Diesel exhaust exposure alters the expression of networks implicated in neurodegeneration in zebrafish brains.

Neurodegenerative diseases are a major cause of disability in the world, but their etiologies largely remain elusive. Genetic factors can only account for a minority of risk for most of these disorders, suggesting environmental factors play a significant role in the development of these diseases. Prolonged exposure to air pollution has recently been identified to increase the risk of Alzheimer's and Parkinson's diseases, but the molecular mechanisms by which it acts are not well understood. Zebrafish embryos exposed to diesel exhaust particle extract (DEPe) lead to dysfunctional autophagy and neuronal loss. Here, we exposed zebrafish embryos to DEPe and performed high throughput proteomic and transcriptomic expression analyses from their brains to identify pathogenic pathways induced by air pollution. DEPe treatment altered several biological processes and signaling pathways relevant to neurodegenerative processes, including xenobiotic metabolism, phagosome maturation, and amyloid processing. The biggest induction of gene expression in brains was in Cyp1A (over 30-fold). The relevance of this expression change was confirmed by blocking induction using CRISPR/Cas9, which resulted in a dramatic increase in sensitivity to DEPe toxicity, confirming that Cyp1A induction was a compensatory protective mechanism. These studies identified disrupted molecular pathways that may contribute to the pathogenesis of neurodegenerative disorders. Ultimately, determining the molecular basis of how air pollution increases the risk of neurodegeneration will help in the development of disease-modifying therapies.

Integrative analysis of gene amplification in Drosophila follicle cells: parameters of origin activation and repression.

In metazoans, how replication origins are specified and subsequently activated is not well understood. Drosophila amplicons in follicle cells (DAFCs) are genomic regions that undergo rereplication to increase DNA copy number. We identified all DAFCs by comparative genomic hybridization, uncovering two new amplicons in addition to four known previously. The complete identification of all DAFCs enabled us to investigate these in vivo replicons with respect to parameters of transcription, localization of the origin recognition complex (ORC), and histone acetylation, yielding important insights into gene amplification as a metazoan replication model. Significantly, ORC is bound across domains spanning 10 or more kilobases at the DAFC rather than at a specific site. Additionally, ORC is bound at many regions that do not undergo amplification, and, in contrast to cell culture, these regions do not correlate with high gene expression. As a developmental strategy, gene amplification is not the predominant means of achieving high expression levels, even in cells capable of amplification. Intriguingly, we found that, in some strains, a new amplicon, DAFC-22B, does not amplify, a consequence of distant repression of ORC binding and origin activation. This repression is alleviated when a fragment containing the origin is placed in different genomic contexts.

Small molecule inhibitors of trans-translation have broad-spectrum antibiotic activity.

The trans-translation pathway for protein tagging and ribosome release plays a critical role for viability and virulence in a wide range of pathogens but is not found in animals. To explore the use of trans-translation as a target for antibiotic development, a high-throughput screen and secondary screening assays were used to identify small molecule inhibitors of the pathway. Compounds that inhibited protein tagging and proteolysis of tagged proteins were recovered from the screen. One of the most active compounds, KKL-35, inhibited the trans-translation tagging reaction with an IC50 = 0.9 µM. KKL-35 and other compounds identified in the screen exhibited broad-spectrum antibiotic activity, validating trans-translation as a target for drug development. This unique target could play a key role in combating strains of pathogenic bacteria that are resistant to existing antibiotics.

Developmental control of gene copy number by repression of replication initiation and fork progression.

Precise DNA replication is crucial for genome maintenance, yet this process has been inherently difficult to study on a genome-wide level in untransformed differentiated metazoan cells. To determine how metazoan DNA replication can be repressed, we examined regions selectively under-replicated in Drosophila polytene salivary glands, and found they are transcriptionally silent and enriched for the repressive H3K27me3 mark. In the first genome-wide analysis of binding of the origin recognition complex (ORC) in a differentiated metazoan tissue, we find that ORC binding is dramatically reduced within these large domains, suggesting reduced initiation as one mechanism leading to under-replication. Inhibition of replication fork progression by the chromatin protein SUUR is an additional repression mechanism to reduce copy number. Although repressive histone marks are removed when SUUR is mutated and copy number restored, neither transcription nor ORC binding is reinstated. Tethering of the SUUR protein to a specific site is insufficient to block replication, however. These results establish that developmental control of DNA replication, at both the initiation and elongation stages, is a mechanism to change gene copy number during differentiation.

Hand-held and integrated single-cell pipettes.

Successful single-cell isolation is a primary step for subsequent chemical and biological analyses of single cells. Conventional single-cell isolation methods often encounter operational complexity, limited efficiency, deterioration of cell viability, incompetence in the isolation of a single-cell into nanoliter liquid, and/or inability to select single adherent cells with specific phenotypes. Here, we develop a hand-held single-cell pipet (hSCP) that is rapid, operationally simple, highly efficient, and inexpensive for unbiased isolation of single viable suspended cells directly from submicroliter cell suspensions into nanoliter droplets without the assistance of any additional equipment. An integrated SCP (iSCP) has also been developed for selective isolation of single suspended and adherent cells according to the fluorescence imaging and morphological features. The isolated single cells can be conveniently transferred into standard 96-/384-well plates, Petri dishes, or vials for cloning, PCR, and other single-cell biochemical assays.

Developmental control of the DNA replication and transcription programs.

Polyploid or polytene cells, which have more than 2C DNA content, are widespread throughout nature and present in most differentiated Drosophila tissues. These cells also can display differential replication, that is, genomic regions of increased or decreased DNA copy number relative to overall genomic ploidy. How frequently differential replication is used as a developmental strategy remains unclear. Here, we use genome-wide array-based comparative genomic hybridization (aCGH) to profile differential DNA replication in isolated and purified larval fat body and midgut tissues of Drosophila, and we compare them with recent aCGH profiles of the larval salivary gland. We identify sites of euchromatic underreplication that are common to all three tissues and others that are tissue specific. We demonstrate that both common and tissue-specific underreplicated sites are dependent on the Suppressor of Underreplication protein, SUUR. mRNA-seq profiling shows that whereas underreplicated regions are generally transcriptionally silent in the larval midgut and salivary gland, transcriptional silencing and underreplication have been uncoupled in the larval fat body. In addition to revealing the prevalence of differential replication, our results show that transcriptional silencing and underreplication can be mechanistically uncoupled.

A Case of Acute Kidney Injury, Proteinuria, and Thrombotic Microangiopathy Associated With Sunitinib Therapy in Metastatic Pancreatic Neuroendocrine Tumor.

There have been rare reports of patients developing nephrotic syndrome and thrombotic microangiopathy (TMA) with tyrosine kinase inhibitors (TKIs). We present the case of a patient with a history of metastatic pancreatic neuroendocrine tumor (pNET), treated with sunitinib, who rapidly developed TMA and acute kidney injury. The patient was successfully treated with cessation of sunitinib and administration of steroids. This case report contributes to the growing body of literature surrounding the rare side effects of TKIs and our experience with the management of these adverse events.

Locally Advanced Gastric Cancer Management: A Cost-Effectiveness Analysis.

Across the nation, patients with locally advanced gastric cancer (LAGC) are managed with modalities including upfront surgery (US) and perioperative chemotherapy (PCT). Preoperative therapies have demonstrated survival benefits over US and thus long-term outcomes are expected to vary between the options. However, as these 2 modalities continue to be regularly employed, we sought to perform a decision analysis comparing the costs and quality-of-life associated with the treatment of patients with LAGC to identify the most cost-effective option. We designed a decision tree model to investigate the survival and costs associated with the most commonly utilized management modalities for LAGC in the United States: US and PCT. The tree described costs and treatment strategies over a 6-month time horizon. Costs were derived from 2022 Medicare reimbursement rates using the third-party payer perspective for physicians and hospitals. Effectiveness was represented using quality-adjusted life-years (QALYs). One-way, two-way, and probabilistic sensitivity analyses were utilized to test the robustness of our findings. PCT was the most cost-effective treatment modality for patients with LAGC over US with a cost of $40,792.16 yielding 3.11 QALYs. US has a cost of $55,575.57 while yielding 3.15 QALYs; the incremental cost-effectiveness ratio (ICER) was $369,585.25. One-way and two-way sensitivity analyses favored PCT in all variations of variables across their standard deviations. Across 100,000 Monte Carlo simulations, 100% of trials favored PCT. In our model simulating patients with LAGC, the most cost-effective treatment strategy was PCT. While US demonstrated improved QALYs over PCT, the associated cost was too great to justify its use.

Multi-domain neurocognitive impairment following definitive intensity-modulated radiotherapy for nasopharyngeal cancer: A cross-sectional study.

INTRODUCTION: Neurocognitive impairment from inadvertent brain irradiation is common following intensity-modulated radiotherapy (IMRT) for nasopharyngeal carcinoma (NPC). This study aimed to determine the prevalence, pattern, and radiation dose-toxicity relationship of this late complication. MATERIALS AND METHODS: We undertook a cross-sectional study of 190 post-IMRT NPC survivors. Neurocognitive function was screened using the Montreal Cognitive Assessment-Hong Kong (HK-MoCA). Detailed assessments of eight distinct neurocognitive domains were conducted: intellectual capacity (WAIS-IV), attention span (Digit Span and Visual Spatial Span), visual memory (Visual Reproduction Span), verbal memory (Auditory Verbal Learning Test), processing speed (Color Trail Test), executive function (Stroop Test), motor dexterity (Grooved Pegboard Test) and language ability (Verbal Fluency Test). The mean percentiles and Z-scores were compared with normative population data. Associations between radiation dose and brain substructures were explored using multivariable logistic regression. RESULTS: The median post-IMRT interval was 7.0 years. The prevalence of impaired HK-MoCA was 25.3 % (48/190). Among the participants, 151 (79.4 %) exhibited impairments in at least one neurocognitive domain. The predominantly impaired domains included verbal memory (short-term: mean Z-score, -0.56, p < 0.001; long-term: mean Z-score, -0.70, p < 0.001), processing speed (basic: mean Z-score, -1.04, p < 0.001; advanced: mean Z-score, -0.38, p < 0.001), executive function (mean Z-score, -1.90, p < 0.001), and motor dexterity (dominant hand: mean Z-score, -0.97, p < 0.001). Radiation dose to the whole brain, hippocampus, and temporal lobe was associated with impairments in executive function, verbal memory, processing speed, and motor dexterity. CONCLUSIONS: Neurocognitive impairment is prevalent and profound in post-IMRT NPC survivors. Cognitive assessment and rehabilitation should be considered part of survivorship care.

Inhibition of tumorigenesis driven by different Wnt proteins requires blockade of distinct ligand-binding regions by LRP6 antibodies.

Disregulated Wnt/beta-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1- and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1- and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wnt-driven cancer.

Pleomorphic Rhabdomyosarcoma: A Systematic Review with Outcome Analysis and Report of a Rare Abdominal Wall Lesion.

Introduction. Pleomorphic rhabdomyosarcoma (RMS) is an aggressive and rare malignant neoplasm with a poor prognosis. As its name suggests, this tumor exhibits extensive pleomorphism with features of skeletal muscle differentiation. Due to its rarity, its diagnosis is often a clinical and pathological challenge. Since only small case series and a few scattered case reports exist in the literature, the impact of different demographic features, tumor site, and/or treatment modality on patient outcomes has yet to be extensively studied. Methods. We report a case of a pleomorphic RMS presenting atypically as an abdominal wall mass. We have also analyzed the National Cancer Institute's Surveillance, Epidemiology and End Results (SEER) database to determine the factors affecting the outcome of this neoplasm. Moreover, we present a review and summary of pleomorphic RMS cases arising from the abdominal wall reported in the English language literature. Results. We found two hundred and forty-two cases of pleomorphic RMS in the SEER database. The majority of the patients were diagnosed after the age of 40, with the age of diagnosis showing a unimodal distribution. The majority of the patients were Caucasian (82%) and male (59%). Age of diagnosis, tumor stage, and surgical management significantly affected the patients' outcome, while patients' ethnicity, sex, or tumor site did not affect the outcome. We only found five previously reported cases of pleomorphic RMS arising from the abdominal wall. Conclusions. Pleomorphic RMS arising from the abdominal wall is extremely rare. Our data sheds light on the factors affecting the outcome of pleomorphic RMS. We have also discussed the challenges involving the histopathological diagnosis of this rare neoplasm and how to best approach this task.

Neoadjuvant Treatment Versus Upfront Surgery in Resectable Pancreatic Cancer: A Cost-Effectiveness Analysis.

PURPOSE: Pancreatic cancer (PC) has an overall 5-year survival rate of 10%. The use of neoadjuvant chemoradiation is debated in resectable disease. The purpose of this study is to evaluate the cost-effectiveness of neoadjuvant chemoradiation followed by pancreaticoduodenectomy (NACRT) versus upfront pancreaticoduodenectomy and adjuvant chemotherapy (USR) in resectable PC. METHODS: A decision tree model was used to estimate the cost-effectiveness of NACRT versus USR. Values from the published literature populate the tree: costs from Medicare (FY2021) reimbursements, and morbidity and survival data for quality-adjusted life-years (QALYs). Patients with resectable pancreatic adenocarcinoma who qualified for resection were included. The ICER was the primary outcome. The model was validated using one-way and two-way deterministic, as well as probabilistic sensitivity analyses. RESULTS: The base case was modeled using a 65-year-old male. NACRT yielded 1.61 QALYs at $45,483.52 USD. USR yielded 1.47 QALYs at a discount of $6,840.96 USD. The ICER was $48,130 USD, which favors NACRT. One-way sensitivity analyses upheld these results except when ≤ 21.0% of NACRT patients proceeded to surgery and when ≤ 85.4% of NACRT patients were resectable at surgery. Two-way sensitivity analyses also favored NACRT except in cases when the proportion of resected disease after NACRT decreased. NACRT was favored in 94.3% of 100,000 random-sampling simulations. CONCLUSION: It is more cost-effective to administer NACRT before surgery for patients with resectable PC. On the basis of sensitivity analyses, USR with adjuvant therapy is only favored if rates of resection and eligibility for resection after NACRT decrease. NACRT should be considered in all patients unless there is an absolute contraindication.

Advancing oncology drug therapies for sub-Saharan Africa.

Cancer incidence is rising across sub-Saharan Africa (SSA), and is often characterized by late-stage presentation, early age of onset and poor survival. While a number of oncology drugs are now improving the length and quality of life for cancer patients in high-income countries, significant disparities in access to a range of oncology therapeutics exist for SSA. A number of challenges to drug access such as drug costs, lack of infrastructure and trained personnel must be urgently addressed to advance oncology therapies for SSA. We present a review of selected oncology drug therapies that are likely to benefit cancer patients with a focus on common malignancies in SSA. We collate available data from seminal clinical trials in high-income countries to highlight the potential for these therapeutics to improve cancer outcomes. In addition, we discuss the need to ensure access to drugs within the WHO Model List of Essential Medicines and highlight therapeutics that require consideration. Available and active oncology clinical trials in the region is tabulated, demonstrating the significant gaps in access to oncology drug trials across much of the region. We issue an urgent call to action to address drug access due to the predicted rise in cancer burden in the region in coming years.

Neurotoxicity of diesel exhaust extracts in zebrafish and its implications for neurodegenerative disease.

Long-term air pollution (AP) exposure, including diesel exhaust exposure, is increasingly being recognized as a major contributor to the development of neurodegenerative diseases such as Parkinson's and Alzheimer's disease. How AP increases the risk of neurodegeneration is not well understood but might include direct neurotoxicity and CNS inflammation. We investigated the impact of diesel exhaust particulate extract (DEPe) exposure on the brain and the mechanisms by which microglia and astroglia might mediate neuronal changes. Zebrafish (ZF) were utilized to determine neuronal toxicity of and microglial response to DEPe and single cell RNA sequencing was employed to study cell type-specific transcriptomic responses within the ZF brain. DEPe exposure induced neuronal injury and microglial activation in vivo. However, preventing the development of microglia did not attenuate DEPe-induced neuron loss, leading us to investigate microglial, astroglial, and neuronal response to DEPe exposure at single-cell resolution. Differentially expressed genes and disease-relevant pathways were identified within glial and neuronal clusters after DEPe exposure. Microglia and astroglia existed in multiple states, some of which appear toxic and others protective to neurons. Neuronal transcriptomic analysis revealed that DEPe exposure reduced expression of autophagy-related genes consistent with direct neurotoxicity. In summary, DEPe exposure was neurotoxic in developing ZF larvae and induced neuroinflammation. The microglial inflammatory response did not contribute to neurotoxicity of DEPe and in fact, some glial clusters upregulated transcriptional pathways that are likely protective. Furthermore, DEPe exposure led to reduced expression of autophagy-related genes in neurons that likely contribute to its toxicity.

High-Content Imaging Phenotypic Screen for Neurogenesis Using Primary Neural Progenitor Cells.

Neurogenesis phenotypic screen of small-molecule library enables the discovery of small-molecule inducers, and identification of associated biological targets and pathways that control neuronal formation from neural progenitor cells (NPCs). Here, we describe protocols for preparing mouse embryonic NPCs, setting up a high-content imaging assay that quantifies the production of Tuj1-labeled neurons, and analysis of high-throughput screens.

The role of transcription in the activation of a Drosophila amplification origin.

The mechanisms that underlie metazoan DNA replication initiation, especially the connection between transcription and replication origin activation, are not well understood. To probe the role of transcription in origin activation, we exploited a specific replication origin in Drosophila melanogaster follicle cells, ori62, which coincides with the yellow-g2 transcription unit and exhibits transcription-dependent origin firing. Within a 10-kb genomic fragment that contains ori62 and is sufficient for amplification, RNA-sequencing analysis revealed that all detected RNAs mapped solely to the yellow-g2 gene. To determine whether transcription is required in cis for ori62 firing, we generated a set of tagged yellow-g2 transgenes in which we could prevent local transcription across ori62 by deletions in the yellow-g2 promoter. Surprisingly, inhibition of yellow-g2 transcription by promoter deletions did not affect ori62 firing. Our results reveal that transcription in cis is not required for ori62 firing, raising the possibility that a trans-acting factor is required specifically for the activation of ori62. This finding illustrates that a diversity of mechanisms can be used in the regulation of metazoan DNA replication initiation.

An antibody that locks HER3 in the inactive conformation inhibits tumor growth driven by HER2 or neuregulin.

HER2/HER3 dimerization resulting from overexpression of HER2 or neuregulin (NRG1) in cancer leads to HER3-mediated oncogenic activation of phosphoinositide 3-kinase (PI3K) signaling. Although ligand-blocking HER3 antibodies inhibit NRG1-driven tumor growth, they are ineffective against HER2-driven tumor growth because HER2 activates HER3 in a ligand-independent manner. In this study, we describe a novel HER3 monoclonal antibody (LJM716) that can neutralize multiple modes of HER3 activation, making it a superior candidate for clinical translation as a therapeutic candidate. LJM716 was a potent inhibitor of HER3/AKT phosphorylation and proliferation in HER2-amplified and NRG1-expressing cancer cells, and it displayed single-agent efficacy in tumor xenograft models. Combining LJM716 with agents that target HER2 or EGFR produced synergistic antitumor activity in vitro and in vivo. In particular, combining LJM716 with trastuzumab produced a more potent inhibition of signaling and cell proliferation than trastuzumab/pertuzumab combinations with similar activity in vivo. To elucidate its mechanism of action, we solved the structure of LJM716 bound to HER3, finding that LJM716 bound to an epitope, within domains 2 and 4, that traps HER3 in an inactive conformation. Taken together, our findings establish that LJM716 possesses a novel mechanism of action that, in combination with HER2- or EGFR-targeted agents, may leverage their clinical efficacy in ErbB-driven cancers.

Mechanisms of rotenone-induced proteasome inhibition.

The etiology of Parkinson's disease is unclear but appears to involve mitochondrial dysfunction, proteasome inhibition, and environmental toxins. It has been shown that pesticides, including the complex I inhibitor rotenone, cause proteasome inhibition but the mechanism of rotenone-induced proteasome dysfunction remains largely unknown. In this study, we examined the role of mitochondrial inhibition, oxidative stress, and microtubule dysfunction as potential mediators of rotenone-induced proteasome inhibition. Proteasome activity (26S) was measured in HEK and SK-N-MC cells expressing an EGFP-U degron fusion protein that is selectively degraded by the proteasome. We found that complexes I and III inhibition led to the production of peroxides and decreased proteasome activity. We also found that rotenone increased nitric oxide production and nitric oxide and peroxynitrites led to proteasome inhibition. The effects of rotenone were attenuated by anti-oxidants and nitric oxide synthase inhibition. Since rotenone can also inhibit microtubule assembly, we tested a specific MT inhibitor and found it led to proteasome dysfunction. Rotenone also led to a decrease in 20S proteasome activity and 20S proteasome subunit immunoreactivity without a change in subunit mRNA. Together, these data suggest that rotenone-induced decreases in proteasome activity are due to increased degradation of proteasome components secondary to oxidative damage and possibly microtubule dysfunction.

Ziram causes dopaminergic cell damage by inhibiting E1 ligase of the proteasome.

The etiology of Parkinson disease (PD) is unclear but may involve environmental toxins such as pesticides leading to dysfunction of the ubiquitin proteasome system (UPS). Here, we measured the relative toxicity of ziram (a UPS inhibitor) and analogs to dopaminergic neurons and examined the mechanism of cell death. UPS (26 S) activity was measured in cell lines after exposure to ziram and related compounds. Dimethyl- and diethyldithiocarbamates including ziram were potent UPS inhibitors. Primary ventral mesencephalic cultures were exposed to ziram, and cell toxicity was assessed by staining for tyrosine hydroxylase (TH) and NeuN antigen. Ziram caused a preferential damage to TH+ neurons and elevated alpha-synuclein levels but did not increase aggregate formation. Mechanistically, ziram altered UPS function through interfering with the targeting of substrates by inhibiting ubiquitin E1 ligase. Sodium dimethyldithiocarbamate administered to mice for 2 weeks resulted in persistent motor deficits and a mild reduction in striatal TH staining but no nigral cell loss. These results demonstrate that ziram causes selective dopaminergic cell damage in vitro by inhibiting an important degradative pathway implicated in the etiology of PD. Chronic exposure to widely used dithiocarbamate fungicides may contribute to the development of PD, and elucidation of its mechanism would identify a new potential therapeutic target.