Bromodomains regulate dynamic targeting of the PBAF chromatin-remodeling complex to chromatin hubs.

Chromatin remodelers actively target arrays of acetylated nucleosomes at select enhancers and promoters to facilitate or shut down the repeated recruitment of RNA polymerase II during transcriptional bursting. It is poorly understood how chromatin remodelers such as PBAF dynamically target different chromatin states inside a live cell. Our live-cell single-molecule fluorescence microscopy study reveals chromatin hubs throughout the nucleus where PBAF rapidly cycles on and off the genome. Deletion of PBAF's bromodomains impairs targeting and stable engagement of chromatin in hubs. Dual color imaging reveals that PBAF targets both euchromatic and heterochromatic hubs with distinct genome-binding kinetic profiles that mimic chromatin stability. Removal of PBAF's bromodomains stabilizes H3.3 binding within chromatin, indicating that bromodomains may play a direct role in remodeling of the nucleosome. Our data suggests that PBAF's dynamic bromodomain-mediated engagement of a nucleosome may reflect the chromatin-remodeling potential of differentially bound chromatin states.

Socioeconomic Status and Individual Personal Responsibility Beliefs Towards Food Access.

Despite worldwide attention given to food access, very little progress has been made under the current model. Recognizing that individual engagement is likely based on individual experiences and perceptions, this research study investigated whether or not a correlation exists between one's socioeconomic status (SES) and perceived personal responsibility for food access. Discussion of results and implications provide fresh insight into the ongoing global debate surrounding food access. Outcomes also provide insight into willing and able participants and point to least-cost solutions which may be better suited to implement and initiate change. Results indicate that the issue of food access is more complex than simply lobbying for better decision-making among individuals and populations, highlighting the importance of unit of analysis considerations.

Micro-Meta App: an interactive tool for collecting microscopy metadata based on community specifications.

For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes.

A DNA repair pathway can regulate transcriptional noise to promote cell fate transitions.

Stochastic fluctuations in gene expression ("noise") are often considered detrimental, but fluctuations can also be exploited for benefit (e.g., dither). We show here that DNA base excision repair amplifies transcriptional noise to facilitate cellular reprogramming. Specifically, the DNA repair protein Apex1, which recognizes both naturally occurring and unnatural base modifications, amplifies expression noise while homeostatically maintaining mean expression levels. This amplified expression noise originates from shorter-duration, higher-intensity transcriptional bursts generated by Apex1-mediated DNA supercoiling. The remodeling of DNA topology first impedes and then accelerates transcription to maintain mean levels. This mechanism, which we refer to as "discordant transcription through repair" ("DiThR," which is pronounced "dither"), potentiates cellular reprogramming and differentiation. Our study reveals a potential functional role for transcriptional fluctuations mediated by DNA base modifications in embryonic development and disease.

Imaging Organization of RNA Processing within the Nucleus.

Within the nucleus, messenger RNA is generated and processed in a highly organized and regulated manner. Messenger RNA processing begins during transcription initiation and continues until the RNA is translated and degraded. Processes such as 5' capping, alternative splicing, and 3' end processing have been studied extensively with biochemical methods and more recently with single-molecule imaging approaches. In this review, we highlight how imaging has helped understand the highly dynamic process of RNA processing. We conclude with open questions and new technological developments that may further our understanding of RNA processing.

Structure of the p53/RNA polymerase II assembly.

The tumor suppressor p53 protein activates expression of a vast gene network in response to stress stimuli for cellular integrity. The molecular mechanism underlying how p53 targets RNA polymerase II (Pol II) to regulate transcription remains unclear. To elucidate the p53/Pol II interaction, we have determined a 4.6 Å resolution structure of the human p53/Pol II assembly via single particle cryo-electron microscopy. Our structure reveals that p53's DNA binding domain targets the upstream DNA binding site within Pol II. This association introduces conformational changes of the Pol II clamp into a further-closed state. A cavity was identified between p53 and Pol II that could possibly host DNA. The transactivation domain of p53 binds the surface of Pol II's jaw that contacts downstream DNA. These findings suggest that p53's functional domains directly regulate DNA binding activity of Pol II to mediate transcription, thereby providing insights into p53-regulated gene expression.

A Single-Molecule Surface-Based Platform to Detect the Assembly and Function of the Human RNA Polymerase II Transcription Machinery.

Single-molecule detection and manipulation is a powerful tool for unraveling dynamic biological processes. Unfortunately, success in such experiments is often challenged by tethering the biomolecule(s) of interest to a biocompatible surface. Here, we describe a robust surface passivation method by dense polymer brush grafting, based on optimized polyethylene glycol (PEG) deposition conditions, exactly at the lower critical point of an aqueous biphasic PEG-salt system. The increased biocompatibility achieved, compared with PEG deposition in sub-optimal conditions away from the critical point, allowed us to successfully detect the assembly and function of a large macromolecular machine, a fluorescent-labeled multi-subunit, human RNA Polymerase II Transcription Pre-Initiation Complex, on single, promoter-containing, surface-immobilized DNA molecules. This platform will enable probing the complex biochemistry and dynamics of large, multi-subunit macromolecular assemblies, such as during the initiation of human RNA Pol II transcription, at the single-molecule level.

Effect of the Antiglaucoma Agent JV-GL1 and Related Compounds in the Canine Eye.

Purpose: JV-GL1 is an efficacious, potent, and long-acting antiglaucoma agent, according to studies in ocular normotensive and hypertensive monkeys. As an obligatory step in the drug development process, studies with exaggerated doses and an accelerated dosing schedule for JV-GL1 were performed in a second species (dog). Methods: Intraocular pressure (IOP) was measured by pneumatonometry in conscious Beagle dogs, which remained conscious throughout the study and gently restrained by hand. Pupil diameter was measured with an Optistick. Ocular surface hyperemia was visually assessed and scored according to a 1-3 assessment scale. Results: JV-GL1, as a 0.01% eye drop, produced significantly greater reductions in IOP than the original clinical dose of bimatoprost (0.03%). JV-GL1 and its free acid enzymatic hydrolysis product PGN 9856, over a 0.01%-0.1% dose range, reduced IOP to ≤10 mm Hg. JV-GL1 and PGN 9856 produced no miosis but a similar degree of ocular surface hyperemia to bimatoprost. Although PGN 9862, a close congener of PGN 9856, was very active as the free acid, esterification essentially abolished its ocular hypotensive activity and ocular surface redness. Conclusion: JV-GL1 was confirmed as a highly effective and potent ocular hypotensive, exceeding the activity of bimatoprost. A similar degree of ocular surface redness was apparent for both compounds, given as eye drops, but no other effects occurred. Results with PGN 9862 and its isopropyl ester confirmed that PGN 9862-isopropyl ester is not bioavailable in the eye and not susceptible to enzymatic hydrolysis in ocular tissues, a first for C1 ester prodrugs in the eye.

Dynamic Assembly and Disassembly of the Human DNA Polymerase δ Holoenzyme on the Genome In Vivo.

Human DNA polymerase delta (Pol δ) forms a holoenzyme complex with the DNA sliding clamp proliferating cell nuclear antigen (PCNA) to perform its essential roles in genome replication. Here, we utilize live-cell single-molecule tracking to monitor Pol δ holoenzyme interaction with the genome in real time. We find holoenzyme assembly and disassembly in vivo are highly dynamic and ordered. PCNA generally loads onto the genome before Pol δ. Once assembled, the holoenzyme has a relatively short lifetime on the genome, implying multiple Pol δ binding events may be needed to synthesize an Okazaki fragment. During disassembly, Pol δ dissociation generally precedes PCNA unloading. We also find that Pol δ p125, the catalytic subunit of the holoenzyme, is maintained at a constant cellular level, indicating an active mechanism for control of Pol δ levels in vivo. Collectively, our studies reveal that Pol δ holoenzyme assembly and disassembly follow a predominant pathway in vivo; however, alternate pathways are observed.

A Highly Effective and Ultra-Long-Acting Anti-Glaucoma Drug, with a Novel Periorbital Delivery Method.

Purpose: Two features define the future of glaucoma therapeutics: (1) greatly improved ocular hypotensive efficacy and (2) a delivery method that improves patient convenience and compliance. A highly efficacious and extraordinarily long-acting ocular hypotensive agent PGN 9856-isopropyl ester represents a potential next-generation anti-glaucoma drug. A new periorbital drug delivery route was also investigated. Methods: PGN 9856-isopropyl ester pharmacology was determined by employing human cells, including prostanoid receptor transfectants, and FLIPr or cellular dielectric spectroscopy technology. Intraocular pressure (IOP) was measured in conscious cynomolgus monkeys trained to accept pneumatonometry when under gentle restraint. For periorbital application, the compound was applied radially using a roller-ball device connected to a cylindrical reservoir. Pharmacokinetic data were obtained using LC/MS/MS instrumentation. Results: Single doses of PGN 9856-isopropyl ester, administered over a 0.001%-0.01% dose range, produced profound decreases in monkey IOP that persisted for at least 5 days, which was long after the drug was detectable in ocular tissues. It was not uncommon for a single eye drop to reduce IOP to the level of 4-7 mm Hg. Drug application to the periorbital dermis of ocular normotensive monkeys produced a similarly profound reduction in IOP, which was well maintained. Conclusions: PGN 9856-isopropyl ester appears to possess efficacy and duration of action properties unmatched by currently prescribed anti-glaucoma agents and by those currently undergoing clinical evaluation. In addition, application to the periorbital skin using a roller-ball device offers a more convenient method of ophthalmic drug delivery than eye drops and is noninvasive, unlike other "dropless" technologies.

Dual inhibition of MDMX and MDM2 as a therapeutic strategy in leukemia.

The tumor suppressor p53 is often inactivated via its interaction with endogenous inhibitors mouse double minute 4 homolog (MDM4 or MDMX) or mouse double minute 2 homolog (MDM2), which are frequently overexpressed in patients with acute myeloid leukemia (AML) and other cancers. Pharmacological disruption of both of these interactions has long been sought after as an attractive strategy to fully restore p53-dependent tumor suppressor activity in cancers with wild-type p53. Selective targeting of this pathway has thus far been limited to MDM2-only small-molecule inhibitors, which lack affinity for MDMX. We demonstrate that dual MDMX/MDM2 inhibition with a stapled α-helical peptide (ALRN-6924), which has recently entered phase I clinical testing, produces marked antileukemic effects. ALRN-6924 robustly activates p53-dependent transcription at the single-cell and single-molecule levels and exhibits biochemical and molecular biological on-target activity in leukemia cells in vitro and in vivo. Dual MDMX/MDM2 inhibition by ALRN-6924 inhibits cellular proliferation by inducing cell cycle arrest and apoptosis in cell lines and primary AML patient cells, including leukemic stem cell-enriched populations, and disrupts functional clonogenic and serial replating capacity. Furthermore, ALRN-6924 markedly improves survival in AML xenograft models. Our study provides mechanistic insight to support further testing of ALRN-6924 as a therapeutic approach in AML and other cancers with wild-type p53.

A new era of studying p53-mediated transcription activation.

To prevent tumorigenesis, p53 stimulates transcription by facilitating the recruitment of the transcription machinery on target gene promoters. Cryo-Electron Microscopy studies on p53-bound RNA Polymerase II (Pol II) reveal that p53 structurally regulates Pol II to affect its DNA binding and elongation, providing new insights into p53-mediated transcriptional regulation.

Brain and Brown Adipose Tissue Metabolism in Transgenic Tg2576 Mice Models of Alzheimer Disease Assessed Using 18F-FDG PET Imaging.

OBJECTIVE: Imaging animal models of Alzheimer disease (AD) is useful for the development of therapeutic drugs and understanding AD. Transgenic Swedish hAPPswe Tg2576 mice are a good model of ß-amyloid plaques. We report 18F-fluoro-2-deoxyglucose (18F-FDG) positron emission tomography (PET) imaging of brain and intrascapular brown adipose tissue (IBAT) in transgenic mice 2576 (Tg2576) and wild-type (WT) mice. METHODS: Transgenic Tg2576 mice and WT mice, >18 months were injected intraperitonally with ≈ 25 to 30 MBq 18F-FDG while awake. After 60 minutes, they were anesthetized with isoflurane (2.5%) and imaged with Inveon MicroPET. Select mice were killed, imaged ex vivo, and 20 µm sections cut for autoradiography. 18F-FDG uptake in brain and IBAT PET and brain autoradiographs were analyzed. RESULTS: Fasting blood glucose levels averaged 120 mg/dL for WT and 100 mg/dL for Tg2576. Compared to WT, Tg2576 mice exhibited a decrease in SUVglc in the various brain regions. Average reductions in the cerebrum regions were as high as -20%, while changes in cerebellum were -3%. Uptake of 18F-FDG in IBAT decreased by -60% in Tg2576 mice and was found to be significant. Intrascapular brown adipose tissue findings in Tg2576 mice are new and not previously reported. Use of blood glucose for PET data analysis and corpus callosum as reference region for autoradiographic analysis were important to detect change in Tg2576 mice. CONCLUSION: Our results suggest that 18F-FDG uptake in the Tg2576 mice brain show 18F-FDG deficits only when blood glucose is taken into consideration.

Structural visualization of the p53/RNA polymerase II assembly.

The master tumor suppressor p53 activates transcription in response to various cellular stresses in part by facilitating recruitment of the transcription machinery to DNA. Recent studies have documented a direct yet poorly characterized interaction between p53 and RNA polymerase II (Pol II). Therefore, we dissected the human p53/Pol II interaction via single-particle cryo-electron microscopy, structural docking, and biochemical analyses. This study reveals that p53 binds Pol II via the Rpb1 and Rpb2 subunits, bridging the DNA-binding cleft of Pol II proximal to the upstream DNA entry site. In addition, the key DNA-binding surface of p53, frequently disrupted in various cancers, remains exposed within the assembly. Furthermore, the p53/Pol II cocomplex displays a closed conformation as defined by the position of the Pol II clamp domain. Notably, the interaction of p53 and Pol II leads to increased Pol II elongation activity. These findings indicate that p53 may structurally regulate DNA-binding functions of Pol II via the clamp domain, thereby providing insights into p53-regulated Pol II transcription.

A Review of Transplantation Practice of the Urologic Organs: Is It Only Achievable for the Kidney?

Transplantation is a viable treatment option for failure of most major organs. Within urology, transplantation of the kidney and ureter are well documented; however, evidence supporting transplantation of other urologic organs is limited. Failure of these organs carries significant morbidity, and transplantation may have a role in management. This article reviews the knowledge, research, and literature surrounding transplantation of each of the urologic organs. Transplantation of the penis, testicle, urethra, vas deferens, and bladder is discussed. Transplantation attempts have been made individually with each of these organs. Penile transplantation has only been performed once in a human. Testicular transplantation research was intertwined with unethical lucrative pursuits. Interest in urethra, bladder, and vas deferens transplantation has decreased as a result of successful surgical reconstructive techniques. Despite years of effort, transplantations of the penis, testicle, urethra, vas deferens, and bladder are not established in current practice. Recent research has shifted toward techniques of reconstruction, tissue engineering, and regenerative medicine.

Imaging Transcription: Past, Present, and Future.

Transcription, the first step of gene expression, is exquisitely regulated in higher eukaryotes to ensure correct development and homeostasis. Traditional biochemical, genetic, and genomic approaches have proved successful at identifying factors, regulatory sequences, and potential pathways that modulate transcription. However, they typically only provide snapshots or population averages of the highly dynamic, stochastic biochemical processes involved in transcriptional regulation. Single-molecule live-cell imaging has, therefore, emerged as a complementary approach capable of circumventing these limitations. By observing sequences of molecular events in real time as they occur in their native context, imaging has the power to derive cause-and-effect relationships and quantitative kinetics to build predictive models of transcription. Ongoing progress in fluorescence imaging technology has brought new microscopes and labeling technologies that now make it possible to visualize and quantify the transcription process with single-molecule resolution in living cells and animals. Here we provide an overview of the evolution and current state of transcription imaging technologies. We discuss some of the important concepts they uncovered and present possible future developments that might solve long-standing questions in transcriptional regulation.

Spinal cord dopamine D2/D3 receptors: in vivo and ex vivo imaging in the rat using (18)F/(11)C-fallypride.

OBJECTIVES: The spinal cord is known to be innervated with dopaminergic cells with catecholaminergic projections arising from the medulla and pons and dopaminergic transmission in the spinal cord is vital for sensory and motor function. Our goal was to evaluate and compare the imaging capability of dopamine D2/D3 receptors in the rat spinal cord using PET ligands (18)F-fallypride and (11)C-fallypride. METHODS: Male Sprague-Dawley rats were used in all in vitro and in vivo studies. Spinal cord and brain sections were used for in vitro autoradiography and ex vivo autoradiography. For in vivo studies animals received a (18)F-fallypride scan or a (11)C-fallypride PET scan. The spinal cord and the brain were then harvested, flash-frozen and imaged ex vivo. For in vivo analysis Logan plots with cerebellum as a reference was used to evaluate binding potentials (BP). Tissue ratios were used for ex vivo analysis. Drug effects were evaluated using clozapine, haloperidol and dopamine were evaluated on spinal cord sections in vitro. RESULTS: In vitro studies showed (18)F-fallypride binding to superficial dorsal horn (SDH), dorsal horn (DH), ventral horn (VH) and the pars centralis (PC). In the cervical section, the greatest amount of binding appeared to be in the SDH. Ex vivo studies showed approximately 6% of (18)F-fallypride in SDH compared to that observed in the striatum. In vivo analysis of both (18)F-fallypride and (11)C-fallypride in the spinal cord were comparable to that in the extrastriatal regions. Haloperidol and clozapine displaced more than 75% of the (18)F-fallypride in spinal cord sections. CONCLUSIONS: Our studies showed (18)F-fallypride and (11)C-fallypride binding in the spinal cord in vitro and in vivo. The binding pattern correlates well with the known distribution of dopamine D2/D3 receptors in the spinal cord.