by Jihong Hu, Songtao Gui, Zhixuan Zhu, Xiaolei Wang, Weidong Ke, Yi Ding

Genomic resources such as single nucleotide polymorphism (SNPs), insertions and deletions (InDels) and SSRs (simple sequence repeats) are essential for crop improvement and better utilization in genetic breeding. However, the resources for the sacred lotus (Nelumbo nucifera Gaertn.) are still limited. In the present study, to dissect large-scale genomic molecular marker resources for sacred lotus, we re-sequenced a Thailand sacred lotus cultivar ‘Chiang Mai wild lotus’ and compared with the reported lotus genome ‘Middle lake wild lotus’. A total of 3,180,059 SNPs, 328, 251 InDels and 14,191 SVs were found between the two genomes. The functional impact analyses of these SNPs indicated that they may be involved in metabolic processes, binding, catalytic activity, etc. Mining the genome sequences for SSRs showed that 191,657 SSRs were identified with a frequency of one SSR per 4.23 kb and 103,656 SSR primer pairs were designed. Furthermore, 14, 502 EST-SSRs were also indentified using the available RNA-seq data in the NCBI. A subset of 150 SSRs (genomic and EST-SSRs) was randomly selected for validation and genetic diversity analysis. The genotypes could be easily distinguished using these SSR markers and the ‘Chiang Mai wild lotus’ was obviously differentiated from the other Chinese accessions. This study provides considerable amounts of genomic resources and markers for the quantitative trait locus (QTL) identification and molecular selection of the species, which could have a potential role in various applications in sacred lotus breeding.

by Zhen Sheng, Yi Sun, Ruixin Zhu, Na Jiao, Kailin Tang, Zhiwei Cao, Chao Ma

Berberine has been identified with anti-proliferative effects on various cancer cells. Many researchers have been trying to elucidate the anti-cancer mechanisms of berberine based on differentially expressed genes. However, differentially alternative splicing genes induced by berberine might also contribute to its pharmacological actions and have not been reported yet. Moreover, the potential functional cross-talking between the two sets of genes deserves further exploration. In this study, RNA-seq technology was used to detect the differentially expressed genes and differentially alternative spliced genes in BEL-7402 cancer cells induced by berberine. Functional enrichment analysis indicated that these genes were mainly enriched in the p53 and cell cycle signalling pathway. In addition, it was statistically proven that the two sets of genes were locally co-enriched along chromosomes, closely connected to each other based on protein-protein interaction and functionally similar on Gene Ontology tree. These results suggested that the two sets of genes regulated by berberine might be functionally cross-talked and jointly contribute to its cell cycle arresting effect. It has provided new clues for further researches on the pharmacological mechanisms of berberine as well as the other botanical drugs.

by Hui-Yeng Y. Yap, Yit-Heng Chooi, Shin-Yee Fung, Szu-Ting Ng, Chon-Seng Tan, Nget-Hong Tan

Lignosus rhinocerotis (Cooke) Ryvarden (tiger milk mushroom) has long been known for its nutritional and medicinal benefits among the local communities in Southeast Asia. However, the molecular and genetic basis of its medicinal and nutraceutical properties at transcriptional level have not been investigated. In this study, the transcriptome of L. rhinocerotis sclerotium, the part with medicinal value, was analyzed using high-throughput Illumina HiSeqTM platform with good sequencing quality and alignment results. A total of 3,673, 117, and 59,649 events of alternative splicing, novel transcripts, and SNP variation were found to enrich its current genome database. A large number of transcripts were expressed and involved in the processing of gene information and carbohydrate metabolism. A few highly expressed genes encoding the cysteine-rich cerato-platanin, hydrophobins, and sugar-binding lectins were identified and their possible roles in L. rhinocerotis were discussed. Genes encoding enzymes involved in the biosynthesis of glucans, six gene clusters encoding four terpene synthases and one each of non-ribosomal peptide synthetase and polyketide synthase, and 109 transcribed cytochrome P450 sequences were also identified in the transcriptome. The data from this study forms a valuable foundation for future research in the exploitation of this mushroom in pharmacological and industrial applications.

by Dengwei Jue, Xuelian Sang, Shengqiao Lu, Chen Dong, Qiufang Zhao, Hongliang Chen, Liqiang Jia

Background

Ubiquitination is a post-translation modification where ubiquitin is attached to a substrate. Ubiquitin-conjugating enzymes (E2s) play a major role in the ubiquitin transfer pathway, as well as a variety of functions in plant biological processes. To date, no genome-wide characterization of this gene family has been conducted in maize (Zea mays).

Methodology/Principal Findings

In the present study, a total of 75 putative ZmUBC genes have been identified and located in the maize genome. Phylogenetic analysis revealed that ZmUBC proteins could be divided into 15 subfamilies, which include 13 ubiquitin-conjugating enzymes (ZmE2s) and two independent ubiquitin-conjugating enzyme variant (UEV) groups. The predicted ZmUBC genes were distributed across 10 chromosomes at different densities. In addition, analysis of exon-intron junctions and sequence motifs in each candidate gene has revealed high levels of conservation within and between phylogenetic groups. Tissue expression analysis indicated that most ZmUBC genes were expressed in at least one of the tissues, indicating that these are involved in various physiological and developmental processes in maize. Moreover, expression profile analyses of ZmUBC genes under different stress treatments (4°C, 20% PEG6000, and 200 mM NaCl) and various expression patterns indicated that these may play crucial roles in the response of plants to stress.

Conclusions

Genome-wide identification, chromosome organization, gene structure, evolutionary and expression analyses of ZmUBC genes have facilitated in the characterization of this gene family, as well as determined its potential involvement in growth, development, and stress responses. This study provides valuable information for better understanding the classification and putative functions of the UBC-encoding genes of maize.

by Jiří Macas, Petr Novák, Jaume Pellicer, Jana Čížková, Andrea Koblížková, Pavel Neumann, Iva Fuková, Jaroslav Doležel, Laura J. Kelly, Ilia J. Leitch

The differential accumulation and elimination of repetitive DNA are key drivers of genome size variation in flowering plants, yet there have been few studies which have analysed how different types of repeats in related species contribute to genome size evolution within a phylogenetic context. This question is addressed here by conducting large-scale comparative analysis of repeats in 23 species from four genera of the monophyletic legume tribe Fabeae, representing a 7.6-fold variation in genome size. Phylogenetic analysis and genome size reconstruction revealed that this diversity arose from genome size expansions and contractions in different lineages during the evolution of Fabeae. Employing a combination of low-pass genome sequencing with novel bioinformatic approaches resulted in identification and quantification of repeats making up 55–83% of the investigated genomes. In turn, this enabled an analysis of how each major repeat type contributed to the genome size variation encountered. Differential accumulation of repetitive DNA was found to account for 85% of the genome size differences between the species, and most (57%) of this variation was found to be driven by a single lineage of Ty3/gypsy LTR-retrotransposons, the Ogre elements. Although the amounts of several other lineages of LTR-retrotransposons and the total amount of satellite DNA were also positively correlated with genome size, their contributions to genome size variation were much smaller (up to 6%). Repeat analysis within a phylogenetic framework also revealed profound differences in the extent of sequence conservation between different repeat types across Fabeae. In addition to these findings, the study has provided a proof of concept for the approach combining recent developments in sequencing and bioinformatics to perform comparative analyses of repetitive DNAs in a large number of non-model species without the need to assemble their genomes.

by Alexander S. Rose, Ulrich Zachariae, Helmut Grubmüller, Klaus Peter Hofmann, Patrick Scheerer, Peter W. Hildebrand

GPCRs catalyze GDP/GTP exchange in the α-subunit of heterotrimeric G proteins (Gαßγ) through displacement of the Gα C-terminal α5 helix, which directly connects the interface of the active receptor (R*) to the nucleotide binding pocket of G. Hydrogen–deuterium exchange mass spectrometry and kinetic analysis of R* catalysed G protein activation have suggested that displacement of α5 starts from an intermediate GDP bound complex (R*•GGDP). To elucidate the structural basis of receptor-catalysed displacement of α5, we modelled the structure of R*•GGDP. A flexible docking protocol yielded an intermediate R*•GGDP complex, with a similar overall arrangement as in the X-ray structure of the nucleotide free complex (R*•Gempty), however with the α5 C-terminus (GαCT) forming different polar contacts with R*. Starting molecular dynamics simulations of GαCT bound to R* in the intermediate position, we observe a screw-like motion, which restores the specific interactions of α5 with R* in R*•Gempty. The observed rotation of α5 by 60° is in line with experimental data. Reformation of hydrogen bonds, water expulsion and formation of hydrophobic interactions are driving forces of the α5 displacement. We conclude that the identified interactions between R* and G protein define a structural framework in which the α5 displacement promotes direct transmission of the signal from R* to the GDP binding pocket.

by Adnan A. K. Al-Rubaye, M. Brian Couger, Sohita Ojha, Jeff F. Pummill, Joseph A. Koon, Robert F. Wideman, Douglas D. Rhoads

Lameness in broiler chickens is a significant animal welfare and financial issue. Lameness can be enhanced by rearing young broilers on wire flooring. We have identified Staphylococcus agnetis as significantly involved in bacterial chondronecrosis with osteomyelitis (BCO) in proximal tibia and femorae, leading to lameness in broiler chickens in the wire floor system. Administration of S. agnetis in water induces lameness. Previously reported in some cases of cattle mastitis, this is the first report of this poorly described pathogen in chickens. We used long and short read next generation sequencing to assemble single finished contigs for the genome and a large plasmid from the chicken pathogen. Comparison of the S. agnetis genome to those of other pathogenic Staphylococci shows that S.agnetis contains a distinct repertoire of virulence determinants. Additionally, the S. agnetis genome has several regions that differ substantially from the genomes of other pathogenic Staphylococci. Comparison of our finished genome to a recent draft genome for a cattle mastitis isolate suggests that future investigations focus on the evolutionary epidemiology of this emerging pathogen of domestic animals.

by Liron Levin, Dan Bar-Yaacov, Amos Bouskila, Michal Chorev, Liran Carmel, Dan Mishmar

RNA-seq is becoming a preferred tool for genomics studies of model and non-model organisms. However, DNA-based analysis of organisms lacking sequenced genomes cannot rely on RNA-seq data alone to isolate most genes of interest, as DNA codes both exons and introns. With this in mind, we designed a novel tool, LEMONS, that exploits the evolutionary conservation of both exon/intron boundary positions and splice junction recognition signals to produce high throughput splice-junction predictions in the absence of a reference genome. When tested on multiple annotated vertebrate mRNA data, LEMONS accurately identified 87% (average) of the splice-junctions. LEMONS was then applied to our updated Mediterranean chameleon transcriptome, which lacks a reference genome, and predicted a total of 90,820 exon-exon junctions. We experimentally verified these splice-junction predictions by amplifying and sequencing twenty randomly selected genes from chameleon DNA templates. Exons and introns were detected in 19 of 20 of the positions predicted by LEMONS. To the best of our knowledge, LEMONS is currently the only experimentally verified tool that can accurately predict splice-junctions in organisms that lack a reference genome.

by Jennifer Prescott, Veronica W. Setiawan, Nicolas Wentzensen, Fredrick Schumacher, Herbert Yu, Ryan Delahanty, Leslie Bernstein, Stephen J. Chanock, Chu Chen, Linda S. Cook, Christine Friedenreich, Monserrat Garcia-Closas, Christopher A. Haiman, Loic Le Marchand, Xiaolin Liang, Jolanta Lissowska, Lingeng Lu, Anthony M. Magliocco, Sara H. Olson, Harvey A. Risch, Xiao-Ou Shu, Giske Ursin, Hannah P. Yang, Peter Kraft, Immaculata De Vivo

Genome-wide association studies (GWAS) have identified common variants that predispose individuals to a higher body mass index (BMI), an independent risk factor for endometrial cancer. Composite genotype risk scores (GRS) based on the joint effect of published BMI risk loci were used to explore whether endometrial cancer shares a genetic background with obesity. Genotype and risk factor data were available on 3,376 endometrial cancer case and 3,867 control participants of European ancestry from the Epidemiology of Endometrial Cancer Consortium GWAS. A BMI GRS was calculated by summing the number of BMI risk alleles at 97 independent loci. For exploratory analyses, additional GRSs were based on subsets of risk loci within putative etiologic BMI pathways. The BMI GRS was statistically significantly associated with endometrial cancer risk (P = 0.002). For every 10 BMI risk alleles a woman had a 13% increased endometrial cancer risk (95% CI: 4%, 22%). However, after adjusting for BMI, the BMI GRS was no longer associated with risk (per 10 BMI risk alleles OR = 0.99, 95% CI: 0.91, 1.07; P = 0.78). Heterogeneity by BMI did not reach statistical significance (P = 0.06), and no effect modification was noted by age, GWAS Stage, study design or between studies (P≥0.58). In exploratory analyses, the GRS defined by variants at loci containing monogenic obesity syndrome genes was associated with reduced endometrial cancer risk independent of BMI (per BMI risk allele OR = 0.92, 95% CI: 0.88, 0.96; P = 2.1 x 10−5). Possessing a large number of BMI risk alleles does not increase endometrial cancer risk above that conferred by excess body weight among women of European descent. Thus, the GRS based on all current established BMI loci does not provide added value independent of BMI. Future studies are required to validate the unexpected observed relation between monogenic obesity syndrome genetic variants and endometrial cancer risk.

by Luiz H. Araujo, Cynthia Timmers, Konstantin Shilo, Weiqiang Zhao, Jianying Zhang, Lianbo Yu, Thanemozhi G. Natarajan, Clinton J. Miller, Ayse Selen Yilmaz, Tom Liu, Joseph Amann, José Roberto Lapa e Silva, Carlos Gil Ferreira, David P. Carbone

Tumor specimens are often preserved as formalin-fixed paraffin-embedded (FFPE) tissue blocks, the most common clinical source for DNA sequencing. Herein, we evaluated the effect of pre-sequencing parameters to guide proper sample selection for targeted gene sequencing. Data from 113 FFPE lung tumor specimens were collected, and targeted gene sequencing was performed. Libraries were constructed using custom probes and were paired-end sequenced on a next generation sequencing platform. A PCR-based quality control (QC) assay was utilized to determine DNA quality, and a ratio was generated in comparison to control DNA. We observed that FFPE storage time, PCR/QC ratio, and DNA input in the library preparation were significantly correlated to most parameters of sequencing efficiency including depth of coverage, alignment rate, insert size, and read quality. A combined score using the three parameters was generated and proved highly accurate to predict sequencing metrics. We also showed wide read count variability within the genome, with worse coverage in regions of low GC content like in KRAS. Sample quality and GC content had independent effects on sequencing depth, and the worst results were observed in regions of low GC content in samples with poor quality. Our data confirm that FFPE samples are a reliable source for targeted gene sequencing in cancer, provided adequate sample quality controls are exercised. Tissue quality should be routinely assessed for pre-analytical factors, and sequencing depth may be limited in genomic regions of low GC content if suboptimal samples are utilized.

by Silvia Lovera, Maria Morando, Encarna Pucheta-Martinez, Jorge L. Martinez-Torrecuadrada, Giorgio Saladino, Francesco L. Gervasio

Due to its inhibition of the Abl kinase domain in the BCR-ABL fusion protein, imatinib is strikingly effective in the initial stage of chronic myeloid leukemia with more than 90% of the patients showing complete remission. However, as in the case of most targeted anti-cancer therapies, the emergence of drug resistance is a serious concern. Several drug-resistant mutations affecting the catalytic domain of Abl and other tyrosine kinases are now known. But, despite their importance and the adverse effect that they have on the prognosis of the cancer patients harboring them, the molecular mechanism of these mutations is still debated. Here by using long molecular dynamics simulations and large-scale free energy calculations complemented by in vitro mutagenesis and microcalorimetry experiments, we model the effect of several widespread drug-resistant mutations of Abl. By comparing the conformational free energy landscape of the mutants with those of the wild-type tyrosine kinases we clarify their mode of action. It involves significant and complex changes in the inactive-to-active dynamics and entropy/enthalpy balance of two functional elements: the activation-loop and the conserved DFG motif. What is more the T315I gatekeeper mutant has a significant impact on the binding mechanism itself and on the binding kinetics.

by Hisashi Ohtsuki, Yoh Iwasa, Martin A. Nowak

We study the evolution of cooperation in a model of indirect reciprocity where people interact in public and private situations. Public interactions have a high chance to be observed by others and always affect reputation. Private interactions have a lower chance to be observed and only occasionally affect reputation. We explore all second order social norms and study conditions for evolutionary stability of action rules. We observe the competition between “honest” and “hypocritical” strategies. The former cooperate both in public and in private. The later cooperate in public, where many others are watching, but try to get away with defection in private situations. The hypocritical idea is that in private situations it does not pay-off to cooperate, because there is a good chance that nobody will notice it. We find simple and intuitive conditions for the evolution of honest strategies.

by Mikhail Shugay, Dmitriy V. Bagaev, Maria A. Turchaninova, Dmitriy A. Bolotin, Olga V. Britanova, Ekaterina V. Putintseva, Mikhail V. Pogorelyy, Vadim I. Nazarov, Ivan V. Zvyagin, Vitalina I. Kirgizova, Kirill I. Kirgizov, Elena V. Skorobogatova, Dmitriy M. Chudakov

Despite the growing number of immune repertoire sequencing studies, the field still lacks software for analysis and comprehension of this high-dimensional data. Here we report VDJtools, a complementary software suite that solves a wide range of T cell receptor (TCR) repertoires post-analysis tasks, provides a detailed tabular output and publication-ready graphics, and is built on top of a flexible API. Using TCR datasets for a large cohort of unrelated healthy donors, twins, and multiple sclerosis patients we demonstrate that VDJtools greatly facilitates the analysis and leads to sound biological conclusions. VDJtools software and documentation are available at https://github.com/mikessh/vdjtools.

Journal of the American Chemical SocietyDOI: 10.1021/jacs.5b07973

Journal of the American Chemical SocietyDOI: 10.1021/jacs.5b10074

Journal of the American Chemical SocietyDOI: 10.1021/jacs.5b09343

Journal of the American Chemical SocietyDOI: 10.1021/jacs.5b10321

Journal of the American Chemical SocietyDOI: 10.1021/jacs.5b10270

Journal of the American Chemical SocietyDOI: 10.1021/jacs.5b09825