How do bacterial endosymbionts work with so few genes?

The move from a free-living environment to a long-term residence inside a host eukaryotic cell has profound effects on bacterial function. While endosymbioses are found in many eukaryotes, from protists to plants to animals, the bacteria that form these host-beneficial relationships are even more diverse. Endosymbiont genomes can become radically smaller than their free-living relatives, and their few remaining genes show extreme compositional biases. The details of how these reduced and divergent gene sets work, and how they interact with their host cell, remain mysterious. This Unsolved Mystery reviews how genome reduction alters endosymbiont biology and highlights a "tipping point" where the loss of the ability to build a cell envelope coincides with a marked erosion of translation-related genes.

Rewiring of Aminoacyl-tRNA Synthetase Localization and Interactions in Plants With Extensive Mitochondrial tRNA Gene Loss.

The number of tRNAs encoded in plant mitochondrial genomes varies considerably. Ongoing loss of bacterial-like mitochondrial tRNA genes in many lineages necessitates the import of nuclear-encoded counterparts that share little sequence similarity. Because tRNAs are involved in highly specific molecular interactions, this replacement process raises questions about the identity and trafficking of enzymes necessary for the maturation and function of newly imported tRNAs. In particular, the aminoacyl-tRNA synthetases (aaRSs) that charge tRNAs are usually divided into distinct classes that specialize on either organellar (mitochondrial and plastid) or nuclear-encoded (cytosolic) tRNAs. Here, we investigate the evolution of aaRS subcellular localization in a plant lineage (Sileneae) that has experienced extensive and rapid mitochondrial tRNA loss. By analyzing full-length mRNA transcripts (PacBio Iso-Seq), we found predicted retargeting of many ancestrally cytosolic aaRSs to the mitochondrion and confirmed these results with colocalization microscopy assays. However, we also found cases where aaRS localization does not appear to change despite functional tRNA replacement, suggesting evolution of novel interactions and charging relationships. Therefore, the history of repeated tRNA replacement in Sileneae mitochondria reveals that differing constraints on tRNA/aaRS interactions may determine which of these alternative coevolutionary paths is used to maintain organellar translation in plant cells.

Cytonuclear integration and co-evolution.

The partitioning of genetic material between the nucleus and cytoplasmic (mitochondrial and plastid) genomes within eukaryotic cells necessitates coordinated integration between these genomic compartments, with important evolutionary and biomedical implications. Classic questions persist about the pervasive reduction of cytoplasmic genomes via a combination of gene loss, transfer and functional replacement - and yet why they are almost always retained in some minimal form. One striking consequence of cytonuclear integration is the existence of 'chimeric' enzyme complexes composed of subunits encoded in two different genomes. Advances in structural biology and comparative genomics are yielding important insights into the evolution of such complexes, including correlated sequence changes and recruitment of novel subunits. Thus, chimeric cytonuclear complexes provide a powerful window into the mechanisms of molecular co-evolution.

Rapid Shifts in Mitochondrial tRNA Import in a Plant Lineage with Extensive Mitochondrial tRNA Gene Loss.

In most eukaryotes, transfer RNAs (tRNAs) are one of the very few classes of genes remaining in the mitochondrial genome, but some mitochondria have lost these vestiges of their prokaryotic ancestry. Sequencing of mitogenomes from the flowering plant genus Silene previously revealed a large range in tRNA gene content, suggesting rapid and ongoing gene loss/replacement. Here, we use this system to test longstanding hypotheses about how mitochondrial tRNA genes are replaced by importing nuclear-encoded tRNAs. We traced the evolutionary history of these gene loss events by sequencing mitochondrial genomes from key outgroups (Agrostemma githago and Silene [=Lychnis] chalcedonica). We then performed the first global sequencing of purified plant mitochondrial tRNA populations to characterize the expression of mitochondrial-encoded tRNAs and the identity of imported nuclear-encoded tRNAs. We also confirmed the utility of high-throughput sequencing methods for the detection of tRNA import by sequencing mitochondrial tRNA populations in a species (Solanum tuberosum) with known tRNA trafficking patterns. Mitochondrial tRNA sequencing in Silene revealed substantial shifts in the abundance of some nuclear-encoded tRNAs in conjunction with their recent history of mt-tRNA gene loss and surprising cases where tRNAs with anticodons still encoded in the mitochondrial genome also appeared to be imported. These data suggest that nuclear-encoded counterparts are likely replacing mitochondrial tRNAs even in systems with recent mitochondrial tRNA gene loss, and the redundant import of a nuclear-encoded tRNA may provide a mechanism for functional replacement between translation systems separated by billions of years of evolutionary divergence.

Small-molecule inhibition of aging-associated chromosomal instability delays cellular senescence.

Chromosomal instability (CIN) refers to the rate at which cells are unable to properly segregate whole chromosomes, leading to aneuploidy. Besides its prevalence in cancer cells and postulated implications in promoting tumorigenesis, studies in aneuploidy-prone mouse models uncovered an unanticipated link between CIN and aging. Using young to old-aged human dermal fibroblasts, we observed a dysfunction of the mitotic machinery arising with age that mildly perturbs chromosome segregation fidelity and contributes to the generation of fully senescent cells. Here, we investigated mitotic mechanisms that contribute to age-associated CIN. We found that elderly cells have an increased number of stable kinetochore-microtubule (k-MT) attachments and decreased efficiency in the correction of improper k-MT interactions. Chromosome mis-segregation rates in old-aged cells decreased upon both genetic and small-molecule enhancement of MT-depolymerizing kinesin-13 activity. Notably, restored chromosome segregation accuracy inhibited the phenotypes of cellular senescence. Therefore, we provide mechanistic insight into age-associated CIN and disclose a strategy for the use of a small-molecule to inhibit age-associated CIN and to delay the cellular hallmarks of aging.

Combining tRNA sequencing methods to characterize plant tRNA expression and post-transcriptional modification.

Differences in tRNA expression have been implicated in a remarkable number of biological processes. There is growing evidence that tRNA genes can play dramatically different roles depending on both expression and post-transcriptional modification, yet sequencing tRNAs to measure abundance and detect modifications remains challenging. Their secondary structure and extensive post-transcriptional modifications interfere with RNA-seq library preparation methods and have limited the utility of high-throughput sequencing technologies. Here, we combine two modifications to standard RNA-seq methods by treating with the demethylating enzyme AlkB and ligating with tRNA-specific adapters in order to sequence tRNAs from four species of flowering plants, a group that has been shown to have some of the most extensive rates of post-transcriptional tRNA modifications. This protocol has the advantage of detecting full-length tRNAs and sequence variants that can be used to infer many post-transcriptional modifications. We used the resulting data to produce a modification index of almost all unique reference tRNAs in Arabidopsis thaliana, which exhibited many anciently conserved similarities with humans but also positions that appear to be 'hot spots' for modifications in angiosperm tRNAs. We also found evidence based on northern blot analysis and droplet digital PCR that, even after demethylation treatment, tRNA-seq can produce highly biased estimates of absolute expression levels most likely due to biased reverse transcription. Nevertheless, the generation of full-length tRNA sequences with modification data is still promising for assessing differences in relative tRNA expression across treatments, tissues or subcellular fractions and help elucidate the functional roles of tRNA modifications.

The multi-faceted regulation of nuclear tRNA gene transcription.

Transfer RNAs are among the most ancient molecules of life on earth. Beyond their crucial role in protein synthesis as carriers of amino acids, they are also important players in a plethora of other biological processes. Many debates in term of biogenesis, regulation and function persist around these fascinating non-coding RNAs. Our review focuses on the first step of their biogenesis in eukaryotes, i.e. their transcription from nuclear genes. Numerous and complementary ways have emerged during evolution to regulate transfer RNA gene transcription. Here, we will summarize the different actors implicated in this process: cis-elements, trans-factors, genomic contexts, epigenetic environments and finally three-dimensional organization of nuclear genomes. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1099-1108, 2019.

Partitioned coalescence support reveals biases in species-tree methods and detects gene trees that determine phylogenomic conflicts.

Genomic datasets sometimes support conflicting phylogenetic relationships when different tree-building methods are applied. Coherent interpretations of such results are enabled by partitioning support for controversial relationships among the constituent genes of a phylogenomic dataset. For the supermatrix (=concatenation) approach, several methods that measure the distribution of support and conflict among loci were introduced over 15 years ago. More recently, partitioned coalescence support (PCS) was developed for phylogenetic coalescence methods that account for incomplete lineage sorting and use the summed fits of gene trees to estimate the species tree. Here, we automate computation of PCS to permit application of this index to genome-scale matrices that include hundreds of loci. Reanalyses of four phylogenomic datasets for amniotes, land plants, skinks, and angiosperms demonstrate how PCS scores can be used to: (1) compare conflicting results favored by alternative coalescence methods, (2) identify outlier gene trees that have a disproportionate influence on the resolution of contentious relationships, (3) assess the effects of missing data in species-tree analysis, and (4) clarify biases in commonly-implemented coalescence methods and support indices. We show that key phylogenomic conclusions from these analyses often hinge on just a few gene trees and that results can be driven by specific biases of a particular coalescence method and/or the differential weight placed on gene trees with high versus low taxon sampling. The attribution of exceptionally high weight to some gene trees and very low weight to other gene trees counters the basic logic of phylogenomic coalescence analysis; even clades in species trees with high support according to commonly used indices (likelihood-ratio test, bootstrap, Bayesian local posterior probability) can be unstable to the removal of only one or two gene trees with high PCS. Computer simulations cannot adequately describe all of the contingencies and complexities of empirical genetic data. PCS scores complement simulation work by providing specific insights into a particular dataset given the assumptions of the phylogenetic coalescence method that is applied. In combination with standard measures of nodal support, PCS provides a more complete understanding of the overall genomic evidence for contested evolutionary relationships in species trees.

Olivine anisotropy suggests Gutenberg discontinuity is not the base of the lithosphere.

Tectonic plates are a key feature of Earth's structure, and their behavior and dynamics are fundamental drivers in a wide range of large-scale processes. The operation of plate tectonics, in general, depends intimately on the manner in which lithospheric plates couple to the convecting interior. Current debate centers on whether the transition from rigid lithosphere to flowing asthenosphere relates to increases in temperature or to changes in composition such as the presence of a small amount of melt or an increase in water content below a specified depth. Thus, the manner in which the rigid lithosphere couples to the flowing asthenosphere is currently unclear. Here we present results from laboratory-based torsion experiments on olivine aggregates with and without melt, yielding an improved database describing the crystallographic alignment of olivine grains. We combine this database with a flow model for oceanic upper mantle to predict the structure of the seismic anisotropy beneath ocean basins. Agreement between our model and seismological observations supports the view that the base of the lithosphere is thermally controlled. This model additionally supports the idea that discontinuities in velocity and anisotropy, often assumed to be the base of the lithosphere, are, instead, intralithospheric features reflecting a compositional boundary established at midocean ridges, not a rheological boundary.

Sleep disturbances associated with cigarette smoking.

Sleep disturbances resulting in insufficient sleep have been linked to negative physical, cognitive, and public health outcomes. Despite this, there has yet a study that examines the impact of smoking on sleep in a US based national sample. The current study sought to observe sleep disturbances associated with smoking status. Sleep disturbances in adults aged 20 years and above, from the 2005-2006 National Health and Nutrition Examination Survey, were measured among current, former, and never smokers (NS). Current smokers (CS) reported significantly less total sleep time, longer sleep onset latency, increased difficulty falling asleep, maintaining sleep, and waking up earlier than desired when compared to NS. Former smokers reported disturbances similar to NS and CS experienced poorer sleep than nonsmokers. Our study is the first to observe sleep difficulty by smoking status in a large, population-based, nationally representative sample. Recommendations for smoking cessation programs are discussed.

Aminoacyl-tRNA Synthetase Evolution within the Dynamic Tripartite Translation System of Plant Cells.

Eukaryotes maintain separate protein translation systems for nuclear and organellar genes, including distinct sets of tRNAs and aminoacyl-tRNA synthetases (aaRSs). In animals, mitochondrial-targeted aaRSs are expressed at lower levels and are less conserved in sequence than cytosolic aaRSs involved in translation of nuclear mRNAs, likely reflecting lower translational demands in mitochondria. In plants, translation is further complicated by the presence of plastids, which share most aaRSs with mitochondria. In addition, plant mitochondrial tRNA pools have a dynamic history of gene loss and functional replacement by tRNAs from other compartments. To investigate the consequences of these distinctive features of translation in plants, we analyzed sequence evolution in angiosperm aaRSs. In contrast to previously studied eukaryotic systems, we found that plant organellar and cytosolic aaRSs exhibit only a small difference in expression levels, and organellar aaRSs are slightly more conserved than cytosolic aaRSs. We hypothesize that these patterns result from high translational demands associated with photosynthesis in mature chloroplasts. We also investigated aaRS evolution in Sileneae, an angiosperm lineage with extensive mitochondrial tRNA replacement and aaRS retargeting. We predicted positive selection for changes in aaRS sequence resulting from these recent changes in subcellular localization and tRNA substrates but found little evidence for accelerated sequence divergence. Overall, the complex tripartite translation system in plant cells appears to have imposed more constraints on the long-term evolutionary rates of organellar aaRSs compared with other eukaryotic lineages, and plant aaRS protein sequences appear largely robust to more recent perturbations in subcellular localization and tRNA interactions.

Incompatibility and Interchangeability in Molecular Evolution.

There is remarkable variation in the rate at which genetic incompatibilities in molecular interactions accumulate. In some cases, minor changes-even single-nucleotide substitutions-create major incompatibilities when hybridization forces new variants to function in a novel genetic background from an isolated population. In other cases, genes or even entire functional pathways can be horizontally transferred between anciently divergent evolutionary lineages that span the tree of life with little evidence of incompatibilities. In this review, we explore whether there are general principles that can explain why certain genes are prone to incompatibilities while others maintain interchangeability. We summarize evidence pointing to four genetic features that may contribute to greater resistance to functional replacement: (1) function in multisubunit enzyme complexes and protein-protein interactions, (2) sensitivity to changes in gene dosage, (3) rapid rate of sequence evolution, and (4) overall importance to cell viability, which creates sensitivity to small perturbations in molecular function. We discuss the relative levels of support for these different hypotheses and lay out future directions that may help explain the striking contrasts in patterns of incompatibility and interchangeability throughout the history of molecular evolution.

Outcomes of Parent-Child Interaction Therapy (PCIT) for families presenting with child maltreatment: A systematic review.

BACKGROUND: The developmental consequences of childhood trauma for young children are extensive and impact a diverse range of areas. Young children require treatments that consider their developmental stage and are inclusive of caregiver involvement. Parent-Child Interaction Therapy (PCIT), with its dyadic focus and developmental sensitivity, is uniquely positioned to offer therapeutic support to young children and their families. AIM: The current study aimed to conduct a systematic review of the current literature on PCIT and trauma and determine treatment outcomes for children and caregivers. METHOD: A systematic review of five electronic databases was undertaken. Studies that utilized PCIT to treat a population who had experienced trauma were included in the review regardless of study design. RESULTS: PCIT was used to treat a population who had experienced trauma in 40 studies. PCIT was an effective treatment in improving a variety of child and parent outcomes in this population including reduced parenting stress, child behavior problems, child trauma symptoms, parental mental health concerns, negative parenting strategies, and reducing potential risk of recidivism of abuse and neglect. These findings should be taken with caution given attrition rates and potential for bias in the study samples. DISCUSSION: Clinicians should consider PCIT as a potential treatment for children who have experienced trauma and their families. Future research should incorporate corroborative sources of information, assessment of caregiver and child trauma symptoms, examination of permanency outcomes, and consider standardization of PCIT modifications for child trauma to determine treatment in this population of children.

Hopeful monsters: unintended sequencing of famously malformed mite mitochondrial tRNAs reveals widespread expression and processing of sense-antisense pairs.

Although tRNA structure is one of the most conserved and recognizable shapes in molecular biology, aberrant tRNAs are frequently found in the mitochondrial genomes of metazoans. The extremely degenerate structures of several mitochondrial tRNAs (mt-tRNAs) have led to doubts about their expression and function. Mites from the arachnid superorder Acariformes are predicted to have some of the shortest mt-tRNAs, with a complete loss of cloverleaf-like shape. While performing mitochondrial isolations and recently developed tRNA-seq methods in plant tissue, we inadvertently sequenced the mt-tRNAs from a common plant pest, the acariform mite Tetranychus urticae, to a high enough coverage to detect all previously annotated T. urticae tRNA regions. The results not only confirm expression, CCA-tailing and post-transcriptional base modification of these highly divergent tRNAs, but also revealed paired sense and antisense expression of multiple T. urticae mt-tRNAs. Mirrored expression of mt-tRNA genes has been hypothesized but not previously demonstrated to be common in any system. We discuss the functional roles that these divergent tRNAs could have as both decoding molecules in translation and processing signals in transcript maturation pathways, as well as how sense-antisense pairs add another dimension to the bizarre tRNA biology of mitochondrial genomes.

A comparative analysis of methods to measure kinetochore-microtubule attachment stability.

During mitosis, spindle microtubules dynamically attach to and detach from kinetochores in a precise and regulated fashion. To ensure mitotic fidelity, kinetochore-microtubule (k-MT) attachments must be stable enough to satisfy the spindle assembly checkpoint (SAC), but sufficiently unstable to facilitate the correction of maloriented attachments. Different methods are available to assess k-MT stability in both live and fixed cells, but a comparative survey of these methods has not yet been reported. Here, we evaluate several quantitative and semiquantitative methods for determining k-MT stability and apply each technique to illustrate changes in spindle microtubule dynamics upon perturbation with physiologically relevant concentrations of microtubule stabilizing (Taxol) and destabilizing (UMK57 and nocodazole) compounds. We discuss the utility of each technique for defining specific features of spindle microtubule dynamics and k-MT attachment stability.

Interchangeable parts: The evolutionarily dynamic tRNA population in plant mitochondria.

Transfer RNAs (tRNAs) remain one of the very few classes of genes still encoded in the mitochondrial genome. These key components of the protein translation system must interact with a large enzymatic network of nuclear-encoded gene products to maintain mitochondrial function. Plants have an evolutionarily dynamic mitochondrial tRNA population, including ongoing tRNA gene loss and replacement by both horizontal gene transfer from diverse sources and import of nuclear-expressed tRNAs from the cytosol. Thus, plant mitochondria represent an excellent model for understanding how anciently divergent genes can act as "interchangeable parts" during the evolution of complex molecular systems. In particular, understanding the integration of the mitochondrial translation system with elements of the corresponding machinery used in cytosolic protein synthesis is a key area for eukaryotic cellular evolution. Here, we review the increasingly detailed phylogenetic data about the evolutionary history of mitochondrial tRNA gene loss, transfer, and functional replacement that has created extreme variation in mitochondrial tRNA populations across plant species. We describe emerging tRNA-seq methods with promise for refining our understanding of the expression and subcellular localization of tRNAs. Finally, we summarize current evidence and identify open questions related to coevolutionary changes in nuclear-encoded enzymes that have accompanied turnover in mitochondrial tRNA populations.

Characterisation of Listeria monocytogenes isolates from cattle using a bovine caruncular epithelial cell model.

Listeria monocytogenes is an important foodborne pathogen in human and veterinary health, causing significant morbidity and mortality including abortion. It has a particular tropism for the gravid uterus, however, the route of infection in reproductive tissues of ruminants (i.e. placentome), is much less clear. In this study, we aimed to investigate a bovine caruncular epithelial cell (BCEC) line as a model for L. monocytogenes infection of the bovine reproductive tract. The BCEC infection model was used to assess the ability of 14 different L. monocytogenes isolates to infect these cells. Lysozyme sensitivity and bacterial survival in 580 µg lysozyme/ml correlated with attenuated ability to proliferate in BCEC (p = 0.004 and p = 0.02, respectively). Four isolates were significantly attenuated compared to the control strain 10403S. One of these strains (AR008) showed evidence of compromised cell wall leading to increased sensitivity to ß-lactam antibiotics, and another (7644) had compromised cell membrane integrity leading to increased sensitivity to cationic peptides. Whole genome sequencing followed by Multi Locus Sequence Type analysis identified that five invasive isolates had the same sequence type, ST59, despite originating from three different clinical conditions. Virulence gene analysis showed that the attenuated isolate LM4 was lacking two virulence genes (uhpT, virR) known to be involved in intracellular growth and virulence. In conclusion, the BCEC model was able to differentiate between the infective potential of different isolates. Moreover, resistance to lysozyme correlated with the ability to invade and replicate within BCEC, suggesting co-selection for surviving challenging environments as the abomasum.

CyMIRA: The Cytonuclear Molecular Interactions Reference for Arabidopsis.

The function and evolution of eukaryotic cells depend upon direct molecular interactions between gene products encoded in nuclear and cytoplasmic genomes. Understanding how these cytonuclear interactions drive molecular evolution and generate genetic incompatibilities between isolated populations and species is of central importance to eukaryotic biology. Plants are an outstanding system to investigate such effects because of their two different genomic compartments present in the cytoplasm (mitochondria and plastids) and the extensive resources detailing subcellular targeting of nuclear-encoded proteins. However, the field lacks a consistent classification scheme for mitochondrial- and plastid-targeted proteins based on their molecular interactions with cytoplasmic genomes and gene products, which hinders efforts to standardize and compare results across studies. Here, we take advantage of detailed knowledge about the model angiosperm Arabidopsis thaliana to provide a curated database of plant cytonuclear interactions at the molecular level. CyMIRA (Cytonuclear Molecular Interactions Reference for Arabidopsis) is available at http://cymira.colostate.edu/ and https://github.com/dbsloan/cymira and will serve as a resource to aid researchers in partitioning evolutionary genomic data into functional gene classes based on organelle targeting and direct molecular interaction with cytoplasmic genomes and gene products. It includes 11 categories (and 27 subcategories) of different cytonuclear complexes and types of molecular interactions, and it reports residue-level information for cytonuclear contact sites. We hope that this framework will make it easier to standardize, interpret, and compare studies testing the functional and evolutionary consequences of cytonuclear interactions.

Safety and Feasibility of a First-Person View, Full-Body Interaction Game for Telerehabilitation Post-Stroke.

This study explored the feasibility and safety of pairing the Microsoft Kinect® sensor with the Oculus Rift® Head Mounted Display (HMD) as a telerehabilitation technology platform for persons post-stroke. To test initial safety, fourteen participants without disabilities (age 30 ± 8.8 years) engaged in a game-based task using the Microsoft Kinect® with a first-person view using the Oculus Rift®. These tasks were repeated for five participants post-stroke (age 56 ± 3.0 years). No significant adverse events occurred in either study population. When using the Oculus Rift® HMD, three participants without disabilities reported dizziness and nausea. All of the participants post-stroke required hands-on assistance for balance and fall prevention. The intensive nature of physical support necessary for this type of interaction limits the application as a telerehabilitation intervention. Given the increasing availability of HMDs for commercial use, it is crucial that the safety of immersive games and technologies for telerehabilitation is fully explored.

Initial Findings From an Acute Hospital Care at Home Waiver Initiative.

This cohort study assesses outcomes of patients treated during the initial 16 months of the Centers for Medicare & Medicaid Services Acute Hospital Care at Home initiative.