Reduced Levels of Lagging Strand Polymerases Shape Stem Cell Chromatin.

Stem cells display asymmetric histone inheritance while non-stem progenitor cells exhibit symmetric patterns in the Drosophila male germline lineage. Here, we report that components involved in lagging strand synthesis, such as DNA polymerase α and δ (Polα and Polδ), have significantly reduced levels in stem cells compared to progenitor cells. Compromising Polα genetically induces the replication-coupled histone incorporation pattern in progenitor cells to be indistinguishable from that in stem cells, which can be recapitulated using a Polα inhibitor in a concentration-dependent manner. Furthermore, stem cell-derived chromatin fibers display a higher degree of old histone recycling by the leading strand compared to progenitor cell-derived chromatin fibers. However, upon reducing Polα levels in progenitor cells, the chromatin fibers now display asymmetric old histone recycling just like GSC-derived fibers. The old versus new histone asymmetry is comparable between stem cells and progenitor cells at both S-phase and M-phase. Together, these results indicate that developmentally programmed expression of key DNA replication components is important to shape stem cell chromatin. Furthermore, manipulating one crucial DNA replication component can induce replication-coupled histone dynamics in non-stem cells in a manner similar to that in stem cells.

Comparison Between REVEAL Lite 2 and COMPERA 2.0 for Risk Stratification in Pulmonary Arterial Hypertension.

BACKGROUND: Risk stratification is the cornerstone of the management of pulmonary arterial hypertension (PAH). Current European Society of Cardiology/European Respiratory Society guidelines recommend using the Comparative, Prospective Registry of Newly Initiated Therapies for Pulmonary Hypertension (COMPERA) three-strata risk model at baseline and the COMPERA 2.0 four-strata model at follow-up. However, the guidelines did not take into consideration other available risk scores such as the Registry to Evaluate Early and Long-Term PAH Disease Management (REVEAL) Lite 2. RESEARCH QUESTION: Is REVEAL Lite 2 better at discriminating risk than the COMPERA risk assessment models at baseline or follow-up evaluations? STUDY DESIGN AND METHODS: This study analyzed data from patients with PAH consecutively enrolled between June 2011 and February 2022 in the PAH registry at our expert Pulmonary Hypertension Center. Patients were stratified according to REVEAL Lite 2 and COMPERA three- and four-strata risk scores at baseline and follow-up to predict the composite outcome for lung transplantation or death. Receiver-operating characteristic curves in predicting the binary outcome at 3, 5, and 7 years were plotted. Areas under the curve of the scores were compared by using the χ2 test. The performance of the scores was determined according to the Harrel C statistic. RESULTS: A total of 296 patients were included for baseline and 196 for follow-up evaluation. The overall transplant-free survival in the patient population at 1, 3, 5, and 7 years was 93.6%, 81.3%, 75.1%, and 68.8%, respectively. At baseline, the C statistic of REVEAL Lite 2 was 0.74 (95% CI, 0.69-0.80), compared with 0.68 (95% CI, 0.63-0.74) for the COMPERA four-strata model and 0.63 (95% CI, 0.58-0.69) for the COMPERA three-strata model. All C statistic differences between REVEAL Lite 2 and the other models were statistically significant at baseline. INTERPRETATION: Our analysis showed that REVEAL Lite 2 was better at baseline at discriminating risk in this patient population. Future guidelines should consider including REVEAL Lite 2 in the management algorithm to help clinicians make informed decisions. Further analysis in larger cohorts could help validate these findings.

Aclarubicin stimulates RNA polymerase II elongation at closely spaced divergent promoters.

Anthracyclines are a class of widely prescribed anticancer drugs that disrupt chromatin by intercalating into DNA and enhancing nucleosome turnover. To understand the molecular consequences of anthracycline-mediated chromatin disruption, we used Cleavage Under Targets and Tagmentation (CUT&Tag) to profile RNA polymerase II during anthracycline treatment in Drosophila cells. We observed that treatment with the anthracycline aclarubicin leads to elevated levels of RNA polymerase II and changes in chromatin accessibility. We found that promoter proximity and orientation affect chromatin changes during aclarubicin treatment, as closely spaced divergent promoter pairs show greater chromatin changes when compared to codirectionally oriented tandem promoters. We also found that aclarubicin treatment changes the distribution of noncanonical DNA G-quadruplex structures both at promoters and at G-rich pericentromeric repeats. Our work suggests that the cancer-killing activity of aclarubicin is driven by the disruption of nucleosomes and RNA polymerase II.

Aclarubicin stimulates RNA polymerase II elongation at closely spaced divergent promoters.

Anthracyclines are a class of widely prescribed anti-cancer drugs that disrupt chromatin by intercalating into DNA and enhancing nucleosome turnover. To understand the molecular consequences of anthracycline-mediated chromatin disruption, we utilized CUT&Tag to profile RNA polymerase II during anthracycline treatment in Drosophila cells. We observed that treatment with the anthracycline aclarubicin leads to elevated levels of elongating RNA polymerase II and changes in chromatin accessibility. We found that promoter proximity and orientation impacts chromatin changes during aclarubicin treatment, as closely spaced divergent promoter pairs show greater chromatin changes when compared to codirectionally-oriented tandem promoters. We also found that aclarubicin treatment changes the distribution of non-canonical DNA G-quadruplex structures both at promoters and at G-rich pericentromeric repeats. Our work suggests that the anti-cancer activity of aclarubicin is driven by the effects of nucleosome disruption on RNA polymerase II, chromatin accessibility and DNA structures.

Differential condensation of sister chromatids acts with Cdc6 to ensure asynchronous S-phase entry in Drosophila male germline stem cell lineage.

During Drosophila melanogaster male germline stem cell (GSC) asymmetric division, preexisting old versus newly synthesized histones H3 and H4 are asymmetrically inherited. However, the biological outcomes of this phenomenon have remained unclear. Here, we tracked old and new histones throughout the GSC cell cycle through the use of high spatial and temporal resolution microscopy. We found unique features that differ between old and new histone-enriched sister chromatids, including differences in nucleosome density, chromosomal condensation, and H3 Ser10 phosphorylation. These distinct chromosomal features lead to their differential association with Cdc6, a pre-replication complex component, and subsequent asynchronous DNA replication initiation in the resulting daughter cells. Disruption of asymmetric histone inheritance abolishes differential Cdc6 association and asynchronous S-phase entry, demonstrating that histone asymmetry acts upstream of these critical cell-cycle progression events. Furthermore, disruption of these GSC-specific chromatin features leads to GSC defects, indicating a connection between histone inheritance, cell-cycle progression, and cell fate determination.

A pairwise distance distribution correction (DDC) algorithm to eliminate blinking-caused artifacts in SMLM.

Single-molecule localization microscopy (SMLM) relies on the blinking behavior of a fluorophore, which is the stochastic switching between fluorescent and dark states. Blinking creates multiple localizations belonging to the same fluorophore, confounding quantitative analyses and interpretations. Here we present a method, termed distance distribution correction (DDC), to eliminate blinking-caused repeat localizations without any additional calibrations. The approach relies on obtaining the true pairwise distance distribution of different fluorophores naturally from the imaging sequence by using distances between localizations separated by a time much longer than the average fluorescence survival time. We show that, using the true pairwise distribution, we can define and maximize the likelihood, obtaining a set of localizations void of blinking artifacts. DDC results in drastic improvements in obtaining the closest estimate of the true spatial organization and number of fluorescent emitters in a wide range of applications, enabling accurate reconstruction and quantification of SMLM images.

Pruriception and neuronal coding in nociceptor subtypes in human and nonhuman primates.

In humans, intradermal administration of ß-alanine (ALA) and bovine adrenal medulla peptide 8-22 (BAM8-22) evokes the sensation of itch. Currently, it is unknown which human dorsal root ganglion (DRG) neurons express the receptors of these pruritogens, MRGPRD and MRGPRX1, respectively, and which cutaneous afferents these pruritogens activate in primate. In situ hybridization studies revealed that MRGPRD and MRGPRX1 are co-expressed in a subpopulation of TRPV1+ human DRG neurons. In electrophysiological recordings in nonhuman primates (Macaca nemestrina), subtypes of polymodal C-fiber nociceptors are preferentially activated by ALA and BAM8-22, with significant overlap. When pruritogens ALA, BAM8-22, and histamine, which activate different subclasses of C-fiber afferents, are administered in combination, human volunteers report itch and nociceptive sensations similar to those induced by a single pruritogen. Our results provide evidence for differences in pruriceptive processing between primates and rodents, and do not support the spatial contrast theory of coding of itch and pain.

Identification of novel synaptonemal complex components in C. elegans.

The synaptonemal complex (SC) is a tripartite protein scaffold that forms between homologous chromosomes during meiosis. Although the SC is essential for stable homologue pairing and crossover recombination in diverse eukaryotes, it is unknown how individual components assemble into the highly conserved SC structure. Here we report the biochemical identification of two new SC components, SYP-5 and SYP-6, in Caenorhabditis elegans. SYP-5 and SYP-6 are paralogous to each other and play redundant roles in synapsis, providing an explanation for why these genes have evaded previous genetic screens. Superresolution microscopy reveals that they localize between the chromosome axes and span the width of the SC in a head-to-head manner, similar to the orientation of other known transverse filament proteins. Using genetic redundancy and structure-function analyses to truncate C-terminal tails of SYP-5/6, we provide evidence supporting the role of SC in both limiting and promoting crossover formation.

Superresolution imaging of chromatin fibers to visualize epigenetic information on replicative DNA.

During DNA replication, the genetic information of a cell is copied. Subsequently, identical genetic information is segregated reliably to the two daughter cells through cell division. Meanwhile, DNA replication is intrinsically linked to the process of chromatin duplication, which is required for regulating gene expression and establishing cell identities. Understanding how chromatin is established, maintained or changed during DNA replication represents a fundamental question in biology. Recently, we developed a method to directly visualize chromatin components at individual replication forks undergoing DNA replication. This method builds upon the existing chromatin fiber technique and combines it with cell type-specific chromatin labeling and superresolution microscopy. In this method, a short pulse of nucleoside analog labels replicative regions in the cells of interest. Chromatin fibers are subsequently isolated and attached to a glass slide, after which a laminar flow of lysis buffer extends the lysed chromatin fibers parallel with the direction of the flow. Fibers are then immunostained for different chromatin-associated proteins and mounted for visualization using superresolution microscopy. Replication foci, or 'bubbles,' are identified by the presence of the incorporated nucleoside analog. For researchers experienced in molecular biology and superresolution microscopy, this protocol typically takes 2-3 d from sample preparation to data acquisition, with an additional day for data processing and quantification.

Asymmetric Histone Inheritance in Asymmetrically Dividing Stem Cells.

Epigenetic mechanisms play essential roles in determining distinct cell fates during the development of multicellular organisms. Histone proteins represent crucial epigenetic components that help specify cell identities. Previous work has demonstrated that during the asymmetric cell division of Drosophila male germline stem cells (GSCs), histones H3 and H4 are asymmetrically inherited, such that pre-existing (old) histones are segregated towards the self-renewing GSC whereas newly synthesized (new) histones are enriched towards the differentiating daughter cell. In order to further understand the molecular mechanisms underlying this striking phenomenon, two key questions must be answered: when and how old and new histones are differentially incorporated by sister chromatids, and how epigenetically distinct sister chromatids are specifically recognized and segregated. Here, we discuss recent advances in our understanding of the molecular mechanisms and cellular bases underlying these fundamental and important biological processes responsible for generating two distinct cells through one cell division.

Asymmetric histone inheritance via strand-specific incorporation and biased replication fork movement.

Many stem cells undergo asymmetric division to produce a self-renewing stem cell and a differentiating daughter cell. Here we show that, similarly to H3, histone H4 is inherited asymmetrically in Drosophila melanogaster male germline stem cells undergoing asymmetric division. In contrast, both H2A and H2B are inherited symmetrically. By combining super-resolution microscopy and chromatin fiber analyses with proximity ligation assays on intact nuclei, we find that old H3 is preferentially incorporated by the leading strand, whereas newly synthesized H3 is enriched on the lagging strand. Using a sequential nucleoside analog incorporation assay, we detect a high incidence of unidirectional replication fork movement in testes-derived chromatin and DNA fibers. Biased fork movement coupled with a strand preference in histone incorporation would explain how asymmetric old and new H3 and H4 are established during replication. These results suggest a role for DNA replication in patterning epigenetic information in asymmetrically dividing cells in multicellular organisms.

The Inherent Asymmetry of DNA Replication.

Semiconservative DNA replication has provided an elegant solution to the fundamental problem of how life is able to proliferate in a way that allows cells, organisms, and populations to survive and replicate many times over. Somewhat lost, however, in our admiration for this mechanism is an appreciation for the asymmetries that occur in the process of DNA replication. As we discuss in this review, these asymmetries arise as a consequence of the structure of the DNA molecule and the enzymatic mechanism of DNA synthesis. Increasing evidence suggests that asymmetries in DNA replication are able to play a central role in the processes of adaptation and evolution by shaping the mutagenic landscape of cells. Additionally, in eukaryotes, recent work has demonstrated that the inherent asymmetries in DNA replication may play an important role in the process of chromatin replication. As chromatin plays an essential role in defining cell identity, asymmetries generated during the process of DNA replication may play critical roles in cell fate decisions related to patterning and development.

Breaking Symmetry - Asymmetric Histone Inheritance in Stem Cells.

Asymmetric cell division (ACD) gives rise to two daughter cells with distinct fates. ACD is widely used during development and by many types of adult stem cells during tissue homeostasis and regeneration. ACD can be regulated by extrinsic cues, such as signaling molecules, as well as by intrinsic factors, such as organelles and cortex proteins. The recent discovery of asymmetric histone inheritance during stem cell ACD has revealed another intrinsic mechanism by which ACD produces two distinct daughters. In this review we discuss these findings in the context of cell-cycle regulation, as well as other studies of ACD, to begin understanding the underlying mechanisms and biological relevance of this phenomenon.

DETENTION OUTLET RETROFIT IMPROVES THE FUNCTIONALITY OF EXISTING DETENTION BASINS BY REDUCING EROSIVE FLOWS IN RECEIVING CHANNELS.

By discharging excess stormwater at rates that more frequently exceed the critical flow for stream erosion, conventional detention basins often contribute to increased channel instability in urban and suburban systems that can be detrimental to aquatic habitat and water quality, as well as adjacent property and infrastructure. However, these ubiquitous assets, valued at approximately $600,000 per km2 in a representative suburban watershed, are ideal candidates to aid in reversing such cycles of channel degradation because improving their functionality would not necessarily require property acquisition or heavy construction. The objective of this research was to develop a simple, cost-effective device that could be installed in detention basin outlets to reduce the erosive power of the relatively frequent storm events (~ < two-year recurrence) and provide a passive bypass to maintain flood control performance during infrequent storms (such as the 100-year recurrence). Results from a pilot installation show that the Detain H2O device reduced the cumulative sediment transport capacity of the preretrofit condition by greater than 40%, and contributed to reduced flashiness and prolonged baseflows in receiving streams. When scaling the strategy across a watershed, these results suggest that potential gains in water quality and stream channel stability could be achieved at costs that are orders of magnitude less than comparable benefits from newly constructed stormwater control measures.

Laser speckle imaging to improve clinical outcomes for patients with trigeminal neuralgia undergoing radiofrequency thermocoagulation.

OBJECTIVE: Percutaneous treatments for trigeminal neuralgia are safe, simple, and effective for achieving good pain control. Procedural risks could be minimized by using noninvasive imaging techniques to improve the placement of the radiofrequency thermocoagulation probe into the trigeminal ganglion. Positioning of a probe is crucial to maximize pain relief and to minimize unwanted side effects, such as denervation in unaffected areas. This investigation examined the use of laser speckle imaging during probe placement in an animal model. METHODS: This preclinical safety study used nonhuman primates, Macaca nemestrina (pigtail monkeys), to examine whether real-time imaging of blood flow in the face during the positioning of a coagulation probe could monitor the location and guide the positioning of the probe within the trigeminal ganglion. RESULTS: Data from 6 experiments in 3 pigtail monkeys support the hypothesis that laser imaging is safe and improves the accuracy of probe placement. CONCLUSIONS: Noninvasive laser speckle imaging can be performed safely in nonhuman primates. Because improved probe placement may reduce morbidity associated with percutaneous rhizotomies, efficacy trials of laser speckle imaging should be conducted in humans.

Histone H3 Threonine Phosphorylation Regulates Asymmetric Histone Inheritance in the Drosophila Male Germline.

A long-standing question concerns how stem cells maintain their identity through multiple divisions. Previously, we reported that pre-existing and newly synthesized histone H3 are asymmetrically distributed during Drosophila male germline stem cell (GSC) asymmetric division. Here, we show that phosphorylation at threonine 3 of H3 (H3T3P) distinguishes pre-existing versus newly synthesized H3. Converting T3 to the unphosphorylatable residue alanine (H3T3A) or to the phosphomimetic aspartate (H3T3D) disrupts asymmetric H3 inheritance. Expression of H3T3A or H3T3D specifically in early-stage germline also leads to cellular defects, including GSC loss and germline tumors. Finally, compromising the activity of the H3T3 kinase Haspin enhances the H3T3A but suppresses the H3T3D phenotypes. These studies demonstrate that H3T3P distinguishes sister chromatids enriched with distinct pools of H3 in order to coordinate asymmetric segregation of "old" H3 into GSCs and that tight regulation of H3T3 phosphorylation is required for male germline activity.

Three functionally distinct classes of C-fibre nociceptors in primates.

In primates, C-fibre polymodal nociceptors are broadly classified into two groups based on mechanosensitivity. Here we demonstrate that mechanically sensitive polymodal nociceptors that respond either quickly (QC) or slowly (SC) to a heat stimulus differ in responses to a mild burn, heat sensitization, conductive properties and chemosensitivity. Superficially applied capsaicin and intradermal injection of ß-alanine, an MrgprD agonist, excite vigorously all QCs. Only 40% of SCs respond to ß-alanine, and their response is only half that of QCs. Mechanically insensitive C-fibres (C-MIAs) are ß-alanine insensitive but vigorously respond to capsaicin and histamine with distinct discharge patterns. Calcium imaging reveals that ß-alanine and histamine activate distinct populations of capsaicin-responsive neurons in primate dorsal root ganglion. We suggest that histamine itch and capsaicin pain are peripherally encoded in C-MIAs, and that primate polymodal nociceptive afferents form three functionally distinct subpopulations with ß-alanine responsive QC fibres likely corresponding to murine MrgprD-expressing, non-peptidergic nociceptive afferents.

Local loperamide injection reduces mechanosensitivity of rat cutaneous, nociceptive C-fibers.

Loperamide reverses signs of mechanical hypersensitivity in an animal model of neuropathic pain suggesting that peripheral opioid receptors may be suitable targets for the treatment of neuropathic pain. Since little is known about loperamide effects on the responsiveness of primary afferent nerve fibers, in vivo electrophysiological recordings from unmyelinated afferents innervating the glabrous skin of the hind paw were performed in rats with an L5 spinal nerve lesion or sham surgery. Mechanical threshold and responsiveness to suprathreshold stimulation were tested before and after loperamide (1.25, 2.5 and 5 µg in 10 µl) or vehicle injection into the cutaneous receptive field. Loperamide dose-dependently decreased mechanosensitivity in unmyelinated afferents of nerve-injured and sham animals, and this effect was not blocked by naloxone pretreatment. We then investigated loperamide effects on nerve conduction by recording compound action potentials in vitro during incubation of the sciatic nerve with increasing loperamide concentrations. Loperamide dose-dependently decreased compound action potentials of myelinated and unmyelinated fibers (ED50 = 8 and 4 µg/10 µl, respectively). This blockade was not prevented by pre-incubation with naloxone. These results suggest that loperamide reversal of behavioral signs of neuropathic pain may be mediated, at least in part, by mechanisms independent of opioid receptors, most probably by local anesthetic actions.