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September 22, 2019  |  

Single-cell multiomics: multiple measurements from single cells.

Single-cell sequencing provides information that is not confounded by genotypic or phenotypic heterogeneity of bulk samples. Sequencing of one molecular type (RNA, methylated DNA or open chromatin) in a single cell, furthermore, provides insights into the cell’s phenotype and links to its genotype. Nevertheless, only by taking measurements of these phenotypes and genotypes from the same single cells can such inferences be made unambiguously. In this review, we survey the first experimental approaches that assay, in parallel, multiple molecular types from the same single cell, before considering the challenges and opportunities afforded by these and future technologies. Copyright © 2016. Published by Elsevier Ltd.

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September 22, 2019  |  

Single-cell RNAseq for the study of isoforms-how is that possible?

Single-cell RNAseq and alternative splicing studies have recently become two of the most prominent applications of RNAseq. However, the combination of both is still challenging, and few research efforts have been dedicated to the intersection between them. Cell-level insight on isoform expression is required to fully understand the biology of alternative splicing, but it is still an open question to what extent isoform expression analysis at the single-cell level is actually feasible. Here, we establish a set of four conditions that are required for a successful single-cell-level isoform study and evaluate how these conditions are met by these technologies in published research.

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September 22, 2019  |  

The small peptide world in long noncoding RNAs.

Long noncoding RNAs (lncRNAs) are a group of transcripts that are longer than 200 nucleotides (nt) without coding potential. Over the past decade, tens of thousands of novel lncRNAs have been annotated in animal and plant genomes because of advanced high-throughput RNA sequencing technologies and with the aid of coding transcript classifiers. Further, a considerable number of reports have revealed the existence of stable, functional small peptides (also known as micropeptides), translated from lncRNAs. In this review, we discuss the methods of lncRNA classification, the investigations regarding their coding potential and the functional significance of the peptides they encode.

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September 22, 2019  |  

Recent insights into the tick microbiome gained through next-generation sequencing.

The tick microbiome comprises communities of microorganisms, including viruses, bacteria and eukaryotes, and is being elucidated through modern molecular techniques. The advent of next-generation sequencing (NGS) technologies has enabled the genes and genomes within these microbial communities to be explored in a rapid and cost-effective manner. The advantages of using NGS to investigate microbiomes surpass the traditional non-molecular methods that are limited in their sensitivity, and conventional molecular approaches that are limited in their scalability. In recent years the number of studies using NGS to investigate the microbial diversity and composition of ticks has expanded. Here, we provide a review of NGS strategies for tick microbiome studies and discuss the recent findings from tick NGS investigations, including the bacterial diversity and composition, influential factors, and implications of the tick microbiome.

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September 22, 2019  |  

Novel molecules lncRNAs, tRFs and circRNAs deciphered from next-generation sequencing/RNA sequencing: computational databases and tools.

Powerful next-generation sequencing (NGS) technologies, more specifically RNA sequencing (RNA-seq), have been pivotal toward the detection and analysis and hypotheses generation of novel biomolecules, long noncoding RNAs (lncRNAs), tRNA-derived fragments (tRFs) and circular RNAs (circRNAs). Experimental validation of the occurrence of these biomolecules inside the cell has been reported. Their differential expression and functionally important role in several cancers types as well as other diseases such as Alzheimer’s and cardiovascular diseases have garnered interest toward further studies in this research arena. In this review, starting from a brief relevant introduction to NGS and RNA-seq and the expression and role of lncRNAs, tRFs and circRNAs in cancer, we have comprehensively analyzed the current landscape of databases developed and computational software used for analyses and visualization for this emerging and highly interesting field of these novel biomolecules. Our review will help the end users and research investigators gain information on the existing databases and tools as well as an understanding of the specific features which these offer. This will be useful for the researchers in their proper usage thereby guiding them toward novel hypotheses generation and saving time and costs involved in extensive experimental processes in these three different novel functional RNAs.© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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September 22, 2019  |  

Functional mitochondria in health and disease.

The ability to rapidly adapt cellular bioenergetic capabilities to meet rapidly changing environmental conditions is mandatory for normal cellular function and for cancer progression. Any loss of this adaptive response has the potential to compromise cellular function and render the cell more susceptible to external stressors such as oxidative stress, radiation, chemotherapeutic drugs, and hypoxia. Mitochondria play a vital role in bioenergetic and biosynthetic pathways and can rapidly adjust to meet the metabolic needs of the cell. Increased demand is met by mitochondrial biogenesis and fusion of individual mitochondria into dynamic networks, whereas a decrease in demand results in the removal of superfluous mitochondria through fission and mitophagy. Effective communication between nucleus and mitochondria (mito-nuclear cross talk), involving the generation of different mitochondrial stress signals as well as the nuclear stress response pathways to deal with these stressors, maintains bioenergetic homeostasis under most conditions. However, when mitochondrial DNA (mtDNA) mutations accumulate and mito-nuclear cross talk falters, mitochondria fail to deliver critical functional outputs. Mutations in mtDNA have been implicated in neuromuscular and neurodegenerative mitochondriopathies and complex diseases such as diabetes, cardiovascular diseases, gastrointestinal disorders, skin disorders, aging, and cancer. In some cases, drastic measures such as acquisition of new mitochondria from donor cells occurs to ensure cell survival. This review starts with a brief discussion of the evolutionary origin of mitochondria and summarizes how mutations in mtDNA lead to mitochondriopathies and other degenerative diseases. Mito-nuclear cross talk, including various stress signals generated by mitochondria and corresponding stress response pathways activated by the nucleus are summarized. We also introduce and discuss a small family of recently discovered hormone-like mitopeptides that modulate body metabolism. Under conditions of severe mitochondrial stress, mitochondria have been shown to traffic between cells, replacing mitochondria in cells with damaged and malfunctional mtDNA. Understanding the processes involved in cellular bioenergetics and metabolic adaptation has the potential to generate new knowledge that will lead to improved treatment of many of the metabolic, degenerative, and age-related inflammatory diseases that characterize modern societies.

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September 22, 2019  |  

The Santa Pola saltern as a model for studying the microbiota of hypersaline environments.

Multi-pond salterns constitute an excellent model for the study of the microbial diversity and ecology of hypersaline environments, showing a wide range of salt concentrations, from seawater to salt saturation. Accumulated studies on the Santa Pola (Alicante, Spain) multi-pond solar saltern during the last 35 years include culture-dependent and culture-independent molecular methods and metagenomics more recently. These approaches have permitted to determine in depth the microbial diversity of the ponds with intermediate salinities (from 10 % salts) up to salt saturation, with haloarchaea and bacteria as the two main dominant groups. In this review, we describe the main results obtained using the different methodologies, the most relevant contributions for understanding the ecology of these extreme environments and the future perspectives for such studies.

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September 22, 2019  |  

Vertebrate genome evolution in the light of fish cytogenomics and rDNAomics.

To understand the cytogenomic evolution of vertebrates, we must first unravel the complex genomes of fishes, which were the first vertebrates to evolve and were ancestors to all other vertebrates. We must not forget the immense time span during which the fish genomes had to evolve. Fish cytogenomics is endowed with unique features which offer irreplaceable insights into the evolution of the vertebrate genome. Due to the general DNA base compositional homogeneity of fish genomes, fish cytogenomics is largely based on mapping DNA repeats that still represent serious obstacles in genome sequencing and assembling, even in model species. Localization of repeats on chromosomes of hundreds of fish species and populations originating from diversified environments have revealed the biological importance of this genomic fraction. Ribosomal genes (rDNA) belong to the most informative repeats and in fish, they are subject to a more relaxed regulation than in higher vertebrates. This can result in formation of a literal ‘rDNAome’ consisting of more than 20,000 copies with their high proportion employed in extra-coding functions. Because rDNA has high rates of transcription and recombination, it contributes to genome diversification and can form reproductive barrier. Our overall knowledge of fish cytogenomics grows rapidly by a continuously increasing number of fish genomes sequenced and by use of novel sequencing methods improving genome assembly. The recently revealed exceptional compositional heterogeneity in an ancient fish lineage (gars) sheds new light on the compositional genome evolution in vertebrates generally. We highlight the power of synergy of cytogenetics and genomics in fish cytogenomics, its potential to understand the complexity of genome evolution in vertebrates, which is also linked to clinical applications and the chromosomal backgrounds of speciation. We also summarize the current knowledge on fish cytogenomics and outline its main future avenues.

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September 22, 2019  |  

Androgen and estrogen sensitivity of bird song: a comparative view on gene regulatory levels.

Singing of songbirds is sensitive to testosterone and its androgenic and estrogenic metabolites in a species-specific way. The hormonal effects on song pattern are likely mediated by androgen receptors (AR) and estrogen receptor alpha (ERa), ligand activated transcription factors that are expressed in neurons of various areas of the songbirds’ vocal control circuit. The distribution of AR in this circuit is rather similar between species while that of ERa is species variant and concerns a key vocal control area, the HVC (proper name). We discuss the regulation of the expression of the cognate AR and ERa and putative splice variants. In particular, we suggest that transcription factor binding sites in the promoter of these receptors differ between bird species. Further, we suggest that AR- and ERa-dependent gene regulation in vocal areas differs between species due to species-specific DNA binding sites of putative target genes that are required for the transcriptional activity of the receptors. We suggest that species differences in the distribution of AR and ERa in vocal areas and in the genomic sensitivity to these receptors contribute to species-specific hormonal regulation of the song.

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September 22, 2019  |  

By land, air, and sea: hemipteran diversity through the genomic lens

Thanks to a recent spate of sequencing projects, the Hemiptera are the first hemimetabolous insect order to achieve a critical mass of species with sequenced genomes, establishing the basis for comparative genomics of the bugs. However, as the most speciose hemimetabolous order, there is still a vast swathe of the hemipteran phylogeny that awaits genomic representation across subterranean, terrestrial, and aquatic habitats, and with lineage-specific and developmentally plastic cases of both wing polyphenisms and flightlessness. In this review, we highlight opportunities for taxonomic sampling beyond obvious pest species candidates, motivated by intriguing biological features of certain groups as well as the rich research tradition of ecological, physiological, developmental, and particularly cytogenetic investigation that spans the diversity of the Hemiptera.

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September 22, 2019  |  

Bat biology, genomes, and the Bat1K project: To generate chromosome-level genomes for all living bat species.

Bats are unique among mammals, possessing some of the rarest mammalian adaptations, including true self-powered flight, laryngeal echolocation, exceptional longevity, unique immunity, contracted genomes, and vocal learning. They provide key ecosystem services, pollinating tropical plants, dispersing seeds, and controlling insect pest populations, thus driving healthy ecosystems. They account for more than 20% of all living mammalian diversity, and their crown-group evolutionary history dates back to the Eocene. Despite their great numbers and diversity, many species are threatened and endangered. Here we announce Bat1K, an initiative to sequence the genomes of all living bat species (n~1,300) to chromosome-level assembly. The Bat1K genome consortium unites bat biologists (>148 members as of writing), computational scientists, conservation organizations, genome technologists, and any interested individuals committed to a better understanding of the genetic and evolutionary mechanisms that underlie the unique adaptations of bats. Our aim is to catalog the unique genetic diversity present in all living bats to better understand the molecular basis of their unique adaptations; uncover their evolutionary history; link genotype with phenotype; and ultimately better understand, promote, and conserve bats. Here we review the unique adaptations of bats and highlight how chromosome-level genome assemblies can uncover the molecular basis of these traits. We present a novel sequencing and assembly strategy and review the striking societal and scientific benefits that will result from the Bat1K initiative.

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September 22, 2019  |  

A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants.

Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) ‘Hongyang’ draft genome has 164 Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models.A second genome of an A. chinensis (genotype Red5) was fully sequenced. This new sequence resulted in a 554.0 Mb assembly with all but 6 Mb assigned to pseudo-chromosomes. Pseudo-chromosomal comparisons showed a considerable number of translocation events have occurred following a whole genome duplication (WGD) event some consistent with centromeric Robertsonian-like translocations. RNA sequencing data from 12 tissues and ab initio analysis informed a genome-wide manual annotation, using the WebApollo tool. In total, 33,044 gene loci represented by 33,123 isoforms were identified, named and tagged for quality of evidential support. Of these 3114 (9.4%) were identical to a protein within ‘Hongyang’ The Kiwifruit Information Resource (KIR v2). Some proportion of the differences will be varietal polymorphisms. However, as most computationally predicted Red5 models required manual re-annotation this proportion is expected to be small. The quality of the new gene models was tested by fully sequencing 550 cloned ‘Hort16A’ cDNAs and comparing with the predicted protein models for Red5 and both the original ‘Hongyang’ assembly and the revised annotation from KIR v2. Only 48.9% and 63.5% of the cDNAs had a match with 90% identity or better to the original and revised ‘Hongyang’ annotation, respectively, compared with 90.9% to the Red5 models.Our study highlights the need to take a cautious approach to draft genomes and computationally predicted genes. Our use of the manual annotation tool WebApollo facilitated manual checking and correction of gene models enabling improvement of computational prediction. This utility was especially relevant for certain types of gene families such as the EXPANSIN like genes. Finally, this high quality gene set will supply the kiwifruit and general plant community with a new tool for genomics and other comparative analysis.

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September 22, 2019  |  

Developing collaborative works for faster progress on fungal respiratory infections in cystic fibrosis.

Cystic fibrosis (CF) is the major genetic inherited disease in Caucasian populations. The respiratory tract of CF patients displays a sticky viscous mucus, which allows for the entrapment of airborne bacteria and fungal spores and provides a suitable environment for growth of microorganisms, including numerous yeast and filamentous fungal species. As a consequence, respiratory infections are the major cause of morbidity and mortality in this clinical context. Although bacteria remain the most common agents of these infections, fungal respiratory infections have emerged as an important cause of disease. Therefore, the International Society for Human and Animal Mycology (ISHAM) has launched a working group on Fungal respiratory infections in Cystic Fibrosis (Fri-CF) in October 2006, which was subsequently approved by the European Confederation of Medical Mycology (ECMM). Meetings of this working group, comprising both clinicians and mycologists involved in the follow-up of CF patients, as well as basic scientists interested in the fungal species involved, provided the opportunity to initiate collaborative works aimed to improve our knowledge on these infections to assist clinicians in patient management. The current review highlights the outcomes of some of these collaborative works in clinical surveillance, pathogenesis and treatment, giving special emphasis to standardization of culture procedures, improvement of species identification methods including the development of nonculture-based diagnostic methods, microbiome studies and identification of new biological markers, and the description of genotyping studies aiming to differentiate transient carriage and chronic colonization of the airways. The review also reports on the breakthrough in sequencing the genomes of the main Scedosporium species as basis for a better understanding of the pathogenic mechanisms of these fungi, and discusses treatment options of infections caused by multidrug resistant microorganisms, such as Scedosporium and Lomentospora species and members of the Rasamsonia argillacea species complex.

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September 22, 2019  |  

Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions.

Biobutanol production by clostridia via the acetone-butanol-ethanol (ABE) pathway is a promising future technology in bioenergetics , but identifying key regulatory mechanisms for this pathway is essential in order to construct industrially relevant strains with high tolerance and productivity. We have applied flow cytometric analysis to C. beijerinckii NRRL B-598 and carried out comparative screening of physiological changes in terms of viability under different cultivation conditions to determine its dependence on particular stages of the life cycle and the concentration of butanol.Dual staining by propidium iodide (PI) and carboxyfluorescein diacetate (CFDA) provided separation of cells into four subpopulations with different abilities to take up PI and cleave CFDA, reflecting different physiological states. The development of a staining pattern during ABE fermentation showed an apparent decline in viability, starting at the pH shift and onset of solventogenesis, although an appreciable proportion of cells continued to proliferate. This was observed for sporulating as well as non-sporulating phenotypes at low solvent concentrations, suggesting that the increase in percentage of inactive cells was not a result of solvent toxicity or a transition from vegetative to sporulating stages. Additionally, the sporulating phenotype was challenged with butanol and cultivation with a lower starting pH was performed; in both these experiments similar trends were obtained-viability declined after the pH breakpoint, independent of the actual butanol concentration in the medium. Production characteristics of both sporulating and non-sporulating phenotypes were comparable, showing that in C. beijerinckii NRRL B-598, solventogenesis was not conditional on sporulation.We have shown that the decline in C. beijerinckii NRRL B-598 culture viability during ABE fermentation was not only the result of accumulated toxic metabolites, but might also be associated with a special survival strategy triggered by pH change.

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September 22, 2019  |  

Mycobacterial biomaterials and resources for researchers.

There are many resources available to mycobacterial researchers, including culture collections around the world that distribute biomaterials to the general scientific community, genomic and clinical databases, and powerful bioinformatics tools. However, many of these resources may be unknown to the research community. This review article aims to summarize and publicize many of these resources, thus strengthening the quality and reproducibility of mycobacterial research by providing the scientific community access to authenticated and quality-controlled biomaterials and a wealth of information, analytical tools and research opportunities.

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