Biomedical Informatics Commons^™

Protocol To Detect M22g Modification On Single Trna Iso-Decoders By Immuno-Northern Blotting, Sseu-Pei Hwang, Katherine Barondeau, Catherine Denicourt

Faculty, Staff and Student Publications

tRNA modifications are highly dynamic and play crucial roles in regulating gene expression. Here, we present an assay for analyzing N2, N2-dimethylguanosine (m22G) modifications on tRNAs using an antibody-based detection approach. We describe steps for isolating tRNAs from human cells via ion-exchange chromatography, capturing specific tRNA iso-decoders on streptavidin magnetic beads, and detecting modifications through immuno-northern blotting. This approach enables researchers to investigate other tRNA modifications using specific antibodies or downstream approaches such as mass spectrometry.

Mechanisms Of Experience-Dependent Place-Cell Referencing In Hippocampal Area Ca1, Fish Kunxun Qian, Yiding Li, Jeffrey C Magee

Faculty, Staff and Students Publications

Hippocampal CA1 place cells (PCs) encode both space- and goal-referenced information to support a cognitive map. The mechanism of this referencing and the role of experience remain poorly understood. Here we longitudinally recorded PC activity while head-fixed mice performed a spatial learning task on a treadmill. In a familiar environment, the CA1 representation consisted of PCs that were referenced to either specific spatial locations or a reward goal in approximately equal proportions; however, the CA1 representation became predominately goal-referenced upon exposure to a novel environment, as space-referenced PCs adaptively switched reference frames. Intracellular membrane potential recordings revealed that individual CA1 …

A Recipe For Disaster - Sodium Bicarbonate Overdose, Adeola A Kosoko, Amara Ogoke, Kyle Vogt

Faculty, Staff and Student Publications

Audience: Emergency medicine residents post-graduate years 1-4.

Introduction: Sodium bicarbonate is a compound found commonplace in many households and in many products. It lends itself to countless everyday uses including cooking and cleaning. Physicians prescribe sodium bicarbonate regularly both on- and off-label regularly for various ailments.1 Due to the ubiquitous way both emergency physicians and the general public use and are exposed to sodium bicarbonate, we wanted to prepare learners to identify and appropriately manage sodium bicarbonate toxicity.Obtaining a thorough history not just of medications prescribed but of significant ingestions or infusions is critical in diagnosing this toxicity. Furthermore, acute …

Preventing Acute Kidney Injury And Its Longer-Term Impact In The Critically Ill, Alexander Zarbock, Lui Forni, Jay L Koyner, Hernando Gómez, Neesh Pannu, Marlies Ostermann, Rinaldo Bellomo, John A Kellum, Thilo Von Groote

Faculty, Staff and Student Publications

Acute kidney injury (AKI) is a heterogeneous syndrome that not only affects short-term morbidity and mortality but also influences long-term outcomes. AKI is part of acute kidney disease (AKD) that encompasses a range of different conditions and is characterized by a kidney dysfunction lasting 90 days or less after which time the term chronic kidney disease (CKD) applies. AKD may result in irreversible loss of nephrons and may lead to CKD. In this narrative review, an update on different aspects of AKI in critically ill patients will be provided. We discuss biomarkers for early diagnosis of AKI, sub-clinical AKI, as …

Long-Term Exposure To Multiple Air Pollutants And Multi-Level Socioeconomic Status: Joint Effects On Age-Related Macular Degeneration, Subsequent Ocular Comorbidity, And Death In Middle-Aged And Older Adults, Yang Yang, Liangkai Chen, Qichen Liu, Min Mu, Jing Huang, Guangming Zhang, Qianqian Song

Faculty, Staff and Student Publications

Background: Both air pollution and socioeconomic status (SES) are recognized as significant determinants of health outcomes. However, no study has explored the combined effects of air pollutants and SES on (1) age-related macular degeneration (AMD) incidence; (2) trajectories from baseline to AMD, subsequent ocular comorbidity (OCMD), and mortality; and (3) life expectancy in middle-aged and older adults.

Methods: Using UK Biobank data, we created two composite air pollution scores (APS) and assessed SES at individual and neighborhood levels. OCMD was defined as glaucoma or cataract occurrence after AMD diagnosis. Cox proportional hazard regression models, multistate models, and life tables were …

Switching From Adalimumab Reference Product To And Among Adalimumab Biosimilars Outside The Usa: Insights For Us Clinicians, John R P Tesser, Aline Charabaty, Adelaide A Hebert

Faculty, Staff and Student Publications

Ten adalimumab biosimilars have been introduced in the United States (USA) since 2023, while adalimumab biosimilars have been available for several years in other countries. These experiences of biosimilar uptake outside the USA can inform US-based healthcare professionals on switching in real-life practice settings. Considerations include how healthcare professionals might meaningfully address patient concerns about outcomes to improve patient satisfaction. A search of the MEDLINE database was used to identify publications on switching to and among adalimumab biosimilars in an ex-US setting, with no restriction on publication language and using a time frame of 1 January 2017 through 12 December …

Integrative Analysis Reveals The Prognostic Effects Of Epigenetic Regulators In Bladder Cancer, Venugopalareddy Mekala, Yupei Lin, Xiang Wang, Naail Chowdhury, Jianrong Li, Chao Cheng

Faculty, Staff and Students Publications

Background: Epigenetic regulatory genes (epiRG) are pivotal in the epigenetic regulation of the human genome, primarily through DNA and histone modifications. These genes are frequently mutated in human cancers, particularly bladder cancer (BC). However, the functional impact of epiRG mutations on patient outcomes remains poorly understood.

Methods: In this study, we developed gene signatures for the most frequent genomic aberrations of epiRG using The Cancer Genome Atlas Bladder Carcinoma (TCGA-BLCA) dataset and validated these signatures with independent tumor expression profiles for prognostic relevance. Furthermore, we evaluated the role of these signature scores in the immune system within the tumor microenvironment …

Pediatric Cancer Predisposition And Surveillance Update: Summary Perspective And Future Directions, Garrett M Brodeur, Lisa R Diller, Kim E Nichols, Sharon E Plon, Christopher C Porter, David Malkin

Faculty, Staff and Students Publications

An increasing number of studies suggest that a significant proportion of children with cancer harbor an underlying predisposition to malignancy, and it is likely that this proportion will only increase. Targeted surveillance for these individuals would likely improve outcomes. Historically, however, for most predisposition syndromes, there were no standardized surveillance protocols for early detection of cancer in predisposed individuals. Therefore, the Pediatric Cancer Working Group of the American Association for Cancer Research convened a workshop in 2016 to develop consensus surveillance recommendations (published in 2017) for children and adolescents with the most common cancer predisposition syndromes. These recommendations provided a …

Functional Regulation Of Macrophages By Ces1d-Mediated Lipid Signaling In Immunometabolism, Long J Shao, Fathima Elizondo, Feng Gao, Rabie Habib, Xin Li, Katherine Pham, Jazmin Ysaguirre, Maryam Elizondo, Shirindokht Shirazi, Kristin L Eckel-Mahan, Sean Hartig, Huaizhu Wu, Kai Sun

Faculty, Staff and Student Publications

Objective: Macrophage accumulation in metabolically active tissues during obesity is common in both animals and humans, but the lipid signaling mechanisms that trigger macrophage inflammation remain unclear. This study investigates the role of Ces1d, an unconventional lipase, in regulating macrophage inflammation under nutritional stress.

Methods: A myeloid-specific Ces1d knockout (LysM-Cre-Ces1d floxed/floxed, KO) mouse model was used for the studies. For in vitro tests, bone marrow-derived macrophages (BMDMs) from control (Ces1d floxed/floxed, WT) and KO mice were assessed for migration, polarization, and activation. For in vivo experiments, WT and KO mice were induced to obesity via a high-fat diet (HFD) and …

Coordinated Regulation By Lncrnas Results In Tight Lncrna-Target Couplings, Hua-Sheng Chiu, Sonal Somvanshi, Chung-Te Chang, Eric James De Bony De Lavergne, Zhaowen Wei, Chih-Hung Hsieh, Wim Trypsteen, Kathleen A Scorsone, Ektaben Patel, Tien T Tang, David B Flint, Mohammad Javad Najaf Panah, Hyunjae Ryan Kim, Purva Rathi, Yan-Hwa Wu Lee, Sarah E Woodfield, Sanjeev A Vasudevan, Andras Attila Heczey, M Waleed Gaber, Gabriel O Sawakuchi, Ting-Wen Chen, Pieter Mestdagh, Xuerui Yang, Pavel Sumazin

Faculty, Staff and Student Publications

The determination of long non-coding RNA (lncRNA) function is a major challenge in RNA biology with applications to basic, translational, and medical research. We developed BigHorn to computationally infer lncRNA-DNA interactions that mediate transcription and chromatin-remodeling factor activity. Its accurate inference enabled the identification of lncRNAs that coordinately regulate both the transcriptional and post-transcriptional processing of their targets. These lncRNAs may act as molecular chaperones, regulating the stability and translation of mRNAs they helped transcribe, leading to tightly coupled expression profiles. Our analysis suggests that lncRNAs regulate cancer genes across tumor contexts, thus propagating the effects of non-coding alterations to …

Clonal Evolution Of Hematopoietic Stem Cells After Autologous Stem Cell Transplantation, Hidetaka Uryu, Koichi Saeki, Hiroshi Haeno, Chiraag Deepak Kapadia, Ken Furudate, Jyoti Nangalia, Michael Spencer Chapman, Linda Zhang, Jennifer Padilla, Li Zhao, Joanne I Hsu, Chong Zhao, Shujuan Chen, Tomoyuki Tanaka, Zongrui Li, Satoko Ogata, Sarah Hanache, Hui Yang, Courtney Dinardo, Naval Daver, Naveen Pemmaraju, Nitin Jain, Farhad Ravandi, Jianhua Zhang, Xingzhi Song, Erika Thompson, Hongli Tang, Latasha Little, Curtis Gumbs, Robert Z Orlowski, Muzaffar Qazilbash, Kapil Bhalla, Simona Colla, Hagop Kantarjian, Rashmi Kanagal-Shamanna, Carlos Bueso-Ramos, Daisuke Nakada, Gheath Al-Atrash, Jeffery Molldrem, P Andrew Futreal, Elizabeth Shpall, Margaret Goodell, Guillermo Garcia-Manero, Koichi Takahashi

Faculty, Staff and Student Publications

The impact of exogenous stressors, such as cancer chemotherapies, on the genomic integrity and clonal dynamics of normal hematopoiesis is not well defined. We conducted whole-genome sequencing on 1,276 single-cell-derived hematopoietic stem and progenitor cell (HSPC) colonies from ten patients with multiple myeloma treated with chemotherapies and six normal donors. Melphalan treatment significantly increased the mutational burden, producing a distinctive mutation signature, whereas other chemotherapeutic agents had minimal effects. Consequently, the clonal diversity and architecture of post-treatment HSPCs resemble those observed in normal elderly individuals, particularly through the progression of oligoclonal hematopoiesis, thereby suggesting that chemotherapy accelerates clonal aging. Integrated …

The Long-Term Effects Of Chemotherapy On Normal Blood Cells, Emily Mitchell, My H Pham, Anna Clay, Rashesh Sanghvi, Nicholas Williams, Sandra Pietsch, Joanne I Hsu, Nina Friesgaard Øbro, Hyunchul Jung, Aditi Vedi, Sarah Moody, Jingwei Wang, Daniel Leonganmornlert, Michael Spencer Chapman, Ellie Dunstone, Anna Santarsieri, Alex Cagan, Heather E Machado, E Joanna Baxter, George Follows, Daniel J Hodson, Ultan Mcdermott, Gary J Doherty, Inigo Martincorena, Laura Humphreys, Krishnaa Mahbubani, Kourosh Saeb Parsy, Koichi Takahashi, Margaret A Goodell, David Kent, Elisa Laurenti, Peter J Campbell, Raheleh Rahbari, Jyoti Nangalia, Michael R Stratton

Faculty, Staff and Student Publications

Several chemotherapeutic agents act by increasing DNA damage in cancer cells, triggering cell death. However, there is limited understanding of the extent and long-term consequences of collateral DNA damage in normal tissues. To investigate the impact of chemotherapy on mutation burdens and the cell population structure of normal tissue, we sequenced blood cell genomes from 23 individuals aged 3-80 years who were treated with a range of chemotherapy regimens. Substantial additional somatic mutation loads with characteristic mutational signatures were imposed by some chemotherapeutic agents, but the effects were dependent on the drug and blood cell types. Chemotherapy induced premature changes …

Crispr-Cas9 Mediated Proteinase 3 Autoantigen Deletion As A Treatment Strategy For Anti-Neutrophil Cytoplasmic Autoantibody-Associated Vasculitis, Uwe Jerke, Claudia Eulenberg-Gustavus, Dimitrios Laurin Wagner, Adrian Schreiber, Ralph Kettritz

Faculty, Staff and Students Publications

Introduction: Proteinase 3 (PR3) is a major autoantigen in patients with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV). Here, we performed a proof-of-principle study using ex vivo CRISPR-Cas9 guided gene editing to eliminate the PR3 autoantigen as an alternative to suppressing the autoimmune response to PR3.

Methods: A ribonucleoprotein (RNP) complex of Cas9 protein and a PR3-specific single guide-RNA was transfected into human CD34+ hematopoietic stem and progenitor cells (HSPC) by electroporation. Effects on PR3 protein abundance, neutrophil differentiation, and ANCA-dependent and -independent neutrophil responses were assessed.

Results: Gene editing introduced a frame shift in exon 2 of PRTN3. Consequently, PR3 …

Bayesian-Weighted Mendelian Randomization Reveals Sleep Apnea Syndrome Mediates The Bmi-Chronic Pain Link, Wenbao Shi, Jing Luo, Liu Yang, Dalin Chen, Yongqin Zhang, Jun Peng, Kurt Ruetzler, Manyun Sun, Hua Jin

Faculty, Staff and Student Publications

Background: Body mass index (BMI) has a complex association with a variety of chronic pain conditions, which may be influenced by sleep apnea syndrome (SAS). However, the role of SAS within the causal pathway linking BMI to chronic pain remains unconfirmed. This study explored whether and to what extent SAS serves as a mediator between BMI and chronic pain using Bayesian-weighted Mendelian randomization (BWMR) techniques.

Methods: This study utilized genome-wide association study (GWAS) summary data for BMI (N=461,460) and SAS (N=476,853; based on ICD-10 code diagnostic registries) from the IEU OpenGWAS database, along with chronic pain GWAS data (ICD-10 code-based …

Phase Ii Basket Trial Of Dual Anti-Ctla-4 And Anti-Pd-1 Blockade In Rare Tumors (Dart) Swog S1609: Pancreatic Neuroendocrine Neoplasm (Pnen) Cohort, Sandip Pravin Patel, Jillian Fisher, Young Kwang Chae, Luisa Solis Soto, Anup Kasi, Bhavana Konda, Mark Walshauser, Edwin Parra, Jiexin Zhang, Caroline Duault, Edgar Gonzalez-Kozlova, Ganiraju Manyam, Jianhua Zhang, Hong Chen, Dzifa Yawa Duose, Caddie Laberiano Fernandez, Raja Luthra, Gheath Al-Atrash, Seunghee Kim-Schulze, Holden T Maecker, Ignacio I Wistuba, Sacha Gnjatic, J Jack Lee, Jianjun Zhang, Christine M Magner, Helen X Chen, Elad Sharon, Megan Othus, Christopher W Ryan, Charles Blanke, Cara L Haymaker, Razelle Kurzrock

Faculty, Staff and Student Publications

Purpose: SWOG S1609 Dual Anti-CTLA-4 and anti-PD-1 blockade in Rare Tumors (DART) studied the efficacy of ipilimumab combined with nivolumab across multiple rare tumor types. We report the results of the pancreatic neuroendocrine neoplasm (PNEN) cohort.

Experimental design: Treatment consisted of ipilimumab 1 mg/kg intravenously every 6 weeks with nivolumab 240 mg intravenously every 2 weeks. The primary endpoint was overall response rate (ORR) (Response Evaluation Criteria In Solid TumorsRECIST V.1.1). Secondary endpoints include progression-free survival (PFS), overall survival (OS), and toxicity. Clinical benefit rate (includes ORR plus stable disease (SD)>6 months was examined. Correlative …

Incidental Findings In Female Pelvis Mri Performed For Gynaecological Malignancies, Silvia Bottazzi, Roberta V Ninkova, Luca Russo, Andrea Ponsiglione, Benedetta Gui, Daniela Demundo, Massimo Imbriaco, Aradhana M Venkatesan, Evis Sala, Stephanie Nougaret, Lucia Manganaro, Stefania Rizzo

Faculty, Staff and Student Publications

Incidental findings on female pelvic MRI present diagnostic challenges and may have significant clinical implications. Defined as abnormalities unrelated to the primary imaging indication, these findings have become increasingly prevalent with the expanded use of MRI in gynaecological practice. Standard gynaecological MRI protocols, incorporating T1- and T2-weighted sequences, diffusion-weighted imaging, and contrast-enhanced sequences, facilitate the characterisation of numerous extra-gynaecological abnormalities, ranging from benign to critical lesions. This review proposes a compartment-based approach for identifying extra-gynaecological findings, discussing their imaging characteristics and differential diagnoses. This approach may help radiologists systematically assess incidental findings, potentially improving the recognition of clinically relevant abnormalities …

Letter Of Concern From The Association Of Academic Chairs Of Emergency Medicine Regarding Acgme Proposed Changes, Richard J Hamilton, Lance B Becker, Richard E Wolfe, D Adam Algren, Thomas Arnold, Michael Baumann, Ross P Berkeley, Terrell S Caffery, Chad M Cannon, Theodore J Corbin, Michael E Chansky, Harinder S Dhindsa, Charles L Emerman, David A Farcy, Chris Fox, Michael A Gibbs, Christopher S Goode, Steven Andy Godwin, Dietrich Jehle, David Johnson, Samuel M Keim, Babak Khazaeni, Barry J Knapp, Clint Hawthorne, John D Hoyle, Michael Christopher Kurz, Evan Leibner, Robert Mcnamara, Robert F Mccormack, Edward A Michelson, Chadwick Miller, Ashley Norse, Andrew Nugent, Brian J O'Neil, David T Overton, Edward A Panacek, William F Paolo, Denis R Pauzé, Amanda L Perez, Ralph J Riviello, Scott W Rodi, Peter S Pang, Juan A Gonzalez Sanchez, David Seaberg, Adam Schwartz, Stephen A Shiver, David P Sklar, Ben C Smith, Jeffrey R Stowell, Marc D Squillante, J Jeremy Thomas, Terry Vanden Hoek, Gregory A Volturo, E Lea Walters, Thomas E Wyatt, Donald M Yealy

Faculty, Staff and Student Publications

This letter, signed by over 50 academic chairs of emergency medicine, urges the ACGME to reconsider a proposed mandate requiring all emergency medicine residency programs to adopt a four-year training model. The authors argue that current three-year programs are supported by data demonstrating equivalent educational and clinical outcomes compared to four-year formats. They criticize the flawed survey methodology underpinning the proposal, note the loss of milestone-based training flexibility, and highlight the lack of added scholarly or clinical value in the fourth year. The letter also outlines negative consequences for fellowship participation, workforce development, trainee debt, and diversity. The signatories advocate …

Mis-Splicing-Derived Neoantigens And Cognate Tcrs In Splicing Factor Mutant Leukemias, Won Jun Kim, Edie I Crosse, Emma De Neef, Inaki Etxeberria, Erich Y Sabio, Eric Wang, Jan Philipp Bewersdorf, Kuan-Ting Lin, Sydney X Lu, Andrea Belleville, Nina Fox, Cynthia Castro, Pu Zhang, Takeshi Fujino, Jennifer Lewis, Jahan Rahman, Beatrice Zhang, Jacob H Winick, Alexander M Lewis, Robert F Stanley, Susan Dewolf, Brigita Meškauskaitė Urben, Meril Takizawa, Tobias Krause, Henrik Molina, Ronan Chaligne, Priya Koppikar, Jeffrey Molldrem, Mathieu Gigoux, Taha Merghoub, Anthony Daniyan, Smita S Chandran, Benjamin D Greenbaum, Christopher A Klebanoff, Robert K Bradley, Omar Abdel-Wahab

Faculty, Staff and Student Publications

Mutations in RNA splicing factors are prevalent across cancers and generate recurrently mis-spliced mRNA isoforms. Here we identified a series of bona fide neoantigens translated from highly stereotyped splicing alterations promoted by neomorphic, leukemia-associated somatic splicing machinery mutations. We utilized feature-barcoded peptide-MHC dextramers to isolate neoantigen-reactive T cell receptors (TCRs) from healthy donors, patients with active myeloid malignancy, and following curative allogeneic stem cell transplant. Neoantigen-reactive CD8+ T cells were present in the blood of patients with active cancer and had a distinct phenotype from virus-reactive T cells with evidence of impaired cytotoxic function. T cells engineered with TCRs recognizing …

Encorafenib, Cetuximab, And Mfolfox6 In Braf-Mutated Colorectal Cancer, Elena Elez, Takayuki Yoshino, Lin Shen, Sara Lonardi, Eric Van Cutsem, Cathy Eng, Tae Won Kim, Harpreet Singh Wasan, Jayesh Desai, Fortunato Ciardiello, Rona Yaeger, Timothy S Maughan, Van K Morris, Christina Wu, Tiziana Usari, Robert Laliberte, Samuel S Dychter, Xiaosong Zhang, Josep Tabernero, Scott Kopetz, Breakwater Trial Investigators

Faculty, Staff and Student Publications

Background: First-line treatment with encorafenib plus cetuximab (EC) with or without chemotherapy (oxaliplatin, leucovorin, and fluorouracil [mFOLFOX6]) for BRAF V600E-mutated metastatic colorectal cancer, an aggressive subtype with a poor prognosis, was compared with standard care (chemotherapy with or without bevacizumab) in an open-label, phase 3 trial, which showed significance regarding one of the two primary end points, objective response according to blinded independent central review (odds ratio for EC+mFOLFOX6 vs. standard care, 2.44; one-sided P< 0.001). This result led to accelerated Food and Drug Administration approval of this investigational combination therapy for BRAF V600E-mutated metastatic colorectal cancer, including as first-line therapy. Data on progression-free survival (the second primary end point) and an updated interim analysis of overall …

A Conditional Point Cloud Diffusion Model For Deformable Liver Motion Tracking Via A Single Arbitrarily-Angled X-Ray Projection, Jiacheng Xie, Hua-Chieh Shao, Yunxiang Li, Shunyu Yan, Chenyang Shen, Jing Wang, You Zhang

Faculty, Staff and Student Publications

Objective. Deformable liver motion tracking using a single x-ray projection enables real-time motion monitoring and treatment intervention. We introduce a conditional point cloud diffusion (PCD) model-based framework for accurate and robust liver motion tracking from arbitrarily angled single x-ray projections. Approach. We propose a conditional PCD model for liver motion tracking (PCD-Liver), which estimates volumetric liver motion by solving deformable vector fields (DVFs) of a prior liver surface point cloud, based on a single x-ray image. It is a patient-specific model of two main components: a rigid alignment model to estimate the liver’s overall shifts, and a conditional PCD …

The Carsr Two-Component System Directly Controls Radd Expression As A Global Regulator That Senses Bacterial Coaggregation In Fusobacterium Nucleatum, Bibek G C, Chenggang Wu

Faculty, Staff and Student Publications

Two-component systems (TCS) enable bacteria to sense and respond to environmental signals, facilitating rapid adaptation. Fusobacterium nucleatum, a key oral pathobiont, employs the CarSR TCS to modulate coaggregation with various gram-positive partners by regulating the expression of radD, encoding a surface adhesion protein, as revealed by RNA-Seq analysis. However, the direct regulation of the radD-containing operon (radABCD) by the response regulator CarR, the broader CarR regulon, and the signals sensed by this system remain unclear. In this study, chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) identified approximately 161 CarR-enriched loci across the genome and …

Intratumoral Neutrophil-To-Lymphocyte Ratio Is Mirrored By Circulating Neutrophil-To-Lymphocyte Ratio In Non-Small Cell Lung Cancer, Kyle G Mitchell, Younghee Lee, Nathaniel Deboever, Marcelo V Negrao, Hai T Tran, Edwin Parra, Lauren Byers, Alexandre Reuben, Lorenzo Federico, Chantale Bernatchez, Jing Wang, Mara B Antonoff, Ara A Vaporciyan, Stephen G Swisher, Tina Cascone, Ignacio I Wistuba, John V Heymach, Don L Gibbons, Jianjun Zhang, Daniel J Mcgrail, Boris Sepesi, Cara L Haymaker

Faculty, Staff and Student Publications

Tumor-initiated emergency granulopoiesis results in expansion of the circulating neutrophil compartment and neutrophil recruitment into the tumor microenvironment (TME), which may in turn promote tumor progression. Although an elevated circulating neutrophil-to-lymphocyte ratio (cNLR) has repeatedly been demonstrated to be an adverse prognostic factor in patients with non-small cell lung cancer (NSCLC), whether this neutrophil expansion in circulation reflects a similar relative neutrophil abundance in the TME remains unclear. We sought to characterize the relationships between cNLR and the intratumoral neutrophil-to-lymphocyte ratio (tNLR), between tNLR and proteogenomic and immune features of NSCLC tumors, and between tNLR and prognosis.We analyzed tNLR (transcriptomic …

Differences In Arterial Events In Vascular Ehlers-Danlos, Loeys-Dietz, And Marfan Syndrome, Ernesto Calderon-Martinez, Walter V Velasco, Dongchuan Guo, Ellen H Hostetler, Zhang Xun, Sara Stephens, Sherene Shalhub, Julie De Backer, Maral Ouzounian, Scott A Lemaire, Olivier Milleron, Nadine Hanna, Pauline Arnaud, Maria Tchitchinadze, Siddharth K Prakash, Mark Lindsay, Julien Marcadier, Richmond Jeremy, Shaine A Morris, Anji T Yetman, Catherine Boileau, Alan C Braverman, Guillaume Jondeau, Dianna M Milewicz

Faculty, Staff and Students Publications

Background: Heritable thoracic aortic disease is due to altered genes that confer a highly penetrant risk for thoracic aortic aneurysm and dissection, and a subset of these genes also cause aneurysms and dissections of peripheral arteries beyond the aorta. Arterial aneurysms, dissections, and ruptures are associated with pathogenic variants (PVs) in COL3A1, which is responsible for vascular Ehlers-Danlos syndrome, but arterial events are rare in Marfan syndrome due to PVs in FBN1, and poorly characterized in Loeys-Dietz syndrome due to PVs in the transforming growth factor (TGF)-β pathway genes.

Objectives: This study sought to define the relative risk of arterial …

Cholesterol Metabolism Regulated By Camkk2-Creb Signaling Promotes Castration-Resistant Prostate Cancer, Chenchu Lin, Thomas L Pulliam, Jenny J Han, Jiaqian Xu, Carlos Vera Recio, Sandi R Wilkenfeld, Yan Shi, Manoj Kushwaha, Sarah Bench, Eduardo Ruiz, Sanjanaa Senthilkumar, Jayasurya Dileep, Peter D A Shepherd, Nora M Navone, Albert R Klekers, Elizabeth M Whitley, Michael M Ittmann, Livia S Eberlin, Wenyi Wang, Daniel E Frigo

Faculty, Staff and Student Publications

Castration-resistant prostate cancer (CRPC) remains an incurable disease in need of improved treatments. CAMKK2 is an emerging therapeutic target whose oncogenic effects in prostate cancer have, to date, been largely attributed to its activation of AMP-activated protein kinase (AMPK). Here, we demonstrate that CAMKK2 promotes prostate cancer growth through an alternative downstream pathway involving CAMKI and CREB. Unbiased transcriptomics identify CREB-mediated transcription as a CAMKK2-regulated process, findings that we validate using diverse molecular, genetic, and pharmacological approaches in vitro and in vivo. CAMKK2 promotes CREB phosphorylation/activation through CAMKIα independently of AMPK, CAMKIV, or other CAMKI isoforms. Functionally, the CREB family …

Protocol For Assessing Mobilization Of Peritoneal B Cells To The Pre-Metastatic Omentum In An Orthotopic Mouse Model Of Ovarian Cancer, Wonjae Lee, Hironari Akasaka, Honami Naora

Faculty, Staff and Student Publications

The omentum is a visceral adipose tissue that undergoes dynamic immunological changes prior to and following metastasis. Here, we present a protocol for assessing the mobilization of peritoneal B cells to the pre-metastatic omentum in a mouse ovarian cancer model. We describe steps for isolation and adoptive transfer of peritoneal donor B cells and their detection in the omentum of recipient mice. This protocol could be utilized to study the mobilization of peritoneal B cells to the omentum in other pathological contexts. For complete details on the use and execution of this protocol, please refer to Lee et al.

Protocol For Establishing Genetically Engineered Murine Lung Organoids Mimicking Cell Plasticity And Regeneration, Bongjun Kim, Jae-Il Park

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Lung endothelial cells (LuECs) play crucial roles in lung regeneration and support lung organoids (LOs). However, detailed guidelines for LO culture using LuECs have not been reported yet. Here, we present an efficient protocol for generating and culturing murine LOs that mimic regeneration. We describe steps for the isolation of LuECs and lung epithelial cells, followed by LO culture. We also describe how to establish genetically engineered LOs using viral transduction. For complete details on the use and execution of this protocol, please refer to Kim et al.

Characterization And Clinical Implications Of P53 Dysfunction In Patients With Myelodysplastic Syndromes, Matteo Zampini, Elena Riva, Luca Lanino, Elisabetta Sauta, Rita Antunes Dos Reis, Rosa Maria Andres Ejarque, Giulia Maggioni, Alberto Termanini, Alessandra Merlotti, Alessia Campagna, Lorenzo Dall'olio, Austin Kulasekararaj, Michela Calvi, Clara Di Vito, Arturo Bonometti, Daoud Rahal, Giorgio Croci, Emanuela Boveri, Umberto Gianelli, Maurilio Ponzoni, Rossella Caselli, Serena Albertazzi, Gabriele Todisco, Marta Ubezio, Laura Crisafulli, Alessandro Frigo, Enrico Lugli, Ettore Mosca, Pamela Acha, Serena Ghisletti, Francesco Nicassio, Armando Santoro, Maria Diez-Campelo, Francesc Solé, Lionel Ades, Uwe Platzbecker, Valeria Santini, Pierre Fenaux, Torsten Haferlach, David Sallman, Guillermo Garcia-Manero, Domenico Mavilio, Daniel Remondini, Gastone Castellani, Saverio D'Amico, Amer M Zeidan, Rami Komrokji, Shahram Kordasti, Francesca Ficara, Matteo Giovanni Della Porta, Calr Consortium

Faculty, Staff and Student Publications

Purpose: Tumor Protein 53 (p53) expressed from gene TP53 is a seminal tumor suppressor. We aimed to characterize mutational and nonmutational mechanisms of p53 dysfunction in myelodysplastic syndromes (MDS) and to investigate their clinical effect.

Patients and methods: We analyzed a cohort of 6,204 patients with MDS and subsets of patients with available information on RNA sequencing of tumor cells (n = 109), high-dimensional phenotype of immune cells (n = 77), and multiomics analysis (RNA sequencing and proteomics) on single cells (n = 15). An independent validation was performed on 914 patients.

Results: Biallelic TP53 inactivation was a powerful driver …

Usp37 Counteracts Hltf To Protect Damaged Replication Forks And Promote Survival Of Brca1-Deficient Cells And Parp Inhibitor Resistance, Mengfan Tang, Siting Li, Ziqiang Zhu, Chao Wang, Shuai Ding, Huimin Zhang, Min Huang, Sarah Keast, Zhen Chen, Ling Yin, Litong Nie, Dan Su, Xu Feng, Junjie Chen

Faculty, Staff and Student Publications

Poly(ADP-ribose) polymerase inhibitors (PARPi) have greatly improved survival of cancer patients harboring BRCA1 mutations. However, therapy resistance develops via either restoration of homologous recombination or replication fork stabilization. Therapeutic targets to overcome PARPi resistance are critically needed. We identified the deubiquitinase USP37 as a key determinant of PARPi toxicity in BRCA1-deficient cells via whole-genome CRISPR screens. USP37 ablation enhanced PARPi sensitivity in BRCA1-deficient cells and also overcame PARPi resistance due to 53BP1 loss. USP37 interacts with and deubiquitinates replication protein A (RPA) at stalled replication forks to limit excessive RPA accumulation, progressive RPA exhaustion, and the conversion of RPA-coated single-stranded …

Protocol To Analyze Trna And Rrna Processing Using Biotin-Labeled Probes, Sseu-Pei Hwang, Katherine Barondeau, Catherine Denicourt

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Mature tRNAs and rRNAs are derived from larger precursor transcripts through intricate endo- and exonuclease processing steps. Here, we present a protocol to analyze rRNA and tRNA processing using total RNA extracted from human cells. We describe steps for separating RNA by denaturing electrophoresis, followed by northern blotting. We detail procedures for detecting pre-rRNA intermediates or specific tRNA species using biotin-labeled probes imaged via fluorescence or chemiluminescence. This non-radioactive, high-sensitivity assay enables the investigation of rRNA and tRNA expression dynamics. For complete details on the use and execution of this protocol, please refer to Hwang et al.

Primary Mouse Choroidal Endothelial Cell Culture, Qiuhua Yang, Yongfeng Cai, Qian Ma, Yuqing Huo

Faculty, Staff and Students Publications

The study of choroidal endothelial cells is essential for understanding the pathological mechanisms underlying choroidal neovascularization and other vision-threatening disorders. Traditional methods for isolating and culturing primary endothelial cells often yield mixed populations or require specialized equipment, limiting their widespread use. Here, we present a straightforward protocol for isolating and culturing primary mouse choroidal endothelial cells. This protocol involves enzymatic digestion of choroidal tissue, magnetic-activated cell sorting (MACS) to enrich CD31+ endothelial cells, and optimized culture conditions to promote cell proliferation and maintain endothelial phenotype. The protocol is strategic, reproducible, and requires minimal specialized equipment, making it accessible for researchers …