Historical Temperature Variability Affects Coral Response to Heat Stress

Coral bleaching is the breakdown of symbiosis between coral animal hosts and their dinoflagellate algae symbionts in response to environmental stress. On large spatial scales, heat stress is the most common factor causing bleaching, which is predicted to increase in frequency and severity as the climate warms. There is evidence that the temperature threshold at which bleaching occurs varies with local environmental conditions and background climate conditions. We investigated the influence of past temperature variability on coral susceptibility to bleaching, using the natural gradient in peak temperature variability in the Gilbert Islands, Republic of Kiribati. The spatial pattern in skeletal growth rates and partial mortality scars found in massive Porites sp. across the central and northern islands suggests that corals subject to larger year-to-year fluctuations in maximum ocean temperature were more resistant to a 2004 warm-water event. In addition, a subsequent 2009 warm event had a disproportionately larger impact on those corals from the island with lower historical heat stress, as indicated by lower concentrations of triacylglycerol, a lipid utilized for energy, as well as thinner tissue in those corals. This study indicates that coral reefs in locations with more frequent warm events may be more resilient to future warming, and protection measures may be more effective in these regions

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

CDK8: A positive regulator of transcription

CDK8 belongs to a group of cyclin-dependent kinases involved in transcriptional regulation from yeast to mammals. CDK8 associates with the Mediator complex, but functions outside of Mediator are also likely. Historically, CDK8 has been described mostly as a transcriptional repressor, but a growing body of research provides unequivocal evidence for various roles of CDK8 in gene activation. Several transcriptional programs of biomedical importance employ CDK8 as a coactivator, including the p53 network, the Wnt/β-catenin pathway, the serum response network, and those governed by SMADs and the thyroid hormone receptor, thus highlighting the importance of further investigation into this enigmatic transcriptional regulator

Stabilin-Mediated Cellular Internalization of Phosphorothioate-Modified Antisense Oligonucleotides (ASOs)

Introduction: Antisense oligonucleotides (ASOs) are short chemically modified oligonucleotides (5-7.4 kDa) that can produce a pharmacological effect by binding to RNA and affecting intermediary metabolism. Over 35 phosphorothioate (PS) ASOs are at various stages of clinical development for use as therapeutic agents and pharmacological tools. Antisense therapy is a progressing area of research, as these small strands of nucleotide oligomers can be produced to silence genes that aggravate chronic disorders or infections. An important distinction for ASOs compared to DNA is the substitution of the phosphodiester (PO) backbone with the PS modification. This sulfur substitution allows for these polar polyanionic molecules to have high stability in biological fluids and selective binding to cell surfaces. Although there is a premium for clinical development of these short chain molecules, the current understanding of the pathways for their ability to traverse plasma membranes remains unresolved. Injected gymnotic ASOs have been shown to accumulate in the liver via the organ’s functional scavenging mechanism in highly endocytically active sinusoidal endothelial cells (SECs) and Kupffer cells (KC) compared to hepatocytes. Our work outlines how a non-DNA binding class of scavenger receptors known as Stabilins binds to, and internalizes these small PS ASOs. Methods: Primary cells from rat and mouse were isolated and cultured with 125I-ASOs. Stable cell lines expressing Stabilin-1 and two Stabilin-2 isoforms (315-HARE and 190-HARE), were used to analyze binding affinity, endocytosis and degradation of 125I-ASOs in the cell. Co-localization was also done to analyze trafficking of ASOs once internalized within the cell by use of fluorescent ASO and lysotracker. Results: It was determined that PS ASOs bind with high affinity to the Stabilin class receptors with the majority of internalization performed by clathrin-mediated endocytosis. Binding was determined to be dependent on proper folding of the receptor, along with relying on salt-bridge formation. Once inside the cell via the Stabilin receptors, co-localization analysis showed ASOs being trafficked for degradation in the lysosome. Increased internalization rates of an ASO targeting the non-coding RNA of malat-1, in the Stabilin-expressing cell lines reduced malat-1 expression more efficiently, indicating not all ASOs are trafficked to the lysosome after internalization. Conclusion: Our work shows that ASOs are internalized into the cells of the liver through clathrin-mediated endocytosis. The understanding of the pathway(s) for chemically modified ASO internalization and trafficking with cell surface receptors will aid in future clinical design of ASOs as therapeutic agents

The Human CDK8 Subcomplex Is a Histone Kinase That Requires Med12 for Activity and Can Function Independently of Mediator▿

The four proteins CDK8, cyclin C, Med12, and Med13 can associate with Mediator and are presumed to form a stable “CDK8 subcomplex” in cells. We describe here the isolation and enzymatic activity of the 600-kDa CDK8 subcomplex purified directly from human cells and also via recombinant expression in insect cells. Biochemical analysis of the recombinant CDK8 subcomplex identifies predicted (TFIIH and RNA polymerase II C-terminal domain [Pol II CTD]) and novel (histone H3, Med13, and CDK8 itself) substrates for the CDK8 kinase. Notably, these novel substrates appear to be metazoan-specific. Such diverse targets imply strict regulation of CDK8 kinase activity. Along these lines, we observe that Mediator itself enables CDK8 kinase activity on chromatin, and we identify Med12—but not Med13—to be essential for activating the CDK8 kinase. Moreover, mass spectrometry analysis of the endogenous CDK8 subcomplex reveals several associated factors, including GCN1L1 and the TRiC chaperonin, that may help control its biological function. In support of this, electron microscopy analysis suggests TRiC sequesters the CDK8 subcomplex and kinase assays reveal the endogenous CDK8 subcomplex—unlike the recombinant submodule—is unable to phosphorylate the Pol II CTD

Stabilin-1 and Stabilin-2 are specific receptors for the cellular internalization of phosphorothioate-modified antisense oligonucleotides (ASOs) in the liver

Phosphorothioate (PS)-modified antisense oligonucleotides (ASOs) have been extensively investigated over the past three decades as pharmacological and therapeutic agents. One second generation ASO, KynamroTM, was recently approved by the FDA for the treatment of homozygous familial hypercholesterolemia and over 35 second generation PS ASOs are at various stages of clinical development. In this report, we show that the Stabilin class of scavenger receptors, which were not previously thought to bind DNA, do bind and internalize PS ASOs. With the use of primary cells from mouse and rat livers and recombinant cell lines each expressing Stabilin-1 and each isoform of Stabilin-2 (315-HARE and 190-HARE), we have determined that PS ASOs bind with high affinity and these receptors are responsible for bulk, clathrin-mediated endocytosis within the cell. Binding is primarily dependent on salt-bridge formation and correct folding of the intact protein receptor. Increased internalization rates also enhanced ASO potency for reducing expression of the non-coding RNA Malat-1, in Stabilin-expressing cell lines. A more thorough understanding of mechanisms by which ASOs are internalized in cells and their intracellular trafficking pathways will aid in the design of next generation antisense agents with improved therapeutic properties

CD40 Generation 2.5 Antisense Oligonucleotide Treatment Attenuates Doxorubicin-induced Nephropathy and Kidney Inflammation

Preclinical and clinical data suggest CD40 activation contributes to renal inflammation and injury. We sought to test whether upregulation of CD40 in the kidney is a causative factor of renal pathology and if reduction of renal CD40 expression, using antisense oligonucleotides (ASOs) targeting CD40, would be beneficial in mouse models of glomerular injury and unilateral ureter obstruction. Administration of a Generation 2.5 CD40 ASO reduced CD40 mRNA and protein levels 75–90% in the kidney. CD40 ASO treatment mitigated functional, transcriptional, and pathological endpoints of doxorubicin-induced nephropathy. Experiments using an activating CD40 antibody revealed CD40 is primed in kidneys following doxorubicin injury or unilateral ureter obstruction and CD40 ASO treatment blunted CD40-dependent renal inflammation. Suborgan fractionation and imaging studies demonstrated CD40 in glomeruli before and after doxorubicin administration that becomes highly enriched within interstitial and glomerular foci following CD40 activation. Such foci were also sites of ASO distribution and activity and may be predominately comprised from myeloid cells as bone marrow CD40 deficiency sharply attenuated CD40 antibody responses. These studies suggest an important role of interstitial renal and/or glomerular CD40 to augment kidney injury and inflammation and demonstrate that ASO treatment could be an effective therapy in such disorders

Cooperative activity of cdk8 and GCN5L within Mediator directs tandem phosphoacetylation of histone H3

The human Mediator complex is generally required for expression of protein-coding genes. Here, we show that the GCN5L acetyltransferase stably associates with Mediator together with the TRRAP polypeptide. Yet, contrary to expectations, TRRAP/GCN5L does not associate with the transcriptionally active core Mediator but rather with Mediator that contains the cdk8 subcomplex. Consequently, this derivative ‘T/G-Mediator' complex does not directly activate transcription in a reconstituted human transcription system. However, within T/G-Mediator, cdk8 phosphorylates serine-10 on histone H3, which in turn stimulates H3K14 acetylation by GCN5L within the complex. Tandem phosphoacetylation of H3 correlates with transcriptional activation, and ChIP assays demonstrate co-occupancy of T/G-Mediator components at several activated genes in vivo. Moreover, cdk8 knockdown causes substantial reduction of global H3 phosphoacetylation, suggesting that T/G-Mediator is a major regulator of this H3 mark. Cooperative H3 modification provides a mechanistic basis for GCN5L association with cdk8-Mediator and also identifies a biochemical means by which cdk8 can indirectly activate gene expression. Indeed our results suggest that T/G-Mediator directs early events—such as modification of chromatin templates—in transcriptional activation

A role for Chk1 in blocking transcriptional elongation of p21 RNA during the S-phase checkpoint

We reported previously that when cells are arrested in S phase, a subset of p53 target genes fails to be strongly induced despite the presence of high levels of p53. When DNA replication is inhibited, reduced p21 mRNA accumulation is correlated with a marked reduction in transcription elongation. Here we show that ablation of the protein kinase Chk1 rescues the p21 transcription elongation defect when cells are blocked in S phase, as measured by increases in both p21 mRNA levels and the presence of the elongating form of RNA polymerase II (RNAPII) toward the 3′ end of the p21 gene. Recruitment of specific elongation and 3′ processing factors (DSIF, CstF-64, and CPSF-100) is also restored. While additional components of the RNAPII transcriptional machinery, such as TFIIB and CDK7, are recruited more extensively to the p21 locus after DNA damage than after replication stress, their recruitment is not enhanced by ablation of Chk1. Significantly, ablating Chk2, a kinase closely related in substrate specificity to Chk1, does not rescue p21 mRNA levels during S-phase arrest. Thus, Chk1 has a direct and selective role in the elongation block to p21 observed during S-phase arrest. These findings demonstrate for the first time a link between the replication checkpoint mediated by ATR/Chk1 and the transcription elongation/3′ processing machinery