Cancer immunotherapy with enveloped self-amplifying mRNA CARG-2020 that modulates IL-12, IL-17 and PD-L1 pathways to prevent tumor recurrence

Targeting multiple immune mechanisms may overcome therapy resistance and further improve cancer immunotherapy for humans. Here, we describe the application of virus-like vesicles (VLV) for delivery of three immunomodulators alone and in combination, as a promising approach for cancer immunotherapy. VLV vectors were designed to deliver single chain interleukin (IL)-12, short-hairpin RNA (shRNA) targeting programmed death ligand 1 (PD-L1), and a dominant-negative form of IL-17 receptor A (dn-IL17RA) as a single payload or as a combination payload. Intralesional delivery of the VLV vector expressing IL-12 alone, as well as the trivalent vector (designated CARG-2020) eradicated large established tumors. However, only CARG-2020 prevented tumor recurrence and provided long-term survival benefit to the tumor-bearing mice, indicating a benefit of the combined immunomodulation. The abscopal effects of CARG-2020 on the non-injected contralateral tumors, as well as protection from the tumor cell re-challenge, suggest immune-mediated mechanism of protection and establishment of immunological memory. Mechanistically, CARG-2020 potently activates Th1 immune mechanisms and inhibits expression of genes related to T cell exhaustion and cancer-promoting inflammation. The ability of CARG-2020 to prevent tumor recurrence and to provide survival benefit makes it a promising candidate for its development for human cancer immunotherapy.

Examining the Association between Medical Marijuana Legalization and Criminal Behaviors: Evidence from a U.S. Representative Sample.

Background: Medical marijuana legalization (MML) has been widely implemented in the past decade. However, the debates regarding the consequences of MML persist, especially criminal behaviors. Objectives: We examined the association between MML and criminal behaviors among adults in the United States. The criminal behaviors measured three past-year offenses: whether the adult (1) have sold illegal drugs, (2) have stolen anything worth > $50 USD, or (3) have attacked someone. Methods: Using the 2015-2020 National Survey of Drug Use and Health, we included 214,505 adults in our primary analysis for 2015-2019 and 27,170 adults in 2020 for supplemental analysis (age > = 18). Weighted multivariable logistic regression models were used to examine the association between MML and three criminal behaviors. Results: In our primary analysis, we observed no statistically significant association between MML and the three outcomes of criminal behavior. Nevertheless, our supplemental analysis of the 2020 data showed MML was associated with increasing odds of the three criminal behaviors (have sold illegal drugs: AOR [adjusted odds ratio] = 1.7; have stolen anything worth > $50 USD: AOR = 1.9; have attacked someone: AOR = 1.8; all p < 0.05). Conclusion: Surveys from 2015 to 2019 did not suggest MML as a risk factor for higher incidence of criminal behaviors. However, 2020 data showed statistically significant association between MML and selected criminal behaviors. Issues related to the COVID-19 pandemic, such as the U.S. economic downturn, could potentially explain this discrepancy. Further research efforts may be warranted.

State-Level Variation in Medicaid Managed Care Enrollment and Specialty Care for Publicly Insured Children.

Importance: Medicaid and Children's Health Insurance Program cover almost 50% of children with special health care needs (CSHCN). CSHCN often require specialty services and have been increasingly enrolled in Medicaid managed care (MMC) plans, but there is a dearth of recent national studies on specialty care access among publicly insured children and particularly CSHCN. Objective: To provide recent, nationwide evidence on the association of MMC penetration with specialty care access among publicly insured children, with a special focus on CSHCN. Design, Setting, and Participants: This cross-sectional study used nationally representative data from the 2016 to 2019 National Survey of Children's Health to identify publicly insured children in 41 states that administered comprehensive managed care organizations for Medicaid. Data analysis was performed from May 2022 to March 2023. Exposure: Form CMS-416 data were used to measure state-year level share of Medicaid-enrolled children who were covered by MMC (ie, MMC penetration). Main Outcomes and Measures: Measures of specialty care access included whether, in the past year, the child had (1) any visit to non-mental health (MH) specialists, (2) any visit to MH professionals, and (3) any unmet health care needs and (4) whether the caregiver ever felt frustrated getting services for their child. Logistic regression models were used to examine the association of MMC penetration with specialty care access among all publicly insured children, and separately for CSHCN and non-CSHCN. Results: Among 20 029 publicly insured children, 7164 (35.8%) were CSHCN, 9537 (48.2%) were female, 4110 (37.2%) were caregiver-reported Hispanic, and 2812 (21.4%) were caregiver-reported non-Hispanic Black (all percentages are weighted). MMC was not associated with significant changes in any visit to non-MH specialists and unmet health care needs. In addition, MMC penetration was positively associated with caregiver frustration among all children (adjusted odds ratio, 1.23; 95% CI, 1.03-1.48; P = .02) and was negatively associated with any visit to MH professionals among CSHCN (adjusted odds ratio, 0.75; 95% CI, 0.58-0.98; P = .04). Conclusions and Relevance: In this cross-sectional study evaluating MMC and specialty care access for publicly insured children, increased MMC enrollment was not associated with improved specialty care access for publicly insured children, including CSHCN. MMC was associated with less access to specialties like MH and increased frustrations among caregivers seeking services for their children.

Comprehensive Analysis and Drug Modulation of Human Endogenous Retrovirus in Hepatocellular Carcinomas.

BACKGROUND: Human endogenous retroviruses (HERVs) play an important role in the development of cancer and many diseases. Here, we comprehensively explored the impact of HERVs on hepatocellular carcinomas (HCCs). METHODS: We employed Telescope to identify HERVs and quantify their expression in the total RNA sequencing data obtained from 254 HCC samples, comprising 254 tumor tissues and 34 matched normal tissues. RESULTS: In total, 3357 locus-specific activations of HERVs were differentially expressed, and 180 were correlated with patient survival. Using these 180 HERVs for classification, we found four subgroups with survival correlation. Higher expression levels of the 180 HERVs were correlated with poorer survival, while age, AFP, some mutations, and copy and structural variants differed among subgroups. The differential expression of host genes in high expression of these 180 HERVs primarily involved the activation of pathways related to immunity and infection, lipid and atherosclerosis, MAPK and NF-kB signaling, and cytokine-cytokine receptor interactions. Conversely, there was a suppression of pathways associated with RNA processing, including nucleocytoplasmic transport, surveillance and ribosome biogenesis, and transcriptional misregulation in cancer pathways. Almost all genes involved in HERV activation restriction, KRAB zinc finger proteins, RNA nucleocytoplasmic transport, stemness, HLA and antigen processing and presentation, and immune checkpoints were overexpressed in cancerous tissues, and many over-expressed HERV-related nearby genes were correlated with high HERV activation and poor survival. Twenty-three immune and stromal cells showed higher expression in non-cancerous than cancerous tissues, and seven were correlated with HERV activation. Small-molecule modulation of alternative splicing (AS) altered the expression of survival-related HERVs and their activation-related genes, as well as nearby genes. CONCLUSION: Comprehensive and integrated approaches for evaluating HERV expression and their correlation with specific pathways have the potential to provide new companion diagnostics and therapeutic strategies for HCC.

Whole genome and RNA sequencing analyses for 254 Taiwanese hepatocellular carcinomas.

BACKGROUND: Comprehensive and integrative analysis of hepatocellular carcinoma (HCC) is important. In this study, we explored Taiwanese HCCs using multi-omics analyses. METHODS: We analyzed 254 HCCs by whole genome sequencing and total RNA sequencing, and then used bioinformatic tools to analyze genomic and transcriptomic alterations in coding and non-coding sequences to explore the clinical importance of each sequence. RESULTS: The frequencies of the five most commonly mutated cancer-related genes were TERT, TP53, CTNNB1, RB1, and ARID1A. Genetic alteration frequencies influenced the etiology of HCC; some alterations were also correlated with clinicopathological conditions. Many cancer-related genes had copy number alterations (CNAs) and structure variants (SVs) that changed according to etiology and exhibited potential associations with survival. We also identified several alterations in histone-related genes, HCC-related long non-coding RNAs, and non-coding driver genes that may contribute to the onset and progression of HCC. Transcriptomic analysis revealed that 229 differentially expressed and 148 novel alternative splicing (AS) genes, as well as the presence of fusion genes, were associated with patient survival. Moreover, somatic mutations, CNAs, and SVs were associated with immune checkpoint gene expression and tumor microenvironment. Finally, we identified relationships among AS, immune checkpoint gene expression and tumor microenvironment. CONCLUSIONS: This study shows that genomic alterations are associated with survival, including DNA-based and RNA-based data. Moreover, genomic alterations and their associations with immune checkpoint genes and the tumor microenvironment may provide novel insights for the diagnosis and treatment of HCC.

Evaluating Medicaid Managed Care Network Adequacy Standards And Associations With Specialty Care Access For Children.

Medicaid managed care plans cover more than 80 percent of Medicaid-enrolled children, including many children with special health care needs (CSHCN). Federal rules require states to set network adequacy standards to improve specialty care access for Medicaid managed care enrollees. Using a quasi-experimental design and 2016-19 National Survey of Children's Health data, we examined the association between quantitative network adequacy standards and access to specialty care among 8,614 Medicaid-enrolled children, including 3,157 with special health care needs, in eighteen states. Outcomes included whether the child had any visit to non-mental health specialists, any visit to mental health professionals, or any unmet health care needs and whether the caregiver ever felt frustrated in getting services for the child in the past year. We observed no association between the adoption of any quantitative network adequacy standard and the above outcomes among Medicaid-enrolled children. Among CSHCN, however, adopting any quantitative standard was positively associated with caregivers feeling frustrated in getting services for the child, especially among CSHCN who visited non-mental health specialists. Without additional interventions, adopting new network adequacy standards may have unintended consequences for CSHCN.

Dysregulation of Immune Cell Subpopulations in Atypical Hemolytic Uremic Syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a rare, life-threatening thrombotic microangiopathy. Definitive biomarkers for disease diagnosis and activity remain elusive, making the exploration of molecular markers paramount. We conducted single-cell sequencing on peripheral blood mononuclear cells from 13 aHUS patients, 3 unaffected family members of aHUS patients, and 4 healthy controls. We identified 32 distinct subpopulations encompassing 5 B-cell types, 16 T- and natural killer (NK) cell types, 7 monocyte types, and 4 other cell types. Notably, we observed a significant increase in intermediate monocytes in unstable aHUS patients. Subclustering analysis revealed seven elevated expression genes, including NEAT1, MT-ATP6, MT-CYB, VIM, ACTG1, RPL13, and KLRB1, in unstable aHUS patients, and four heightened expression genes, including RPS27, RPS4X, RPL23, and GZMH genes, in stable aHUS patients. Additionally, an increase in the expression of mitochondria-related genes suggested a potential influence of cell metabolism on the clinical progression of the disease. Pseudotime trajectory analysis revealed a unique immune cell differentiation pattern, while cell-cell interaction profiling highlighted distinctive signaling pathways among patients, family members, and controls. This single-cell sequencing study is the first to confirm immune cell dysregulation in aHUS pathogenesis, offering valuable insights into molecular mechanisms and potential new diagnostic and disease activity markers.

Visualizing Metal Distribution in Plants Using Synchrotron X-Ray Fluorescence Microscopy Techniques.

Recent improvements in synchrotron-based X-ray fluorescence (SXRF) microscopy established it as an advanced analytical tool for analyzing 2D- and 3D distribution of mineral elements in plants. Among existing imaging techniques, SXRF microscopy offers several unique capabilities, including in situ metal quantification in plant tissues and high sensitivity, as low as 1 mg kg-1, at the nanoscale spatial resolution. SXRF is increasingly utilized in different plant science disciplines to provide a fundamental understanding of metal homeostasis, and the function of trace elements in plant metabolism and development. Here, we describe methods for SXRF imaging, including sample preparation, the optimization of conventional SXRF for analyzing trace elements, and the development of confocal SXRF (C-SXRF).

RNA recruitment switches the fate of protein condensates from autophagic degradation to accumulation.

Protein condensates can evade autophagic degradation under stress or pathological conditions. However, the underlying mechanisms are unclear. Here, we demonstrate that RNAs switch the fate of condensates in Caenorhabditis elegans. PGL granules undergo autophagic degradation in embryos laid under normal conditions and accumulate in embryos laid under heat stress conditions to confer stress adaptation. In heat-stressed embryos, mRNAs and RNA control factors partition into PGL granules. Depleting proteins involved in mRNA biogenesis and stability suppresses PGL granule accumulation and triggers their autophagic degradation, while loss of activity of proteins involved in RNA turnover facilitates accumulation. RNAs facilitate LLPS of PGL granules, enhance their liquidity, and also inhibit recruitment of the gelation-promoting scaffold protein EPG-2 to PGL granules. Thus, RNAs are important for controlling the susceptibility of phase-separated protein condensates to autophagic degradation. Our work provides insights into the accumulation of ribonucleoprotein aggregates associated with the pathogenesis of various diseases.

Loss of OPT3 function decreases phloem copper levels and impairs crosstalk between copper and iron homeostasis and shoot-to-root signaling in Arabidopsis thaliana.

Copper (Cu) and iron (Fe) are essential micronutrients that are toxic when accumulating in excess in cells. Thus, their uptake by roots is tightly regulated. While plants sense and respond to local Cu availability, the systemic regulation of Cu uptake has not been documented in contrast to local and systemic control of Fe uptake. Fe abundance in the phloem has been suggested to act systemically, regulating the expression of Fe uptake genes in roots. Consistently, shoot-to-root Fe signaling is disrupted in Arabidopsis thaliana mutants lacking the phloem companion cell-localized Fe transporter, OLIGOPEPTIDE TRANSPORTER 3 (AtOPT3). We report that AtOPT3 also transports Cu in heterologous systems and contributes to its delivery from sources to sinks in planta. The opt3 mutant contained less Cu in the phloem, was sensitive to Cu deficiency and mounted a transcriptional Cu deficiency response in roots and young leaves. Feeding the opt3 mutant and Cu- or Fe-deficient wild-type seedlings with Cu or Fe via the phloem in leaves downregulated the expression of both Cu- and Fe-deficiency marker genes in roots. These data suggest the existence of shoot-to-root Cu signaling, highlight the complexity of Cu/Fe interactions, and the role of AtOPT3 in fine-tuning root transcriptional responses to the plant Cu and Fe needs.

Integrated genomic analyses of hepatocellular carcinoma.

BACKGROUND: Genomic alterations play important roles in the development of cancer. We explored the impact of protein-coding genes and transcriptomic changes on clinical and molecular alterations in Taiwanese hepatocellular carcinoma (HCC) patients. METHODS: We analyzed 147 whole-exome sequencing and 100 RNA sequencing datasets of HCC and compared them with The Cancer Genome Atlas (TCGA)-Liver Hepatocellular Carcinoma cohort and develop a panel of 81 apoptosis-related genes for molecular classification. RESULTS: TERT (50%), TP53 (25%), CTNNB1 (14%), ARID1A (12%), and KMT2C (11%) were the most common genetic alterations of cancer-related genes. ALDH2 and KMT2C mutated at much higher frequencies in our cohort than in TCGA, whereas CTNNB1 was found only in 14% of our Taiwanese patients. A high germline mutation rate of ALDH2 in the APOBEC mutational signature and herb drug-related aristolochic acid-associated signature was also observed. Groups A and B of HCC were identified when we used apoptosis-related genes for molecular classification. The latter group, which had poorer survival outcomes, had significantly more aDC, CD4+ Tem, macrophages M2, NKT, plasma cells, and Th1 cells, and less CD4+ memory T cells, CD8+ Tcm, cDC, iDC, and Th2 cells, as well as more inter-chromosome fusion genes. Metatranscriptomic analysis revealed 54 cases of HBV infection. Moreover, we found that the main target gene of HBV integration is ALB. CONCLUSIONS: Unique genomic alterations were observed in our Taiwanese HCC patients. Molecular classification using apoptosis-related genes could lead to new therapeutic approaches for HCC.

Application of microfluidic assays for cardiovascular disease markers in early warning and rapid diagnosis / 中华预防医学杂志

Cardiovascular disease is a major threat to human health and has become the leading cause of death worldwide; therefore, early diagnosis and treatment are of great value. Due to its miniaturization, integration, and ease of operation, microfluidic technology enables the rapid, multi-target detection of cardiovascular disease markers and significantly facilitates the early and rapid diagnosis of cardiovascular disease. This article reviews the research progress of microfluidics in cardiovascular disease detection, analyzes its advantages and weaknesses in the rapid detection of protein, lipid, and nucleic acid biomarkers, hopes to provide a reference to promote the quick detection technology of cardiovascular disease, and thus proposes new considerations for the early management of cardiovascular disease.

Single-cell RNA sequencing of immune cells in patients with acute gout.

Cell subpopulations in the blood and joint fluid of patients with gout are poorly understood. Single-cell RNA sequencing and bioinformatic tools were used to identify cell subsets and their gene signatures in blood and synovial fluid (SF) cells, determine their relationships, characterize the diversity, and evaluate interactions among specific cell types. We identified 34 subpopulations (5 types of B cells, 16 types of T and natural killer cells, 9 types of monocytes, and 4 other cell types) in the blood of five healthy subjects and seven patients with acute gouty, and the SF of three patients with acute gout. We found that naïve CD4 T cells and classical monocytes cell populations were enriched in patients with gout, whereas plasmacytoid dendritic cells and intermediate monocytes were more abundant in healthy subjects. SF was enriched in Th1/Th17 cells, effector memory CD8 T cells, mucosal-associated invariant T cells, and macrophages. Subclusters of these cell subpopulations showed different compositions between healthy subjects and those with acute gout, according to blood and SF samples. At the cellular level, the inflammation score of a subpopulation or subcluster was highest in SF, following by the blood of acute gout patients and healthy person, whereas energy score showed the opposite trend. We also detected specific cell-cell interactions for interleukin-1, tumor necrosis factor-α, and transforming growth factor-ß1 expression in the cells of patients with acute gout. Our study reveals cellular and molecular insights on inflammatory responses to hyperuricemia or uric crystal and may provide therapeutic guidance to improve treatments for gout.

Molecular Classification of Hepatocellular Carcinoma Using Wnt-Hippo Signaling Pathway-Related Genes.

In Taiwan, a combination of hepatitis B and C infection, economic boom-related food and alcohol overconsumption, and Chinese medicine prescriptions has led to a high rate of hepatocellular carcinoma (HCC). However, the causative factors and underlying tumor biology for this unique HCC environment have not been identified. Wnt and Hippo signaling pathways play an important regulatory role in HCC development, and their functions are generally considered as positive and negative regulators of cell proliferation, respectively. In this study, we characterized the molecular features of HCC using a newly developed classification system based on the expression of the Wnt-Hippo signaling pathway-related genes. RNA sequencing (RNA-Seq) was performed on liver tumor tissues from 100 patients with liver cancer. RNA-Seq data for 272 previously characterized Wnt-Hippo signaling pathway-related genes were used for hierarchical clustering. We analyzed the data in terms of prognostic value, transcriptome features, immune infiltration, and clinical characteristics, and compared the resulting subclasses with previously published classifications. Four subclasses of HCC (HCCW1-4) were identified. Subclass HCCW1 displayed the highest PCDHB4 expression. Subclass HCCW2 displayed lower Edmondson-Steiner grades (I and II) and CTNNB1 mutation frequencies. Subclass HCCW3 was associated with a good prognosis, the highest PCDHGB7 expression, high CD8+ naïve T cells abundance, and relatively low TP53 mutation rates. Subclass HCCW4 was associated with a poor prognosis, the highest PCDHB2 and PCDHB6 expression, a relatively high abundance of Th1 cells, NKT and class-switched memory B cells, relatively low enrichment of cDC, iDC, and CD4+ memory T cells, and high Edmondson-Steiner grades (III and IV). We also identified Wnt-Hippo signaling pathway-related genes that may influence immune cell infiltration. We developed a panel of 272 Wnt-Hippo signaling pathway-related genes to classify HCC into four groups based on Taiwanese HCC and The Cancer Genome Atlas Liver Hepatocellular Carcinoma datasets. This novel molecular classification system may aid the treatment of HCC.

Exploring RNA modifications, editing, and splicing changes in hyperuricemia and gout.

Hyperuricemia and gout are two of the most common metabolic disorders worldwide; their incidence is increasing with changes in lifestyle, and they are correlated with many diseases, including renal and cardiovascular diseases. The majority of studies on hyperuricemia and gout have focused on the discovery of the associated genes and their functions and on the roles of monocytes and neutrophils in the development of gout. Virtually no studies investigating the epigenomics of gout disease or exploring the clinical significance of such research have been conducted. In this study, we observed that the expression of enzymes involved in RNA modifications or RNA editing was affected in uric acid (UA)- or monosodium urate (MSU)-treated cell lines. RNA alternative splicing and splicing factors were also affected by UA or MSU treatment. We used transcriptome sequencing to analyze genome-wide RNA splicing and RNA editing and found significant changes in RNA splicing and RNA editing in MSU- or UA-treated THP-1 and HEK293 cells. We further found significant changes of RNA modifications, editing, and splicing in patients with gout. The data indicate that RNA modifications, editing, and splicing play roles in gout. The findings of this study may help to understand the mechanism of RNA splicing and modifications in gout, facilitating the development of new diagnostic and therapeutic strategies.

National Estimates of Mental Health Needs Among Adults With Self-Reported CKD in the United States.

Introduction: Among adults with chronic kidney disease (CKD), comorbid mental illness is associated with poorer health outcomes and can impede access to transplantation. We provide the first US nationally representative estimates of the prevalence of mental illness and mental health (MH) treatment receipt among adults with self-reported CKD. Methods: Using 2015 to 2019 National Survey on Drug Use and Health (NSDUH) data, we conducted an observational study of 152,069 adults (age ≥22 years) reporting CKD (n = 2544), with no reported chronic conditions (n = 117,235), or reporting hypertension (HTN) or diabetes mellitus (DM) but not CKD (HTN/DM, n = 32,290). We compared prevalence of (past-year) any mental illness, serious mental illness (SMI), MH treatment, and unmet MH care needs across the groups using logistic regression models. Results: Approximately 26.6% of US adults reporting CKD also had mental illness, including 7.1% with SMI. When adjusting for individual characteristics, adults reporting CKD were 15.4 percentage points (PPs) and 7.3 PPs more likely than adults reporting no chronic conditions or HTN/DM to have any mental illness (P < 0.001) and 5.6 PPs (P < 0.001) and 2.2 PPs (P = 0.01) more likely to have SMI, respectively. Adults reporting CKD were also more likely to receive any MH treatment (21% vs. 12%, 18%, respectively) and to have unmet MH care needs (6% vs. 3%, 5%, respectively). Conclusion: Mental illness is common among US adults reporting CKD. Enhanced management of MH needs could improve treatment outcomes and quality-of-life downstream.

Mutation Analysis of Second Primary Tumors in Oral Cancer in Taiwanese Patients through Next-Generation Sequencing.

Head and neck cancer has poor overall survival. Patients with head and neck cancer more frequently develop second primary tumors than do patients with other cancers, leading to a poor prognosis. In this study, we used next-generation sequencing to analyze and compare mutations between first tumors and second tumors in oral cancer. We retrieved tumor tissues collected from 13 patients who were diagnosed twice as having cancer. We used driver gene and trunk mutations to distinguish between recurrent cancer and primary cancer in oral cancer. We observed unique driver gene mutations in three patients with an initial clinical diagnosis of recurrent cancer; hence, we believe that the corresponding patients had primary cancer. Four patients with an initial clinical diagnosis of primary cancer were found to actually have recurrent cancer according to our results. Genetic testing can be used to enhance the accuracy of clinical diagnosis.