CX-5461 Preferentially Induces Top2α-Dependent DNA Breaks at Ribosomal DNA Loci.

While genotoxic chemotherapeutic agents are among the most effective tools to combat cancer, they are often associated with severe adverse effects caused by indiscriminate DNA damage in non-tumor tissue as well as increased risk of secondary carcinogenesis. This study builds on our previous work demonstrating that the RNA Polymerase I (Pol I) transcription inhibitor CX-5461 elicits a non-canonical DNA damage response and our discovery of a critical role for Topoisomerase 2α (Top2α) in the initiation of Pol I-dependent transcription. Here, we identify Top2α as a mediator of CX-5461 response in the murine Eµ-Myc B lymphoma model whereby sensitivity to CX-5461 is dependent on cellular Top2α expression/activity. Most strikingly, and in contrast to canonical Top2α poisons, we found that the Top2α-dependent DNA damage induced by CX-5461 is preferentially localized at the ribosomal DNA (rDNA) promoter region, thereby highlighting CX-5461 as a loci-specific DNA damaging agent. This mechanism underpins the efficacy of CX-5461 against certain types of cancer and can be used to develop effective non-genotoxic anticancer drugs.

Moving From Transactional to Relational: How Funders Can Work in Partnership With Black, Indigenous and People of Color Communities.

BACKGROUND: The Racial Equity Coalition (REC) formed to address persistent educational disparities. The coalition was composed of 14 Black, Indigenous and People of Color (BIPOC) organizations that provide culturally integrative youth services. OBJECTIVES: REC, with support from United Way of King County, engaged in participatory research to identify commonalities and shared struggles to inform collective action. Participatory research aligns with REC's commitment to equitable participatory processes. This article focuses on REC's experiences with funders. The objective was to understand what creates positive and challenging experiences with funders, and to identify recommendations for funders to become more culturally responsive. METHODS: A research committee was formed including representatives of nine REC organizations and United Way of King County staff. The committee conducted interviews with each of the 14 REC organizations and conducted thematic analysis of interview transcripts. Through participatory analysis, the committee drafted narratives that were further refined through a series of research retreats attended by all REC organizations. RESULTS: Recommendations were to incentivize collaboration, listen to communities to create culturally responsive definitions of success and measurement strategies, arrive at mutually agreed upon approaches with organizations, honor the connections BIPOC organizations have with their communities, and provide unrestricted funding to allow BIPOC organizations greater agency. CONCLUSIONS: A major challenge for BIPOC organizations is navigating White dominant culture that too often shows up in funding requirements. Having to fit dominant culture standards stifles BIPOC organizations' abilities to meet community needs and the responsiveness of their approaches. REC identified recommendations for funders to be more culturally responsive and community centered.

Clinical outcomes of immunoglobulin treatment for patients with secondary antibody deficiency: Data from the Ontario immunoglobulin treatment case registry.

BACKGROUND: Despite the increasing number of cases of secondary antibody deficiency (SAD) and immunoglobulin (Ig) utilization, there is a paucity of data in the literature on clinical and patient-reported outcomes in this population. OBJECTIVE: To describe immunoglobulin utilization patterns, clinical and patient-reported outcomes in patients with SAD on immunoglobulin replacement therapy (IgRT). METHODS: A cross-sectional study of patients with secondary antibody deficiency enrolled in the Ontario Immunoglobulin Treatment (ONIT) Case Registry from June 2020 to September 2022 was completed. Demographics, comorbidities, indications for immunoglobulin treatment, clinical infections at baseline and post IgRT, and patient-reported outcomes were collected and analyzed. RESULTS: There were 140 patients (58 males; 82 females; median age 68) with SAD during the study period; 131 were on subcutaneous Ig (SCIG) and 9 were on intravenous Ig (IVIG). The most common indication was chronic lymphocytic leukemia (CLL) (N = 52). IgRT reduced the average annual number of infections by 82.6%, emergency room (ER) visits by 84.6%, and hospitalizations by 83.3%. Overall, 84.6% of patients reported their health as better compared to before IgRT. Among those patients who switched from IVIG to SCIG (N = 35), 33.3% reported their health as the same, and 62.9% reported their health as better. CONCLUSIONS: This study demonstrates that IgRT significantly improved clinical outcomes and patient-reported general health state in patients with SAD. This study also further supports the use of SCIG in patients with SAD.

Coinhibition of topoisomerase 1 and BRD4-mediated pause release selectively kills pancreatic cancer via readthrough transcription.

Pancreatic carcinoma lacks effective therapeutic strategies resulting in poor prognosis. Transcriptional dysregulation due to alterations in KRAS and MYC affects initiation, development, and survival of this tumor type. Using patient-derived xenografts of KRAS- and MYC-driven pancreatic carcinoma, we show that coinhibition of topoisomerase 1 (TOP1) and bromodomain-containing protein 4 (BRD4) synergistically induces tumor regression by targeting promoter pause release. Comparing the nascent transcriptome with the recruitment of elongation and termination factors, we found that coinhibition of TOP1 and BRD4 disrupts recruitment of transcription termination factors. Thus, RNA polymerases transcribe downstream of genes for hundreds of kilobases leading to readthrough transcription. This occurs during replication, perturbing replisome progression and inducing DNA damage. The synergistic effect of TOP1 + BRD4 inhibition is specific to cancer cells leaving normal cells unaffected, highlighting the tumor's vulnerability to transcriptional defects. This preclinical study provides a mechanistic understanding of the benefit of combining TOP1 and BRD4 inhibitors to treat pancreatic carcinomas addicted to oncogenic drivers of transcription and replication.

T-SPOT.TB Reactivity in Southern African Children With and Without in Utero Human Immunodeficiency Virus Exposure.

Infants who are human immunodeficiency virus (HIV)-exposed uninfected (iHEU) experience higher risk of infectious morbidity than infants HIV-unexposed uninfected (iHUU). We compared tuberculosis (TB) infection prevalence in 418 Bacillus Calmette-Guérin vaccinated sub-Saharan African iHEU and iHUU aged 9-18 months using T-SPOT.TB. Prevalence of TB infection was low and did not differ by HIV exposure status.

1-Octanol emitted by Oecophylla smaragdina weaver ants repels and deters oviposition in Queensland fruit fly.

Humans have used weaver ants, Oecophylla smaragdina, as biological control agents to control insect pests in orchards for many centuries. Over recent decades, the effectiveness of weaver ants as biological control agents has been attributed in part to deterrent and oviposition inhibiting effects of kairomones produced by the ants, but the chemical identity of these kairomones has remained unknown. We have identified the kairomone responsible for deterrence and oviposition inhibition by O. smaragdina, providing a significant advance in understanding the chemical basis of their predator/prey interactions. Olfactometer assays with extracts from weaver ants demonstrated headspace volatiles to be highly repellent to Queensland fruit fly, Bactrocera tryoni. Using electrophysiology and bioassays, we demonstrate that this repellence is induced by a single compound, 1-octanol. Of 16 compounds identified in O. smaragdina headspace, only 1-octanol evoked an electrophysiological response from B. tryoni antennae. Flies had greatly reduced oviposition and spent significantly less time in an olfactometer arm in the presence of 1-octanol or a synthetic blend of headspace volatiles containing 1-octanol than in the presence of a synthetic blend of headspace volatiles without 1-octanol, or clean air. Taken together, our results demonstrate that 1-octanol is the functional kairomone component of O. smaragdina headspace that explains repellence and oviposition deterrence, and is hence an important contributor to the effectiveness of these ants as biological control agents.

TOP1 CAD-seq: A protocol to map catalytically engaged topoisomerase 1 in human cells.

TOP1 CAD-seq enables mapping of TOP1 sites of covalent engagement with DNA. The procedure depends upon enrichment of DNA-covalent adducts using chaotropic salts and immunoprecipitation with an antibody specific for TOP1. Here, we describe a step-by-step protocol compatible with Illumina sequencing and bioinformatic pipeline for preliminary data analysis. Compared to other approaches for the genomic study of topoisomerases, TOP1 CAD-seq provides information about active TOP1 engaged on the DNA, taking advantage of low background due to absence of crosslinking. For complete details on the use and execution of this protocol, please refer to Das et al. (2022).

Sap flow monitoring of two Australian native tree species in a suburban setting: Implications for tree selection and management.

Sap flow, the transport of fluid in the water-conducting xylem tissues of plants, is commonly measured in studies of plant-water relationships by the heat pulse velocity method. Publications have been rare of long-term sap flow measurements for individual trees in a suburban environment. Plant-water relations in urban settings are essential for promoting urban greening where there is a perceived danger to infrastructure and buildings from planting trees in streets on clay sites. The function of residential houses built on reactive clays can be significantly impaired and walls of buildings cracked if considerable amounts of water are extracted from the soil by the root system of a tree or a group of trees in close proximity, leading to localised soil shrinkage settlement. This part of the wider study aimed to monitor sap flow of eight individual Australian native trees from two species using the heat ratio method (HRM) in the field over 12 months. The analysis of monthly sap flow volume showed a similar pattern for all monitored trees, although daily water demand varied substantially. Methods for estimating tree leaf surface area, crown shape and crown volume were investigated and the equation for calculating thermal diffusivity (k) and sap flow velocity on the basis of the HRM was reviewed. It has been proposed that k may vary substantially depending on how thermal conductivity (K) is estimated, which could lead to significant discrepancies for estimations of plant transpiration. Two K models (KHog and KVan) were investigated and it was found that the impact on mean daily sap volume was negligible for the trees in this study.

Impact of establishing a specialized hepatobiliary unit on liver resections in a non-specialized tertiary centre in regional Australia.

BACKGROUND: In April 2018, a dedicated hepatobiliary unit was established in a tertiary hospital in North Queensland. Changes included the employment of a hepatobiliary-trained surgeon, centralized referrals, and formalized multidisciplinary team meetings. This study aimed to evaluate the impact of establishing a hepatobiliary unit on outcomes after liver resection, in a regional centre where such procedures were previously performed by non-specialist general surgeons. METHODS: Adult patients who underwent elective liver resection in Townsville from 2013 to 2020 were included in the study. Outcomes after liver resection were collected across two study periods - before and after the hepatobiliary unit was established. The primary end points were a before and after comparison of the 90-day morbidity and mortality and the R1 margin rates. RESULTS: Across the two study periods, 76 and 77 patients, respectively, underwent liver resection. Rates of R1 resection, 90-day mortality and major complications were not significantly different between the two study periods. Primary tumours (14.5% before versus 50.6% after) and cirrhosis (1.3% before versus 14.3% after) were significantly higher in the latter period, as was the median length of stay (4 days before versus 6 days after). Annual surgical volume increased by 75% in the period after 2018 compared to the 5 years preceding it. CONCLUSION: Establishing a centralized hepatobiliary unit in a tertiary regional centre resulted in increased surgical volume and case complexity, with no change in early outcomes after liver resection. Overall, this dedicated unit improved the accessibility of a subspecialty surgical service in regional Australia.

MYC assembles and stimulates topoisomerases 1 and 2 in a "topoisome".

High-intensity transcription and replication supercoil DNA to levels that can impede or halt these processes. As a potent transcription amplifier and replication accelerator, the proto-oncogene MYC must manage this interfering torsional stress. By comparing gene expression with the recruitment of topoisomerases and MYC to promoters, we surmised a direct association of MYC with topoisomerase 1 (TOP1) and TOP2 that was confirmed in vitro and in cells. Beyond recruiting topoisomerases, MYC directly stimulates their activities. We identify a MYC-nucleated "topoisome" complex that unites TOP1 and TOP2 and increases their levels and activities at promoters, gene bodies, and enhancers. Whether TOP2A or TOP2B is included in the topoisome is dictated by the presence of MYC versus MYCN, respectively. Thus, in vitro and in cells, MYC assembles tools that simplify DNA topology and promote genome function under high output conditions.

MYCMI-7: A Small MYC-Binding Compound that Inhibits MYC: MAX Interaction and Tumor Growth in a MYC-Dependent Manner.

Deregulated expression of MYC family oncogenes occurs frequently in human cancer and is often associated with aggressive disease and poor prognosis. While MYC is a highly warranted target, it has been considered "undruggable," and no specific anti-MYC drugs are available in the clinic. We recently identified molecules named MYCMIs that inhibit the interaction between MYC and its essential partner MAX. Here we show that one of these molecules, MYCMI-7, efficiently and selectively inhibits MYC:MAX and MYCN:MAX interactions in cells, binds directly to recombinant MYC, and reduces MYC-driven transcription. In addition, MYCMI-7 induces degradation of MYC and MYCN proteins. MYCMI-7 potently induces growth arrest/apoptosis in tumor cells in a MYC/MYCN-dependent manner and downregulates the MYC pathway on a global level as determined by RNA sequencing. Sensitivity to MYCMI-7 correlates with MYC expression in a panel of 60 tumor cell lines and MYCMI-7 shows high efficacy toward a collection of patient-derived primary glioblastoma and acute myeloid leukemia (AML) ex vivo cultures. Importantly, a variety of normal cells become G1 arrested without signs of apoptosis upon MYCMI-7 treatment. Finally, in mouse tumor models of MYC-driven AML, breast cancer, and MYCN-amplified neuroblastoma, treatment with MYCMI-7 downregulates MYC/MYCN, inhibits tumor growth, and prolongs survival through apoptosis with few side effects. In conclusion, MYCMI-7 is a potent and selective MYC inhibitor that is highly relevant for the development into clinically useful drugs for the treatment of MYC-driven cancer. Significance: Our findings demonstrate that the small-molecule MYCMI-7 binds MYC and inhibits interaction between MYC and MAX, thereby hampering MYC-driven tumor cell growth in culture and in vivo while sparing normal cells.

Topoisomerase 1 activity during mitotic transcription favors the transition from mitosis to G1.

As cells enter mitosis, chromatin compacts to facilitate chromosome segregation yet remains transcribed. Transcription supercoils DNA to levels that can impede further progression of RNA polymerase II (RNAPII) unless it is removed by DNA topoisomerase 1 (TOP1). Using ChIP-seq on mitotic cells, we found that TOP1 is required for RNAPII translocation along genes. The stimulation of TOP1 activity by RNAPII during elongation allowed RNAPII clearance from genes in prometaphase and enabled chromosomal segregation. Disruption of the TOP1-RNAPII interaction impaired RNAPII spiking at promoters and triggered defects in the post-mitotic transcription program. This program includes factors necessary for cell growth, and cells with impaired TOP1-RNAPII interaction are more sensitive to inhibitors of mTOR signaling. We conclude that TOP1 is necessary for assisting transcription during mitosis with consequences for growth and gene expression long after mitosis is completed. In this sense, TOP1 ensures that cellular memory is preserved in subsequent generations.

The Epstein-Barr virus deubiquitinating enzyme BPLF1 regulates the activity of topoisomerase II during productive infection.

Topoisomerases are essential for the replication of herpesviruses but the mechanisms by which the viruses hijack the cellular enzymes are largely unknown. We found that topoisomerase-II (TOP2) is a substrate of the Epstein-Barr virus (EBV) ubiquitin deconjugase BPLF1. BPLF1 co-immunoprecipitated and deubiquitinated TOP2, and stabilized SUMOylated TOP2 trapped in cleavage complexes (TOP2ccs), which halted the DNA damage response to TOP2-induced double strand DNA breaks and promoted cell survival. Induction of the productive virus cycle in epithelial and lymphoid cell lines carrying recombinant EBV encoding the active enzyme was accompanied by TOP2 deubiquitination, accumulation of TOP2ccs and resistance to Etoposide toxicity. The protective effect of BPLF1 was dependent on the expression of tyrosyl-DNA phosphodiesterase 2 (TDP2) that releases DNA-trapped TOP2 and promotes error-free DNA repair. These findings highlight a previously unrecognized function of BPLF1 in supporting a non-proteolytic pathway for TOP2ccs debulking that favors cell survival and virus production.

TLR-4 Agonist Induces IFN-γ Production Selectively in Proinflammatory Human M1 Macrophages through the PI3K-mTOR- and JNK-MAPK-Activated p70S6K Pathway.

IFN-γ, a proinflammatory cytokine produced primarily by T cells and NK cells, activates macrophages and engages mechanisms to control pathogens. Although there is evidence of IFN-γ production by murine macrophages, IFN-γ production by normal human macrophages and their subsets remains unknown. Herein, we show that human M1 macrophages generated by IFN-γ and IL-12- and IL-18-stimulated monocyte-derived macrophages (M0) produce significant levels of IFN-γ. Further stimulation of IL-12/IL-18-primed macrophages or M1 macrophages with agonists for TLR-2, TLR-3, or TLR-4 significantly enhanced IFN-γ production in contrast to the similarly stimulated M0, M2a, M2b, and M2c macrophages. Similarly, M1 macrophages generated from COVID-19-infected patients' macrophages produced IFN-γ that was enhanced following LPS stimulation. The inhibition of M1 differentiation by Jak inhibitors reversed LPS-induced IFN-γ production, suggesting that differentiation with IFN-γ plays a key role in IFN-γ induction. We subsequently investigated the signaling pathway(s) responsible for TLR-4-induced IFN-γ production in M1 macrophages. Our results show that TLR-4-induced IFN-γ production is regulated by the ribosomal protein S6 kinase (p70S6K) through the activation of PI3K, the mammalian target of rapamycin complex 1/2 (mTORC1/2), and the JNK MAPK pathways. These results suggest that M1-derived IFN-γ may play a key role in inflammation that may be augmented following bacterial/viral infections. Moreover, blocking the mTORC1/2, PI3K, and JNK MAPKs in macrophages may be of potential translational significance in preventing macrophage-mediated inflammatory diseases.

Deficiency of Polη in Saccharomyces cerevisiae reveals the impact of transcription on damage-induced cohesion.

The structural maintenance of chromosome (SMC) complex cohesin mediates sister chromatid cohesion established during replication, and damage-induced cohesion formed in response to DSBs post-replication. The translesion synthesis polymerase Polη is required for damage-induced cohesion through a hitherto unknown mechanism. Since Polη is functionally associated with transcription, and transcription triggers de novo cohesion in Schizosaccharomyces pombe, we hypothesized that transcription facilitates damage-induced cohesion in Saccharomyces cerevisiae. Here, we show dysregulated transcriptional profiles in the Polη null mutant (rad30Δ), where genes involved in chromatin assembly and positive transcription regulation were downregulated. In addition, chromatin association of RNA polymerase II was reduced at promoters and coding regions in rad30Δ compared to WT cells, while occupancy of the H2A.Z variant (Htz1) at promoters was increased in rad30Δ cells. Perturbing histone exchange at promoters inactivated damage-induced cohesion, similarly to deletion of the RAD30 gene. Conversely, altering regulation of transcription elongation suppressed the deficient damage-induced cohesion in rad30Δ cells. Furthermore, transcription inhibition negatively affected formation of damage-induced cohesion. These results indicate that the transcriptional deregulation of the Polη null mutant is connected with its reduced capacity to establish damage-induced cohesion. This also suggests a linkage between regulation of transcription and formation of damage-induced cohesion after replication.

Analysis of Myc Chromatin Binding by Calibrated ChIP-Seq Approach.

Here, we present a strategy to map and quantify the interactions between Myc and chromatin using a calibrated Myc ChIP-seq approach. We recommend the use of an internal spike-in control for post-sequencing normalization to enable detection of broad changes in Myc binding as can occur under conditions with varied Myc abundance. We also highlight a range of bioinformatic analyses that can dissect the downstream effects of Myc binding. These methods include peak calling, mapping Myc onto an integrated metagenome, juxtaposing ChIP-seq data with matching RNA-seq data, and identifying gene ontologies enriched for genes with high Myc binding. Our aim is to provide a guided strategy, from cell harvest through to bioinformatic analysis, to elucidate the global effects of Myc on transcription.

A High-Throughput NanoBiT-Based Serological Assay Detects SARS-CoV-2 Seroconversion.

High-throughput detection strategies for antibodies against SARS-CoV-2 in patients recovering from COVID-19, or in vaccinated individuals, are urgently required during this ongoing pandemic. Serological assays are the most widely used method to measure antibody responses in patients. However, most of the current methods lack the speed, stability, sensitivity, and specificity to be selected as a test for worldwide serosurveys. Here, we demonstrate a novel NanoBiT-based serological assay for fast and sensitive detection of SARS-CoV-2 RBD-specific antibodies in sera of COVID-19 patients. This assay can be done in high-throughput manner at 384 samples per hour and only requires a minimum of 5 µL of serum or 10 ng of antibody. The stability of our NanoBiT reporter in various temperatures (4-42 °C) and pH (4-12) settings suggests the assay will be able to withstand imperfect shipping and handling conditions for worldwide seroepidemiologic surveillance in the post-vaccination period of the pandemic. Our newly developed rapid assay is highly accessible and may facilitate a more cost-effective solution for seroconversion screening as vaccination efforts progress.

History of paediatric gastroenterology in Australia.

Paediatric gastroenterology in Australia has undergone remarkable changes over the more than six decades since Charlotte Anderson's pioneering work, and is now a well-established specialty in its own right. Australian paediatric gastroenterologists have made important contributions nationally and internationally.