date show diverse mechanisms of gene regulation, most of which remain poorly understood. In this review, we discuss the role of identified putative lncRNAs that may act as eTMs for nutrient-responsive microRNAs (miRNAs) in plants. If functionally validated, these putative lncRNAs would enhance current understanding of the role of lncRNAs in nutrient homeostasis in plants.

Description: Genes, Vol. 9, Pages 223: Divergent Roles of RPA Homologs of the Model Archaeon Halobacterium salinarum in Survival of DNA Damage Genes doi: 10.3390/genes9040223 Authors: Jessica J. Evans Patrick E. Gygli Julienne McCaskill Linda C. DeVeaux The haloarchaea are unusual in possessing genes for multiple homologs to the ubiquitous single-stranded DNA binding protein (SSB or replication protein A, RPA) found in all three domains of life. Halobacterium salinarum contains five homologs: two are eukaryotic in organization, two are prokaryotic and are encoded on the minichromosomes, and one is uniquely euryarchaeal. Radiation-resistant mutants previously isolated show upregulation of one of the eukaryotic-type RPA genes. Here, we have created deletions in the five RPA operons. These deletion mutants were exposed to DNA-damaging conditions: ionizing radiation, UV radiation, and mitomycin C. Deletion of the euryarchaeal homolog, although not lethal as in Haloferax volcanii, causes severe sensitivity to all of these agents. Deletion of the other RPA/SSB homologs imparts a variable sensitivity to these DNA-damaging agents, suggesting that the different RPA homologs have specialized roles depending on the type of genomic insult encountered.

Description: Genes, Vol. 9, Pages 222: Erratum: Jimena Soledad Cadona, et al.; Pathogenicity Islands Distribution in Non-O157 Shiga Toxin-Producing Escherichia coli (STEC). Genes 2018, 9, 81 Genes doi: 10.3390/genes9040222 Authors: Jimena Cadona Ana Bustamante Juliana González Andrea Sanso We wish to make the following correction to the paper by Soledad-Cadona et al.[...]

Description: Genes, Vol. 9, Pages 224: Genome Wide Identification, Evolutionary, and Expression Analysis of VQ Genes from Two Pyrus Species Genes doi: 10.3390/genes9040224 Authors: Yunpeng Cao Dandan Meng Muhammad Abdullah Qing Jin Yi Lin Yongping Cai The VQ motif-containing gene, a member of the plant-specific genes, is involved in the plant developmental process and various stress responses. The VQ motif-containing gene family has been studied in several plants, such as rice (Oryza sativa), maize (Zea mays), and Arabidopsis (Arabidopsis thaliana). However, no systematic study has been performed in Pyrus species, which have important economic value. In our study, we identified 41 and 28 VQ motif-containing genes in Pyrus bretschneideri and Pyrus communis, respectively. Phylogenetic trees were calculated using A. thaliana and O. sativa VQ motif-containing genes as a template, allowing us to categorize these genes into nine subfamilies. Thirty-two and eight paralogous of VQ motif-containing genes were found in P. bretschneideri and P. communis, respectively, showing that the VQ motif-containing genes had a more remarkable expansion in P. bretschneideri than in P. communis. A total of 31 orthologous pairs were identified from the P. bretschneideri and P. communis VQ motif-containing genes. Additionally, among the paralogs, we found that these duplication gene pairs probably derived from segmental duplication/whole-genome duplication (WGD) events in the genomes of P. bretschneideri and P. communis, respectively. The gene expression profiles in both P. bretschneideri and P. communis fruits suggested functional redundancy for some orthologous gene pairs derived from a common ancestry, and sub-functionalization or neo-functionalization for some of them. Our study provided the first systematic evolutionary analysis of the VQ motif-containing genes in Pyrus, and highlighted the diversification and duplication of VQ motif-containing genes in both P. bretschneideri and P. communis.

Description: Genes, Vol. 9, Pages 220: Full Mitogenomes in the Critically Endangered Kākāpō Reveal Major Post-Glacial and Anthropogenic Effects on Neutral Genetic Diversity Genes doi: 10.3390/genes9040220 Authors: Nicolas Dussex Johanna von Seth Bruce C. Robertson Love Dalén Understanding how species respond to population declines is a central question in conservation and evolutionary biology. Population declines are often associated with loss of genetic diversity, inbreeding and accumulation of deleterious mutations, which can lead to a reduction in fitness and subsequently contribute to extinction. Using temporal approaches can help us understand the effects of population declines on genetic diversity in real time. Sequencing pre-decline as well as post-decline mitogenomes representing all the remaining mitochondrial diversity, we estimated the loss of genetic diversity in the critically endangered kākāpō (Strigops habroptilus). We detected a signal of population expansion coinciding with the end of the Pleistocene last glacial maximum (LGM). Also, we found some evidence for northern and southern lineages, supporting the hypothesis that the species may have been restricted to isolated northern and southern refugia during the LGM. We observed an important loss of neutral genetic diversity associated with European settlement in New Zealand but we could not exclude a population decline associated with Polynesian settlement in New Zealand. However, we did not find evidence for fixation of deleterious mutations. We argue that despite high pre-decline genetic diversity, a rapid and range-wide decline combined with the lek mating system, and life-history traits of kākāpō contributed to a rapid loss of genetic diversity following severe population declines.

Description: Genes, Vol. 9, Pages 221: Genome-Wide Analyses of Calcium Sensors Reveal Their Involvement in Drought Stress Response and Storage Roots Deterioration after Harvest in Cassava Genes doi: 10.3390/genes9040221 Authors: Wei Hu Yan Yan Weiwei Tie Zehong Ding Chunlai Wu Xupo Ding Wenquan Wang Zhiqiang Xia Jianchun Guo Ming Peng Calcium (Ca2+) plays a crucial role in plant development and responses to environmental stimuli. Currently, calmodulins (CaMs), calmodulin-like proteins (CMLs), and calcineurin B-like proteins (CBLs), such as Ca2+ sensors, are not well understood in cassava (Manihot esculenta Crantz), an important tropical crop. In the present study, 8 CaMs, 48 CMLs, and 9 CBLs were genome-wide identified in cassava, which were divided into two, four, and four groups, respectively, based on evolutionary relationship, protein motif, and gene structure analyses. Transcriptomic analysis revealed the expression diversity of cassava CaMs-CMLs-CBLs in distinct tissues and in response to drought stress in different genotypes. Generally, cassava CaMs-CMLs-CBLs showed different expression profiles between cultivated varieties (Arg7 and SC124) and wild ancestor (W14) after drought treatment. In addition, numerous CaMs-CMLs-CBLs were significantly upregulated at 6 h, 12 h, and 48 h after harvest, suggesting their possible role during storage roots (SR) deterioration. Further interaction network and co-expression analyses suggested that a CBL-mediated interaction network was widely involved in SR deterioration. Taken together, this study provides new insights into CaMs-CMLs-CBLs-mediated drought adaption and SR deterioration at the transcription level in cassava, and identifies some candidates for the genetic improvement of cassava.

Description: Genes, Vol. 9, Pages 235: The Most Important Virulence Markers of Yersinia enterocolitica and Their Role during Infection Genes doi: 10.3390/genes9050235 Authors: Agata Bancerz-Kisiel Marta Pieczywek Piotr Łada Wojciech Szweda Yersinia enterocolitica is the causative agent of yersiniosis, a zoonotic disease of growing epidemiological importance with significant consequences for public health. This pathogenic species has been intensively studied for many years. Six biotypes (1A, 1B, 2, 3, 4, 5) and more than 70 serotypes of Y. enterocolitica have been identified to date. The biotypes of Y. enterocolitica are divided according to their pathogenic properties: the non-pathogenic biotype 1A, weakly pathogenic biotypes 2–5, and the highly pathogenic biotype 1B. Due to the complex pathogenesis of yersiniosis, further research is needed to expand our knowledge of the molecular mechanisms involved in the infection process and the clinical course of the disease. Many factors, both plasmid and chromosomal, significantly influence these processes. The aim of this study was to present the most important virulence markers of Y. enterocolitica and their role during infection.

Description: Genes, Vol. 9, Pages 243: The Oncojanus Paradigm of Respiratory Complex I Genes doi: 10.3390/genes9050243 Authors: Giulia Leone Houda Abla Giuseppe Gasparre Anna Maria Porcelli Luisa Iommarini Mitochondrial respiratory function is now recognized as a pivotal player in all the aspects of cancer biology, from tumorigenesis to aggressiveness and chemotherapy resistance. Among the enzymes that compose the respiratory chain, by contributing to energy production, redox equilibrium and oxidative stress, complex I assumes a central role. Complex I defects may arise from mutations in mitochondrial or nuclear DNA, in both structural genes or assembly factors, from alteration of the expression levels of its subunits, or from drug exposure. Since cancer cells have a high-energy demand and require macromolecules for proliferation, it is not surprising that severe complex I defects, caused either by mutations or treatment with specific inhibitors, prevent tumor progression, while contributing to resistance to certain chemotherapeutic agents. On the other hand, enhanced oxidative stress due to mild complex I dysfunction drives an opposite phenotype, as it stimulates cancer cell proliferation and invasiveness. We here review the current knowledge on the contribution of respiratory complex I to cancer biology, highlighting the double-edged role of this metabolic enzyme in tumor progression, metastasis formation, and response to chemotherapy.

Description: Genes, Vol. 9, Pages 244: Chitinase mRNA Levels Determined by QPCR in Crab-Eating Monkey (Macaca fascicularis) Tissues: Species-Specific Expression of Acidic Mammalian Chitinase and Chitotriosidase Genes doi: 10.3390/genes9050244 Authors: Maiko Uehara Eri Tabata Kazuhiro Ishii Akira Sawa Misa Ohno Masayoshi Sakaguchi Vaclav Matoska Peter Bauer Fumitaka Oyama Mice and humans express two active chitinases: acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT1). Both chitinases are thought to play important roles in specific pathophysiological conditions. The crab-eating monkey (Macaca fascicularis) is one of the most frequently used nonhuman primate models in basic and applied biomedical research. Here, we performed gene expression analysis of two chitinases in normal crab-eating monkey tissues by way of quantitative real-time polymerase chain reaction (qPCR) using a single standard DNA molecule. Levels of AMCase and CHIT1 messenger RNAs (mRNAs) were highest in the stomach and the lung, respectively, when compared to other tissues. Comparative gene expression analysis of mouse, monkey, and human using monkey–mouse–human hybrid standard DNA showed that the AMCase mRNA levels were exceptionally high in mouse and monkey stomachs while very low in the human stomach. As for the CHIT1 mRNA, we detected higher levels in the monkey lung when compared with those of mouse and human. The differences of mRNA expression between the species in the stomach tissues were basically reflecting the levels of the chitinolytic activities. These results indicate that gene expression of AMCase and CHIT1 differs between mammalian species and requiring special attention in handling data in chitinase-related studies in particular organisms.

Description: Genes, Vol. 9, Pages 246: tRNA-Derived Small RNA: A Novel Regulatory Small Non-Coding RNA Genes doi: 10.3390/genes9050246 Authors: Siqi Li Zhengping Xu Jinghao Sheng Deep analysis of next-generation sequencing data unveils numerous small non-coding RNAs with distinct functions. Recently, fragments derived from tRNA, named as tRNA-derived small RNA (tsRNA), have attracted broad attention. There are mainly two types of tsRNAs, including tRNA-derived stress-induced RNA (tiRNA) and tRNA-derived fragment (tRF), which differ in the cleavage position of the precursor or mature tRNA transcript. Emerging evidence has shown that tsRNAs are not merely tRNA degradation debris but have been recognized to play regulatory roles in many specific physiological and pathological processes. In this review, we summarize the biogeneses of various tsRNAs, present the emerging concepts regarding functions and mechanisms of action of tsRNAs, highlight the potential application of tsRNAs in human diseases, and put forward the current problems and future research directions.

Description: Genes, Vol. 9, Pages 245: Transcription Factor Binding Site Enrichment Analysis in Co-Expression Modules in Celiac Disease Genes doi: 10.3390/genes9050245 Authors: Irati Romero-Garmendia Koldo Garcia-Etxebarria Hector Hernandez-Vargas Izortze Santin Amaia Jauregi-Miguel Leticia Plaza-Izurieta Marie-Pierre Cros Maria Legarda Iñaki Irastorza Zdenko Herceg Nora Fernandez-Jimenez Jose Ramon Bilbao The aim of this study was to construct celiac co-expression patterns at a whole genome level and to identify transcription factors (TFs) that could drive the gliadin-related changes in coordination of gene expression observed in celiac disease (CD). Differential co-expression modules were identified in the acute and chronic responses to gliadin using expression data from a previous microarray study in duodenal biopsies. Transcription factor binding site (TFBS) and Gene Ontology (GO) annotation enrichment analyses were performed in differentially co-expressed genes (DCGs) and selection of candidate regulators was performed. Expression of candidates was measured in clinical samples and the activation of the TFs was further characterized in C2BBe1 cells upon gliadin challenge. Enrichment analyses of the DCGs identified 10 TFs and five were selected for further investigation. Expression changes related to active CD were detected in four TFs, as well as in several of their in silico predicted targets. The activation of TFs was further characterized in C2BBe1 cells upon gliadin challenge, and an increase in nuclear translocation of CAMP Responsive Element Binding Protein 1 (CREB1) and IFN regulatory factor-1 (IRF1) in response to gliadin was observed. Using transcriptome-wide co-expression analyses we are able to propose novel genes involved in CD pathogenesis that respond upon gliadin stimulation, also in non-celiac models.

Description: Genes, Vol. 9, Pages 251: Erratum: Dan Li et al.; Transcription Factor and lncRNA Regulatory Networks Identify Key Elements in Lung Adenocarcinoma. Genes 2018, 9, 12 Genes doi: 10.3390/genes9050251 Authors: Dan Li William Yang Jialing Zhang Jack Yang Renchu Guan Mary Yang The authors wish to make the following change to their paper [...]

Description: Genes, Vol. 9, Pages 252: Investigating the Epigenetic Discrimination of Identical Twins Using Buccal Swabs, Saliva, and Cigarette Butts in the Forensic Setting Genes doi: 10.3390/genes9050252 Authors: Athina Vidaki Vivian Kalamara Elena Carnero-Montoro Timothy Spector Jordana Bell Manfred Kayser Monozygotic (MZ) twins are typically indistinguishable via forensic DNA profiling. Recently, we demonstrated that epigenetic differentiation of MZ twins is feasible; however, proportions of twin differentially methylated CpG sites (tDMSs) identified in reference-type blood DNA were not replicated in trace-type blood DNA. Here we investigated buccal swabs as typical forensic reference material, and saliva and cigarette butts as commonly encountered forensic trace materials. As an analog to a forensic case, we analyzed one MZ twin pair. Epigenome-wide microarray analysis in reference-type buccal DNA revealed 25 candidate tDMSs with >0.5 twin-to-twin differences. MethyLight quantitative PCR (qPCR) of 22 selected tDMSs in trace-type DNA revealed in saliva DNA that six tDMSs (27.3%) had >0.1 twin-to-twin differences, seven (31.8%) had smaller (<0.1) but robustly detected differences, whereas for nine (40.9%) the differences were in the opposite direction relative to the microarray data; for cigarette butt DNA, results were 50%, 22.7%, and 27.3%, respectively. The discrepancies between reference-type and trace-type DNA outcomes can be explained by cell composition differences, method-to-method variation, and other technical reasons including bisulfite conversion inefficiency. Our study highlights the importance of the DNA source and that careful characterization of biological and technical effects is needed before epigenetic MZ twin differentiation is applicable in forensic casework.

Description: Genes, Vol. 9, Pages 260: Identification and Characterization of the WOX Family Genes in Five Solanaceae Species Reveal Their Conserved Roles in Peptide Signaling Genes doi: 10.3390/genes9050260 Authors: Xiaoxu Li Madiha Hamyat Cheng Liu Ahmad Salman Xiaoming Gao Cun Guo Yuanying Wang Yongfeng Guo Members of the plant-specific WOX (WUSCHEL-related homeobox) transcription factor family have been reported to play important roles in peptide signaling that regulates stem cell maintenance and cell fate specification in various developmental processes. Even though remarkable advances have been made in studying WOX genes in Arabidopsis, little is known about this family in Solanaceae species. A total of 45 WOX members from five Solanaceae species were identified, including eight members from Solanum tuberosum, eight from Nicotiana tomentosiformis, 10 from Solanum lycopersicum, 10 from Nicotiana sylvestris and nine from Nicotiana tabacum. The newly identified WOX members were classified into three clades and nine subgroups based on phylogenetic analysis using three different methods. The patterns of exon-intron structure and motif organization of the WOX proteins agreed with the phylogenetic results. Gene duplication events and ongoing evolution were revealed by additional branches on the phylogenetic tree and the presence of a partial WUS-box in some non-WUS clade members. Gene expression with or without CLE (clavata3 (clv3)/embryo surrounding region-related) peptide treatments revealed that tobacco WOX genes showed similar or distinct expression patterns compared with their Arabidopsis homologues, suggesting either functional conservation or divergence. Expression of Nicotiana tabacum WUSCHEL (NtabWUS) in the organizing center could rescue the wus-1 mutant phenotypes in Arabidopsis, implying conserved roles of the Solanaceae WOX proteins in peptide-mediated regulation of plant development.

Description: Genes, Vol. 9, Pages 264: The Guppy Sex Chromosome System and the Sexually Antagonistic Polymorphism Hypothesis for Y Chromosome Recombination Suppression Genes doi: 10.3390/genes9050264 Authors: Deborah Charlesworth Sex chromosomes regularly evolve suppressed recombination, distinguishing them from other chromosomes, and the reason for this has been debated for many years. It is now clear that non-recombining sex-linked regions have arisen in different ways in different organisms. A major hypothesis is that a sex-determining gene arises on a chromosome and that sexually antagonistic (SA) selection (sometimes called intra-locus sexual conflict) acting at a linked gene has led to the evolution of recombination suppression in the region, to reduce the frequency of low fitness recombinant genotypes produced. The sex chromosome system of the guppy (Poecilia reticulata) is often cited as supporting this hypothesis because SA selection has been demonstrated to act on male coloration in natural populations of this fish, and probably contributes to maintaining polymorphisms for the genetic factors involved. I review classical genetic and new molecular genetic results from the guppy, and other fish, including approaches for identifying the genome regions carrying sex-determining loci, and suggest that the guppy may exemplify a recently proposed route to sex chromosome evolution.

Description: Genes, Vol. 9, Pages 270: Selective Activation of Alternative MYC Core Promoters by Wnt-Responsive Enhancers Genes doi: 10.3390/genes9060270 Authors: Jorge A. Bardales Evin Wieser Hideya Kawaji Yasuhiro Murakawa Xavier Darzacq In Metazoans, transcription of most genes is driven by the use of multiple alternative promoters. Although the precise regulation of alternative promoters is important for proper gene expression, the mechanisms that mediates their differential utilization remains unclear. Here, we investigate how the two alternative promoters (P1, P2) that drive MYC expression are regulated. We find that P1 and P2 can be differentially regulated across cell-types and that their selective usage is largely mediated by distal regulatory sequences. Moreover, we show that in colon carcinoma cells, Wnt-responsive enhancers preferentially upregulate transcription from the P1 promoter using reporter assays and in the context of the endogenous Wnt induction. In addition, multiple enhancer deletions using CRISPR/Cas9 corroborate the regulatory specificity of P1. Finally, we show that preferential activation between Wnt-responsive enhancers and the P1 promoter is influenced by the distinct core promoter elements that are present in the MYC promoters. Taken together, our results provide new insight into how enhancers can specifically target alternative promoters and suggest that formation of these selective interactions could allow more precise combinatorial regulation of transcription initiation.

Description: Genes, Vol. 9, Pages 271: Genetic Association with Subgingival Bacterial Colonization in Chronic Periodontitis Genes doi: 10.3390/genes9060271 Authors: Franco Cavalla Claudia Biguetti Jessica Lima Melchiades Andre Tabanez Michelle de Campos Soriani Azevedo Ana Favaro Trombone Marcelo Faveri Magda Feres Gustavo Pompermaier Garlet Chronic periodontitis is the most prevalent form of inflammatory destructive bone disease and has been affecting humans since antiquity. Evidence suggest that genetic factors can highly influence periodontitis risk, modulating disease elements such as the susceptibility to microbial colonization and the nature of subsequent host-microbe interaction. Several single-nucleotide polymorphisms (SNPs) have been associated with the occurrence of periodontitis, but the full range of genetic influence in periodontitis outcomes remains to be determined. In this context, this study comprises an analysis of possible correlation between periodontitis-related genetic variants with changes in the subgingival microbiological pattern performed in a Brazilian population (n = 167, comprising 76 chronic periodontitis patients and 91 healthy subjects). For the genetic characterization, 19 candidate SNPs were selected based on the top hits of previous large genome wide association studies (GWAS), while the subgingival microbiota was characterized for the presence and relative quantity of 40 bacterial species by DNA-DNA checkerboard. The case/control association test did not demonstrate a significant effect of the target SNPs with the disease phenotype. The polymorphism rs2521634 proved significantly associated with Tannerella. forsythia, Actinomyces gerencseriae, Fusobacterium periodonticum, and Prevotella nigrescens; rs10010758 and rs6667202 were associated with increased counts of Porphyromonas gingivalis; and rs10043775 proved significantly associated with decreased counts of Prevotella intermedia. In conclusion, we present strong evidence supporting a direct connection between the host’s genetic profile, specifically rs2521634, rs10010758, rs6667202, and rs10043775 polymorphisms, and the occurrence of chronic periodontitis-associated bacteria.

Description: Genes, Vol. 9, Pages 273: Genome-Wide Identification and Expression Profiling Analysis of the Xyloglucan Endotransglucosylase/Hydrolase Gene Family in Tobacco (Nicotiana tabacum L.) Genes doi: 10.3390/genes9060273 Authors: Meng Wang Zongchang Xu Anming Ding Yingzhen Kong Xyloglucan endotransglucosylase/hydrolase genes (XTHs) encode enzymes required for the reconstruction and modification of xyloglucan backbones, which will result in changes of cell wall extensibility during growth. A total of 56 NtXTH genes were identified from common tobacco, and 50 cDNA fragments were verified by PCR amplification. The 56 NtXTH genes could be classified into two subfamilies: Group I/II and Group III according to their phylogenetic relationships. The gene structure, chromosomal localization, conserved protein domains prediction, sub-cellular localization of NtXTH proteins and evolutionary relationships among Nicotiana tabacum, Nicotiana sylvestrisis, Nicotiana tomentosiformis, Arabidopsis, and rice were also analyzed. The NtXTHs expression profiles analyzed by the TobEA database and qRT-PCR revealed that NtXTHs display different expression patterns in different tissues. Notably, the expression patterns of 12 NtXTHs responding to environment stresses, including salinity, alkali, heat, chilling, and plant hormones, including IAA and brassinolide, were characterized. All the results would be useful for the function study of NtXTHs during different growth cycles and stresses.

Description: Genes, Vol. 9, Pages 274: Transitions from Single- to Multi-Locus Processes during Speciation with Gene Flow Genes doi: 10.3390/genes9060274 Authors: Martin P. Schilling Sean P. Mullen Marcus Kronforst Rebecca J. Safran Patrik Nosil Jeffrey L. Feder Zachariah Gompert Samuel M. Flaxman During speciation-with-gene-flow, a transition from single-locus to multi-locus processes can occur, as strong coupling of multiple loci creates a barrier to gene flow. Testing predictions about such transitions with empirical data requires building upon past theoretical work and the continued development of quantitative approaches. We simulated genomes under several evolutionary scenarios of gene flow and divergent selection, extending previous work with the additions of neutral sites and coupling statistics. We used these simulations to investigate, in a preliminary way, if and how selected and neutral sites differ in the conditions they require for transitions during speciation. For the parameter combinations we explored, as the per-locus strength of selection grew and/or migration decreased, it became easier for selected sites to show divergence—and thus to rise in linkage disequilibrium (LD) with each other as a statistical consequence—farther in advance of the conditions under which neutral sites could diverge. Indeed, even very low rates of effective gene flow were sufficient to prevent differentiation at neutral sites. However, once strong enough, coupling among selected sites eventually reduced gene flow at neutral sites as well. To explore whether similar transitions might be detectable in empirical data, we used published genome resequencing data from three taxa of Heliconius butterflies. We found that fixation index ( F S T ) outliers and allele-frequency outliers exhibited stronger patterns of within-deme LD than the genomic background, as expected. The statistical characteristics of within-deme LD—likely indicative of the strength of coupling of barrier loci—varied between chromosomes and taxonomic comparisons. Qualitatively, the patterns we observed in the empirical data and in our simulations suggest that selection drives rapid genome-wide transitions to multi-locus coupling, illustrating how divergence and gene flow interact along the speciation continuum.

Description: Genes, Vol. 9, Pages 278: RECTA: Regulon Identification Based on Comparative Genomics and Transcriptomics Analysis Genes doi: 10.3390/genes9060278 Authors: Xin Chen Anjun Ma Adam McDermaid Hanyuan Zhang Chao Liu Huansheng Cao Qin Ma Regulons, which serve as co-regulated gene groups contributing to the transcriptional regulation of microbial genomes, have the potential to aid in understanding of underlying regulatory mechanisms. In this study, we designed a novel computational pipeline, regulon identification based on comparative genomics and transcriptomics analysis (RECTA), for regulon prediction related to the gene regulatory network under certain conditions. To demonstrate the effectiveness of this tool, we implemented RECTA on Lactococcus lactis MG1363 data to elucidate acid-response regulons. A total of 51 regulons were identified, 14 of which have computational-verified significance. Among these 14 regulons, five of them were computationally predicted to be connected with acid stress response. Validated by literature, 33 genes in Lactococcus lactis MG1363 were found to have orthologous genes which were associated with six regulons. An acid response related regulatory network was constructed, involving two trans-membrane proteins, eight regulons (llrA, llrC, hllA, ccpA, NHP6A, rcfB, regulons #8 and #39), nine functional modules, and 33 genes with orthologous genes known to be associated with acid stress. The predicted response pathways could serve as promising candidates for better acid tolerance engineering in Lactococcus lactis. Our RECTA pipeline provides an effective way to construct a reliable gene regulatory network through regulon elucidation, and has strong application power and can be effectively applied to other bacterial genomes where the elucidation of the transcriptional regulation network is needed.

Description: Genes, Vol. 9, Pages 333: MicroRNA-106a-5p Inhibited C2C12 Myogenesis via Targeting PIK3R1 and Modulating the PI3K/AKT Signaling Genes doi: 10.3390/genes9070333 Authors: Xiao Li Youbo Zhu Huifang Zhang Guangjun Ma Guofang Wu Aoqi Xiang Xin’E. Shi Gong She Yang Shiduo Sun The microRNA (miR)-17 family is widely expressed in mammalian tissues and play important roles in various physiological and pathological processes. Here, the functions of miR-106a-5p, a member of miR-17 family, were explored during myogenic differentiation in C2C12 cell line. First, miR-106a-5p was found to be relatively lower expressed in two-month skeletal muscle tissues and gradually decreased upon myogenic stimuli. Forced expression of miR-106a-5p significantly reduced the differentiation index, fusion index as well as the expression of myogenic markers (MyoD, MyoG, MyHC, Myomixer, Myomarker). Meanwhile, the levels of phosphorylated AKT were reduced by overexpression of miR-106a-5p, and administration of insulin-like growth factor 1 (IGF1), a booster of myogenic differentiation, could recover all the inhibitory effects above of miR-106a-5p. Furthermore, miR-106a-5p was elevated in aged muscles and dexamethasone (DEX)-treated myotubes, and up-regulation of miR-106a-5p significantly reduced the diameters of myotubes accompanied with increased levels of muscular atrophy genes and decreased PI3K/AKT activities. Finally, miR-106a-5p was demonstrated to directly bind to the 3’-UTR of PIK3R1, thus, repress the PI3K/AKT signaling.

Description: Genes, Vol. 9, Pages 332: Host-Pathogen Interactions Mediated by MDR Transporters in Fungi: As Pleiotropic as it Gets! Genes doi: 10.3390/genes9070332 Authors: Mafalda Cavalheiro Pedro Pais Mónica Galocha Miguel C. Teixeira Fungal infections caused by Candida, Aspergillus, and Cryptococcus species are an increasing problem worldwide, associated with very high mortality rates. The successful prevalence of these human pathogens is due to their ability to thrive in stressful host niche colonization sites, to tolerate host immune system-induced stress, and to resist antifungal drugs. This review focuses on the key role played by multidrug resistance (MDR) transporters, belonging to the ATP-binding cassette (ABC), and the major facilitator superfamilies (MFS), in mediating fungal resistance to pathogenesis-related stresses. These clearly include the extrusion of antifungal drugs, with C. albicans CDR1 and MDR1 genes, and corresponding homologs in other fungal pathogens, playing a key role in this phenomenon. More recently, however, clues on the transcriptional regulation and physiological roles of MDR transporters, including the transport of lipids, ions, and small metabolites, have emerged, linking these transporters to important pathogenesis features, such as resistance to host niche environments, biofilm formation, immune system evasion, and virulence. The wider view of the activity of MDR transporters provided in this review highlights their relevance beyond drug resistance and the need to develop therapeutic strategies that successfully face the challenges posed by the pleiotropic nature of these transporters.

Description: Genes, Vol. 9, Pages 331: SEGF: A Novel Method for Gene Fusion Detection from Single-End Next-Generation Sequencing Data Genes doi: 10.3390/genes9070331 Authors: Hai Xu Xiaojin Wu Dawei Sun Shijun Li Siwen Zhang Miao Teng Jianlong Bu Xizhe Zhang Bo Meng Weitao Wang Geng Tian Huixin Lin Dawei Yuan Jidong Lang Shidong Xu With the development and application of next-generation sequencing (NGS) and target capture technology, the demand for an effective analysis method to accurately detect gene fusion from high-throughput data is growing. Hence, we developed a novel fusion gene analyzing method called single-end gene fusion (SEGF) by starting with single-end DNA-seq data. This approach takes raw sequencing data as input, and integrates the commonly used alignment approach basic local alignment search tool (BLAST) and short oligonucleotide analysis package (SOAP) with stringent passing filters to achieve successful fusion gene detection. To evaluate SEGF, we compared it with four other fusion gene discovery analysis methods by analyzing sequencing results of 23 standard DNA samples and DNA extracted from 286 lung cancer formalin fixed paraffin embedded (FFPE) samples. The results generated by SEGF indicated that it not only detected the fusion genes from standard samples and clinical samples, but also had the highest accuracy and sensitivity among the five compared methods. In addition, SEGF was capable of detecting complex gene fusion types from single-end NGS sequencing data compared with other methods. By using SEGF to acquire gene fusion information at DNA level, more useful information can be retrieved from the DNA panel or other DNA sequencing methods without generating RNA sequencing information to benefit clinical diagnosis or medication instruction. It was a timely and cost-effective measure with regard to research or diagnosis. Considering all the above, SEGF is a straightforward method without manipulating complicated arguments, providing a useful approach for the precise detection of gene fusion variation.

Description: Genes, Vol. 9, Pages 335: Combining Targeted Metabolites Analysis and Transcriptomics to Reveal Chemical Composition Difference and Underlying Transcriptional Regulation in Maca (Lepidium Meyenii Walp.) Ecotypes Genes doi: 10.3390/genes9070335 Authors: Qiansi Chen Meng Li Chen Wang Zefeng Li Jiayang Xu Qingxia Zheng Pingping Liu Huina Zhou Maca (Lepidium meyenii Walp.) is a traditional Andean crop with great potential for various sanitarian and medical functions, which is attracting increased research attention. The majority of previous Maca studies were focused on biochemistry and pharmacodynamics, while the genetic basis of its unique characteristics lagged due to a lack of genome information. The authors perform gas chromatography-mass spectrometry (GC/MS) analysis in the hypocotyls of three Maca ecotypes and identify 79 compounds. Among them, 62 compounds have distinct profiles among Maca ecotypes. To reveal the underlying regulatory mechanism of the chemical composition differences, de novo transcriptome sequencing is performed and the transcription profiles of three Maca ecotypes are comparatively analyzed. Functional analysis indicates several key pathways, including “starch and sucrose metabolism,” “phenylpropanoid biosynthesis,” “phenylalanine metabolism” and “plant-pathogen interaction,” are involved in regulating the chemical compositions of Maca. Combining metabolomics and transcriptomics analysis indicates transcription factors such as MYB and WRKY and mediators such as protein kinase and bifunctional inhibitors might be critical regulators of chemical composition in Maca. The transcriptome reference genome and differentially expressed genes (DEGs) obtained in this study might serve as an initial step to illustrate the genetic differences in nutrient component, secondary metabolites content, medicinal function and stress resistance in Maca.

Description: Genes, Vol. 9, Pages 334: Functional Analysis of Promoters of Genes in Lipid Metabolism and Their Transcriptional Response to STAT3 under Leptin Signals Genes doi: 10.3390/genes9070334 Authors: Kun Wu Xiao-Ying Tan Yi-Huan Xu Guang-Hui Chen Mei-Qin Zhuo We characterized the promoters of target genes of the signal transducer and activator of transcription 3, STAT3 (carnitine palmitoyltransferase I, CPT Iα1b, acetyl-CoA carboxylase alpha, ACCα; fatty acid synthase, FAS; and peroxisome proliferator-activated receptor gamma, PPARγ) in a teleost Pelteobagrus fulvidraco. Binding sites of STAT3 were predicted on these promoters, indicating that STAT3 probably mediated their transcriptional activities. Leptin had no effect on the activity of ACCα and PPARγ promoters, but increased CPT Iα1b promoter activity and decreased FAS promoter activity. The −979/−997 STAT3 binding site of CPT Iα1b and the −794/−812 STAT3 binding site of FAS were functional binding loci responsible for leptin-induced transcriptional activation. The study provided direct evidence that STAT3 regulated the expression of CPT Iα1b and FAS at the transcription level, and determined the STAT3 response element on promoters of CPT Iα1b and FAS under leptin signal.

Description: Genes, Vol. 9, Pages 347: Possible Role of Envelope Components in the Extreme Copper Resistance of the Biomining Acidithiobacillus ferrooxidans Genes doi: 10.3390/genes9070347 Authors: Nia Oetiker Rodrigo Norambuena Cristóbal Martínez-Bussenius Claudio A. Navarro Fernando Amaya Sergio A. Álvarez Alberto Paradela Carlos A. Jerez Acidithiobacillus ferrooxidans resists extremely high concentrations of copper. Strain ATCC 53993 is much more resistant to the metal compared with strain ATCC 23270, possibly due to the presence of a genomic island in the former one. The global response of strain ATCC 53993 to copper was analyzed using iTRAQ (isobaric tag for relative and absolute quantitation) quantitative proteomics. Sixty-seven proteins changed their levels of synthesis in the presence of the metal. On addition of CusCBA efflux system proteins, increased levels of other envelope proteins, such as a putative periplasmic glucan biosynthesis protein (MdoG) involved in the osmoregulated synthesis of glucans and a putative antigen O polymerase (Wzy), were seen in the presence of copper. The expression of A. ferrooxidansmdoG or wzy genes in a copper sensitive Escherichia coli conferred it a higher metal resistance, suggesting the possible role of these components in copper resistance of A. ferrooxidans. Transcriptional levels of genes wzy, rfaE and wzz also increased in strain ATCC 23270 grown in the presence of copper, but not in strain ATCC 53993. Additionally, in the absence of this metal, lipopolysaccharide (LPS) amounts were 3-fold higher in A. ferrooxidans ATCC 53993 compared with strain 23270. Nevertheless, both strains grown in the presence of copper contained similar LPS quantities, suggesting that strain 23270 synthesizes higher amounts of LPS to resist the metal. On the other hand, several porins diminished their levels in the presence of copper. The data presented here point to an essential role for several envelope components in the extreme copper resistance by this industrially important acidophilic bacterium.

Description: Genes, Vol. 9, Pages 346: The Genetics of a Behavioral Speciation Phenotype in an Island System Genes doi: 10.3390/genes9070346 Authors: Thomas Blankers Kevin P. Oh Kerry L. Shaw Mating behavior divergence can make significant contributions to reproductive isolation and speciation in various biogeographic contexts. However, whether the genetic architecture underlying mating behavior divergence is related to the biogeographic history and the tempo and mode of speciation remains poorly understood. Here, we use quantitative trait locus (QTL) mapping to infer the number, distribution, and effect size of mating song rhythm variations in the crickets Laupala eukolea and Laupala cerasina, which occur on different islands (Maui and Hawaii). We then compare these results with a similar study of an independently evolving species pair that diverged within the same island. Finally, we annotate the L. cerasina transcriptome and test whether the QTL fall in functionally enriched genomic regions. We document a polygenic architecture behind the song rhythm divergence in the inter-island species pair that is remarkably similar to that previously found for an intra-island species pair in the same genus. Importantly, the QTL regions were significantly enriched for potential homologs of the genes involved in pathways that may be modulating the cricket song rhythm. These clusters of loci could constrain the spatial genomic distribution of the genetic variation underlying the cricket song variation and harbor several candidate genes that merit further study.

Description: Genes, Vol. 9, Pages 349: Description of Genetic Variants in BRCA Genes in Mexican Patients with Ovarian Cancer: A First Step towards Implementing Personalized Medicine Genes doi: 10.3390/genes9070349 Authors: Jesus Rolando Delgado-Balderas Maria Lourdes Garza-Rodriguez Gabriela Sofia Gomez-Macias Alvaro Barboza-Quintana Oralia Barboza-Quintana Ricardo M. Cerda-Flores Ivett Miranda-Maldonado Hugo Mauricio Vazquez-Garcia Lezmes Dionicio Valdez-Chapa Mauro Antonio-Macedo Michael Dean Hugo A. Barrera-Saldaña Gynecologic cancers are among the leading causes of death worldwide, ovarian cancer being the one with the highest mortality rate. Olaparib is a targeted therapy used in patients presenting mutations in BRCA1 and BRCA2 genes. The aim of this study was to describe BRCA1 and BRCA2 gene variants in Mexican patients with ovarian cancer. Sequencing of BRCA1 and BRCA2 genes from tumors of 50 Mexican patients with ovarian cancer was made in a retrospective, non-randomized, and exploratory study. We found genetic variants in 48 of 50 cases. A total of 76 polymorphic variants were found in BRCA1, of which 50 (66%) had not been previously reported. Furthermore, 104 polymorphic variants were found in BRCA2, of which 63 (60%) had not been reported previously. Of these polymorphisms, 5/76 (6.6%) and 4/104 (3.8%) were classified as pathogenic in BRCA1 and BRCA2, respectively. We have described the genetic variants in BRCA1 and BRCA2 of tumors from Northeast Mexican patients with sporadic ovarian cancers. Our results showed that the use of genetic testing helps recognize patients that carry pathogenic variants which could be beneficial for personalized medicine treatments.

Description: Genes, Vol. 9, Pages 357: Genetic Targeting of GRP78 in the VMH Improves Obesity Independently of Food Intake Genes doi: 10.3390/genes9070357 Authors: Laura Liñares-Pose Eva Rial-Pensado Ánxela Estévez-Salguero Edward Milbank Ismael González-García Claudia Rodríguez Patricia Seoane-Collazo Noelia Martinez-Sánchez Rubén Nogueiras Dolores Prieto Carlos Diéguez Cristina Contreras Miguel López Recent data have demonstrated that the hypothalamic GRP78/BiP (glucose regulated protein 78 kDa/binding immunoglobulin protein) modulates brown adipose tissue (BAT) thermogenesis by acting downstream on AMP-activated protein kinase (AMPK). Herein, we aimed to investigate whether genetic over-expression of GRP78 in the ventromedial nucleus of the hypothalamus (VMH: a key site regulating thermogenesis) could ameliorate very high fat diet (vHFD)-induced obesity. Our data showed that stereotaxic treatment with adenoviruses harboring GRP78 in the VMH reduced hypothalamic endoplasmic reticulum ER stress and reversed vHFD-induced obesity. Herein, we also demonstrated that this body weight decrease was more likely associated with an increased BAT thermogenesis and browning of white adipose tissue (WAT) than to anorexia. Overall, these results indicate that the modulation of GRP78 in the VMH may be a target against obesity.

Description: Genes, Vol. 9, Pages 367: Over a Thousand Years of Evolutionary History of Domestic Geese from Russian Archaeological Sites, Analysed Using Ancient DNA Genes doi: 10.3390/genes9070367 Authors: Johanna Honka Matti T. Heino Laura Kvist Igor V. Askeyev Dilyara N. Shaymuratova Oleg V. Askeyev Arthur O. Askeyev Marja E. Heikkinen Jeremy B. Searle Jouni Aspi The European domestic goose is a widely farmed species known to have descended from the wild greylag goose (Anser anser). However, the evolutionary history of this domesticate is still poorly known. Ancient DNA studies have been useful for many species, but there has been little such work on geese. We have studied temporal genetic variation among domestic goose specimens excavated from Russian archaeological sites (4th–18th centuries) using a 204 base pair fragment of the mitochondrial control region. Specimens fell into three different genetic clades: the domestic D-haplogroup, the F-haplogroup that includes both wild and domestic geese, and a clade comprising another species, the taiga bean goose. Most of the subfossil geese carried typical domestic D-haplotypes. The domestication status of the geese carrying F-haplotypes is less certain, as the haplotypes identified were not present among modern domestic geese and could represent wild geese (misclassified as domestics), introgression from wild geese, or local domestication events. The bones of taiga bean goose were most probably misidentified as domestic goose but the domestication of bean goose or hybridization with domestic goose is also possible. Samples from the 4th to 10th century were clearly differentiated from the later time periods due to a haplotype that was found only in this early period, but otherwise no temporal or geographical variation in haplotype frequencies was apparent.

Description: Genes, Vol. 9, Pages 366: Integrase-Controlled Excision of Metal-Resistance Genomic Islands in Acinetobacter baumannii Genes doi: 10.3390/genes9070366 Authors: Zaaima AL-Jabri Roxana Zamudio Eva Horvath-Papp Joseph D. Ralph Zakariya AL-Muharrami Kumar Rajakumar Marco R. Oggioni Genomic islands (GIs) are discrete gene clusters encoding for a variety of functions including antibiotic and heavy metal resistance, some of which are tightly associated to lineages of the core genome phylogenetic tree. We have investigated the functions of two distinct integrase genes in the mobilization of two metal resistant GIs, G08 and G62, of Acinetobacter baumannii. Real-time PCR demonstrated integrase-dependent GI excision, utilizing isopropyl β-d-1-thiogalactopyranoside IPTG-inducible integrase genes in plasmid-based mini-GIs in Escherichia coli. In A. baumannii, integrase-dependent excision of the original chromosomal GIs could be observed after mitomycin C induction. In both E. coli plasmids and A. baumannii chromosome, the rate of excision and circularization was found to be dependent on the expression level of the integrases. Susceptibility testing in A. baumannii strain ATCC 17978, A424, and their respective ΔG62 and ΔG08 mutants confirmed the contribution of the GI-encoded efflux transporters to heavy metal decreased susceptibility. In summary, the data evidenced the functionality of two integrases in the excision and circularization of the two Acinetobacter heavy-metal resistance GIs, G08 and G62, in E. coli, as well as when chromosomally located in their natural host. These recombination events occur at different frequencies resulting in genome plasticity and may participate in the spread of resistance determinants in A. baumannii.