Search

Background and purpose Based on reported success with staged treatment of distal tibial fractures, we designed a 2-stage protocol including external/internal locked plating. We retrospectively assessed the outcome of open distal tibial fractures treated according to this protocol. Patients and methods From March 2006 through July 2008, 16 patients who sustained open distal tibial fractures were treated by a two-stage protocol. The first stage consisted of low-profile, locked plates for temporary external fixation after debridement and anatomic reduction, followed by soft tissue reconstruction. The second stage consisted of locked plates for definitive internal fixation, using minimally invasive percutaneous osteosynthesis. All fractures were followed for median 2 (1-3) years. Results The reduction was classified as being good in 15 patients and fair in 1 patient. All fractures united at a median of 6 (6-12) months. At the latest follow-up, 7 patients had excellent and 9 had good Iowa ankle scores; ankle motion ranged from a median of 10 (5-20) degrees of dorsiflexion to 40 (20-60) degrees of plantar flexion. Interpretation We believe that the 2-stage external/internal locked plating technique is an effective procedure for treatment of open distal tibial fractures in patients who need a long period of external fixation. We achieved good reduction with immediate ankle-sparing stable fixation. Soft tissue reconstruction and subsequent definitive fixation led to union of all fractures with good function. [ABSTRACT FROM AUTHOR]

The health impacts of the diurnal temperature range (DTR), which may be affected by climate change, have received little attention. The objectives of this study were (1) to evaluate the association of DTR and cardiopulmonary outcomes, (2) to select the proper thresholds for a DTR warning system, and (3) to identify vulnerable groups. The weather and health records in Taiwan from 2000 to 2019, with a maximum DTR of 12.8 °C, were analyzed using generalized additive models. The health outcomes included cardiovascular (CVD) and respiratory disease (RD) categories and several sub-categories, such as ischemic heart disease, stroke, pneumonia, asthma, and chronic obstructive pulmonary disease. The results showed that the associations of DTR and cardiopulmonary outcomes were as significant as, and sometimes even stronger than, those of the daily maximum temperature and daily minimum apparent temperature in the warm and cold seasons, respectively. The significant association began at DTR of 6 °C, lower than previously reported. The identified DTR warning thresholds were 8.5 and 11 °C for the warm and cold seasons, respectively. DTR is statistically significantly associated with a 5-36% and a 9-20% increase in cardiopulmonary emergency and hospitalized cases in the warm season with a 1 °C increase above 8.5 °C, respectively. In the cold season, DTR is significantly associated with 7-41%, 4-30%, and 36-100% increases in cardiopulmonary emergency, hospitalized, and mortality with a 1 °C increase above 11 °C, respectively. People with hypertension, hyperglycemia, and hyperlipidemia had even higher risks. Vulnerable age and sex groups were identified if they had a lower DTR-health threshold than the general population, which can be integrated into a warning system. In conclusion, DTR may be increased on a local or city scale under climate change; a DTR warning system and vulnerable group identification may be warranted in most countries for health risk reduction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The kinome is a dynamic system of kinases regulating signaling networks in cells and dysfunction of protein kinases contributes to many diseases. Regulation of the protein expression of kinases alters cellular responses to environmental changes and perturbations. We configured a library of 672 proteotypic peptides to quantify >300 kinases in a single LC-MS experiment using ten micrograms protein from human tissues including biopsies. This enables absolute quantitation of kinase protein abundance at attomole-femtomole expression levels, requiring no kinase enrichment and less than ten micrograms of starting protein from flash-frozen and formalin fixed paraffin embedded tissues. Breast cancer biopsies, organoids, and cell lines were analyzed using the SureQuant method, demonstrating the heterogeneity of kinase protein expression across and within breast cancer clinical subtypes. Kinome quantitation was coupled with nanoscale phosphoproteomics, providing a feasible method for novel clinical diagnosis and understanding of patient kinome responses to treatment.

The pleiotropic roles of nSMase2-generated ceramide include regulation of intracellular ceramide signaling and exosome biogenesis. We investigated the effects of eliminating nSMase2 on early and advanced PDA, including its influence on the microenvironment. Employing the KPC mouse model of pancreatic cancer, we demonstrate that pancreatic epithelial nSMase2 ablation reduces neoplasia and promotes a PDA subtype switch from aggressive basal-like to classical. nSMase2 elimination prolongs survival of KPC mice, hinders vasculature development, and fosters a robust immune response. nSMase2 loss leads to recruitment of cytotoxic T cells, N1-like neutrophils, and abundant infiltration of anti-tumorigenic macrophages in the pancreatic preneoplastic microenvironment. Mechanistically, we demonstrate that nSMase2-expressing PDA cell small extracellular vesicles (sEVs) reduce survival of KPC mice; PDA cell sEVs generated independently of nSMase2 prolong survival of KPC mice and reprogram macrophages to a proinflammatory phenotype. Collectively, our study highlights previously unappreciated opposing roles for exosomes, based on biogenesis pathway, during PDA progression.

Here, we introduce 'TICIT', targeted integration by CRISPR-Cas9 and integrase technologies, which utilizes the site-specific DNA recombinase - phiC31 integrase - to insert large DNA fragments into CRISPR-Cas9 target loci. This technique, which relies on first knocking in a 39-basepair phiC31 landing site via CRISPR-Cas9, enables researchers to repeatedly perform site-specific transgenesis at the exact genomic location with high precision and efficiency. We applied this approach to devise a method for the instantaneous determination of a zebrafish's genotype simply by examining its color. When a zebrafish mutant line must be propagated as heterozygotes due to homozygous lethality, employing this method allows facile identification of a population of homozygous mutant embryos even before the mutant phenotypes manifest. Thus, it should facilitate various downstream applications, such as large-scale chemical screens. We demonstrated that TICIT could also create reporter fish driven by an endogenous promoter. Further, we identified a landing site in the tyrosinase gene that could support transgene expression in a broad spectrum of tissue and cell types. In sum, TICIT enables site-specific DNA integration without requiring complex donor DNA construction. It can yield consistent transgene expression, facilitate diverse applications in zebrafish, and may be applicable to cells in culture and other model organisms., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Numerous aspects of cellular signaling are regulated by the kinome-the network of over 500 protein kinases that guides and modulates information transfer throughout the cell. The key role played by both individual kinases and assemblies of kinases organized into functional subnetworks leads to kinome dysregulation driving many diseases, particularly cancer. In the case of pancreatic ductal adenocarcinoma (PDAC), a variety of kinases and associated signaling pathways have been identified for their key role in the establishment of disease as well as its progression. However, the identification of additional relevant therapeutic targets has been slow and is further confounded by interactions between the tumor and the surrounding tumor microenvironment. In this work, we attempt to link the state of the human kinome, or kinotype, with cell viability in treated, patient-derived PDAC tumor and cancer-associated fibroblast cell lines. We applied classification models to independent kinome perturbation and kinase inhibitor cell screen data, and found that the inferred kinotype of a cell has a significant and predictive relationship with cell viability. We further find that models are able to identify a set of kinases whose behavior in response to perturbation drive the majority of viability responses in these cell lines, including the understudied kinases CSNK2A1/3, CAMKK2, and PIP4K2C. We next utilized these models to predict the response of new, clinical kinase inhibitors that were not present in the initial dataset for model devlopment and conducted a validation screen that confirmed the accuracy of the models. These results suggest that characterizing the perturbed state of the human protein kinome provides significant opportunity for better understanding of signaling behavior and downstream cell phenotypes, as well as providing insight into the broader design of potential therapeutic strategies for PDAC., Competing Interests: Shawn M. Gomez is an Academic Editor for PeerJ., (©2024 Berginski et al.)

Background: Cholangiocarcinoma (CCA) features highly desmoplastic stroma that promotes structural and functional resistance to therapy. Lysyl oxidases (LOX, LOXL1-4) catalyze collagen cross-linking, thereby increasing stromal rigidity and facilitating therapeutic resistance. Here, we evaluate the role of lysyl oxidases in stromal desmoplasia and the effects of pan-lysyl oxidase (pan-LOX) inhibition in CCA., Methods: Resected CCA and normal liver specimens were analyzed from archival tissues. Spontaneous and orthotopic murine models of intrahepatic CCA (iCCA) were used to assess the impact of the pan-LOX inhibitor PXS-5505 in treatment and correlative studies. The functional role of pan-LOX inhibition was interrogated through in vivo and ex vivo assays., Results: All 5 lysyl oxidases are upregulated in CCA and reduced lysyl oxidase expression is correlated with an improved prognosis in resected patients with CCA. Spontaneous and orthotopic murine models of intrahepatic cholangiocarcinoma upregulate all 5 lysyl oxidase isoforms. Pan-LOX inhibition reversed mechanical compression of tumor vasculature, resulting in improved chemotherapeutic penetrance and cytotoxic efficacy. The combination of chemotherapy with pan-LOX inhibition increased damage-associated molecular pattern release, which was associated with improved antitumor T-cell responses. Pan-LOX inhibition downregulated macrophage invasive signatures in vitro, rendering tumor-associated macrophages more susceptible to chemotherapy. Mice bearing orthotopic and spontaneously occurring intrahepatic cholangiocarcinoma tumors exhibited delayed tumor growth and improved survival following a combination of pan-LOX inhibition with chemotherapy., Conclusions: CCA upregulates all 5 lysyl oxidase isoforms, and pan-LOX inhibition reverses tumor-induced mechanical forces associated with chemotherapy resistance to improve chemotherapeutic efficacy and reprogram antitumor immune responses. Thus, combination therapy with pan-LOX inhibition represents an innovative therapeutic strategy in CCA., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

CRISPR prime editing ( PE ) requires a Cas9 nickase-reverse transcriptase fusion protein (known as PE2) and a prime editing guide RNA ( pegRNA ), an extended version of a standard guide RNA ( gRNA ) that both specifies the intended target genomic sequence and encodes the desired genetic edit. Here, we show that sequence complementarity between the 5' and the 3' regions of a pegRNA can negatively impact its ability to complex with Cas9, thereby potentially reducing PE efficiency. We demonstrate this limitation can be overcome by a simple pegRNA refolding procedure, which improved ribonucleoprotein-mediated PE efficiencies in zebrafish embryos by up to nearly 25-fold. Further gains in PE efficiencies of as much as sixfold could also be achieved by introducing point mutations designed to disrupt internal interactions within the pegRNA. Our work defines simple strategies that can be implemented to improve the efficiency of PE., Competing Interests: WZ, JM, HL, CT, JY No competing interests declared, KP has a financial interest in SeQure Dx, Inc. KP's interests and relationships have been disclosed to Massachusetts General Hospital and Mass General Brigham in accordance with their conflict of interest policies, JJ has, or had during the course of this research, financial interests in several companies developing gene editing technology: Beam Therapeutics, Blink Therapeutics, Chroma Medicine, Editas Medicine, EpiLogic Therapeutics, Excelsior Genomics, Hera Biolabs, Monitor Biotechnologies, Nvelop Therapeutics (f/k/a ETx, Inc), Pairwise Plants, Poseida Therapeutics, SeQure Dx, Inc, and Verve Therapeutics. JKJ's interests were reviewed and are managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict of interest policies. JKJ is a co-inventor on various patents and patent applications that describe gene editing and epigenetic editing technologies, (© 2023, Zhang et al.)

Cancer-associated fibroblast (CAF) subpopulations in pancreatic ductal adenocarcinoma (PDAC) have been identified using single-cell RNA sequencing (scRNAseq) with divergent characteristics, but their clinical relevance remains unclear. We translate scRNAseq-derived CAF cell-subpopulation-specific marker genes to bulk RNAseq data, and develop a single- sample classifier, DeCAF, for the classification of clinically rest raining and perm issive CAF subtypes. We validate DeCAF in 19 independent bulk transcriptomic datasets across four tumor types (PDAC, mesothelioma, bladder and renal cell carcinoma). DeCAF subtypes have distinct histology features, immune landscapes, and are prognostic and predict response to therapy across cancer types. We demonstrate that DeCAF is clinically replicable and robust for the classification of CAF subtypes in patients for multiple tumor types, providing a better framework for the future development and translation of therapies against permissive CAF subtypes and preservation of restraining CAF subtypes., Significance: We introduce a replicable and robust classifier, DeCAF, that delineates the significance of the role of permissive and restraining CAF subtypes in cancer patients. DeCAF is clinically tractable, prognostic and predictive of treatment response in multiple cancer types and lays the translational groundwork for the preclinical and clinical development of CAF subtype specific therapies.

Background: To determine the effect of colchicine on cancer risk in patients with the immune-mediated inflammatory diseases (IMIDs)-related to colchicine use., Methods: This is a time-dependent propensity-matched general population study based on the National Health Insurance Research Database (NHIRD) of Taiwan. We identified the IMIDs patients (n = 111,644) newly diagnosed between 2000 and 2012 based on the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM)-274,712, 135, 136.1, 279.49, 518.3, 287.0, 696.0, 696.1, 696.8, 420, 429.4, 710.0, 710.1, 710.3, 710.4, 714.0, 720, 55.0, 55.1, 55.9, 556., Inclusion Criteria: aged ≧ 20 years, if a patient had at least these disease diagnosis requirements within 1 year of follow-up, and, these patients had at least two outpatient visits or an inpatient visit. After propensity-matched according to age, sex, comorbidities, medications and index date, the IMIDs patients enter into colchicine users (N = 16,026) and colchicine nonusers (N = 16,026). Furthermore, time-dependent Cox models were used to analyze cancer risk in propensity-matched colchicine users compared with the nonusers. The cumulative cancer incidence was analyzed using Cox proportional regression analysis. We calculated adjusted hazard ratios (aHRs) and their 95% confidence intervals (95% CIs) for cancer after adjusting for sex, age, comorbidities, and use of medicine including acetylcysteine, medication for smoking cessation such as nicotine replacement medicines (the nicotine patch) and pill medicines (varenicline), anti-inflammatory drugs and immunosuppressant drugs., Results: Comparing the colchicine nonusers, all cancer risk were mildly attenuated, the (aHR (95% CI)) of all cancer is (0.84 (0.55, 0.99)). Meanwhile, the colchicine users were associated with the lower incidence of the colorectal cancer, the (aHRs (95% CI)) is (0.22 (0.19, 0.89)). Those aged < 65 years and male/female having the colchicine users were associated with lower risk the colorectal cancer also. Moreover, the colchicine > 20 days use with the lower aHR for colorectal cancer., Conclusion: Colchicine was associated with the lower aHR of the all cancer and colorectal cancer formation in patients with the IMIDs., (© 2024. The Author(s).)

Alternatively spliced tissue factor (asTF) promotes the progression of pancreatic ductal adenocarcinoma (PDAC) by activating β1-integrins on PDAC cell surfaces. hRabMab1, a first-in-class humanized inhibitory anti-asTF antibody we recently developed, can suppress PDAC primary tumor growth as a single agent. Whether hRabMab1 has the potential to suppress metastases in PDAC is unknown. Following in vivo screening of three asTF-proficient human PDAC cell lines, we chose to make use of KRAS G12V-mutant human PDAC cell line PaCa-44, which yields aggressive primary orthotopic tumors with spontaneous spread to PDAC-relevant anatomical sites, along with concomitant severe leukocytosis. The experimental design featured orthotopic tumors formed by luciferase labeled PaCa-44 cells; administration of hRabMab1 alone or in combination with gemcitabine/paclitaxel (gem/PTX); and the assessment of the treatment outcomes on the primary tumor tissue as well as systemic spread. When administered alone, hRabMab1 exhibited poor penetration of tumor tissue; however, hRabMab1 was abundant in tumor tissue when co-administered with gem/PTX, which resulted in a significant decrease in tumor cell proliferation; leukocyte infiltration; and neovascularization. Gem/PTX alone reduced primary tumor volume, but not metastatic spread; only the combination of hRabMab1 and gem/PTX significantly reduced metastatic spread. RNA-seq analysis of primary tumors showed that the addition of hRabMab1 to gem/PTX enhanced the downregulation of tubulin binding and microtubule motor activity. In the liver, hRabMab1 reduced liver metastasis as a single agent. Only the combination of hRabMab1 and gem/PTX eliminated tumor cell-induced leukocytosis. We here demonstrate for the first time that hRabMab1 may help suppress metastasis in PDAC. hRabMab1's ability to improve the efficacy of chemotherapy is significant and warrants further investigation.

The purpose of this paper is to assess the effect of glucagon-like peptide-1 receptor agonists (GLP-1RAs) on stroke or heart disease in patients having chronic respiratory disease and diabetes (CD) with underlying diseases related to COVID-19. From 1998 to 2019, we adjusted competing risk by assessing the effect of GLP-1RAs on stroke or heart disease in a CD cohort after propensity matching based on the Taiwan National Health Insurance Research Database. We also used the time-dependent method to examine the results. GLP-1 RA and non-GLP-1 RA user groups included 15,801 patients (53% women and 46% men with a mean age of 52.6 ± 12.8 years). The time between the diagnoses of DM and the initial use of the GLP-1 RA among the stroke subcohort (<2000 days) was shorter than that of the heart disease subcohort (>2000 days) (all p -values < 0.05). The overall risks of stroke, ischemic, and hemorrhagic stroke were significantly lower in GLP-1 RA users than nonusers. The adjusted subhazard ratio (aSHR) was 0.76 [95% CI 0.65-0.90], 0.77 [95% CI 0.64-0.92], and 0.69 [95% CI 0.54-0.88] ( p < 0.05 for all). Furthermore, a ≥351-day use had a significantly lower stroke risk than GLP-1 RA nonusers (aSHR 0.35 [95% CI 0.26-0.49]). The time-dependent method revealed the same result, such as lower stroke, and ischemic or hemorrhagic stroke risk. In contrast, the cardiac arrhythmia incidence was higher in GLP-1 RA users with an aSHR of 1.36 [95% CI 1.16-1.59]. However, this risk disappeared after the ≥351-day use with 1.21 (0.98, 1.68) aSHR. Longer GLP-1 RA use was associated with a decreased risk of ischemic or hemorrhagic stroke and the risk of cardiac arrhythmia disappears in a CD cohort. Both a shorter lag time use of the GLP-1 RA and a longer time use of GLP-1 RA were associated with a decreased risk of ischemic or hemorrhagic stroke in the CD cohort. The GLP-1 RA use in the early stage and optimal time use in the CD cohort may avoid the stroke risk.

Protein kinase activity forms the backbone of cellular information transfer, acting both individually and as part of a broader network, the kinome. Their central role in signaling leads to kinome dysfunction being a common driver of disease, and in particular cancer, where numerous kinases have been identified as having a causal or modulating role in tumor development and progression. As a result, the development of therapies targeting kinases has rapidly grown, with over 70 kinase inhibitors approved for use in the clinic and over double this number currently in clinical trials. Understanding the relationship between kinase inhibitor treatment and their effects on downstream cellular phenotype is thus of clear importance for understanding treatment mechanisms and streamlining compound screening in therapy development. In this work, we combine two large-scale kinome profiling data sets and use them to link inhibitor-kinome interactions with cell line treatment responses (AUC/IC 50 ). We then built computational models on this data set that achieve a high degree of prediction accuracy (R 2 of 0.7 and RMSE of 0.9) and were able to identify a set of well-characterized and understudied kinases that significantly affect cell responses. We further validated these models experimentally by testing predicted effects in breast cancer cell lines and extended the model scope by performing additional validation in patient-derived pancreatic cancer cell lines. Overall, these results demonstrate that broad quantification of kinome inhibition state is highly predictive of downstream cellular phenotypes., Competing Interests: Shawn M. Gomez is an Academic Editor for PeerJ., (©2023 Joisa et al.)

Significant heat-related casualties underlie the urgency of establishing a heat-health warning system (HHWS). This paper presents an evidence-based pilot HHWS developed for Taipei City, Taiwan, through a co-design process engaging stakeholders. In the co-design process, policy concerns related to biometeorology, epidemiology and public health, and risk communication aspects were identified, with knowledge gaps being filled by subsequent findings. The biometeorological results revealed that Taipei residents were exposed to wet-bulb globe temperature (WBGT) levels of health concern for at least 100 days in 2016. The hot spots and periods identified using WBGT would be missed out if using temperature, underlining the importance of adopting an appropriate heat indicator. Significant increases in heat-related emergency were found in Taipei at WBGT exceeding 36°C with reference-adjusted risk ratio (RaRR) of 2.42, taking 30°C as the reference; and residents aged 0-14 had the highest risk enhancement (RaRR = 7.70). As for risk communication, occurring frequency was evaluated to avoid too frequent warnings, which would numb the public and exhaust resources. After integrating knowledge and reconciling the different preferences and perspectives, the pilot HHWS was co-implemented in 2018 by the science team and Taipei City officials; accompanying responsive measures were formulated for execution by ten city government departments/offices. The results of this pilot served as a useful reference for establishing a nationwide heat-alert app in 2021/2022. The lessons learnt during the interactive co-design processes provide valuable insights for establishing HHWSs worldwide., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lung et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Motivation: Single-cell RNA-sequencing (scRNA-seq) has enabled the molecular profiling of thousands to millions of cells simultaneously in biologically heterogenous samples. Currently, the common practice in scRNA-seq is to determine cell type labels through unsupervised clustering and the examination of cluster-specific genes. However, even small differences in analysis and parameter choosing can greatly alter clustering results and thus impose great influence on which cell types are identified. Existing methods largely focus on determining the optimal number of robust clusters, which can be problematic for identifying cells of extremely low abundance due to their subtle contributions toward overall patterns of gene expression., Results: Here, we present a carefully designed framework, SCISSORS, which accurately profiles subclusters within broad cluster(s) for the identification of rare cell types in scRNA-seq data. SCISSORS employs silhouette scoring for the estimation of heterogeneity of clusters and reveals rare cells in heterogenous clusters by a multi-step semi-supervised reclustering process. Additionally, SCISSORS provides a method for the identification of marker genes of high specificity to the cell type. SCISSORS is wrapped around the popular Seurat R package and can be easily integrated into existing Seurat pipelines., Availability and Implementation: SCISSORS, including source code and vignettes, are freely available at https://github.com/jr-leary7/SCISSORS., (© The Author(s) 2023. Published by Oxford University Press.)

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease for which potent therapies have limited efficacy. Several studies have described the transcriptomic landscape of PDAC tumors to provide insight into potentially actionable gene expression signatures to improve patient outcomes. Despite centralization efforts from multiple organizations and increased transparency requirements from funding agencies and publishers, analysis of public PDAC data remains difficult. Bioinformatic pitfalls litter public transcriptomic data, such as subtle inclusion of low-purity and non-adenocarcinoma cases. These pitfalls can introduce non-specificity to gene signatures without appropriate data curation, which can negatively impact findings. To reduce barriers to analysis, we have created pdacR ( http://pdacR.bmi.stonybrook.edu , github.com/rmoffitt/pdacR), an open-source software package and web-tool with annotated datasets from landmark studies and an interface for user-friendly analysis in clustering, differential expression, survival, and dimensionality reduction. Using this tool, we present a multi-dataset analysis of PDAC transcriptomics that confirms the basal-like/classical model over alternatives., (© 2023. The Author(s).)

Little is understood about the embryonic development of sociality. We screened 1120 known drugs and found that embryonic inhibition of topoisomerase IIα (Top2a) resulted in lasting social deficits in zebrafish. In mice, prenatal Top2 inhibition caused defects in social interaction and communication, which are behaviors that relate to core symptoms of autism. Mutation of Top2a in zebrafish caused down-regulation of a set of genes highly enriched for genes associated with autism in humans. Both the Top2a-regulated and autism-associated gene sets have binding sites for polycomb repressive complex 2 (PRC2), a regulatory complex responsible for H3K27 trimethylation (H3K27me3). Moreover, both gene sets are highly enriched for H3K27me3. Inhibition of the PRC2 component Ezh2 rescued social deficits caused by Top2 inhibition. Therefore, Top2a is a key component of an evolutionarily conserved pathway that promotes the development of social behavior through PRC2 and H3K27me3.

Plasma cell responses are associated with anti-tumor immunity and favorable response to immunotherapy. B cells can amplify anti-tumor immune responses through antibody production; yet B cells in patients and tumor-bearing mice often fail to support this effector function. We identify dysregulated transcriptional program in B cells that disrupts differentiation of naive B cells into anti-tumor plasma cells. The signaling network contributing to this dysfunction is driven by interleukin (IL) 35 stimulation of a STAT3-PAX5 complex that upregulates the transcriptional regulator BCL6 in naive B cells. Transient inhibition of BCL6 in tumor-educated naive B cells is sufficient to reverse the dysfunction in B cell differentiation, stimulating the intra-tumoral accumulation of plasma cells and effector T cells and rendering pancreatic tumors sensitive to anti-programmed cell death protein 1 (PD-1) blockade. Our findings argue that B cell effector dysfunction in cancer can be due to an active systemic suppression program that can be targeted to synergize with T cell-directed immunotherapy., Competing Interests: Declaration of interests B.G.V. declares equity in GeneCentric Therapeutics., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: This study investigated the effect of colchicine use on the risks of heart disease (HD), pericarditis, endocarditis, myocarditis, cardiomyopathy, cardiac arrhythmia, and cardiac failure in patients having interstitial lung disease (ILD) with virus infection (ILD cohort)., Methods: We retrospectively enrolled ILD cohort between 2000 and 2013 from the Longitudinal Health Insurance Database and divided them into colchicine users ( n = 12,253) and colchicine non-users ( n = 12,253) through propensity score matching. The event of interest was the diagnosis of HD. The incidence of HD was analyzed using multivariate Cox proportional hazards models between colchicine users and the comparison cohort after adjustment for age, sex, medication, comorbidities, and index date based on the time-dependent analysis., Results: Colchicine users had a significantly lower risk of HD ( aHR = 0.87, 95% confidence interval (CI]) = 0.82-0.92) than did the colchicine non-user. For colchicine non-users as the reference, the aHR (95% CI) of the patients who received colchicine of 2-7, 8-30, 31-150, and > 150 days were 0.89 (0.81-0.98), 0.84 (0.76-0.94), 090 (0.80-0.99), and 0.83 (0.74-0.93), respectively; regardless of duration use, the lower risk of HD persisted in colchicine users. The cumulative incidence of HD in colchicine users was significantly lower than that in the colchicine non-users (log-rank p < 0.001)., Conclusion: The addition of short-term or long-term colchicine to standard medical therapy may have benefits to prevent the HD among the ILD patients concurrent with a virus infection or comorbidities even in elderly patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yeh, Hung, Lin, Chen, Liw, Yu and Kao.)

This study aimed to determine the effect of colchicine use on the risk of stroke among patients with diabetes mellitus (DM). We retrospectively enrolled patients with DM between 2000 and 2013 from the Longitudinal Health Insurance Database and divided them into a colchicine cohort (n = 8761) and noncolchicine cohort (n = 8761) by using propensity score matching (PSM). The event of interest was a stroke, including ischemic stroke and hemorrhagic stroke. The incidence of stroke was analyzed using multivariate Cox proportional hazards models between the colchicine cohort and the comparison cohort after adjustment for several confounding factors. The subdistribution hazard model was also performed for examination of the competing risk. The colchicine cohort had a significantly lower incidence of stroke [adjusted hazard ratios (aHR), 95% confidence intervals (95%CI)] (aHR = 0.61, 95%CI = 0.55-0.67), ischemic stroke (aHR = 0.59, 95%CI = 0.53-0.66), and hemorrhagic stroke (aHR = 0.66, 95%CI = 0.53-0.82) compared with the noncolchicine cohort. Drug analysis indicated that patients in the colchicine cohort who received colchicine of cumulative daily defined dose (cDDD) > 14 and duration > 28 days had a lower risk of stroke and ischemic stroke compared with nonusers. The colchicine cohort (cDDD > 150, duration > 360 days) also had a lower risk of stroke, ischemic stroke, and hemorrhagic stroke. The cumulative incidence of stroke, ischemic stroke, and hemorrhagic stroke in the colchicine cohort was significantly lower than that in the noncolchicine cohort (log-rank P < 0.001). However, the subdistribution hazard model reveal the colchicine was not associated with the hemorrhagic stroke in DM patients without gout (aHR = 0.69, 95%CI = 0.47-1.00). Colchicine use with cDDD > 14 and duration > 28 days was associated with lower risk of stroke and ischemic stroke, and colchicine use with cDDD > 150 and duration > 360 days played an auxiliary role in the prevention of stroke, ischemic stroke, and hemorrhagic stroke in patients with DM. The colchicine for the hemorrhagic stroke in DM patients without gout seem to be null effect., (© 2022. The Author(s).)

To investigate the effects of hydroxychloroquine (HCQ) drug use on the risk of pulmonary vascular disease (PVD) in an interstitial lung disease cohort (ILD cohort, ILD+ virus infection), we retrospectively enrolled the ILD cohort with HCQ (HCQ users, N = 4703) and the ILD cohort without HCQ (non-HCQ users, N = 4703) by time-dependence after propensity score matching. Cox models were used to analyze the risk of PVD. We calculated the adjusted hazard ratios (aHRs) and their 95% confidence intervals (CIs) for PVD after adjusting for sex, age, comorbidities, index date and immunosuppressants, such as steroids, etc. Compared with the HCQ nonusers, in HCQ users, the aHRs (95% CIs) for PVD were (2.24 (1.42, 3.54)), and the women’s aHRs for PVD were (2.54, (1.49, 4.35)). The aHRs based on the days of HCQ use for PVD of 28−30 days, 31−120 days, and >120 days were (1.27 (0.81, 1.99)), (3.00 (1.81, 4.87)) and (3.83 (2.46, 5.97)), respectively. The medium or long-term use of HCQ or young women receiving HCQ were associated with a higher aHR for PVD in the ILD cohort. These findings indicated interplay of the primary immunologic effect of ILD, comorbidities, women, age and virus in the HCQ users.

Elevated levels of Mucin-16 (MUC16) in conjunction with a high expression of truncated O-glycans is implicated in playing crucial roles in the malignancy of pancreatic ductal adenocarcinoma (PDAC). However, the mechanisms by which such aberrant glycoforms present on MUC16 itself promote an increased disease burden in PDAC are yet to be elucidated. This study demonstrates that the CRISPR/Cas9-mediated genetic deletion of MUC16 in PDAC cells decreases tumor cell migration. We found that MUC16 enhances tumor malignancy by activating the integrin-linked kinase and focal adhesion kinase (ILK/FAK)-signaling axis. These findings are especially noteworthy in truncated O-glycan (Tn and STn antigen)-expressing PDAC cells. Activation of these oncogenic-signaling pathways resulted in part from interactions between MUC16 and integrin complexes (α4β1), which showed a stronger association with aberrant glycoforms of MUC16. Using a monoclonal antibody to functionally hinder MUC16 significantly reduced the migratory cascades in our model. Together, these findings suggest that truncated O-glycan containing MUC16 exacerbates malignancy in PDAC by activating FAK signaling through specific interactions with α4 and β1 integrin complexes on cancer cell membranes. Targeting these aberrant glycoforms of MUC16 can aid in the development of a novel platform to study and treat metastatic pancreatic cancer.

Background: It is sometimes difficult to distinguish between asthma and bronchiectasis as their symptoms overlap, and these two diseases are associated with pulmonary tuberculosis (PTB) or pneumonia. Objective: The purpose of this study is to determine the effects of bronchodilator drugs, steroids, antidepressants drugs, and antianxiety drugs on the risks of PTB or pneumonia in patients with bronchiectasis-asthma combination or bronchiectasis-asthma-chronic obstructive pulmonary disease combination-BCAS cohort. Methods: After propensity score matching, we retrospectively studied patients with BCAS ( N = 620) and without BCAS ( N = 2,314) through an analysis. The cumulative incidence of PTB or pneumonia was analyzed through Cox proportional regression. After adjustment for sex, age, comorbidities, and medications [including long-acting beta2 agonist/muscarinic antagonists (LABAs/LAMAs), short-acting beta2 agonist/muscarinic antagonists (SABAs/SAMAs), leukotriene receptor antagonist, montelukast, steroids (inhaled corticosteroids, ICSs; oral steroids, OSs), anti-depressants (fluoxetine), and anti-anxiety drugs (benzodiazepines, BZDs)], we calculated the adjusted hazard ratios (aHR) and their 95% confidence intervals (95% CI) for these risks. Similar to OSs, ICSs are associated with an increased risk of PTB or pneumonia, lumping these two as steroids (ICSs/OSs). Results: For the aHR (95% CI), with non-LABAs/non-OSs as the reference 1, the use of LABAs [0.70 (0.52-0.94)]/OSs [0.35 (0.29-0.44)] was associated with a lower risk of PTB or pneumonia. However, the current use of LABAs [2.39 (1.31-4.34)]/SABAs [1.61 (1.31-1.96)], steroids [ICSs 3.23 (1.96-5.29)]/OSs 1.76 (1.45-2.14)], and BZDs [alprazolam 1.73 (1.08-2.75)/fludiazepam 7.48 (1.93-28.9)] was associated with these risks. The current use of LAMAs [0.52 (0.14-1.84)]/SAMAs [1.45 (0.99-2.11)] was not associated with these risks. Conclusion: The current use of LAMAs/SAMAs is relatively safe with respect to PTB or pneumonia risks, but LABAs/SABAs, steroids, and BZDs could be used after evaluation of the benefit for the BCAS cohort. However, we must take the possible protopathic bias into account., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yeh, Lin, Yang, Hsu and Kao.)

Objective: We investigated the effects of medication on heart disease and ischemic stroke (HDS) risk in patients with predominant bronchiectasis-asthma combination (BCAS)., Methods: BCAS and non-BCAS cohorts ( N = 588 and 1,118, respectively) were retrospectively enrolled. The cumulative incidence of HDS was analyzed using Cox proportional regression; propensity scores were estimated using non-parsimonious multivariable logistic regression. Adjusted hazard ratios (aHRs) and 95% confidence intervals (CIs) for HDS were calculated, adjusting for sex, age, comorbidities, and medication {long- and short-acting β2 agonists and muscarinic antagonists (LABAs/SABAs and LAMAs/SAMAs), steroids [inhaled corticosteroid steroids (ICSs), oral steroids (OSs)], antiarrhythmics, antidepressants (fluoxetine), benzodiazepines (alprazolam, fludiazepam), statins and antihypertensive drugs (diuretics, cardioselective beta blockers, calcium channel blockers (CCBs) and angiotensin converting enzyme inhibitors (ACEi), angiotensin II blockers)}., Results: Compared with the non-BCAS cohort, the BCAS cohort taking LABAs, SABAs, SAMAs, ICSs, OSs, antiarrhythmics, and alprazolam had an elevated HDS risk [aHRs (95% CIs): 2.36 (1.25-4.33), 2.65 (1.87-3.75), 2.66 (1.74-4.05), 2.53 (1.61-3.99), 1.76 (1.43-2.18), 9.88 (3.27-30.5), and 1.73 (1.15-2.58), respectively except fludiazepam 1.33 (0.73-2.40)]. The aHRs (95% CIs) for LABAs ≤ 30 days, DDDs <415, ICSs ≤ 30 days were 1.10 (0.38-3.15), 2.95 (0.22-38.8), 1.45 (0.76-2.77). The aHRs (95% CIs) for current and recent alprazolam were 1.78 (1.09-2.93) and 777.8 (1.34-451590.0); for current and past fludiazepam were 1.39 (0.75-2.59) and 1.29 (0.42-4.01) and for past alprazolam was 1.57 (0.55-4.46); respectively. The aHRs (95% CIs) for alprazolam >30 DDDs, fludiazepam >20 DDDs, ICSs ≦415 DDDs, and OSs DDDs ≦15 were 1.60 (0.78-3.29), 2.43 (0.90-6.55), 5.02 (1.76-14.3), and 2.28 (1.43-3.62), respectively., Conclusion: The bronchodilators, steroids, and antiarrhythmics were associated with higher risk of HDS, even low dose use of steroids. However, the current use of LABAs/ICSs were not associated with HDS. Benzodiazepines were relatively safe, except for current or recent alprazolam use. Notably, taking confounders into account is crucial in observational studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yeh, Lai, Yang, Hsu and Kao.)

Prime editors have been delivered using DNA or RNA vectors. Here we demonstrate prime editing with purified ribonucleoprotein complexes. We introduced somatic mutations in zebrafish embryos with frequencies as high as 30% and demonstrate germline transmission. We also observed unintended insertions, deletions and prime editing guide RNA (pegRNA) scaffold incorporations. In HEK293T and primary human T cells, prime editing with purified ribonucleoprotein complexes introduced desired edits with frequencies of up to 21 and 7.5%, respectively., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

ECT2 is an activator of RHO GTPases that is essential for cytokinesis. In addition, ECT2 was identified as an oncoprotein when expressed ectopically in NIH/3T3 fibroblasts. However, oncogenic activation of ECT2 resulted from N-terminal truncation, and such truncated ECT2 proteins have not been found in patients with cancer. In this study, we observed elevated expression of full-length ECT2 protein in preneoplastic colon adenomas, driven by increased ECT2 mRNA abundance and associated with APC tumor-suppressor loss. Elevated ECT2 levels were detected in the cytoplasm and nucleus of colorectal cancer tissue, suggesting cytoplasmic mislocalization as one mechanism of early oncogenic ECT2 activation. Importantly, elevated nuclear ECT2 correlated with poorly differentiated tumors, and a low cytoplasmic:nuclear ratio of ECT2 protein correlated with poor patient survival, suggesting that nuclear and cytoplasmic ECT2 play distinct roles in colorectal cancer. Depletion of ECT2 reduced anchorage-independent cancer cell growth and invasion independent of its function in cytokinesis, and loss of Ect2 extended survival in a Kras G12D Apc -null colon cancer mouse model. Expression of ECT2 variants with impaired nuclear localization or guanine nucleotide exchange catalytic activity failed to restore cancer cell growth or invasion, indicating that active, nuclear ECT2 is required to support tumor progression. Nuclear ECT2 promoted ribosomal DNA transcription and ribosome biogenesis in colorectal cancer. These results support a driver role for both cytoplasmic and nuclear ECT2 overexpression in colorectal cancer and emphasize the critical role of precise subcellular localization in dictating ECT2 function in neoplastic cells. SIGNIFICANCE: ECT2 overexpression and mislocalization support its role as a driver in colon cancer that is independent from its function in normal cell cytokinesis., (©2021 American Association for Cancer Research.)

A better understanding of the metabolic constraints of a tumor may lead to more effective anticancer treatments. Evidence has emerged in recent years shedding light on a crucial aspartate dependency of many tumor types. As a precursor for nucleotide synthesis, aspartate is indispensable for cell proliferation. Moreover, the malate-aspartate shuttle plays a key role in redox balance, and a deficit in aspartate can lead to oxidative stress. It is now recognized that aspartate biosynthesis is largely governed by mitochondrial metabolism, including respiration and glutaminolysis in cancer cells. Therefore, under conditions that suppress mitochondrial metabolism, including mutations, hypoxia, or chemical inhibitors, aspartate can become a limiting factor for tumor growth and cancer cell survival. Notably, aspartate availability has been associated with sensitivity or resistance to various therapeutics that are presently in the clinic or in clinical trials, arguing for a critical need for more effective aspartate-targeting approaches. In this review, we present current knowledge of the metabolic roles of aspartate in cancer cells and describe how cancer cells maintain aspartate levels under different metabolic states. We also highlight several promising aspartate level-modulating agents that are currently under investigation.

Cancer cells bearing distinct KRAS mutations exhibit variable sensitivity to SHP2 inhibitors (SHP2i). Here we show that cells harboring KRAS Q61H are uniquely resistant to SHP2i, and investigate the underlying mechanisms using biophysics, molecular dynamics, and cell-based approaches. Q61H mutation impairs intrinsic and GAP-mediated GTP hydrolysis, and impedes activation by SOS1, but does not alter tyrosyl phosphorylation. Wild-type and Q61H-mutant KRAS are both phosphorylated by Src on Tyr32 and Tyr64 and dephosphorylated by SHP2, however, SHP2i does not reduce ERK phosphorylation in KRAS Q61H cells. Phosphorylation of wild-type and Gly12-mutant KRAS, which are associated with sensitivity to SHP2i, confers resistance to regulation by GAP and GEF activities and impairs binding to RAF, whereas the near-complete GAP/GEF-resistance of KRAS Q61H remains unaltered, and high-affinity RAF interaction is retained. SHP2 can stimulate KRAS signaling by modulating GEF/GAP activities and dephosphorylating KRAS, processes that fail to regulate signaling of the Q61H mutant., (© 2021. The Author(s).)

Purpose: To determine the effects of statins and steroids on the risk of coronary artery disease (CAD) and stroke in patients with interstitial lung disease and pulmonary fibrosis (ILD-PF)., Methods: We retrospectively enrolled patients with ILD-PF who were using statins (statin cohort, N = 11,567) and not using statins (nonstatin cohort, N = 26,159). Cox proportional regression was performed to analyze the cumulative incidence of CAD and stroke. Adjusted hazard ratios (aHRs) and 95% confidence intervals (CIs) of CAD and stroke were determined after sex, age, and comorbidities, as well as the use of inhaler corticosteroids (ICSs), oral steroids (OSs), and statins, were controlled for., Results: Compared with those of patients without statin use, the aHRs (95% CIs) of patients with statin use for CAD and ischemic stroke were 0.72 (0.65-0.79) and 0.52 (0.38-0.72), respectively. For patients taking single-use statins but not ICSs/OSs, the aHRs (95% CIs) for CAD and ischemic stroke were 0.72 (0.65-0.79)/0.69 (0.61-0.79) and 0.54 (0.39-0.74)/0.50 (0.32-0.79), respectively. For patients using ICSs/OSs, the aHRs (95% CIs) for CAD and ischemic stroke were 0.71 (0.42-1.18)/0.74 (0.64-0.85) and 0.23 (0.03-1.59)/0.54 (0.35-0.85), respectively., Conclusions: The findings demonstrate that statin use, either alone or in combination with OS use, plays an auxiliary role in the management of CAD and ischemic stroke in patients with ILD-PF., Competing Interests: The authors have declared that no competing interests exist.

Objective: To determine the effect of statins on risk of cancer in patients with interstitial lung disease (ILD) and pulmonary fibrosis., Setting: We retrospectively enrolled patients with ILD and pulmonary fibrosis and divided them into two cohorts by statin use (statin users (n=10 036) and statin non-users (n=10 036))., Participants: We selected patients with ILD and pulmonary fibrosis (N=53 862) from Taiwan's National Health Insurance Research Database. Time-dependent Cox models were used to compare risk of cancer of propensity-matched statin users and non-users. Cumulative cancer incidence was analysed through Cox proportional regression. We calculated adjusted HRs (aHRs) and their 95% CIs for cancer after adjusting for sex, age, comorbidities, and use of inhaled corticosteroids, oral steroids and statins., Results: Compared with statin non-users, the aHRs (95% CIs) for statin users were 0.60 (0.55 to 0.65) for cancer, 0.52 (0.35 to 0.78) for haematological malignancy, 0.52 (0.38 to 0.72) for cancer of the head and neck, 0.73 (0.59 to 0.89) for colorectal cancer, 0.34 (0.26 to 0.43) for liver cancer, 0.39 (0.23 to 0.67) for pancreatic cancer, 0.40 (0.17 to 0.96) for skin cancer, 0.67 (0.52 to 0.87) for breast cancer, 0.27 (0.14 to 0.54) for cervical cancer, 0.37 (0.30 to 0.46) for other immunological cancers, 0.73 (0.54 to 0.98) for bladder/kidney cancer and 0.88 (0.71 to 1.09) for lung cancer., Conclusion: Statin use is associated with lower risk of cancer in the ILD and pulmonary fibrosis cohort., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Pancreatic ductal adenocarcinoma (PDAC) is notorious for a dense fibrotic stroma that is interlaced with a collagen-based extracellular matrix (ECM) that plays an important role in tumor biology. Traditionally thought to only provide a physical barrier from host responses and systemic chemotherapy, new studies have demonstrated that the ECM maintains biomechanical and biochemical properties of the tumor microenvironment (TME) and restrains tumor growth. Recent studies have shown that the ECM augments tumor stiffness, interstitial fluid pressure, cell-to-cell junctions, and microvascularity using a mix of biomechanical and biochemical signals to influence tumor fate for better or worse. In addition, PDAC tumors have been shown to use ECM-derived peptide fragments as a nutrient source in nutrient-poor conditions. While collagens are the most abundant proteins found in the ECM, several studies have identified growth factors, integrins, glycoproteins, and proteoglycans in the ECM. This review focuses on the dichotomous nature of the PDAC ECM, the types of collagens and other proteins found in the ECM, and therapeutic strategies targeting the PDAC ECM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Perez, Kearney and Yeh.)

Most heat-health studies identified thresholds just outside human comfort zones, which are often too low to be used in heat-warning systems for reducing climate-related health risks. We refined a generalized additive model for selecting thresholds with substantial health risk enhancement, based on Taiwan population records of 2000-2017, considering lag effects and different spatial scales. Reference-adjusted risk ratio (RaRR) is proposed, defined as the ratio between the relative risk of an essential health outcome for a threshold candidate against that for a reference; the threshold with the highest RaRR is potentially the optimal one. It was found that the wet-bulb globe temperature (WBGT) is a more sensitive heat-health indicator than temperature. At lag 0, the highest RaRR (1.66) with WBGT occurred in emergency visits of children, while that in hospital visits occurred for the working-age group (1.19), presumably due to high exposure while engaging in outdoor activities. For most sex, age, and sub-region categories, the RaRRs of emergency visits were higher than those of hospital visits and all-cause mortality; thus, emergency visits should be employed (if available) to select heat-warning thresholds. This work demonstrates the applicability of this method to facilitate the establishment of heat-warning systems at city or country scales by authorities worldwide.

Background: Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy. Redox factor-1 (Ref-1), a redox signaling protein, regulates the conversion of several transcription factors (TFs), including HIF-1α, STAT3 and NFκB from an oxidized to reduced state leading to enhancement of their DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia., Methods: scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model and validated using proteomics and qRT-PCR. The identified Ref-1's role in mitochondrial function was confirmed using mitochondrial function assays, qRT-PCR, western blotting and NADP assay. Further, the effect of Ref-1 redox function inhibition against pancreatic cancer metabolism was assayed using 3D co-culture in vitro and xenograft studies in vivo., Results: Distinct transcriptional variation in central metabolism, cell cycle, apoptosis, immune response, and genes downstream of a series of signaling pathways and transcriptional regulatory factors were identified in Ref-1 knockdown vs Scrambled control from the scRNA-seq data. Mitochondrial DEG subsets downregulated with Ref-1 knockdown were significantly reduced following Ref-1 redox inhibition and more dramatically in combination with Devimistat in vitro. Mitochondrial function assays demonstrated that Ref-1 knockdown and Ref-1 redox signaling inhibition decreased utilization of TCA cycle substrates and slowed the growth of pancreatic cancer co-culture spheroids. In Ref-1 knockdown cells, a higher flux rate of NADP + consuming reactions was observed suggesting the less availability of NADP + and a higher level of oxidative stress in these cells. In vivo xenograft studies demonstrated that tumor reduction was potent with Ref-1 redox inhibitor similar to Devimistat., Conclusion: Ref-1 redox signaling inhibition conclusively alters cancer cell metabolism by causing TCA cycle dysfunction while also reducing the pancreatic tumor growth in vitro as well as in vivo., (© 2021. The Author(s).)

To study disease development, an inventory of an organ's cell types and understanding of physiologic function is paramount. Here, we performed single-cell RNA-sequencing to examine heterogeneity of murine pancreatic duct cells, pancreatobiliary cells, and intrapancreatic bile duct cells. We describe an epithelial-mesenchymal transitory axis in our three pancreatic duct subpopulations and identify osteopontin as a regulator of this fate decision as well as human duct cell dedifferentiation. Our results further identify functional heterogeneity within pancreatic duct subpopulations by elucidating a role for geminin in accumulation of DNA damage in the setting of chronic pancreatitis. Our findings implicate diverse functional roles for subpopulations of pancreatic duct cells in maintenance of duct cell identity and disease progression and establish a comprehensive road map of murine pancreatic duct cell, pancreatobiliary cell, and intrapancreatic bile duct cell homeostasis., Competing Interests: AH, AR, LL, SA, JS, SG, XP, HJ, DB, MP, LL, JL, EC, MA, GF, JY, CY, GK, VW, MH No competing interests declared, (© 2021, Hendley et al.)

Profiling of whole transcriptomes has become a cornerstone of molecular biology and an invaluable tool for the characterization of clinical phenotypes and the identification of disease subtypes. Analyses of these data are becoming ever more sophisticated as we move beyond simple comparisons to consider networks of higher-order interactions and associations. Gene regulatory networks (GRNs) model the regulatory relationships of transcription factors and genes and have allowed the identification of differentially regulated processes in disease systems. In this perspective, we discuss gene targeting scores, which measure changes in inferred regulatory network interactions, and their use in identifying disease-relevant processes. In addition, we present an example analysis for pancreatic ductal adenocarcinoma (PDAC), demonstrating the power of gene targeting scores to identify differential processes between complex phenotypes, processes that would have been missed by only performing differential expression analysis. This example demonstrates that gene targeting scores are an invaluable addition to gene expression analysis in the characterization of diseases and other complex phenotypes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Weighill, Ben Guebila, Glass, Platig, Yeh and Quackenbush.)

Aberrant expression of CA125/MUC16 is associated with pancreatic ductal adenocarcinoma (PDAC) progression and metastasis. However, knowledge of the contribution of MUC16 to pancreatic tumorigenesis is limited. Here, we show that MUC16 expression is associated with disease progression, basal-like and squamous tumor subtypes, increased tumor metastasis, and short-term survival of PDAC patients. MUC16 enhanced tumor malignancy through the activation of AKT and GSK3β oncogenic signaling pathways. Activation of these oncogenic signaling pathways resulted in part from increased interactions between MUC16 and epidermal growth factor (EGF)-type receptors, which were enhanced for aberrant glycoforms of MUC16. Treatment of PDAC cells with monoclonal antibody (mAb) AR9.6 significantly reduced MUC16-induced oncogenic signaling. mAb AR9.6 binds to a unique conformational epitope on MUC16, which is influenced by O-glycosylation. Additionally, treatment of PDAC tumor-bearing mice with either mAb AR9.6 alone or in combination with gemcitabine significantly reduced tumor growth and metastasis. We conclude that the aberrant expression of MUC16 enhances PDAC progression to an aggressive phenotype by modulating oncogenic signaling through ErbB receptors. Anti-MUC16 mAb AR9.6 blocks oncogenic activities and tumor growth and could be a novel immunotherapeutic agent against MUC16-mediated PDAC tumor malignancy., Competing Interests: Declaration of interests M.A.H. and P.R. have an equity interest in OncoCare Therapeutics. R.M. is employed by Quest PharmaTech and has an equity interest in this company. All other authors declare no competing interests., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

The complexity of human cancer often results in significant heterogeneity in response to treatment. Precision medicine offers the potential to improve patient outcomes by leveraging this heterogeneity. Individualized treatment rules (ITRs) formalize precision medicine as maps from the patient covariate space into the space of allowable treatments. The optimal ITR is that which maximizes the mean of a clinical outcome in a population of interest. Patient-derived xenograft (PDX) studies permit the evaluation of multiple treatments within a single tumor, and thus are ideally suited for estimating optimal ITRs. PDX data are characterized by correlated outcomes, a high-dimensional feature space, and a large number of treatments. Here we explore machine learning methods for estimating optimal ITRs from PDX data. We analyze data from a large PDX study to identify biomarkers that are informative for developing personalized treatment recommendations in multiple cancers. We estimate optimal ITRs using regression-based (Q-learning) and direct-search methods (outcome weighted learning). Finally, we implement a superlearner approach to combine multiple estimated ITRs and show that the resulting ITR performs better than any of the input ITRs, mitigating uncertainty regarding user choice. Our results indicate that PDX data are a valuable resource for developing individualized treatment strategies in oncology. Supplementary materials for this article are available online.

Desmoplasia describes the deposition of extensive extracellular matrix and defines primary pancreatic ductal adenocarcinoma (PDA). The acellular component of this stroma has been implicated in PDA pathogenesis and is being targeted therapeutically in clinical trials. By analyzing the stromal content of PDA samples from numerous annotated PDA data sets and correlating stromal content with both anatomic site and clinical outcome, we found PDA metastases in the liver, the primary cause of mortality to have less stroma, have higher tumor cellularity than primary tumors. Experimentally manipulating stromal matrix with an anti-lysyl oxidase like-2 (anti-LOXL2) antibody in syngeneic orthotopic PDA mouse models significantly decreased matrix content, led to lower tissue stiffness, lower contrast retention on computed tomography, and accelerated tumor growth, resulting in diminished overall survival. These studies suggest an important protective role of stroma in PDA and urge caution in clinically deploying stromal depletion strategies.

Under conditions of inherent or induced mitochondrial dysfunction, cancer cells manifest overlapping metabolic phenotypes, suggesting that they may be targeted via a common approach. Here, we use multiple oxidative phosphorylation (OXPHOS)-competent and incompetent cancer cell pairs to demonstrate that treatment with α-ketoglutarate (aKG) esters elicits rapid death of OXPHOS-deficient cancer cells by elevating intracellular aKG concentrations, thereby sequestering nitrogen from aspartate through glutamic-oxaloacetic transaminase 1 (GOT1). Exhaustion of aspartate in these cells resulted in immediate depletion of adenylates, which plays a central role in mediating mTOR inactivation and inhibition of glycolysis. aKG esters also conferred cytotoxicity in a variety of cancer types if their cell respiration was obstructed by hypoxia or by chemical inhibition of the electron transport chain (ETC), both of which are known to increase aspartate and GOT1 dependencies. Furthermore, preclinical mouse studies suggested that cell-permeable aKG displays a good biosafety profile, eliminates aspartate only in OXPHOS-incompetent tumors, and prevents their growth and metastasis. This study reveals a novel cytotoxic mechanism for the metabolite aKG and identifies cell-permeable aKG, either by itself or in combination with ETC inhibitors, as a potential anticancer approach. SIGNIFICANCE: These findings demonstrate that OXPHOS deficiency caused by either hypoxia or mutations, which can significantly increase cancer virulence, renders tumors sensitive to aKG esters by targeting their dependence upon GOT1 for aspartate synthesis. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/17/3492/F1.large.jpg., (©2020 American Association for Cancer Research.)

Phosphatidylserine (PS) is often externalized in viable pancreatic cancer cells and is therapeutically targetable using PS-selective drugs. One of the first-line treatments for advanced pancreatic cancer disease, gemcitabine (GEM), provides only marginal benefit to patients. We therefore investigated the therapeutic benefits of combining GEM and the PS-targeting drug, saposin C-dioleoylphosphatidylserine (SapC-DOPS), for treating pancreatic ductal adenocarcinoma (PDAC). Using cell-cycle analyses and a cell surface PS-based sorting method in vitro, we observed an increase in surface PS as cells progress through the cell cycle from G1 to G2/M. We also observed that GEM treatment preferentially targets G1 phase cells that have low surface PS, resulting in an increased median surface PS level of PDAC cells. Inversely, SapC-DOPS preferentially targets high surface PS cells that are predominantly in the G2/M phase. Finally, combination therapy in subcutaneous and orthotopic PDAC tumors in vivo with SapC-DOPS and GEM or Abraxane (Abr)/GEM (one of the current standards of care) significantly inhibits tumor growth and increases survival compared with individual treatments. Our studies confirm a surface PS and cell cycle-based enhancement of cancer cytotoxicity following SapC-DOPS treatment in combination with GEM or Abr/GEM. Thus, PDAC patients treated with Abr/GEM may benefit from concurrent administration of SapC-DOPS., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Over 55,000 people in the United States are diagnosed with pancreatic ductal adenocarcinoma (PDAC) yearly, and fewer than 20% of these patients survive a year beyond diagnosis. Chemotherapies are considered or used in nearly every PDAC case, but there is limited understanding of the complex signaling responses underlying resistance to these common treatments. Here, we take an unbiased approach to study protein kinase network changes following chemotherapies in patient-derived xenograft (PDX) models of PDAC to facilitate design of rational drug combinations. Proteomics profiling following chemotherapy regimens reveals that activation of JNK-JUN signaling occurs after 5-fluorouracil plus leucovorin (5-FU + LEU) and FOLFOX (5-FU + LEU plus oxaliplatin [OX]), but not after OX alone or gemcitabine. Cell and tumor growth assays with the irreversible inhibitor JNK-IN-8 and genetic manipulations demonstrate that JNK and JUN each contribute to chemoresistance and cancer cell survival after FOLFOX. Active JNK1 and JUN are specifically implicated in these effects, and synergy with JNK-IN-8 is linked to FOLFOX-mediated JUN activation, cell cycle dysregulation, and DNA damage response. This study highlights the potential for JNK-IN-8 as a biological tool and potential combination therapy with FOLFOX in PDAC and reinforces the need to tailor treatment to functional characteristics of individual tumors.

Background: Studies on the association between polymyositis/dermatomyositis (PM/DM) and acute respiratory failure (ARF) are considerably limited. We investigated whether ARF is associated with PM/DM using a nationwide cohort study., Methods: We identified 1,374 patients with newly diagnosed PM/DM and 13,740 comparison individuals without PM/DM (non-PM/DM) randomly selected from the general population; frequency matched by age, sex, and index year using the National Health Insurance Research Database; and followed up until the end of 2011 to measure the incidence of ARF. Cox proportional hazards regression analysis was used to measure the hazard ratio (HR) of ARF for the PM/DM cohort in comparison with the non-PM/DM cohort., Results: The adjusted HR of ARF was 5.05 for the PM/DM cohort compared with the non-PM/DM cohort after adjusting for sex, age, comorbidities, Charlson comorbidity index (CCI) score and medicine. The risk of ARF significantly increased irrespective of age, sex, comorbidities and medicine. Meanwhile, the PM/DM cohort with comorbidities, such as cardiac disease (hypertension), pulmonary disease (chronic obstructive pulmonary disease and pneumonia), and pulmonary vascular diseases had additive effects on the incident ARF., Conclusions: This study determined the cross-reaction of pulmonary heart disease in the PM/DM cohort with incident ARF even without comorbidities., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare., (2020 Annals of Translational Medicine. All rights reserved.)

Purpose: To evaluate the association among chronic obstructive pulmonary disease (COPD) with asthma, steroid use, and pneumonia in the general population., Methods: Using Taiwan's National Health Insurance Research Database to identify patients with incident pneumonia, we established a COPD with asthma cohort of 12,538 patients and a COPD cohort of 25,069 patients. In both cohorts, the risk of incident pneumonia was assessed using multivariable Cox proportional hazards models., Results: The adjusted hazard ratio (aHR) with 95% confidence interval (CI) for incident pneumonia was 2.38 (2.14, 2.66) in the COPD with asthma cohort, regardless of age, sex, comorbidities, and drug use. COPD cohort without inhaled corticosteroids (ICSs) use served as a reference. The aHR (95% CI) for COPD cohort with ICSs use was 1.34 (0.98, 1.83); that for COPD with asthma cohort without ICSs use was 2.46 (2.20, 2.76); and that for COPD with asthma cohort with ICSs use was 2.32 (1.99, 2.72). COPD cohort without oral steroids (OSs) use served as a reference; the aHR (95% CI) for COPD with asthma cohort without OSs use and with OSs use was 3.25 (2.72, 3.89) and 2.38 (2.07, 2.74), respectively., Conclusions: The COPD with asthma cohort had a higher risk of incident pneumonia, regardless of age, sex, comorbidities, and ICSs or OSs use. COPD cohort with ICSs use did not have a notable risk of incident pneumonia. The COPD with asthma cohort had a higher risk of incident pneumonia, even without ICSs/OSs use., Competing Interests: The authors have declared that no competing interests exist.

Background: Intrinsically photosensitive retinal ganglion cells (ipRGCs) control non-visual light responses (e.g. pupillary light reflex and circadian entrainment). Patients with diabetic retinopathy (DR) show reduced ipRGC function, as inferred by abnormalities in the post illumination pupil response (PIPR). We explored whether ipRGC function in DR is associated with circadian outputs and sleep/wake behavior., Methods: Forty-five participants (15 without diabetes, 15 with type 2 diabetes (T2D) and no DR, 15 with T2D and DR) participated. ipRGC function was inferred from the PIPR (pupil size following stimulus offset). Circadian outputs were melatonin amplitude (overnight urinary 6-sulfatoxymelatonin (aMT6s)) and timing (dim light melatonin onset (DLMO)), and evening salivary cortisol levels. Sleep/wake patterns were measured with wrist actigraphy and insomnia symptoms were assessed subjectively., Results: Patients with T2D and DR had smaller PIPR and lower urinary aMT6s than other groups (p < 0.001). In adjusted regression models, smaller PIPR was associated with lower urinary aMT6s (β = 4.552, p = 0.005). Patients with DR were more likely to have no detectable DLMO (p = 0.049), higher evening salivary cortisol, greater insomnia symptoms and greater sleep variability compared to other groups. Sleep duration, efficiency and rest-activity rhythms were similar., Conclusion: Reduced ipRGC function in DR is associated with circadian dysregulation and sleep disturbances, although a causal relationship cannot be established in this cross-sectional study. Prospective mechanistic and intervention studies examining circadian and sleep health in these patients are warranted.

Purpose: Molecular subtyping for pancreatic cancer has made substantial progress in recent years, facilitating the optimization of existing therapeutic approaches to improve clinical outcomes in pancreatic cancer. With advances in treatment combinations and choices, it is becoming increasingly important to determine ways to place patients on the best therapies upfront. Although various molecular subtyping systems for pancreatic cancer have been proposed, consensus regarding proposed subtypes, as well as their relative clinical utility, remains largely unknown and presents a natural barrier to wider clinical adoption., Experimental Design: We assess three major subtype classification schemas in the context of results from two clinical trials and by meta-analysis of publicly available expression data to assess statistical criteria of subtype robustness and overall clinical relevance. We then developed a single-sample classifier (SSC) using penalized logistic regression based on the most robust and replicable schema., Results: We demonstrate that a tumor-intrinsic two-subtype schema is most robust, replicable, and clinically relevant. We developed Purity Independent Subtyping of Tumors (PurIST), a SSC with robust and highly replicable performance on a wide range of platforms and sample types. We show that PurIST subtypes have meaningful associations with patient prognosis and have significant implications for treatment response to FOLIFIRNOX., Conclusions: The flexibility and utility of PurIST on low-input samples such as tumor biopsies allows it to be used at the time of diagnosis to facilitate the choice of effective therapies for patients with pancreatic ductal adenocarcinoma and should be considered in the context of future clinical trials., (©2019 American Association for Cancer Research.)

In the genomic era, the identification of gene signatures associated with disease is of significant interest. Such signatures are often used to predict clinical outcomes in new patients and aid clinical decision-making. However, recent studies have shown that gene signatures are often not replicable. This occurrence has practical implications regarding the generalizability and clinical applicability of such signatures. To improve replicability, we introduce a novel approach to select gene signatures from multiple datasets whose effects are consistently non-zero and account for between-study heterogeneity. We build our model upon some rank-based quantities, facilitating integration over different genomic datasets. A high dimensional penalized Generalized Linear Mixed Model (pGLMM) is used to select gene signatures and address data heterogeneity. We compare our method to some commonly used strategies that select gene signatures ignoring between-study heterogeneity. We provide asymptotic results justifying the performance of our method and demonstrate its advantage in the presence of heterogeneity through thorough simulation studies. Lastly, we motivate our method through a case study subtyping pancreatic cancer patients from four gene expression studies.

Background: To determine the effects of bronchodilator, steroid, and anti-arrhythmia drug use on the risk of heart disease/stroke (HDS) in patients with bronchiectasis-chronic obstructive pulmonary disease overlap syndrome (BCOS). Methods: We retrospectively enrolled patients with BCOS (BCOS cohort, n = 1,493) and patients without bronchiectasis and chronic obstructive pulmonary disease (COPD) (non-BCOS cohort, n = 5,972). The cumulative incidence of HDS was analyzed through Cox proportional regression. We calculated adjusted hazard ratios (aHRs) and their 95% confidence intervals (CIs) for HDS after adjustments for sex, age, comorbidities, long-acting β2-agonist or long-acting muscarinic antagonist (LABAs/LAMAs) use, short-acting β2-agonist or short-acting muscarinic antagonist (SABAs/SAMAs) use, oral steroid (OSs) or inhaled corticosteroid steroid (ICSs) use, and anti-arrhythmia drugs use. Results: The aHR (95% CI) for HDS was 1.08 (0.28-4.06) for patients using LAMAs compared with those not using drugs. Regarding drug use days, the aHRs (95% CIs) were 32.2 (1.79-773.0), 1.85 (1.01-3.39), and 31.1 (3.25-297.80) for those with recent SABAs use, past ICSs use, and past anti-arrythmia drugs use, respectively. Regarding cumulative drug dose, the aHRs (95% CIs) were 2.12 (1.46-3.10), 3.48 (1.13-10.6), 3.19 (2.04-4.99), 28.1 (1.42-555.7), 2.09 (1.32-3.29), 2.28 (1.53-3.40), and 1.93 (1.36-2.74) for those with a low dose of SABAs, medium dose of SABAs, low dose of SAMAs, low dose of ICSs, medium dose of ICSs, low dose of OSs, and medium dose of OSs, respectively. Conclusions: Compared with patients without bronchiectasis and COPD, BCOS patients with recent SABAs, past ICSs, and past anti-arrhythmia drugs use; a low or medium SABAs ICSs, and OSs dose; and a low SAMAs dose had a higher risk of HDS. LAMAs were not associated with HDS., (Copyright © 2019 Yeh, Yang, Hsu and Kao.)