The CDK9-SPT5 axis in control of transcription elongation by RNAPII.

The RNA polymerase II (RNAPII) transcription cycle is regulated at every stage by a network of cyclin-dependent protein kinases (CDKs) and protein phosphatases. Progression of RNAPII from initiation to termination is marked by changing patterns of phosphorylation on the highly repetitive carboxy-terminal domain (CTD) of RPB1, its largest subunit, suggesting the existence of a CTD code. In parallel, the conserved transcription elongation factor SPT5, large subunit of the DRB sensitivity-inducing factor (DSIF), undergoes spatiotemporally regulated changes in phosphorylation state that may be directly linked to the transitions between transcription-cycle phases. Here we review insights gained from recent structural, biochemical, and genetic analyses of human SPT5, which suggest that two of its phosphorylated regions perform distinct functions at different points in transcription. Phosphorylation within a flexible, RNA-binding linker promotes release from the promoter-proximal pause-frequently a rate-limiting step in gene expression-whereas modifications in a repetitive carboxy-terminal region are thought to favor processive elongation, and are removed just prior to termination. Phosphorylations in both motifs depend on CDK9, catalytic subunit of positive transcription elongation factor b (P-TEFb); their different timing of accumulation on chromatin and function during the transcription cycle might reflect their removal by different phosphatases, different kinetics of phosphorylation by CDK9, or both. Perturbations of SPT5 regulation have profound impacts on viability and development in model organisms through largely unknown mechanisms, while enzymes that modify SPT5 have emerged as potential therapeutic targets in cancer; elucidating a putative SPT5 code is therefore a high priority.

Structural basis of Cdk7 activation by dual T-loop phosphorylation.

Cyclin-dependent kinase 7 (Cdk7) is required in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of transcription factor TFIIH. Cdk7 forms active complexes by associating with Cyclin H and Mat1, and is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates basic residues conserved in other CDKs, pS164 nucleates an arginine network unique to the ternary Cdk7 complex, involving all three subunits. We identify differential dependencies of kinase activity and substrate recognition on the individual phosphorylations. CAK function is unaffected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by T170 phosphorylation. Moreover, dual T-loop phosphorylation stimulates multisite phosphorylation of the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7 activation is a two-step process wherein S164 phosphorylation precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing processivity, while pT170 enhances activity towards key transcriptional substrates.

Unmet supportive care needs among survivors of stroke in Australia: A cross-sectional study.

PURPOSE/OBJECTIVE: To examine, among survivors of stroke: (a) the prevalence of and most frequently reported unmet needs; and (b) the sociodemographic and clinical factors associated with higher counts of unmet needs. RESEARCH METHOD/DESIGN: A cross-sectional study was undertaken with survivors of stroke recently discharged from eight hospitals in Australia, with institutional board approval. Survivors were mailed one survey for completion after their discharge from hospital. Unmet needs were measured by the Longer-Term Unmet Needs After Stroke (LUNS) tool. The number and percentage of participants who reported unmet needs were calculated. The association of sociodemographic factors, type of stroke, and thrombolysis treatment to total LUNS scores was examined using mixed ordinal logistic regression. RESULTS: A total of 402 survivors (35% of those approached) between April 2018 to December 2019 returned a completed survey. 83% reported at least one unmet need. The most frequently reported unmet need was needing more information about their stroke (n = 239, 61%). Those who identified as Aboriginal and/or Torres Strait Islander had approximately 5.6-fold higher odds (OR = 5.59, p = .025) of having more longer-term unmet needs compared to those who did not identify as Aboriginal and/or Torres Strait Islander. CONCLUSIONS/IMPLICATIONS: Unmet needs are common in recently discharged survivors of stroke. These findings may be used to inform strategies that support recovery. Providing more information may help reduce unmet needs among survivors of stroke. Enhanced hospital discharge planning and enhanced community services for survivors may help better prepare them and their caregivers for the return home. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

A New Isosporan (Apicomplexa: Eimeriidae) from the Pacific Blue-Tailed Skink, Emoia caeruleocauda (Sauria: Scincidae: Eugongylinae), from Guam, U.S. Territory.

PURPOSE: Nothing is known about coccidians (Apicomplexa: Eimeriidae) from the Pacific blue-tailed skink, Emoia caeruleocauda. Here, we report mensural and morphometric data on a new species of Isospora from E. caeruleocauda from Guam, US Territory. METHODS: Feces from four E. caeruleocauda collected by hand in November 2023 were placed in individual vials containing 2.5% potassium dichromate. They were examined for sporulated oocysts after flotation in Sheather's sugar solution, measured, and photographed. RESULTS: A single (25%) E. caeruleocauda was found to be passing oocysts representing a new species of Isospora. Oocysts of Isospora guamensis n. sp. are ellipsoidal to ovoidal with a bi-layered wall, measure (L × W) 16.5 × 11.8 µm, and have a length/width (L/W) ratio of 1.4; a micropyle and an oocyst residuum were absent but a polar granule was present. Sporocysts are ovoidal and measure 9.4 × 6.5 µm, L/W 1.4; Stieda and sub-Stieda bodies were present but a para-Stieda body was absent. The sporocyst residuum is composed various-sized granules in a compact rounded or irregular mass, sometimes dispersed between the sporozoites. The new species can be differentiated from all other isosporans from skinks by possessing the smallest oocysts known from this host family. CONCLUSION: This is the first time an isosporan coccidian has been reported from E. caeruleocauda as well as the first report of a coccidian from a Guam-inhabiting skink.

Neuromodulatory Focused Ultrasound for Epilepsy: Are Animal Models Useful?

Ultrasound neuromodulation is a potential alternative therapy for suppressing epileptic discharges. Recently, several human clinical trials have reported promising results from repeated focused ultrasound (FUS) treatments for temporal lobe epilepsy. In this Viewpoint, we highlight the valuable guidance of preclinical validation methods for choosing the optimal FUS parameters, thus ensuring consistency with the outcomes of clinical trials and leading human trials to the safest and most effective approaches.

Corrigendum to Contractility defects hinder glycoprotein VI-mediated platelet activation and affect platelet functions beyond clot contraction [Research and Practice in Thrombosis and Haemostasis Volume 8, Issue 1, January 2024, 102322].

[This corrects the article DOI: 10.1016/j.rpth.2024.102322.].

Contractility defects hinder glycoprotein VI-mediated platelet activation and affect platelet functions beyond clot contraction.

Background: Active and passive biomechanical properties of platelets contribute substantially to thrombus formation. Actomyosin contractility drives clot contraction required for stabilizing the hemostatic plug. Impaired contractility results in bleeding but is difficult to detect using platelet function tests. Objectives: To determine how diminished myosin activity affects platelet functions, including and beyond clot contraction. Methods: Using the myosin IIA-specific pharmacologic inhibitor blebbistatin, we modulated myosin activity in platelets from healthy donors and systematically characterized platelet responses at various levels of inhibition by interrogating distinct platelet functions at each stage of thrombus formation using a range of complementary assays. Results: Partial myosin IIA inhibition neither affected platelet von Willebrand factor interactions under arterial shear nor platelet spreading and cytoskeletal rearrangements on fibrinogen. However, it impacted stress fiber formation and the nanoarchitecture of cell-matrix adhesions, drastically reducing and limiting traction forces. Higher blebbistatin concentrations impaired platelet adhesion under flow, altered mechanosensing at lamellipodia edges, and eliminated traction forces without affecting platelet spreading, α-granule secretion, or procoagulant platelet formation. Unexpectedly, myosin IIA inhibition reduced calcium influx, dense granule secretion, and platelet aggregation downstream of glycoprotein (GP)VI and limited the redistribution of GPVI on the cell membrane, whereas aggregation induced by adenosine diphosphate or arachidonic acid was unaffected. Conclusion: Our findings highlight the importance of both active contractile and passive crosslinking roles of myosin IIA in the platelet cytoskeleton. They support the hypothesis that highly contractile platelets are needed for hemostasis and further suggest a supportive role for myosin IIA in GPVI signaling.

Comparing the efficacy of anti-seizure medications using matched cohorts on a large insurance claims database.

Epilepsy is a severe chronic neurological disease affecting 60 million people worldwide. Primary treatment is with anti-seizure medicines (ASMs), but many patients continue to experience seizures. We used retrospective insurance claims data on 280,587 patients with uncontrolled epilepsy (UE), defined as status epilepticus, need for a rescue medicine, or admission or emergency visit for an epilepsy code. We conducted a computational risk ratio analysis between pairs of ASMs using a causal inference method, in order to match 1034 clinical factors and simulate randomization. Data was extracted from the MarketScan insurance claims Research Database records from 2011 to 2015. The cohort consisted of individuals over 18 years old with a diagnosis of epilepsy who took one of eight ASMs and had more than a year of history prior to the filling of the drug prescription. Seven ASM exposures were analyzed: topiramate, phenytoin, levetiracetam, gabapentin, lamotrigine, valproate, and carbamazepine or oxcarbazepine (treated as the same exposure). We calculated the risk ratio of UE between pairs of ASM after controlling for bias with inverse propensity weighting applied to 1034 factors, such as demographics, confounding illnesses, non-epileptic conditions treated by ASMs, etc. All ASMs exhibited a significant reduction in the prevalence of UE, but three drugs showed pair-wise differences compared to other ASMs. Topiramate consistently was associated with a lower risk of UE, with a mean risk ratio range of 0.68-0.93 (average 0.82, CI: 0.56-1.08). Phenytoin and levetiracetam were consistently associated with a higher risk of UE with mean risk ratio ranges of 1.11 to 1.47 (average 1.13, CI 0.98-1.65) and 1.15 to 1.43 (average 1.2, CI 0.72-1.69), respectively. Large-scale retrospective insurance claims data - combined with causal inference analysis - provides an opportunity to compare the effect of treatments in real-world data in populations 1,000-fold larger than those in typical randomized trials. Our causal analysis identified the clinically unexpected finding of topiramate as being associated with a lower risk of UE; and phenytoin and levetiracetam as associated with a higher risk of UE (compared to other studied drugs, not to baseline). However, we note that our data set for this study only used insurance claims events, which does not comprise actual seizure frequencies, nor a clear picture of side effects. Our results do not advocate for any change in practice but demonstrate that conclusions from large databases may differ from and supplement those of randomized trials and clinical practice and therefore may guide further investigation.

The CoREST repressor complex mediates phenotype switching and therapy resistance in melanoma.

Virtually all patients with BRAF-mutant melanoma develop resistance to MAPK inhibitors largely through nonmutational events. Although the epigenetic landscape is shown to be altered in therapy-resistant melanomas and other cancers, a specific targetable epigenetic mechanism has not been validated. Here, we evaluated the corepressor for element 1-silencing transcription factor (CoREST) epigenetic repressor complex and the recently developed bivalent inhibitor corin within the context of melanoma phenotype plasticity and therapeutic resistance. We found that CoREST was a critical mediator of the major distinct melanoma phenotypes and that corin treatment of melanoma cells led to phenotype reprogramming. Global assessment of transcript and chromatin changes conferred by corin revealed specific effects on histone marks connected to epithelial-mesenchymal transition-associated (EMT-associated) transcription factors and the dual-specificity phosphatases (DUSPs). Remarkably, treatment of BRAF inhibitor-resistant (BRAFi-R) melanomas with corin promoted resensitization to BRAFi therapy. DUSP1 was consistently downregulated in BRAFi-R melanomas, which was reversed by corin treatment and associated with inhibition of p38 MAPK activity and resensitization to BRAFi therapies. Moreover, this activity was recapitulated by the p38 MAPK inhibitor BIRB 796. These findings identify the CoREST repressor complex as a central mediator of melanoma phenotype plasticity and resistance to targeted therapy and suggest that CoREST inhibitors may prove beneficial for patients with BRAFi-resistant melanoma.

Structural basis of Cdk7 activation by dual T-loop phosphorylation.

Cyclin-dependent kinase 7 (Cdk7) occupies a central position in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of the general transcription factor TFIIH. Cdk7 forms an active complex upon association with Cyclin H and Mat1, and its catalytic activity is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the fully activated human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates a set of basic residues conserved in other CDKs, pS164 nucleates an arginine network involving all three subunits that is unique to the ternary Cdk7 complex. We identify differential dependencies of kinase activity and substrate recognition on individual phosphorylations within the Cdk7 T loop. The CAK function of Cdk7 is not affected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by phosphorylation at T170. Moreover, dual T-loop phosphorylation at both T170 and S164 stimulates multi-site phosphorylation of transcriptional substrates-the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and the SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7-regulatory phosphorylation is a two-step process in which phosphorylation of S164 precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing Cdk7 processivity, while the canonical pT170 enhances kinase activity towards critical substrates involved in transcription.

A Dataset of Amphibian Species in U.S. National Parks.

National parks and other protected areas are important for preserving landscapes and biodiversity worldwide. An essential component of the mission of the United States (U.S.) National Park Service (NPS) requires understanding and maintaining accurate inventories of species on protected lands. We describe a new, national-scale synthesis of amphibian species occurrence in the NPS system. Many park units have a list of amphibian species observed within their borders compiled from various sources and available publicly through the NPSpecies platform. However, many of the observations in NPSpecies remain unverified and the lists are often outdated. We updated the amphibian dataset for each park unit by collating old and new park-level records and had them verified by regional experts. The new dataset contains occurrence records for 292 of the 424 NPS units and includes updated taxonomy, international and state conservation rankings, hyperlinks to a supporting reference for each record, specific notes, and related fields which can be used to better understand and manage amphibian biodiversity within a single park or group of parks.