Predicting transfer RNA gene activity from sequence and genome context.

Transfer RNA (tRNA) genes are among the most highly transcribed genes in the genome owing to their central role in protein synthesis. However, there is evidence for a broad range of gene expression across tRNA loci. This complexity, combined with difficulty in measuring transcript abundance and high sequence identity across transcripts, has severely limited our collective understanding of tRNA gene expression regulation and evolution. We establish sequence-based correlates to tRNA gene expression and develop a tRNA gene classification method that does not require, but benefits from, comparative genomic information and achieves accuracy comparable to molecular assays. We observe that guanine + cytosine (G + C) content and CpG density surrounding tRNA loci is exceptionally well correlated with tRNA gene activity, supporting a prominent regulatory role of the local genomic context in combination with internal sequence features. We use our tRNA gene activity predictions in conjunction with a comprehensive tRNA gene ortholog set spanning 29 placental mammals to estimate the evolutionary rate of functional changes among orthologs. Our method adds a new dimension to large-scale tRNA functional prediction and will help prioritize characterization of functional tRNA variants. Its simplicity and robustness should enable development of similar approaches for other clades, as well as exploration of functional diversification of members of large gene families.

ARM-seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments.

High-throughput RNA sequencing has accelerated discovery of the complex regulatory roles of small RNAs, but RNAs containing modified nucleosides may escape detection when those modifications interfere with reverse transcription during RNA-seq library preparation. Here we describe AlkB-facilitated RNA methylation sequencing (ARM-seq), which uses pretreatment with Escherichia coli AlkB to demethylate N(1)-methyladenosine (m(1)A), N(3)-methylcytidine (m(3)C) and N(1)-methylguanosine (m(1)G), all commonly found in tRNAs. Comparative methylation analysis using ARM-seq provides the first detailed, transcriptome-scale map of these modifications and reveals an abundance of previously undetected, methylated small RNAs derived from tRNAs. ARM-seq demonstrates that tRNA fragments accurately recapitulate the m(1)A modification state for well-characterized yeast tRNAs and generates new predictions for a large number of human tRNAs, including tRNA precursors and mitochondrial tRNAs. Thus, ARM-seq provides broad utility for identifying previously overlooked methyl-modified RNAs, can efficiently monitor methylation state and may reveal new roles for tRNA fragments as biomarkers or signaling molecules.

The UCSC Archaeal Genome Browser: 2012 update.

The UCSC Archaeal Genome Browser (http://archaea.ucsc.edu) offers a graphical web-based resource for exploration and discovery within archaeal and other selected microbial genomes. By bringing together existing gene annotations, gene expression data, multiple-genome alignments, pre-computed sequence comparisons and other specialized analysis tracks, the genome browser is a powerful aggregator of varied genomic information. The genome browser environment maintains the current look-and-feel of the vertebrate UCSC Genome Browser, but also integrates archaeal and bacterial-specific tracks with a few graphic display enhancements. The browser currently contains 115 archaeal genomes, plus 31 genomes of viruses known to infect archaea. Some of the recently developed or enhanced tracks visualize data from published high-throughput RNA-sequencing studies, the NCBI Conserved Domain Database, sequences from pre-genome sequencing studies, predicted gene boundaries from three different protein gene prediction algorithms, tRNAscan-SE gene predictions with RNA secondary structures and CRISPR locus predictions. We have also developed a companion resource, the Archaeal COG Browser, to provide better search and display of arCOG gene function classifications, including their phylogenetic distribution among available archaeal genomes.

A sperm-enriched 5'fragment of tRNA-Valine regulates preimplantation embryonic transcriptome and development.

Sperm small RNAs have been implicated in intergenerational epigenetic inheritance of paternal environmental effects; however, their biogenesis and functions remain poorly understood. We previously identified a 5' fragment of tRNA-Valine-CAC-2 (tRFValCAC) as one of the most abundant small RNA in mature sperm. tRFValCAC is specifically enriched in sperm during post-testicular maturation in the epididymis, and we found that it is delivered to sperm from epididymis epithelial cells via extracellular vesicles. Here, we investigated the mechanistic basis of tRFValCAC delivery to sperm and its functions in the early embryo. We show that tRFValCAC interacts with an RNA binding protein, heterogeneous nuclear ribonucleoprotein A/B (hnRNPAB), in the epididymis, and this interaction regulates the sorting and packing of tRFValCAC into extracellular vesicles. In the embryo, we found that tRFValCAC regulates early embryonic mRNA processing and splicing. Inhibition of tRFValCAC in preimplantation embryos altered the transcript abundance of genes involved in RNA splicing and mRNA processing. Importantly, tRFValCAC-inhibited embryos showed altered mRNA splicing, including alternative splicing of various splicing factors and genes important for proper preimplantation embryonic development. Finally, we find that inhibition of tRFValCAC in zygotes delayed preimplantation embryonic development. Together, our results reveal a novel function of a sperm-enriched tRF in regulating alternating splicing and preimplantation embryonic development and shed light on the mechanism of sperm small RNA-mediated epigenetic inheritance.

Epididymis-specific RNase A family genes regulate fertility and small RNA processing.

Sperm small RNAs are implicated in intergenerational transmission of paternal environmental effects. Small RNAs generated by cleavage of tRNAs, known as tRNA fragments (tRFs), are an abundant class of RNAs in mature sperm, and can be modulated by environmental conditions. The ribonuclease(s) responsible for the biogenesis of tRFs in the male reproductive tract remains unknown. Angiogenin, a member of the Ribonuclease A superfamily (RNase A), cleaves tRNAs to generate tRFs in response to cellular stress. Four paralogs of Angiogenin, namely Rnase9, Rnase10, Rnase11, and Rnase12, are specifically expressed in the epididymis-a long, convoluted tubule where sperm mature and acquire fertility and motility. The biological functions of these genes remain largely unknown. Here, by generating mice deleted for all four genes (Rnase9-12-/-, termed "KO" for Knock Out), we report that these genes regulate fertility and RNA processing. KO mice showed complete male sterility. KO sperm fertilized oocytes in vitro but failed to efficiently fertilize oocytes in vivo, likely due to an inability of sperm to pass through the utero-tubular junction. Intriguingly, there were decreased levels of fragments of tRNAs (tRFs) and rRNAs (rRNA-derived small RNAs or rsRNAs) in the KO epididymis and epididymal luminal fluid, implying that Rnase9-12 regulate the biogenesis and/or stability of tRFs and rsRNAs. Importantly, KO sperm showed a dramatic decrease in the levels of tRFs, demonstrating a role of Rnase9-12 in regulating sperm RNA composition. Together, our results reveal an unexpected role of four epididymis-specific non-canonical RNase A family genes in fertility and RNA processing.

Sex-split analysis of pathology and motor-behavioral outcomes in a mouse model of CLN8-Batten disease reveals an increased disease burden and trajectory in female Cln8mnd mice.

BACKGROUND: CLN8-Batten disease (CLN8 disease) is a rare neurodegenerative disorder characterized phenotypically by progressive deterioration of motor and cognitive abilities, visual symptoms, epileptic seizures, and premature death. Mutations in CLN8 results in characteristic Batten disease symptoms and brain-wide pathology including accumulation of lysosomal storage material, gliosis, and neurodegeneration. Recent investigations of other subforms of Batten disease (CLN1, CLN3, CLN6) have emphasized the influence of biological sex on disease and treatment outcomes; however, little is known about sex differences in the CLN8 subtype. To determine the impact of sex on CLN8 disease burden and progression, we utilized a Cln8mnd mouse model to measure the impact and progression of histopathological and behavioral outcomes between sexes. RESULTS: Several notable sex differences were observed in the presentation of brain pathology, including Cln8mnd female mice consistently presenting with greater GFAP+ astrocytosis and CD68+ microgliosis in the somatosensory cortex, ventral posteromedial/ventral posterolateral nuclei of the thalamus, striatum, and hippocampus when compared to Cln8mnd male mice. Furthermore, sex differences in motor-behavioral assessments revealed Cln8mnd female mice experience poorer motor performance and earlier death than their male counterparts. Cln8mnd mice treated with an AAV9-mediated gene therapy were also examined to assess sex differences on therapeutics outcomes, which revealed no appreciable differences between the sexes when responding to the therapy. CONCLUSIONS: Taken together, our results provide further evidence of biologic sex as a modifier of Batten disease progression and outcome, thus warranting consideration when conducting investigations and monitoring therapeutic impact.

Impaired dynamic balance control in adolescents with idiopathic scoliosis and abnormal somatosensory evoked potentials.

BACKGROUND: Both balance control dysfunction and dysfunction of the central nervous system have been proposed as being causative factors in adolescent idiopathic scoliosis (AIS), yet the relationship between these factors has not been investigated in detail. An intergroup comparative study was conducted to investigate the effect of abnormal somatosensory function on the dynamic balance parameters of girls with AIS. METHODS: The relationship between dynamic balance control and abnormal somatosensory function seen in AIS patients was examined by studying the dynamic balance parameters in normal controls, in AIS patients with normal posterior tibial nerve somatosensory cortical evoked potentials (PTN-SCEPs), and in AIS patients with abnormal PTN-SCEPs. Gait parameters were recorded in 18 AIS girls (8 showing abnormal PTN-SCEPs and 10 showing normal PTN-SCEPs). Eight healthy age-matched volunteers served as a control group. RESULTS: No significant left-right asymmetry of gait parameters was found for the controls or the AIS patients with normal PTN-SCEPs, whereas significantly higher gait parameters were found on the side of the curvature in the AIS patients with abnormal PTN-SCEPs. CONCLUSIONS: Somatosensory dysfunction in AIS patients shows to have an impact on dynamic balance control. Further studies to examine the association between somatosensory dysfunction and balance control and how they may be related to the etiology of AIS are recommended. LEVEL OF EVIDENCE: Diagnostic study, level IV (case-control study).

Stress response of a marine ammonia-oxidizing archaeon informs physiological status of environmental populations.

High representation by ammonia-oxidizing archaea (AOA) in marine systems is consistent with their high affinity for ammonia, efficient carbon fixation, and copper (Cu)-centric respiratory system. However, little is known about their response to nutrient stress. We therefore used global transcriptional and proteomic analyses to characterize the response of a model AOA, Nitrosopumilus maritimus SCM1, to ammonia starvation, Cu limitation and Cu excess. Most predicted protein-coding genes were transcribed in exponentially growing cells, and of ~74% detected in the proteome, ~6% were modified by N-terminal acetylation. The general response to ammonia starvation and Cu stress was downregulation of genes for energy generation and biosynthesis. Cells rapidly depleted transcripts for the A and B subunits of ammonia monooxygenase (AMO) in response to ammonia starvation, yet retained relatively high levels of transcripts for the C subunit. Thus, similar to ammonia-oxidizing bacteria, selective retention of amoC transcripts during starvation appears important for subsequent recovery, and also suggests that AMO subunit transcript ratios could be used to assess the physiological status of marine populations. Unexpectedly, cobalamin biosynthesis was upregulated in response to both ammonia starvation and Cu stress, indicating the importance of this cofactor in retaining functional integrity during times of stress.

Reliability of radiographic intervertebral disc height measurement for in vivo rat-tail model.

Quantification of changes in intervertebral disc height is critical for studying intervertebral disc degeneration. Reliability of disc height measurement is therefore especially important for in vivo studies using animal models of disc degeneration. In this study, the effects of image intensity and percentage of disc width used for disc height measurement from radiographic images were evaluated in a rat-tail model. Radiographs were taken for 10 Sprague-Dawley rats using a standardized protocol. Average disc heights of the caudal 8-9 discs were determined using original and intensity adjusted images with different percentages of disc width. The average disc height was found to be significantly affected by both the image intensity and the percentage of disc width measured. A higher reliability was found in the measurement for image with adjusted intensity and using smaller disc width. Image intensity is suggested to be controlled and the disc width should be taken into account in quantifying the disc height.

The effect of backpack weight on the standing posture and balance of schoolgirls with adolescent idiopathic scoliosis and normal controls.

Concerns have been raised regarding the effect of carrying a backpack on adolescent posture and balance, but the effect of backpack loading combined with other factors affecting balance, such as adolescent idiopathic scoliosis (AIS), has not been determined. This study examines the effects of backpack load on the posture and balance of schoolgirls with AIS and normal controls. The standing posture of 26 schoolgirls with mild AIS (mean age 13, Cobb angle 10-25 degrees ) and 20 age-matched normal schoolgirls were recorded without a backpack and while carrying a standard dual-strap backpack loaded at 7.5%, 10%, 12.5% and 15% of the subject's bodyweight (BW). Kinematics of the pelvis, trunk and head were recorded using a motion analysis system and centre of pressure (COP) data were recorded using a force platform. Reliable COP data could only be derived for 13 of the subjects with AIS. Increasing backpack load causes a significantly increased flexion of the trunk in relation to the pelvis and extension of the head in relation to the trunk, as well as increased antero-posterior range of COP motion. While backpack load appears to affect balance predominantly in the antero-posterior direction, differences between groups were more evident in the medio-lateral direction, with AIS subjects showing poor balance in this direction. Overall, carrying a backpack causes similar sagittal plane changes in posture and balance in both normal and AIS groups. Load size or subject group did not influence balance, but the additive effect of backpack carrying and AIS on postural control alters the risk of fall in this population. Therefore, load limit recommendations based on normal subjects should not be applicable to subjects with AIS.

The effect of load carriage on the gait of girls with adolescent idiopathic scoliosis and normal controls.

Adolescent idiopathic scoliosis (AIS) and load-bearing both appear to place similar demands on gait, but no data regarding the combined effects of load-bearing gait in subjects with AIS could be found. The gait patterns of 22 normal adolescent girls and 28 girls with mild AIS (Cobb angle<25 degrees ) were recorded at backpack loads of 0, 7.5, 10, 12.5 and 15% body weight. Temporal-distance and joint kinematic, moment and power parameters were analyzed by repeated measures ANOVA. Findings showed that backpack carriage places an increased demand on the musculature of the lower limb and results in a gait characterized by reduced pelvic motion and greater hip flexion-extension. AIS has a generally similar effect on gait kinematics as backpack carriage, with AIS subjects having significantly longer double support durations, shorter single support durations and lower knee joint power generation and absorption than normal subjects. No interaction between backpack load and AIS was found however, although investigation of parameters indicating a critical response to load showed that this typically occurred at lower backpack loads (7.5% body weight) in the AIS group. Overall, both AIS and load-bearing place increased demands on gait, but carriage of a loaded backpack does not appear to cause any greater demand on subjects with AIS than normal controls.

The effect of prosthesis alignment on the symmetry of gait in subjects with unilateral transtibial amputation.

A high degree of gait symmetry is characteristic of healthy gait. The aim of this study is to examine the symmetry of various gait parameters in subjects with unilateral trans-tibial amputation over a range of acceptable anteroposterior translational and tilt alignments, and further to examine if a consistent alignment of highest symmetry can be found. Acceptable alignments were determined by bench, static and dynamic testing on level and non-level surfaces. A total of 15 kinetic and kinematic parameters were then measured in the seven subjects participating in this study. Results indicate that some parameters show consistently higher symmetries, particularly the vertical ground reaction force parameters and the stance duration, step length and time to full knee flexion during the swing phase. Symmetries in other parameters such as knee flexion at loading response, acceleration impulse, and peak anteroposterior propulsive force seem to have little relevance in determining whether the gait pattern for that prosthetic alignment is acceptable or not. While analysis of the symmetry of more relevant gait parameters may assist the prosthetist in consistently and objectively identifying a most symmetrical alignment within the acceptable range, further clinical study is required before any conclusions can be drawn regarding evaluation of symmetry as a tool in defining any optimum alignment.

The kinematics and intra- and inter-therapist consistencies of lower cervical rotational manipulation.

The aim of this study was to determine the in vivo kinematics of cervical rotational manipulation in normal subjects and examine the consistency of this technique within and between therapists. A four camera motion analysis system operating at 120 Hz was used to measure the head on trunk angular displacements during manipulation performed by three therapists on eight subjects. One of the therapists performed the manipulation twice for each subject over separate sessions. A consistent pattern of de-rotation prior to thrust was found with little motion other than axial rotation during de-rotation and thrust. The pooled mean de-rotation displacement was 4.8 degrees and the pooled mean thrust angle was 11.3 degrees , but these varied widely, and none of the kinematic parameters recorded proved to be consistent within or between therapists. Most of the kinematic parameters were correlated with the exception of thrust duration. Qualitative analysis shows a consistent technique in cervical rotational manipulation. Pre-manipulation positioning of the head relative to the trunk was fairly consistent for a single therapist over separate sessions, but other than this, the kinematic parameters in cervical rotational manipulation are generally inconsistent within and between therapists.

Muscular and centre of pressure response to sudden release of load in symmetric and asymmetric stoop lifting tasks.

Sudden changes in load during asymmetric lifting may be associated with a particularly high risk of loss of balance and spinal injury. Centre of pressure (COP) motions and electromyographic responses of trunk and lower limb muscles were studied in 10 normal male volunteers during sudden release of 20, 40, 60 and 80N stoop lifting loads in symmetric and asymmetric postures. Similar overall COP responses and muscular response strategies to sudden release of load were seen in both postures, although the asymmetric posture showed a larger medio-lateral COP displacements and greater co-contraction asymmetries. While sudden release of load in asymmetric stoop lifting does not seem to involve a greater risk of fall than symmetric lifting, the muscular response results in more complex and asymmetric loading of the trunk, indicating greater localised segmental loading and therefore increased risk of tissue injury.

Sudden release during a pulling task: the effect of release load on stance perturbation and recovery.

Ten subjects performed a horizontal pulling task with sudden release for three randomly allocated loads of 20, 40, and 60 N to examine the effect of sudden release load on centre of pressure (COP) motion and the response strategy with respect to load, and also to determine critical loads at which the response strategy must involve stepping to prevent a fall. Sudden release produces COP deflection directed posteriorly and laterally towards the anteroposterior midline. The magnitude of these deflections increase with release load (P<0.001), as does the time taken to re-establish normal stance following release (P<0.001). Interaction between lateral and anteroposterior shifts in the COP are seen following release, and establishing COP over a favoured limb may be an important factor in the response strategy. This may be so, because if the perturbation is large enough to provoke a protective step, then the COP will have to be located towards one limb before the step can take place. Linear regression of the anteroposterior limit of the COP indicates a wide range of critical loads that will result in a necessary preventive step backwards being taken (80-257 N, mean 153 N), although these values are likely to be overestimated somewhat.

Changes in nuclear composition following cyclic compression of the intervertebral disc in an in vivo rat-tail model.

While in vitro studies have shown that mechanical loading can result in changes in the composition of intervertebral disc matrix, the effects of cyclic loading in vivo have not been considered. The objective of this study was to assess the effect of static and cyclic compression of different frequencies on the nuclear composition of the intervertebral disc. Thirty-six Sprague-Dawley rats were randomly divided into a control group (no pin insertion, no loading), a sham group (pins inserted in sixth and seventh caudal vertebrae, no loading), a static loading group (compression applied via pins) and cyclic loading groups (loading at 0.5, 1.5 or 2.5 Hz). Loading was applied for 1 h each day from the third to 17th day following pin insertion, and the caudal 5-6, 6-7 and 7-8 discs harvested to quantify proteoglycan content, collagen content and chondrocyte density in the nucleus pulposus. Static compression resulted in a significant reduction in total proteoglycan content as compared with the adjacent control disc, but this effect was not seen in any of the cyclic loading groups. However, comparison with the sham group appears to indicate an overall decrease in total proteoglycan content at the targeted and adjacent levels following cyclic loading. The 0.5 Hz loading group showed a significantly greater total proteoglycan content than all other compression groups, and also showed a lower total collagen content than the sham group. Results suggest that frequency dependent changes in composition occur in response to cyclic loading, but are not limited to the directly loaded disc alone. Further studies are required to verify this, but the choice of control appears to need careful consideration in all studies of this nature.

The effect of cyclic compression on the mechanical properties of the inter-vertebral disc: an in vivo study in a rat tail model.

OBJECTIVE: To assess the changes in the mechanical properties of inter-vertebral discs in vivo following static and cyclic compressive loading of different frequencies. DESIGN: An in vivo biomechanical study using a rat-tail model of the inter-vertebral disc.Background. Mechanical loading has been suggested as playing a major role in the etiology of disc degeneration, but the relationship is still not fully understood. METHODS: Sixty Sprague-Dawley rats were subject to daily compressive stress via pins inserted in the 6th and 7th caudal vertebrae over a two-week loading period. Animals were randomly divided into a sham group (pin insertion, no loading), a static loading group, or cyclic loading groups of 0.5, 1.5, or 2.5 Hz. Loading was applied for 1 h each day from the 3rd to 17th day following pin insertion, and the angular compliance, angular laxity, and inter-pin distance were measured in vivo at days 0, 3, 10 and 17. RESULTS: Changes in the inter-vertebral disc height depended on the frequency of loading, with the decrease in disc height in the static compression group significantly greater than that in all other groups, whereas the decrease in the 1.5 Hz cyclic compression group was significantly smaller than that in all other compression groups. CONCLUSIONS: Changes in disc properties depend on both the total load exposure and the frequency of loading. Cyclic loading in general produced less marked changes than static loading, but loading at particular frequencies may result in more severe changes. RELEVANCE: Previous studies have shown the in vivo changes in the mechanical properties of inter-vertebral discs to depend on the magnitude and duration of loading. In this study, a frequency dependent response to cyclic loading is also demonstrated.

Multi-planar bending properties of lumbar intervertebral joints following cyclic bending.

OBJECTIVE: To assess the changes in the multi-planar bending properties of intervertebral joints following cyclic bending along different directions. DESIGN: An in vitro biomechanical study using porcine lumbar motion segments. BACKGROUND: Repeated bending has been suggested as part of the etiology of gradual prolapse of the intervertebral disc, but the multi-planar changes in bending properties following cyclic loading have not been examined in detail. METHODS: Porcine lumbar motion segments were subject to 1500 cycles of bending along directions of 0 degrees (flexion), 30 degrees, 60 degrees, or 90 degrees (right lateral bending). The multi-planar bending moments and hysteresis energies were recorded before loading and after various cycle numbers. RESULTS: Repeated bending at 30 degrees and 60 degrees resulted in greater decreases in mean bending moment and hysteresis energy than bending at 0 degrees or 90 degrees. No significant differences were seen between loading groups for the change in bending moment along the anterior testing directions, but significant differences were observed in the posterior and lateral testing directions, with bending at 30 degrees causing a significantly greater decrease in bending moment in the postero-lateral directions. CONCLUSIONS: The change in mechanical properties of porcine intervertebral joints due to cyclic bending depend on the direction of loading and the direction in which the properties are measured. Loading at 30 degrees provokes the most marked changes in bending moment and hysteresis energy.

The effects of release height on center of pressure and trunk muscle response following sudden release of stoop lifting tasks.

BACKGROUND AND OBJECTIVES: Sudden release of load during lifting threatens postural stability and is countered by trunk muscle response, which can generate high loads on the spine, and may be a cause of tissue injury. The postural threat following sudden release and the consequent muscular response are likely to depend on the posture at the time of release. This study investigates the effects of sudden release of load at two release heights of one- and three-quarters of the knee to shoulder distance during stoop lifting. METHODS: Ten normal southern Chinese male volunteers were subject to sudden release of 20, 40, 60 and 80 N loads during stoop lifting trials. The release was randomly selected to be on the third, fourth or fifth cycle of a trial and was triggered at heights of one- and three-quarters of the total knee to shoulder lifting distance. The subjects stood on a force platform to allow the postural disturbance to be recorded by monitoring the center of pressure (COP), and electromyographic (EMG) data were collected from the rectus abdominus, internal oblique, external oblique, erector spinae and latissimus dorsi muscle groups. RESULTS: The COP excursion moved closer to the posterior limit of stability with increasing release load, and this effect was significantly more marked for release from the lower of the two heights. The minimum posterior COP separation from the posterior limit of stability was significantly less for the lower release height at all loads (p<0.001 in all cases). EMG data showed that the agonist-antagonist co-contraction durations were higher for the lower release height, and unlike sudden release from the higher level, showed a significant increase with increasing load. CONCLUSIONS: Sudden release at lower release height during stoop lifting results in significantly greater postural disturbance and spinal loading. The mean load predicted to result in fall or stumble at the lower release height (133 N) is significantly less than that predicted at the higher of the two release heights (245 N). A more marked effect of release load is also seen in the postural disturbance and trunk muscle co-contraction time for the lower release height, and particular care should therefore be taken when handling potentially unstable loads under these conditions. If the security of the load cannot be guaranteed, storage at a higher level may reduce the risk of injury due to sudden release of the load.