L-Type Calcium Channels: Structure and Functions

Voltage-gated calcium channels (VGCCs) manage the electrical signaling of cells by allowing the selective-diffusion of calcium ions in response to the changes in the cellular membrane potential. Among the different VGCCs, the long-lasting or the L-type calcium channels (LTCCs) are prevalently expressed in a variety of cells, such as skeletal muscle, ventricular myocytes, smooth muscles and dendritic cells and forms the largest family of the VGCCs. Their wide expression pattern and significant role in diverse cellular events, including neurotransmission, cell cycle, muscular contraction, cardiac action potential and gene expression, has made these channels the major targets for drug development. In this book chapter, we aim to provide a comprehensive overview of the different VGCCs and focus on the sequence-structure–function properties of the LTCCs. Our chapter will summarize and review the various experimental and computational analyses performed on the structures of the LTCCs and their implications in drug discovery applications

A minimum reporting standard for multiple sequence alignments

Multiple sequence alignments (MSAs) play a pivotal role in studies of molecular sequence data, but nobody has developed a minimum reporting standard (MRS) to quantify the completeness of MSAs. We present an MRS that relies on four simple completeness metrics. The metrics are implemented in AliStat, a program developed to support the MRS. A survey of published MSAs illustrates the benefits and unprecedented transparency offered by the MRS.CSIRO

Reverse engineering: transaminase biocatalystdevelopment using ancestral sequencereconstruction

The development of new biocatalysts using ancestral sequence reconstruction is reported. When applied to an ω-transaminase, the ancestral proteins demonstrated novel and superior activities with eighty percent of the forty compounds tested compared to the modern day protein, and improvements in activity of up to twenty fold. These included a range of compounds pertinent as feedstocks in polyamide manufacture.MW and SK were supported by CSIRO Research Office Postdoctoral Fellowships

Comparative Genomics of Cyanobacterial Symbionts Reveals Distinct, Specialized Metabolism in Tropical Dysideidae Sponges.

Marine sponges are recognized as valuable sources of bioactive metabolites and renowned as petri dishes of the sea, providing specialized niches for many symbiotic microorganisms. Sponges of the family Dysideidae are well documented to be chemically talented, often containing high levels of polyhalogenated compounds, terpenoids, peptides, and other classes of bioactive small molecules. This group of tropical sponges hosts a high abundance of an uncultured filamentous cyanobacterium, Hormoscilla spongeliae Here, we report the comparative genomic analyses of two phylogenetically distinct Hormoscilla populations, which reveal shared deficiencies in essential pathways, hinting at possible reasons for their uncultivable status, as well as differing biosynthetic machinery for the production of specialized metabolites. One symbiont population contains clustered genes for expanded polybrominated diphenylether (PBDE) biosynthesis, while the other instead harbors a unique gene cluster for the biosynthesis of the dysinosin nonribosomal peptides. The hybrid sequencing and assembly approach utilized here allows, for the first time, a comprehensive look into the genomes of these elusive sponge symbionts.IMPORTANCE Natural products provide the inspiration for most clinical drugs. With the rise in antibiotic resistance, it is imperative to discover new sources of chemical diversity. Bacteria living in symbiosis with marine invertebrates have emerged as an untapped source of natural chemistry. While symbiotic bacteria are often recalcitrant to growth in the lab, advances in metagenomic sequencing and assembly now make it possible to access their genetic blueprint. A cell enrichment procedure, combined with a hybrid sequencing and assembly approach, enabled detailed genomic analysis of uncultivated cyanobacterial symbiont populations in two chemically rich tropical marine sponges. These population genomes reveal a wealth of secondary metabolism potential as well as possible reasons for historical difficulties in their cultivation

Co-circulation of genetically distinct highly pathogenic avian influenza A clade 2.3.4.4 (H5N6) viruses in wild waterfowl and poultry in Europe and East Asia, 2017–18

Highly pathogenic avian influenza (HPAI) H5 clade 2.3.4.4 viruses were first introduced into Europe in late 2014 and re-introduced in late 2016, following detections in Asia and Russia. In contrast to the 2014–15 H5N8 wave, there was substantial local virus amplification in wild birds in Europe in 2016–17 and associated wild bird mortality, with evidence for occasional gene exchange with low pathogenic avian influenza (LPAI) viruses. Since December 2017, several European countries have again reported events or outbreaks with HPAI H5N6 reassortant viruses in both wild birds and poultry, respectively. Previous phylogenetic studies have shown that the two earliest incursions of HPAI H5N8 viruses originated in Southeast Asia and subsequently spread to Europe. In contrast, this study indicates that recent HPAI H5N6 viruses evolved from the H5N8 2016–17 viruses during 2017 by reassortment of a European HPAI H5N8 virus and wild host reservoir LPAI viruses. The genetic and phenotypic differences between these outbreaks and the continuing detections of HPAI viruses in Europe are a cause of concern for both animal and human health. The current co-circulation of potentially zoonotic HPAI and LPAI virus strains in Asia warrants the determination of drivers responsible for the global spread of Asian lineage viruses and the potential threat they pose to public health

Genomic epidemiology reveals transmission patterns and dynamics of SARS-CoV-2 in Aotearoa New Zealand

New Zealand, a geographically remote Pacific island with easily sealable borders, implemented a nationwide 'lockdown' of all non-essential services to curb the spread of COVID-19. Here, we generate 649 SARS-CoV-2 genome sequences from infected patients in New Zealand with samples collected during the 'first wave', representing 56% of all confirmed cases in this time period. Despite its remoteness, the viruses imported into New Zealand represented nearly all of the genomic diversity sequenced from the global virus population. These data helped to quantify the effectiveness of public health interventions. For example, the effective reproductive number, Re of New Zealand's largest cluster decreased from 7 to 0.2 within the first week of lockdown. Similarly, only 19% of virus introductions into New Zealand resulted in ongoing transmission of more than one additional case. Overall, these results demonstrate the utility of genomic pathogen surveillance to inform public health and disease mitigation

TMFoldRec: a statistical potential-based transmembrane protein fold recognition tool.

BACKGROUND: Transmembrane proteins (TMPs) are the key components of signal transduction, cell-cell adhesion and energy and material transport into and out from the cells. For the deep understanding of these processes, structure determination of transmembrane proteins is indispensable. However, due to technical difficulties, only a few transmembrane protein structures have been determined experimentally. Large-scale genomic sequencing provides increasing amounts of sequence information on the proteins and whole proteomes of living organisms resulting in the challenge of bioinformatics; how the structural information should be gained from a sequence. RESULTS: Here, we present a novel method, TMFoldRec, for fold prediction of membrane segments in transmembrane proteins. TMFoldRec based on statistical potentials was tested on a benchmark set containing 124 TMP chains from the PDBTM database. Using a 10-fold jackknife method, the native folds were correctly identified in 77 % of the cases. This accuracy overcomes the state-of-the-art methods. In addition, a key feature of TMFoldRec algorithm is the ability to estimate the reliability of the prediction and to decide with an accuracy of 70 %, whether the obtained, lowest energy structure is the native one. CONCLUSION: These results imply that the membrane embedded parts of TMPs dictate the TM structures rather than the soluble parts. Moreover, predictions with reliability scores make in this way our algorithm applicable for proteome-wide analyses. AVAILABILITY: The program is available upon request for academic use

Whole-genome resequencing of Cucurbita pepo morphotypes to discover genomic variants associated with morphology and horticulturally valuable traits

[EN] Cucurbita pepo contains two cultivated subspecies, each of which encompasses four fruit-shape morphotypes (cultivar groups). The Pumpkin, Vegetable Marrow, Cocozelle, and Zucchini Groups are of subsp. pepo and the Acorn, Crookneck, Scallop, and Straightneck Groups are of subsp. ovifera. Recently, a de novo assembly of the C. pepo subsp. pepo Zucchini genome was published, providing insights into its evolution. To expand our knowledge of evolutionary processes within C. pepo and to identify variants associated with particular morphotypes, we performed whole-genome resequencing of seven of these eight C. pepo morphotypes. We report for the first time whole-genome resequencing of the four subsp. pepo (Pumpkin, Vegetable Marrow, Cocozelle, green Zucchini, and yellow Zucchini) morphotypes and three of the subsp. ovifera (Acorn, Crookneck, and Scallop) morphotypes. A high-depth resequencing approach was followed, using the BGISEQ-500 platform that enables the identification of rare variants, with an average of 33.5X. Approximately 94.5% of the clean reads were mapped against the reference Zucchini genome. In total, 3,823,977 high confidence single-nucleotide polymorphisms (SNPs) were identified. Within each accession, SNPs varied from 636,918 in green Zucchini to 2,656,513 in Crookneck, and were distributed homogeneously along the chromosomes. Clear differences between subspecies pepo and ovifera in genetic variation and linkage disequilibrium are highlighted. In fact, comparison between subspecies pepo and ovifera indicated 5710 genes (22.5%) with Fst > 0.80 and 1059 genes (4.1%) with Fst = 1.00 as potential candidate genes that were fixed during the independent evolution and domestication of the two subspecies. Linkage disequilibrium was greater in subsp. ovifera than in subsp. pepo, perhaps reflective of the earlier differentiation of morphotypes within subsp. ovifera. Some morphotype-specific genes have been localized. Our results offer new clues that may provide an improved understanding of the underlying genomic regions involved in the independent evolution and domestication of the two subspecies. Comparisons among SNPs unique to particular subspecies or morphotypes may provide candidate genes responsible for traits of high economic importance.This work has been supported by Hellenic Agricultural Organization (ELGO) Demeter. Furthermore, we thank the Conselleria de Educacio, Investigacio, Cultura i Esport (Generalitat Valenciana) for funding Project Prometeo 2017/078 "Seleccion de Variedades Tradicionales y Desarrollo de Nuevas Variedades de Cucurbitaceas Adaptadas a la Produccion Ecologica". Also, this work was supported by Chiang Mai University.Xanthopoulou, A.; Montero-Pau, J.; Mellidou, I.; Kissoudis, C.; Blanca Postigo, JM.; Picó Sirvent, MB.; Tsaballa, A.... (2019). Whole-genome resequencing of Cucurbita pepo morphotypes to discover genomic variants associated with morphology and horticulturally valuable traits. Horticulture Research. 6:1-17. https://doi.org/10.1038/s41438-019-0176-9S1176Maynard, D. & Paris, H. in The Encyclopedia of Fruits & Nuts (eds Paull, R. E. & Janick, J.) 276–313 (CABI, New Jersey, U.S.A., 2018).Paris, H. S. in Genetics and Genomics of Cucurbitaceae, Grumet, Rebecca, Katzir, Nurit, Garcia-Mas, Jordi (Eds.) 111–154 (Springer, New York, U.S.A., 2016).Whitaker, T. W. & Davis, G. N. Cucurbits (Leonard Hill (Books) Ltd., London, and Interscience Publishers Inc., New York, 1962).Paris, H. S. History of the cultivar-groups of Cucurbita pepo. Hortic. Rev. 25, 71–170 (2001).Paris, H. S. A proposed subspecific classifiaction for Cucurbita pepo. Phytologia (USA) 61, 133–138 (1986).Lira, R., Andres, T. C. & Nee, M. in Systematic and Ecogeographic Studies on Crop Genepools, Vol. 9, 1–115 (International Plant Genetic Resources Institute, Roma, Italia, 1995).Castellanos-Morales, G. Historical biogeography and phylogeny of Cucurbita: insights from ancestral area reconstruction and niche evolution. Mol. Phylogenet. Evol. 128, 38–54 (2018).Paris, H. S., Lebeda, A., Křistkova, E., Andres, T. C. & Nee, M. H. Parallel evolution under domestication and phenotypic differentiation of the cultivated subspecies of Cucurbita pepo (Cucurbitaceae). Econ. Bot. 66, 71–90 (2012).Dong, W., Wu, D., Li, G., Wu, D. & Wang, Z. Next-generation sequencing from bulked segregant analysis identifies a dwarfism gene in watermelon. Sci. Rep. 8, 2908 (2018).Galpaz, N. et al. Deciphering genetic factors that determine melon fruit‐quality traits using RNA‐Seq‐based high‐resolution QTL and eQTL mapping. Plant J. 94, 169–191 (2018).Gur, A. et al. Genome-wide linkage-disequilibrium mapping to the candidate gene level in melon (Cucumis melo). Sci. Rep. 7, 9770 (2017).Blanca, J. et al. Transcriptome characterization and high throughput SSRs and SNPs discovery in Cucurbita pepo (Cucurbitaceae). BMC Genom. 12, 104 (2011).Esteras, C. et al. High-throughput SNP genotyping in Cucurbita pepo for map construction and quantitative trait loci mapping. BMC Genom. 13, 80 (2012).Montero-Pau, J. et al. An SNP-based saturated genetic map and QTL analysis of fruit-related traits in Zucchini using genotyping-by-sequencing. BMC Genom. 18, 94 (2017).Vicente-Dólera, N. et al. First TILLING platform in Cucurbita pepo: a new mutant resource for gene function and crop improvement. PLoS ONE 9, e112743 (2014).Wyatt, L. E., Strickler, S. R., Mueller, L. A. & Mazourek, M. An acorn squash (Cucurbita pepo ssp. ovifera) fruit and seed transcriptome as a resource for the study of fruit traits in Cucurbita. Hortic. Res. 2, 14070 (2015).Xanthopoulou, A. et al. De novo comparative transcriptome analysis of genes involved in fruit morphology of pumpkin cultivars with extreme size difference and development of EST-SSR markers. Gene 622, 50–66 (2017).Montero‐Pau, J. et al. De novo assembly of the zucchini genome reveals a whole‐genome duplication associated with the origin of the Cucurbita genus. Plant Biotechnol. J. 16, 1161–1171 (2018).Garcia-Mas, J. et al. Cloning and mapping of resistance gene homologues in melon. Plant Sci. 161, 165–172 (2001).Xanthopoulou, A. et al. Comparative analysis of genetic diversity in Greek Genebank collection of summer squash (‘Cucurbita pepo’) landraces using start codon targeted (SCoT) polymorphism and ISSR markers. Aust. J. Crop Sci. 9, 14 (2015).Huang, J. et al. A reference human genome dataset of the BGISEQ-500 sequencer. Gigascience 6, gix024 (2017).Natarajan, K. N. et al. Comparative analysis of sequencing technologies for single-cell transcriptomics. Genome Biol. 20, 70 (2019).Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).Tian, L. et al. Transcript and proteomic analysis of developing white lupin (Lupinus albus L.) roots. BMC Plant Biol. 9, 1 (2009).Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).Bradbury, P. J. et al. TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics 23, 2633–2635 (2007).Chang, C. C. et al. Second-generation PLINK: rising to the challenge of larger and richer datasets. Gigascience 4, 7 (2015).Team, R. C. (2015). http://www.r-project.org/ .Krzywinski, M. I. et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–1645 (2009).Kosman, E. & Leonard, K. J. Similarity coefficients for molecular markers in studies of genetic relationships between individuals for haploid, diploid, and polyploid species. Mol. Ecol. 14, 415–424 (2005).Huson, D. H. & Bryant, D. Estimating Phylogenetic Trees and Networks Using SplitsTree 4. www.splitstree.org (2005).Danecek, P. et al. The variant call format and VCFtools. Bioinformatics 27, 2156–2158 (2011).Cingolani, P. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strainw1118; iso-2; iso-3. Fly 6, 80–92 (2012).Wu, S. et al. A common genetic mechanism underlies morphological diversity in fruits and other plant organs. Nat. Commun. 9, 4734 (2018).Drevensek, S. et al. The Arabidopsis TRM1–TON1 interaction reveals a recruitment network common to plant cortical microtubule arrays and eukaryotic centrosomes. Plant Cell 24, 178–191 (2012).Sievers, F. et al. Fast, scalable generation of high‐quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).Nguyen, L.-T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2014).Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A. & Jermiin, L. S. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods 14, 587 (2017).Hoang, D. T., Chernomor, O., von Haeseler, A., Minh, B. Q. & Vinh, L. S. UFBoot2: improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35, 518–522 (2017).Bailey, T. L. et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 37, W202–W208 (2009).Leida, C. et al. Variability of candidate genes, genetic structure and association with sugar accumulation and climacteric behavior in a broad germplasm collection of melon (Cucumis melo L.). BMC Genet. 16, 28 (2015).Esteras, C. et al. SNP genotyping in melons: genetic variation, population structure, and linkage disequilibrium. Theor. Appl. Genet. 126, 1285–1303 (2013).Maria José Gonzalo et al. Re-evaluation of the role of Indian germplasm as center of melon diversification based on genotyping-by-sequencing analysis. BMC Genom. 20, p. 448 (2019).Nimmakayala, P. et al. Single nucleotide polymorphisms generated by genotyping by sequencing to characterize genome-wide diversity, linkage disequilibrium, and selective sweeps in cultivated watermelon. BMC Genom. 15, 767 (2014).Gonzalo, M. J. & Monforte, A. J. in Genetics and Genomics of Cucurbitaceae, Grumet, Rebecca, Katzir, Nurit, Garcia-Mas, Jordi (Eds.) 269–290 (Springer, New York, U.S.A., 2016).Pomares-Viciana, T. et al. First RNA-seq approach to study fruit set and parthenocarpy in zucchini (Cucurbita pepo L.). BMC Plant Biol. 19, 61 (2019).Lu, S. et al. The cauliflower Or gene encodes a DnaJ cysteine-rich domain-containing protein that mediates high levels of β-carotene accumulation. Plant Cell 18, 3594–3605 (2006).Jin, B., Kim, J., Jung, J., Kim, D. & Park, Y. Characterization of IQ domain gene homologs as common candidate genes for elongated fruit shape in cucurbits. Hortic. Sci. Technol. 36, 85–97 (2018).van der Knaap, E. et al. What lies beyond the eye: the molecular mechanisms regulating tomato fruit weight and shape. Front. Plant Sci. 5, 227 (2014).Xiao, H., Jiang, N., Schaffner, E., Stockinger, E. J. & Van Der Knaap, E. A retrotransposon-mediated gene duplication underlies morphological variation of tomato fruit. Science 319, 1527–1530 (2008).Dou, J. et al. Genetic mapping reveals a candidate gene (ClFS1) for fruit shape in watermelon (Citrullus lanatus L.). Theor. Appl. Genet. 131, 947–958 (2018).Pan, Y. et al. Round fruit shape in WI7239 cucumber is controlled by two interacting quantitative trait loci with one putatively encoding a tomato SUN homolog. Theor. Appl. Genet. 130, 573–586 (2017).Liu, J. et al. Banana Ovate family protein MaOFP1 and MADS-box protein MuMADS1 antagonistically regulated banana fruit ripening. PLoS ONE 10, e0123870 (2015).Liu, J. et al. Mu MADS 1 and Ma OFP 1 regulate fruit quality in a tomato ovate mutant. Plant Biotechnol. J. 16, 989–1001 (2018).Cong, B., Barrero, L. S. & Tanksley, S. D. Regulatory change in YABBY-like transcription factor led to evolution of extreme fruit size during tomato domestication. Nat. Genet. 40, 800 (2008).Huang, Z., Van Houten, J., Gonzalez, G., Xiao, H. & van der Knaap, E. Genome-wide identification, phylogeny and expression analysis of SUN, OFP and YABBY gene family in tomato. Mol. Genet. Genom. 288, 111–129 (2013).Bowman, J. L. The YABBY gene family and abaxial cell fate. Curr. Opin. Plant Biol. 3, 17–22 (2000).Liu, J., Van Eck, J., Cong, B. & Tanksley, S. D. A new class of regulatory genes underlying the cause of pear-shaped tomato fruit. Proc. Natl Acad. Sci. USA 99, 13302–13306 (2002).Tsaballa, A., Pasentsis, K., Darzentas, N. & Tsaftaris, A. S. Multiple evidence for the role of an Ovate-like gene in determining fruit shape in pepper. BMC Plant Biol. 11, 46 (2011).Wang, S., Chang, Y., Guo, J. & Chen, J. G. Arabidopsis Ovate family protein 1 is a transcriptional repressor that suppresses cell elongation. Plant J. 50, 858–872 (2007).Lazzaro, M. D., Wu, S., Snouffer, A., Wang, Y. & Van Der Knaap, E. Plant organ shapes are regulated by protein interactions and associations with microtubules. Front. Plant Sci. 9, 1766 (2018)

Plastome-wide rearrangements and gene losses in carnivorous Droseraceae

The plastid genomes of four related carnivorous plants (Drosera regia, Drosera erythrorhiza, Aldrovanda vesiculosa and Dionaea muscipula) were sequenced to examine changes potentially induced by the transition to carnivory. The plastid genomes of the Droseraceae show multiple rearrangements, gene losses and large expansions or contractions of the inverted repeat. All the ndh genes are lost or non-functional, as well as in some of the species, clpP1, ycf1, ycf2 and some tRNA genes. Uniquely amongst land plants, the trnK gene has no intron. Carnivory in the Droseraceae coincides with changes in plastid gene content similar to those induced by parasitism and mycoheterotrophy, suggesting parallel changes in chloroplast function due to the similar switch from autotrophy to (mixo-) heterotrophy. A molecular phylogeny of the taxa based on all shared plastid genes indicates that the ‘snap-traps’ of Aldrovanda and Dionaea have a common origin

The Genome of Caenorhabditis bovis

The free-living nematode Caenorhabditis elegans is a key laboratory model for metazoan biology. C. elegans has also become a model for parasitic nematodes despite being only distantly related to most parasitic species. All of the ∼65 Caenorhabditis species currently in culture are free-living, with most having been isolated from decaying plant or fungal matter. Caenorhabditis bovis is a particularly unusual species that has been isolated several times from the inflamed ears of Zebu cattle in Eastern Africa, where it is associated with the disease bovine parasitic otitis. C. bovis is therefore of particular interest to researchers interested in the evolution of nematode parasitism. However, as C. bovis is not in laboratory culture, it remains little studied. Here, by sampling livestock markets and slaughterhouses in Western Kenya, we successfully reisolated C. bovis from the ear of adult female Zebu. We sequenced the genome of C. bovis using the Oxford Nanopore MinION platform in a nearby field laboratory and used the data to generate a chromosome-scale draft genome sequence. We exploited this draft genome sequence to reconstruct the phylogenetic relationships of C. bovis to other Caenorhabditis species and reveal the changes in genome size and content that have occurred during its evolution. We also identified expansions in several gene families that have been implicated in parasitism in other nematode species. The high-quality draft genome and our analyses thereof represent a significant advancement in our understanding of this unusual Caenorhabditis species