Lack of affinity signature for germinal center cells that have initiated plasma cell differentiation.

Long-lived plasma cells (PCs) secrete antibodies that can provide sustained immunity against infection. High-affinity cells are proposed to preferentially select into this compartment, potentiating the immune response. We used single-cell RNA-seq to track the germinal center (GC) development of Ighg2A10 B cells, specific for the Plasmodium falciparum circumsporozoite protein (PfCSP). Following immunization with Plasmodium sporozoites, we identified 3 populations of cells in the GC light zone (LZ). One LZ population expressed a gene signature associated with the initiation of PC differentiation and readily formed PCs in vitro. The estimated affinity of these pre-PC B cells was indistinguishable from that of LZ cells that remained in the GC. This remained true when high- or low-avidity recombinant PfCSP proteins were used as immunogens. These findings suggest that the initiation of PC development occurs via an affinity-independent process.

CRMnet: A deep learning model for predicting gene expression from large regulatory sequence datasets.

Recent large datasets measuring the gene expression of millions of possible gene promoter sequences provide a resource to design and train optimized deep neural network architectures to predict expression from sequences. High predictive performance due to the modeling of dependencies within and between regulatory sequences is an enabler for biological discoveries in gene regulation through model interpretation techniques. To understand the regulatory code that delineates gene expression, we have designed a novel deep-learning model (CRMnet) to predict gene expression in Saccharomyces cerevisiae. Our model outperforms the current benchmark models and achieves a Pearson correlation coefficient of 0.971 and a mean squared error of 3.200. Interpretation of informative genomic regions determined from model saliency maps, and overlapping the saliency maps with known yeast motifs, supports that our model can successfully locate the binding sites of transcription factors that actively modulate gene expression. We compare our model's training times on a large compute cluster with GPUs and Google TPUs to indicate practical training times on similar datasets.

Treatment-Refractory Sternocostoclavicular Hyperostosis.

Sternocostoclavicular hyperostosis (SCCH) is an infrequent chronic inflammatory disorder of the axial skeleton of unknown origin. SCCH goes often unrecognized due to a low level of awareness for the disorder. It typically presents with relapsing and remitting pain in the shoulder, neck, and anterior chest wall area with occasional swelling and tenderness of the sternoclavicular area. The diagnosis is confirmed radiologically by sclerosis and hyperostosis of the sternoclavicular joints. There have been several reports in which intravenous bisphosphonates and tumor necrosis factor-inhibitors have shown reasonable efficacy in the treatment of this disorder. We report a patient with a long history of SCCH in whom pamidronate 60 mg intravenously every 3 months for 3 years failed to reduce symptom severity and improve radiologic findings.

Statistical Methods for Transcriptome-Wide Analysis of RNA Methylation by Bisulfite Sequencing.

For the transcriptome-wide detection and quantification of the 5-methylcytosine (m5C) methylation modification of RNA, one experimental approach is via bisulfite conversion. In this chapter we discuss statistical methods, and a corresponding computational pipeline, to perform transcriptome-wide differential m5C methylation analysis between RNA samples, specialized for this assay.

A new link between transcriptional initiation and pre-mRNA splicing: The RNA binding histone variant H2A.B.

The replacement of histone H2A with its variant forms is critical for regulating all aspects of genome organisation and function. The histone variant H2A.B appeared late in evolution and is most highly expressed in the testis followed by the brain in mammals. This raises the question of what new function(s) H2A.B might impart to chromatin in these important tissues. We have immunoprecipitated the mouse orthologue of H2A.B, H2A.B.3 (H2A.Lap1), from testis chromatin and found this variant to be associated with RNA processing factors and RNA Polymerase (Pol) II. Most interestingly, many of these interactions with H2A.B.3 (Sf3b155, Spt6, DDX39A and RNA Pol II) were inhibited by the presence of endogenous RNA. This histone variant can bind to RNA directly in vitro and in vivo, and associates with mRNA at intron-exon boundaries. This suggests that the ability of H2A.B to bind to RNA negatively regulates its capacity to bind to these factors (Sf3b155, Spt6, DDX39A and RNA Pol II). Unexpectedly, H2A.B.3 forms highly decompacted nuclear subdomains of active chromatin that co-localizes with splicing speckles in male germ cells. H2A.B.3 ChIP-Seq experiments revealed a unique chromatin organization at active genes being not only enriched at the transcription start site (TSS), but also at the beginning of the gene body (but being excluded from the +1 nucleosome) compared to the end of the gene. We also uncover a general histone variant replacement process whereby H2A.B.3 replaces H2A.Z at intron-exon boundaries in the testis and the brain, which positively correlates with expression and exon inclusion. Taken together, we propose that a special mechanism of splicing may occur in the testis and brain whereby H2A.B.3 recruits RNA processing factors from splicing speckles to active genes following its replacement of H2A.Z.

Bone and bone marrow involvement in sarcoidosis.

Bone and bone marrow involvement in sarcoidosis have been infrequently reported. We aimed to describe the clinical features, radiological descriptions, pathological examinations, and outcomes of three patients with osseous sarcoidosis and one patient with bone marrow sarcoidosis seen at our institution. Our case series included fluorodeoxyglucose positron emission tomography descriptions in assessing the whole-body extent of sarcoidosis. In the era of advanced imaging, large bone and axial skeleton sarcoidosis lesions are more common than previously reported.

Transcriptome dynamics of the microRNA inhibition response.

We report a high-resolution time series study of transcriptome dynamics following antimiR-mediated inhibition of miR-9 in a Hodgkin lymphoma cell-line-the first such dynamic study of the microRNA inhibition response-revealing both general and specific aspects of the physiological response. We show miR-9 inhibition inducing a multiphasic transcriptome response, with a direct target perturbation before 4 h, earlier than previously reported, amplified by a downstream peak at ∼32 h consistent with an indirect response due to secondary coherent regulation. Predictive modelling indicates a major role for miR-9 in post-transcriptional control of RNA processing and RNA binding protein regulation. Cluster analysis identifies multiple co-regulated gene regulatory modules. Functionally, we observe a shift over time from mRNA processing at early time points to translation at later time points. We validate the key observations with independent time series qPCR and we experimentally validate key predicted miR-9 targets. Methodologically, we developed sensitive functional data analytic predictive methods to analyse the weak response inherent in microRNA inhibition experiments. The methods of this study will be applicable to similar high-resolution time series transcriptome analyses and provides the context for more accurate experimental design and interpretation of future microRNA inhibition studies.

Genome-wide nucleosome map and cytosine methylation levels of an ancient human genome.

Epigenetic information is available from contemporary organisms, but is difficult to track back in evolutionary time. Here, we show that genome-wide epigenetic information can be gathered directly from next-generation sequence reads of DNA isolated from ancient remains. Using the genome sequence data generated from hair shafts of a 4000-yr-old Paleo-Eskimo belonging to the Saqqaq culture, we generate the first ancient nucleosome map coupled with a genome-wide survey of cytosine methylation levels. The validity of both nucleosome map and methylation levels were confirmed by the recovery of the expected signals at promoter regions, exon/intron boundaries, and CTCF sites. The top-scoring nucleosome calls revealed distinct DNA positioning biases, attesting to nucleotide-level accuracy. The ancient methylation levels exhibited high conservation over time, clustering closely with modern hair tissues. Using ancient methylation information, we estimated the age at death of the Saqqaq individual and illustrate how epigenetic information can be used to infer ancient gene expression. Similar epigenetic signatures were found in other fossil material, such as 110,000- to 130,000-yr-old bones, supporting the contention that ancient epigenomic information can be reconstructed from a deep past. Our findings lay the foundation for extracting epigenomic information from ancient samples, allowing shifts in epialleles to be tracked through evolutionary time, as well as providing an original window into modern epigenomics.

Social insect genomes exhibit dramatic evolution in gene composition and regulation while preserving regulatory features linked to sociality.

Genomes of eusocial insects code for dramatic examples of phenotypic plasticity and social organization. We compared the genomes of seven ants, the honeybee, and various solitary insects to examine whether eusocial lineages share distinct features of genomic organization. Each ant lineage contains ∼4000 novel genes, but only 64 of these genes are conserved among all seven ants. Many gene families have been expanded in ants, notably those involved in chemical communication (e.g., desaturases and odorant receptors). Alignment of the ant genomes revealed reduced purifying selection compared with Drosophila without significantly reduced synteny. Correspondingly, ant genomes exhibit dramatic divergence of noncoding regulatory elements; however, extant conserved regions are enriched for novel noncoding RNAs and transcription factor-binding sites. Comparison of orthologous gene promoters between eusocial and solitary species revealed significant regulatory evolution in both cis (e.g., Creb) and trans (e.g., fork head) for nearly 2000 genes, many of which exhibit phenotypic plasticity. Our results emphasize that genomic changes can occur remarkably fast in ants, because two recently diverged leaf-cutter ant species exhibit faster accumulation of species-specific genes and greater divergence in regulatory elements compared with other ants or Drosophila. Thus, while the "socio-genomes" of ants and the honeybee are broadly characterized by a pervasive pattern of divergence in gene composition and regulation, they preserve lineage-specific regulatory features linked to eusociality. We propose that changes in gene regulation played a key role in the origins of insect eusociality, whereas changes in gene composition were more relevant for lineage-specific eusocial adaptations.

Molecular pathways involved in the improvement of non-alcoholic fatty liver disease.

UNLABELLED: Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis are components of the metabolic syndrome. Serum leptin levels are elevated in obesity, but the role of leptin in the pathophysiology of the liver involvement is still unclear. To identify the effects and mechanisms by which leptin influences the pathogenesis of NAFLD, we performed epididymal white adipose tissue (eWAT) transplantation from congenic wild-type mice into the subcutaneous dorsal area of Lep(ob/ob) recipient mice and compared the results with those of the Lep(ob/ob) sham-operated mice. The mice were followed for 102-216 days. During killing, the transplanted mice had significantly lost body weight and exhibited significantly higher leptin levels, improved glucose tolerance, and lower liver injury scores than the sham-operated mice. Liver microarray analysis showed that novel pathways related to GA-binding protein (GABP) transcription factor targets, pheromone binding, and olfactory signaling were differentially expressed in the transplanted mice. Our data also replicate pathways known to be involved in NAFLD, such as those involved in the regulation of microRNAs, lipid, glucose, and glutathione metabolism, peroxisome proliferator-activated receptor signaling, cellular regulation, carboxylic acid processes, iron, heme, and tetrapyrrole binding, immunity and inflammation, insulin signaling, cytochrome P450 function, and cancer. CONCLUSION: wild-type eWAT transplantation into Lep(ob/ob) mice led to improvements in metabolism, body weight, and liver injury, possibly attributed to the production of leptin by the transplanted eWAT. These improvements were accompanied by the differential expression of novel pathways. The causal relationship between GABP downregulation and NAFLD improvement remains to be determined.

Histone H2A.Z inheritance during the cell cycle and its impact on promoter organization and dynamics.

Although it has been clearly established that well-positioned histone H2A.Z-containing nucleosomes flank the nucleosome-depleted region (NDR) at the transcriptional start site (TSS) of active mammalian genes, how this chromatin-based information is transmitted through the cell cycle is unknown. We show here that in mouse trophoblast stem cells, the amount of histone H2A.Z at promoters decreased during S phase, coinciding with homotypic (H2A.Z-H2A.Z) nucleosomes flanking the TSS becoming heterotypic (H2A.Z-H2A). To our surprise these nucleosomes remained heterotypic at M phase. At the TSS, we identified an unstable heterotypic histone H2A.Z-containing nucleosome in G1 phase that was lost after DNA replication. These dynamic changes at the TSS mirror a global expansion of the NDR at S and M phases, which, unexpectedly, is unrelated to transcriptional activity. Coincident with the loss of histone H2A.Z at promoters, histone H2A.Z is targeted to the centromere when mitosis begins.

Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA.

The modified base 5-methylcytosine (m(5)C) is well studied in DNA, but investigations of its prevalence in cellular RNA have been largely confined to tRNA and rRNA. In animals, the two m(5)C methyltransferases NSUN2 and TRDMT1 are known to modify specific tRNAs and have roles in the control of cell growth and differentiation. To map modified cytosine sites across a human transcriptome, we coupled bisulfite conversion of cellular RNA with next-generation sequencing. We confirmed 21 of the 28 previously known m(5)C sites in human tRNAs and identified 234 novel tRNA candidate sites, mostly in anticipated structural positions. Surprisingly, we discovered 10,275 sites in mRNAs and other non-coding RNAs. We observed that distribution of modified cytosines between RNA types was not random; within mRNAs they were enriched in the untranslated regions and near Argonaute binding regions. We also identified five new sites modified by NSUN2, broadening its known substrate range to another tRNA, the RPPH1 subunit of RNase P and two mRNAs. Our data demonstrates the widespread presence of modified cytosines throughout coding and non-coding sequences in a transcriptome, suggesting a broader role of this modification in the post-transcriptional control of cellular RNA function.

New families of human regulatory RNA structures identified by comparative analysis of vertebrate genomes.

Regulatory RNA structures are often members of families with multiple paralogous instances across the genome. Family members share functional and structural properties, which allow them to be studied as a whole, facilitating both bioinformatic and experimental characterization. We have developed a comparative method, EvoFam, for genome-wide identification of families of regulatory RNA structures, based on primary sequence and secondary structure similarity. We apply EvoFam to a 41-way genomic vertebrate alignment. Genome-wide, we identify 220 human, high-confidence families outside protein-coding regions comprising 725 individual structures, including 48 families with known structural RNA elements. Known families identified include both noncoding RNAs, e.g., miRNAs and the recently identified MALAT1/MEN ß lincRNA family; and cis-regulatory structures, e.g., iron-responsive elements. We also identify tens of new families supported by strong evolutionary evidence and other statistical evidence, such as GO term enrichments. For some of these, detailed analysis has led to the formulation of specific functional hypotheses. Examples include two hypothesized auto-regulatory feedback mechanisms: one involving six long hairpins in the 3'-UTR of MAT2A, a key metabolic gene that produces the primary human methyl donor S-adenosylmethionine; the other involving a tRNA-like structure in the intron of the tRNA maturation gene POP1. We experimentally validate the predicted MAT2A structures. Finally, we identify potential new regulatory networks, including large families of short hairpins enriched in immunity-related genes, e.g., TNF, FOS, and CTLA4, which include known transcript destabilizing elements. Our findings exemplify the diversity of post-transcriptional regulation and provide a resource for further characterization of new regulatory mechanisms and families of noncoding RNAs.

Locating protein-coding sequences under selection for additional, overlapping functions in 29 mammalian genomes.

The degeneracy of the genetic code allows protein-coding DNA and RNA sequences to simultaneously encode additional, overlapping functional elements. A sequence in which both protein-coding and additional overlapping functions have evolved under purifying selection should show increased evolutionary conservation compared to typical protein-coding genes--especially at synonymous sites. In this study, we use genome alignments of 29 placental mammals to systematically locate short regions within human ORFs that show conspicuously low estimated rates of synonymous substitution across these species. The 29-species alignment provides statistical power to locate more than 10,000 such regions with resolution down to nine-codon windows, which are found within more than a quarter of all human protein-coding genes and contain ∼2% of their synonymous sites. We collect numerous lines of evidence that the observed synonymous constraint in these regions reflects selection on overlapping functional elements including splicing regulatory elements, dual-coding genes, RNA secondary structures, microRNA target sites, and developmental enhancers. Our results show that overlapping functional elements are common in mammalian genes, despite the vast genomic landscape.

A high-resolution map of human evolutionary constraint using 29 mammals.

The comparison of related genomes has emerged as a powerful lens for genome interpretation. Here we report the sequencing and comparative analysis of 29 eutherian genomes. We confirm that at least 5.5% of the human genome has undergone purifying selection, and locate constrained elements covering ∼4.2% of the genome. We use evolutionary signatures and comparisons with experimental data sets to suggest candidate functions for ∼60% of constrained bases. These elements reveal a small number of new coding exons, candidate stop codon readthrough events and over 10,000 regions of overlapping synonymous constraint within protein-coding exons. We find 220 candidate RNA structural families, and nearly a million elements overlapping potential promoter, enhancer and insulator regions. We report specific amino acid residues that have undergone positive selection, 280,000 non-coding elements exapted from mobile elements and more than 1,000 primate- and human-accelerated elements. Overlap with disease-associated variants indicates that our findings will be relevant for studies of human biology, health and disease.

Systematic clustering of transcription start site landscapes.

Genome-wide, high-throughput methods for transcription start site (TSS) detection have shown that most promoters have an array of neighboring TSSs where some are used more than others, forming a distribution of initiation propensities. TSS distributions (TSSDs) vary widely between promoters and earlier studies have shown that the TSSDs have biological implications in both regulation and function. However, no systematic study has been made to explore how many types of TSSDs and by extension core promoters exist and to understand which biological features distinguish them. In this study, we developed a new non-parametric dissimilarity measure and clustering approach to explore the similarities and stabilities of clusters of TSSDs. Previous studies have used arbitrary thresholds to arrive at two general classes: broad and sharp. We demonstrated that in addition to the previous broad/sharp dichotomy an additional category of promoters exists. Unlike typical TATA-driven sharp TSSDs where the TSS position can vary a few nucleotides, in this category virtually all TSSs originate from the same genomic position. These promoters lack epigenetic signatures of typical mRNA promoters and a substantial subset of them are mapping upstream of ribosomal protein pseudogenes. We present evidence that these are likely mapping errors, which have confounded earlier analyses, due to the high similarity of ribosomal gene promoters in combination with known G addition bias in the CAGE libraries. Thus, previous two-class separations of promoter based on TSS distributions are motivated, but the ultra-sharp TSS distributions will confound downstream analyses if not removed.

MicroRNA transfection and AGO-bound CLIP-seq data sets reveal distinct determinants of miRNA action.

Microarray expression analyses following miRNA transfection/inhibition and, more recently, Argonaute cross-linked immunoprecipitation (CLIP)-seq assays have been used to detect miRNA target sites. CLIP and expression approaches measure differing stages of miRNA functioning-initial binding of the miRNP complex and subsequent message repression. We use nonparametric predictive models to characterize a large number of known target and flanking features, utilizing miRNA transfection, HITS-CLIP, and PAR-CLIP data. In particular, we utilize the precise spatial information provided by CLIP-seq to analyze the predictive effect of target flanking features. We observe distinct target determinants between expression-based and CLIP-based data. Target flanking features such as flanking region conservation are an important AGO-binding determinant-we hypothesize that CLIP experiments have a preference for strongly bound miRNP-target interactions involving adjacent RNA-binding proteins that increase the strength of cross-linking. In contrast, seed-related features are major determinants in expression-based studies, but less so for CLIP-seq studies, and increased miRNA concentrations typical of transfection studies contribute to this difference. While there is a good overlap between miRNA targets detected by miRNA transfection and CLIP-seq, the detection of CLIP-seq targets is largely independent of the level of subsequent mRNA degradation. Also, models built using CLIP-seq data show strong predictive power between independent CLIP-seq data sets, but are not strongly predictive for expression change. Similarly, models built from expression data are not strongly predictive for CLIP-seq data sets, supporting the finding that the determinants of miRNA binding and mRNA degradation differ. Predictive models and results are available at http://servers.binf.ku.dk/antar/.

Predicting microRNA targets in time-series microarray experiments via functional data analysis.

BACKGROUND: MicroRNA (miRNA) target prediction is an important component in understanding gene regulation. One approach is computational: searching nucleotide sequences for miRNA complementary base pairing. An alternative approach explored in this paper is the use of gene expression profiles from time-series microarray experiments to aid in miRNA target prediction. This requires distinguishing genuine targets from genes that are secondarily down-regulated as part of the same regulatory module. We use a functional data analytic (FDA) approach, FDA being a subfield of statistics that extends standard multivariate techniques to datasets with predictor and/or response variables that are functional. RESULTS: In a miR-124 transfection experiment spanning 120 hours, for genes with measurably down-regulated mRNA, exploratory functional data analysis showed differences in expression profiles over time between directly and indirectly down-regulated genes, such as response latency and biphasic response for direct miRNA targets. For prediction, an FDA approach was shown to effectively classify direct miR-124 targets from time-series microarray data (accuracy 88%; AUC 0.96), providing better performance than multivariate approaches. CONCLUSION: Exploratory FDA analysis can reveal interesting aspects of dynamic microarray miRNA studies. Predictive FDA models can be applied where computational miRNA target predictors fail or are unreliable, e.g. when there is a lack of evolutionary conservation, and can provide posterior probabilities to provide additional confirmatory evidence to validate candidate miRNA targets computationally predicted using sequence information. This approach would be applicable to the investigation of other miRNAs and suggests that dynamic microarray studies at a higher time resolution could reveal further details on miRNA regulation.

Imaging of shoulder injuries in sports medicine: current protocols and concepts.

The shoulder joint has a wide breadth of derangements that can occur with sports activity. Whether the mechanism of injury is acute or the sequela of repetitive microtrauma, recent advances in musculoskeletal imaging and the understanding of athletic shoulder trauma will hopefully steer the clinician and radiologist alike to the proper diagnosis. Injury is inevitable. When it occurs, the clinician cognizant of the current concepts and protocols in the imaging of shoulder injury will be better prepared to diagnose and subsequently treat these disorders.