RNA Levels Research Articles

Epigenetic landscape reorganisation and reactivation of embryonic development genes are associated with malignancy in IDH-mutant astrocytoma

Accurate grading of IDH-mutant gliomas defines patient prognosis and guides the treatment path. Histological grading is challenging, and aside from CDKN2A/B homozygous deletions in IDH-mutant astrocytomas, there are no other objective molecular markers used for grading. RNA-sequencing was conducted on primary IDH-mutant astrocytomas (n = 138) included in the prospective CATNON trial, which was performed to assess the prognostic effect of adjuvant and concurrent temozolomide. We integrated the RNA-sequencing data with matched DNA-methylation and NGS data. We also used multi-omics data from IDH-mutant astrocytomas included in the TCGA dataset and validated results on matched primary and recurrent samples from the GLASS-NL study. Since discrete classes do not adequately capture grading of these tumours, we utilised DNA-methylation profiles to generate a Continuous Grading Coefficient (CGC) based on classification scores from a CNS-tumour classifier. CGC was an independent predictor of survival outperforming current WHO-CNS5 and methylation-based classification. Our RNA-sequencing analysis revealed four distinct transcription clusters that were associated with (i) upregulation of cell cycling genes; (ii) downregulation of glial differentiation genes; (iii) upregulation of embryonic development genes (e.g. HOX, PAX, and TBX) and (iv) upregulation of extracellular matrix genes. The upregulation of embryonic development genes was associated with a specific increase of CpG island methylation near these genes. Higher grade IDH-mutant astrocytomas have DNA-methylation signatures that, on the RNA level, are associated with increased cell cycling, tumour cell de-differentiation and extracellular matrix remodelling. These combined molecular signatures can serve as an objective marker for grading of IDH-mutant astrocytomas.

Molecular and Clinical Features of Pancreatic Acinar Cell Carcinoma: A Single-Institution Case Series

Objectives: Acinar cell carcinoma (ACC) accounts for about 1% of pancreatic cancers. The molecular and clinical features of ACC are less characterized than those of pancreatic ductal adenocarcinoma. Methods: We retrospectively evaluated the clinical and molecular features of ACC patients who underwent germline and/or somatic molecular testing at The University of Texas MD Anderson Cancer Center from 2008 to 2022 and two cases from 2023–2024 who underwent RNA and TME analysis by Boston Gene. Patient information was extracted from our institutional database with the approval of the Institutional Review Board. Results: We identified 16 patients with available molecular testing results. Fourteen patients had metastatic disease, one had borderline resectable disease, and one had localized resectable disease at diagnosis. Fifteen patients were wild type for KRAS (one patient had unknown KRAS status). Somatic/germline mutations of DNA damage repair genes (BRCA1/2, PALB2, and ATM) were present in 5 of 12 patients tested for these genes. One patient was found to have RET fusion and responded favorably to selpercatinib for over 42 months. The median overall survival (OS) was 24 months for patients with metastatic disease. One of the additional two cases who underwent BostonGene testing was found to have NTRK1 fusion. RNA and TME analysis by Boston Gene of the two cases reported immune desert features and relatively lower RNA levels of CEACAM5, CD47, CD74, and MMP1 and higher RNA levels of CDH6 compared with PDAC.

Optimizing a CRISPR-Cas13d Gene Circuit for Tunable Target RNA Downregulation with Minimal Collateral RNA Cutting.

The invention of RNA-guided DNA cutting systems has revolutionized biotechnology. More recently, RNA-guided RNA cutting by Cas13d entered the scene as a highly promising alternative to RNA interference to engineer cellular transcriptomes for biotechnological and therapeutic purposes. Unfortunately, "collateral damage" by indiscriminate off-target cutting tampered enthusiasm for these systems. Yet, how collateral activity, or even RNA target reduction depends on Cas13d and guide RNA abundance has remained unclear due to the lack of expression-tuning studies to address this question. Here we use precise expression-tuning gene circuits to show that both nonspecific and specific, on-target RNA reduction depend on Cas13d and guide RNA levels, and that nonspecific RNA cutting from trans cleavage might contribute to on-target RNA reduction. Using RNA-level control techniques, we develop new Multi-Level Optimized Negative-Autoregulated Cas13d and crRNA Hybrid (MONARCH) gene circuits that achieve a high dynamic range with low basal on-target RNA reduction while minimizing collateral activity in human kidney cells and green monkey cells most frequently used in human virology. MONARCH should bring RNA-guided RNA cutting systems to the forefront, as easily applicable, programmable tools for transcriptome engineering in biotechnological and medical applications.

CircTSN promotes the proliferation and metastasis of gastric cancer through the miR-1825/SLC38A2 signaling axis.

Comprehensive treatment of gastric cancer (GC) is progressing, but the rapid proliferation and metastasis of GC remains a cause of high recurrence and mortality rates. In this study we investigated GC-associated circRNA tending to yield more insight into the mechanisms of gastric cancer development. We detected the expression levels of circTSN in GC tissues and cell lines using qRT-PCR. The circular structure of circTSN was confirmed by Sanger sequencing, agarose gel electrophoresis and RNase R. A series of cell functional experiments were employed to investigate the implication of circTSN aberrant expression on the proliferation and metastasis of GC cells. The predicted binding domain between circTSN and miR-1825 was analyzed by luciferase reporter gene analysis. Meanwhile, subcutaneous tumor xenografts in nude mice were used to validate the role of circTSN in vivo. It was found that RNA levels of circTSN were significantly elevated in GC tissues and cell lines, which was also confirmed to contain a closed-loop structure. CCK8, clone formation, EdU, transwell and in vivo experiments indicated that the highly expressed circTSN was involved in the proliferation and metastasis process of GC. In addition, circTSN modulates the expression of SLC38A2 by sequence-specific binding to miR-1825. This study identified that circTSN, which is highly expressed in GC, was able to contribute to the proliferation and metastasis of GC cell through miR-1825/SLC38A2 axis and this might provide a new candidate target for the precision treatment of GC.

7877 Adipose Depot Differences In Regenerative Properties Of Exosomes Derived From Human Adipose Stem Cells

Abstract Disclosure: M.A. Krause-Hauch: None. R. Patel: None. B. Osborne: None. P. Albear: None. N.A. Patel: None. Many patients struggle with chronic recalcitrant wounds that are the result of delayed healing. Underlying inflammation is one major risk factor for impaired dermal wound healing that may lead to pathologies such as infection and scarring. Adult human adipose stem cells (hASCs) have shown tremendous potential in regenerative medicine. The regenerative potential of hASCs is dependent on secreted bioactive material which is packaged and released in extracellular exosomes. We previously showed that hASC exosomes from the subcutaneous depot (sc) promoted repair in human dermal fibroblasts using in vitro assays. In this study, we evaluated exosomes derived from the omental depot (om) of hASC and compared its efficacy with sc-hASC. Our results show differences in levels of the long noncoding RNA (lncRNA) cargo between om-hASCexo and sc-hASCexo. Next, we evaluated their repair potential in a wound model in vivo. Male and female F344 rats were dorsally wounded via punch biopsy and silicone scaffolding was attached to prevent contraction. The wounds were then either not treated (control) or treated with 50μg om-hASCexo or sc-hASCexo every alternate day when each wound size was measured for 1 week to monitor healing progress. After 7 days, wounds treated with either om-hASCexo and sc-hASCexo closed significantly faster than untreated control wounds. While no difference was observed in closure percentage after 7 days for wounds treated with either om-hASCexo and sc-hASCexo, sc-hASCexo appeared to act faster earlier (1-2 days post wound) in the wound healing process while om-hASCexo acts faster during later stages (2-4 days post wound) of wound healing. The wound tissue was collected for biochemical and histological analysis. qPCR analysis on day 7 revealed that both om-hASCexo and sc-hASCexo treatment resulted in increased levels of Collagen 1 & 3 and MMP9, while levels of TGF-β decreased with treatment. These results indicate that om-hASCexo and sc-hASCexo enhanced the wound healing process while decreasing inflammation of the wound. To determine the differences between om-hASCexo and sc-hASCexo treatments on wound healing pathways, RNAseq was performed. Results show specificity in pathway activation and gene expression between the om-hASCexo and sc-hASCexo treatments. In conclusion, we have demonstrated that the regenerative mechanisms differ between the hASC depots from which exosomes are secreted. At a broader level, this finding may shed light on the body’s organ health and repair processes in normal or disease states. Presentation: 6/3/2024

7704 Generating pituitary cells from iPSCs of patients with child onset congenital hypopituitarism harboring PROP1 pathogenic allelic variants

Abstract Disclosure: J.M. Marques: None. F. Fattahi: None. L.R. Carvalho: None. Introduction: Reduced or absent levels of pituitary hormones due to genetic or organic causes is known as combined pituitary hormone deficiency (CPHD) and PROP1 is the most frequent cause of CPHD, but more than 85% of the cases are without molecular diagnosis. Novel Techniques such as induced pluripotent stem cells (iPSC), can be used to generate patient’s specific pituitary cells for disease modeling. Aim: To generate iPSC from CO-CH patients’ peripheral blood mononuclear cells (PBMC) and differentiate them into pituitary cells. Methods: Peripheral blood was collected from 2 siblings (HC1 and HC2) harboring compound heterozygous PROP1 allelic variants and their parents (HC3 and HC4). PBMCs were isolated and expanded with MNC medium prior to nucleoporation and transfection with plasmids containing pluripotency genes (OCT4 and SOX2; cMYC and KLF). After 48h, transfected cells were transferred to Geltrex coated plates and fed with E8, NaB and βFGF until pluripotent colonies were formed. iPSCs were differentiated into cranial placode cells using pituitary placode conditioned medium (PPCM) until day 15. For pituitary cell maturation, pituitary conditioned medium (PPCM without SB431542) was used until day 30. qPCR and flow cytometry using the Kit BD Stemflow - Human Pluripotent Stem Cell Transcription Factor Analysis (BD Biosciences) for stem cells and the eBioscience™ Foxp3 / Transcription Factor Fixation/Permeabilization (Thermo Fisher Scientific) for the placode and pituitary markers were used. Pituitary hormonal levels from cell supernatant were measured at the Laboratory of Hormones and Molecular Genetics - HCFMUSP. Results: we generated iPSCs from patient and control blood cells. iPSCs were more than 90% positive for the pluripotency markers SOX2, NANOG, OCT3/4. Pituitary cells were generated with positivity for the placode marker SIX1 (58%) and pituitary markers, as LHX3 (56%) and PROP1 (59%), at day 15. Control cell lineages expressed higher levels of pituitary hormones at days 15 and 30 at RNA and protein levels while patient cells failed to induce hormone-producing cells properly. Pituitary hormonal release was impaired in patients pituitary cells. Conclusion: CO-CH patients’ PBMC were dedifferentiated to a pluripotent state, being able to generate pituitary hormone producing cells. Patient cells were able to mimic human phenotype. These cells can be used, in the future, to study CO-CH basis in patients specific background, to develop pituitary disease modeling as well as test possible new treatments. Presentation: 6/3/2024

12622 Molecular Physiology And Compensatory Response In Maladapted Pancreatic Islets Of Humanized Mice Model For Type 1 Diabetes

Abstract Disclosure: A. Hussain: None. A. Aldasouqi: None. S. Rafiqi: None. S.S. Khan: None. R. Paparodis: None. J.C. Jaume: None. S. Imam: None. Genetic predisposition linked with specific HLA (DR and DQ) alleles, environmental factors, and other uncharacterized physiological aberrations triggers an autoimmune attack and depletion of β cells in pancreatic islets, leading to decreased insulin secretion and diabetes. Our laboratory has generated humanized mice carrying the antigen-presenting HLA-DQ8 diabetes-linked haplotype and expressing the human autoantigen GAD65 in pancreatic beta cells. These humanized mice spontaneously develop type 1 diabetes (T1D) and represent the full spectrum of pathophysiological conditions experienced in T1D. Using humanized mice as a model of study, we studied the molecular physiology and compensatory response in maladapted pancreas under normal physiological conditions and hyperglycemic stress of T1D. We studied the differential expression of some important genes by quantitative PCR in maladapted islets to identify the physiological processes affected during the initial oxidative stress exerted by autoimmune conditions. Our results confirmed that levels of cyclin B, cMyc, Cyc D, Cyc E, PDGFRA, and NOTCH are reduced during autoimmune insult, reflecting the poor regenerative capacity of islet cells. However, Cyc A has an elevated expression, reflecting that islet cells, particularly the β cells of Type 1 diabetic mice, may have regenerative potential that may be explored. The genes for Desmin and Survivin showed decreased expression, whereas genes for Smad and Synuclein displayed increased expression. The genes for insulin and somatostatin showed elevated expression, whereas the gene for glucagon displayed reduced expression. The results suggest that during autoimmune insults, the β cells moderately whip up the synthesis of insulin-specific RNA to maintain the requirements. Among the endoplasmic reticulum-specific molecular chaperones, Calnexin showed an elevated expression, whereas calreticulin displayed a reduced expression. Among the genes involved with the architecture of the islet, Lamin and Integrin both display increased expression, reflecting the dynamic change in islet architecture during the autoimmune depletion of β cells. We also investigated the expression of ER-specific stress genes both at RNA and protein levels. Our preliminary results have confirmed that ER-specific stress genes showed an elevated expression during oxidative stress mediated by autoimmune T1D conditions. Presentation: 6/3/2024

The Serratia sp. strain C2 confers tomato tolerance to high salt, virus infection and both stresses in combination

Besides increasing plant growth, several Plant Growth Promoting Rhizobacteria (PGPR), can enhance tolerance to biotic and/or abiotic stresses of numerous plant species. While cultivated plants are frequently subject to combined stresses in the field, there is limited knowledge of the effect of PGPR on plants undergoing simultaneous stress conditions. Therefore, we tested the beneficial properties of the halotolerant PGPR Serratia sp. strain C2, previously shown to enhance salt stress tolerance in barley, on tomato plants exposed to salinity, to Potato Virus Y (PVY) infection, and both stresses simultaneously. In our experimental conditions, C2 inoculation improved tomato tolerance to salt stress and positively correlated with a 46–68 % decrease in the level of PVY RNA compared to non-inoculated tomato plants. Morphometric, physiological and biochemical analyses (e.g., chlorophyll, sugar and proline accumulation, oxidative stress status and NDVI) indicated that C2 treatments had beneficial effects on tomato growth under simple and combined stress conditions. This is the first report of a PGPR enhancing tolerance not only to individually induced salinity and PVY infection, but also to both stresses in combination. Moreover, the expression analysis of selected genes involved in stress responses and RNA silencing-mediated antiviral immunity suggests that C2 can interfere with distinct defence response pathways to enhance stress tolerance in tomato. These pioneering results support the perspective of using PGPR as multi-spectrum and multi-host biostimulants for improving plant growth and protection from biotic, abiotic, and combined stresses to promote sustainable crop production in the face of environmental changes.

Developmental assembly of multi-component polymer systems through interconnected synthetic gene networks in vitro

Living cells regulate the dynamics of developmental events through interconnected signaling systems that activate and deactivate inert precursors. This suggests that similarly, synthetic biomaterials could be designed to develop over time by using chemical reaction networks to regulate the availability of assembling components. Here we demonstrate how the sequential activation or deactivation of distinct DNA building blocks can be modularly coordinated to form distinct populations of self-assembling polymers using a transcriptional signaling cascade of synthetic genes. Our building blocks are DNA tiles that polymerize into nanotubes, and whose assembly can be controlled by RNA molecules produced by synthetic genes that target the tile interaction domains. To achieve different RNA production rates, we use a strategy based on promoter “nicking” and strand displacement. By changing the way the genes are cascaded and the RNA levels, we demonstrate that we can obtain spatially and temporally different outcomes in nanotube assembly, including random DNA polymers, block polymers, and as well as distinct autonomous formation and dissolution of distinct polymer populations. Our work demonstrates a way to construct autonomous supramolecular materials whose properties depend on the timing of molecular instructions for self-assembly, and can be immediately extended to a variety of other nucleic acid circuits and assemblies.

A Randomized, Double-Blind, Placebo-Controlled, Short-Term Monotherapy Study of MK-6186, an HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitor, in Treatment-Naïve HIV-Infected Participants.

Objective: To assess the antiviral activity, pharmacokinetics, and safety of MK-6186 in HIV-1 non-nucleoside reverse transcriptase inhibitor (NNRTI)-naïve, HIV-1-infected male participants. Design: Double-blind, randomized, two-panel study. Methods: In 2 sequential panels, 18 participants received MK-6186 (40 mg [Panel A] or 150 mg [Panel B]) or matching placebo once daily for 7 days. Plasma samples were collected for measurement of HIV-1 RNA levels and MK-6186 pharmacokinetics. Results: For the mean change from baseline in HIV-1 RNA (log10 copies/mL) at 24 h post Day 7 dose, the mean difference (90% confidence interval) between MK-6186 and placebo was -1.54 (-1.73, -1.34) in the 40-mg group and -1.28 (-1.81, -0.75) in the 150-mg group. One participant in the 150-mg group had viral rebound at 24 h after Day 6 dosing (Day 7 predose) associated with outgrowth of the V106A minority variant. Ultra-deep sequencing confirmed expansion of this predose minority variant from 0.26% to 63.67%. No outgrowth or rebound was seen in another participant in whom a V106A minority variant was also detected. MK-6186 was generally well tolerated. MK-6186 was rapidly absorbed with peak concentrations at 2 h followed by a biphasic decline. The effective t½ of MK-6186 was 43.9 to 48.7 h. Steady state was not achieved. Conclusions: Daily monotherapy with MK-6186 demonstrated robust antiviral activity with maximal antiviral activity at a dose of 40 mg. One participant in the 150-mg group exhibited viral rebound with outgrowth of the resistant V106A minority variant, demonstrating a risk of resistance development typical of NNRTIs. The reason for this outgrowth remains unclear as no outgrowth occurred in a participant in the 40-mg group in whom the V106A minority variant was also detected. MK-6186 may be an alternative next-generation NNRTI in combination therapy, in that combination antiretroviral therapy could prevent outgrowth of resistant minority variants.

Rhesus rotavirus NSP1 mediates extra-intestinal infection and is a contributing factor for biliary obstruction.

We previously demonstrated that in Ifnar1-/-Ifngr1-/- or Stat1-/- suckling mice lacking intact type I and type II interferon (IFN) signaling, rhesus rotavirus (RRV) infection causes a lethal disease with clinical manifestations similar to biliary atresia, including acholic stools, oily fur, growth retardation, and excess mortality. Elevated levels of viral RNA are detected in the bile ducts and liver of diseased pups together with severe inflammatory responses in these tissues. However, the viral determinants and the molecular mechanisms driving this process remain incompletely understood. Using an optimized rotavirus (RV) reverse genetics system, we generated a panel of recombinant RVs that encode non-structural protein 1 (NSP1) derived from different RV strains. We found that compared to the parental simian SA11 strain that is less biliary pathogenic, SA11 containing an RRV-derived NSP1 resulted in severe biliary obstructive disease comparable to that associated with RRV infection, reflected by high levels of viral RNA and inflammation in the biliary tract, liver, and pancreas. In contrast, RRV containing an SA11-originated NSP1 showed only mild biliary obstruction comparable to what was observed during SA11 infection. Infection with a monoreassortant RRV virus carrying NSP1 from the bovine RV UK strain also showed substantially reduced viral replication in extra-intestinal organs and did not develop clinical biliary diseases. Mechanistically, RRV NSP1 seemed to promote active viral replication in hepatocytes and this expanded tropism led to enhanced infiltration of CD4 and CD8 T cells, causing immunopathology and damage in the hepatobiliary system. These results highlight an unexpectedly important role of RV NSP1 in viral replication and disease progression in extra-intestinal tissues.

Ubiquitin-independent degradation of Bim blocks macrophage pyroptosis in sepsis-related tissue injury

Pyroptosis, a typical inflammatory cell death mode, has been increasingly demonstrated to have therapeutic value in inflammatory diseases such as sepsis. However, the mechanisms and therapeutic targets of sepsis remain elusive. Here, we reported that REGγ inhibition promoted pyroptosis by regulating members of the gasdermin family in macrophages. Mechanistically, REGγ directly degraded Bim, a factor of the Bcl-2 family that can inhibit the cleavage of GSDMD/E, ultimately preventing the occurrence of pyroptosis. Furthermore, cecal ligation and puncture (CLP)-induced sepsis model mice showed downregulation of REGγ at both the RNA and protein levels. Gasdermin-mediated pyroptosis was augmented in REGγ-knockout mice, and these mice exhibited more severe sepsis-related tissue injury. More importantly, we found that REGγ expression was downregulated in clinical sepsis samples, such as those from patients with Pseudomonas aeruginosa (PA) infection. Finally, PA-infected mice showed decreased REGγ levels in the lung. In summary, our study reveals that the REGγ-Bim-GSDMD/E pathway is a novel regulatory mechanism of pyroptosis in sepsis-related tissue injury.

Identification of Human Pathways Acting on Nuclear Non-Coding RNAs Using the Mirror Forward Genetic Approach.

Despite critical roles in diseases, human pathways acting on strictly nuclear non-coding RNAs have been refractory to forward genetics. To enable their forward genetic discovery, we developed a single-cell approach that "Mirrors" activities of nuclear pathways with cytoplasmic fluorescence. Application of Mirror to two nuclear pathways targeting MALAT1's 3' end, the pathway of its maturation and the other, the degradation pathway blocked by the triple-helical Element for Nuclear Expression (ENE), identified nearly all components of three complexes: Ribonuclease P and the RNA Exosome, including nuclear DIS3, EXOSC10, and C1D, as well as the Nuclear Exosome Targeting (NEXT) complex. Additionally, Mirror identified DEAD-box helicase DDX59 associated with the genetic disorder Oral-Facial-Digital syndrome (OFD), yet lacking known substrates or roles in nuclear RNA degradation. Knockout of DDX59 exhibits stabilization of the full-length MALAT1 with a stability-compromised ENE and increases levels of such long non-coding RNAs as NEAT1_1 and NIPBL-DT, as well as 3'-extended forms of small nuclear RNAs. It also exhibits extensive retention of minor introns, including in OFD-associated genes, suggesting a mechanism for DDX59 association with OFD. Mirror efficiently identifies pathways acting on strictly nuclear non-coding RNAs, including essential and indirectly-acting components, and, as a result, uncovers unexpected links to human disease.

ALKBH5 Regulates Osteogenic Differentiation via the lncRNA/mRNA Complex

Human adipose-derived stem cells (hASCs) are commonly used in bone tissue regeneration. The N6-methyladenosine (m6A) modification has emerged as a novel regulatory mechanism for gene expression, playing a critical role in osteogenic differentiation of stem cells. However, the precise role and mechanism of alkylation repair homolog 5 (ALKBH5) in hASC osteogenesis remain incompletely elucidated and warrant further investigation. Herein, we employed methylated RNA immunoprecipitation sequencing, RNA sequencing, and weighted gene coexpression network analysis to identify a key long noncoding RNA (lncRNA) in hASCs: lncRNA AK311120. Functional experiments demonstrated that lnc-AK311120 promoted the osteogenic differentiation of hASCs, while a mutation at the m6A central site A of lnc-AK311120 was found to decrease the level of m6A modification. The osteogenic effect of ALKBH5 was confirmed both in vitro and in vivo using a mandibular defect model in nude mice. Subsequent investigations revealed that knockdown of ALKBH5 resulted in a significant increase in the m6A modification level of lnc-AK311120, accompanied by a downregulation in the expression level of lnc-AK311120. Additional rescue experiments demonstrated that overexpression of lnc-AK311120 could restore the phenotype after ALKBH5 knockdown. We observed that AK311120 interacted with the RNA-binding proteins DExH-Box helicase 9 (DHX9) and YTH domain containing 2 (YTHDC2) to form a ternary complex, while mitogen-activated protein kinase kinase 7 (MAP2K7) served as the shared downstream target gene of DHX9 and YTHDC2. Knockdown of AK311120 led to a reduction in the binding affinity between DHX9/YTHDC2 and the target gene MAP2K7. Furthermore, ALKBH5 facilitated the translation of MAP2K7 and activated the downstream JNK signaling pathway through the AK311120–DHX9–YTHDC2 complex, without affecting its messenger RNA level. Collectively, we have investigated the regulatory effect and mechanism of ALKBH5-mediated demethylation of lncRNA in hASC osteogenesis for the first time, offering a promising approach for bone tissue engineering.

Extracellular DNA from the blood plasma of patients with schizophrenia stimulates the TLR9-NF-kB signaling pathway in cultured human lymphocytes

Schizophrenia is a mental illness of complex etiology. Recently, there has been increased interest in the role of the immune system in the pathophysiology of mental disorders. The concept of neuroinflammation in neurodevelopmental disorders is gaining widespread interest, including the role of Toll-like receptors.Schizophrenia is associated with an increase in the concentration of cfDNA in human blood, and the composition of cfDNA fragments changes significantly compared to cellular DNA: GC-rich fragments of the ribosomal repeat accumulate and base oxidation occurs. Similar changes, but less pronounced, also occur for cfDNA from healthy donors.To confirm the hypothesis about the possible participation of cfDNA in the inflammation induction, we studied the effect of cfDNA samples on cultured mononuclear cells.Unlike cellular DNA, cfDNA(SZ) and cfDNA(K) stimulate transcription of the TLR9 gene in mononuclear cells. After 1 hour the amount of TLR9 RNA increases by 2.9 and 3.3 times compared to the control. After 24 hours, the TLR9 RNA level decreases slightly, but is still 2-3 times higher than the control level. After 1 hour, TLR9 protein increases by 1.5 and 1.7 times, respectively, and further increased after 24h of culture.An increase TLR9 protein expression correlates with an increase of the transcription factor NF-kB in lymphocytes and is accompanied by an increase in proinflammatory cytokine IL8 RNA, the transcription of IL8 is controlled by the NF-kB factor.Thus, cfDNA(SZ) and cfDNA(K) stimulate the TLR9-NF-kB-proinflammatory cytokine signaling pathway in lymphocytes. The effect of cfDNA also depends on the concentration of these fragments in the extracellular environment. Since the concentrations of cfDNA in the blood of patients with schizophrenia are significantly increased compared to healthy donors, we should expect a much higher level of activation of the TLR9-NF-kB signaling pathway in the body cells of sick people.Samples of cfDNA from patients with schizophrenia have a pronounced biological effect on cells of the immune system, stimulating the synthesis of pro-inflammatory cytokines by activating the TLR9-NF-kB-proinflammatory cytokines signaling pathway. High levels of cfDNA in blood plasma may be one of the reasons for the induction and maintenance of low-level inflammation in schizophrenia.

Abstract C106: Enhanced characterization of molecular attributes through multi-omics analysis and comprehensive evaluation of global and local genomic ancestry in colorectal cancer among Hispanic-Latinos

Abstract Introduction: Colorectal cancer (CRC) remains a significant public health challenge, representing the second leading cause of cancer-related mortality in the United States. Although mortality rates have generally decreased, the Hispanic/Latino (H/L) population in the Los Angeles area still experiences mortality rates up to 20% higher than those of their Caucasian American counterparts. Additionally, they are often diagnosed at a younger age and with more advanced disease stages, highlighting significant disparities in CRC outcomes. Currently, there are limited studies integrating clinical and multi-omics data from H/L populations, which are crucial for understanding the implications of colorectal tumorigenesis and addressing these disparities effectively. Methods: Clinical and genomic sequencing data were obtained from 60 primary CRC Tumor/Normal (T/N) samples within the Hispanic/Latino (H/L) population in the Los Angeles area through the PE-CGS network. For comparison, we analyzed 3,578 samples from non-Hispanic Whites (NHW) obtained from public databases. Additionally, we retrieved three CRC samples with spatial transcriptomic (ST) data from the 10xGenomics database. Using Whole Exome and RNA sequencing data, we performed somatic mutations, somatic copy number alterations (SCNAs), differential gene expression, cellular pathways, gene fusions, and global & local genomic ancestry analyses. The ST data underwent analysis utilizing newly released bioinformatics tools. Clinical and genomic data were integrated. Results: In these studies, distinct Amerindian (AMR) genomic ancestry proportion, age at diagnosis, and tumor localization patterns were observed, alongside significant mutations in key genes such as APC, TP53, and KRAS. The studies revealed evident cancer heterogeneity, influenced by factors such as mutation type, microsatellite instability, tumor subsite, and ethnicity. Additionally, several previously well-defined SCNAs were detected in the majority of tumors. Comparative analysis of gene expression and cellular pathways between H/L and NHW samples revealed distinct patterns. Furthermore, we identified gene fusions that potentially contribute to oncogenic activity in CRC. Ancestry analysis pointed to a predominant AMR ancestry. Notably, distinct associations of genetic ancestry with CRC tumor clinicopathological characteristics were identified. ST analysis delineated separate cancer, immune, and stroma components within the tumors. Conclusion: Our research has explored the molecular profiling of CRC tumors in H/L patients, providing crucial insights into the characterization of CRC tumors at the DNA and RNA levels, as well as the complex clinical and genomic diversity within our target population. Additionally, it offers valuable insights into the heterogeneity of CRC tumors and the tumor microenvironment. These findings establish the groundwork for subsequent projects, potentially leading to precision medicine approaches. Citation Format: Enrique I. Velazquez-Villarreal, Brigette Waldrup, Yonatan Amzaleg, Yuxin Jin, Mackenzie Postel, Donna Loza, Serina Ovalle, Elizabeth Quino, Carmen Chavez, Julie Culver, Mariana C. Stern, Heinz-Josef Lenz, David W. Craig, John D. Carpten. Enhanced characterization of molecular attributes through multi-omics analysis and comprehensive evaluation of global and local genomic ancestry in colorectal cancer among Hispanic-Latinos [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C106.

What's in a name: the multifaceted function of DNA‐ and RNA‐binding proteins in T cell responses

Cellular differentiation allows cells to transition between different functional states and adapt to various environmental cues. The diversity and plasticity of this process is beautifully exemplified by T cells responding to pathogens, which undergo highly specialized differentiation tailored to the ongoing infection. Such antigen‐induced T cell differentiation is regulated at the transcriptional level by DNA‐binding proteins and at the post‐transcriptional level by RNA‐binding proteins. Although traditionally defined as separate protein classes, a growing body of evidence indicates an overlap between these two groups of proteins, collectively coined DNA/RNA‐binding proteins (DRBPs). In this review, we describe how DRBPs might bind both DNA and RNA, discuss the putative functional relevance of this dual binding, and provide an exploratory analysis into characteristics that are associated with DRBPs. To exemplify the significance of DRBPs in T cell biology, we detail the activity of several established and putative DRBPs during the T cell response. Finally, we highlight several methodologies that allow untangling of the distinct functionalities of DRBPs at the DNA and RNA level, including key considerations to take into account when applying such methods.

High serum growth differentiation factor 15 is a risk factor for the occurrence of hepatocellular carcinoma in chronic hepatitisB patients treated with nucleos(t)ide analogs.

Patients with chronic hepatitisB (CHB) remain at risk for hepatocellular carcinoma (HCC) even with nucleos(t)ide analog therapy. We evaluated risk factors for HCC development, including serum hepatitisB virus (HBV) RNA, hepatitis B core-related antigen level, and growth differentiation factor 15 (GDF15) level, a predictor of HCC development in patients with chronic hepatitisC. We collected clinical data and stored serum from CHB patients without a history of HCC who were receiving nucleos(t)ide analog treatment for more than 1year and whose HBV DNA level was less than 3.0 log IU/mL. We measured the serum levels of HBV RNA and GDF15. Among 242 CHB patients, 57 had detectable HBV RNA, and GDF15 was quantified in all patients. The median GDF15 level was 0.86ng/mL. Cox proportional hazards analysis revealed that male sex and higher GDF15, FIB-4 index, alpha-fetoprotein and gamma-glutamyl transpeptidase were independent risk factors for HCC. The presence of HBV RNA above the lower limit of quantification was not a risk factor. When we set cutoff values based on the Youden index, the cumulative incidence of HCC was significantly higher in the male, AFP ≥3.0ng/mL, gamma-glutamyl transpeptidase ≥22U/L, FIB-4 index≥1.93, and GDF-15≥1.17ng/mL groups. In patients with no or more than three of these five risk factors, the 10-year HCC cumulative incidence rates were 0% and 41.0%, respectively. High serum GDF15 is an independent risk factor for the occurrence of HCC in CHB patients treated with nucleos(t)ide analogs.

A novel dual-epigenetic inhibitor enhances recombinant monoclonal antibody expression in CHO cells

Epigenetic regulation plays a central role in the regulation of a number of cellular processes such as proliferation, differentiation, cell cycle, and apoptosis. In particular, small molecule epigenetic modulators are key elements that can effectively influence gene expression by precisely regulating the epigenetic state of cells. To identify useful small-molecule regulators that enhance the expression of recombinant proteins in Chinese hamster ovary (CHO) cells, we examined a novel dual-HDAC/LSD1 inhibitor I-4 as a supplement for recombinant CHO cells. Treatment with 2 μM I-4 was most effective in increasing monoclonal antibody production. Despite cell cycle arrest at the G1/G0 phase, which inhibits cell growth, the addition of the inhibitor at 2 µM to monoclonal antibody-expressing CHO cell cultures resulted in a 1.94-fold increase in the maximal monoclonal antibody titer and a 2.43-fold increase in specific monoclonal antibody production. In addition, I-4 significantly increased the messenger RNA levels of the monoclonal antibody and histone H3 acetylation and methylation levels. We also investigated the effect on HDAC-related isoforms and found that interference with the HDAC5 gene increased the monoclonal antibody titer by 1.64-fold. The results of this work provide an effective method of using epigenetic regulatory strategies to enhance the expression of recombinant proteins in CHO cells.Key points• HDAC/LSD1 dual-target small molecule inhibitor can increase the expression level of recombinant monoclonal antibodies in CHO cells.• By affecting the acetylation and methylation levels of histones in CHO cells and downregulating HDAC5, the production of recombinant monoclonal antibodies increased.• It provides an effective pathway for applying epigenetic regulation strategies to enhance the expression of recombinant proteins.

Investigation of the expression of long non-coding RNA in Parkinson’s disease

BackgroundParkinson’s disease is the second most common age-related neurodegenerative disease after Alzheimer’s. Pathogenic factors in Parkinson’s include inflammation and oxidative stress, which lead to dopaminergic cell apoptosis. The case–control study aims to determine the expression level of long non-coding RNAs (lncRNAs) of the apoptosis pathway in Parkinson’s patients compared to healthy individuals. In the case–control study, 50 patients with Parkinson’s disease were examined, along with 50 healthy individuals matched in age and sex. In both groups, the expression of long non-coding RNAs, including taurine upregulated 1 (TUG1), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), nuclear-enriched abundant transcript 1 (NEAT1), and growth arrest-specific 5 (GAS5), was compared using real-time polymerase chain reaction (PCR).ResultsThe ratio of MALAT1, NEAT1, and TUG1 gene expression in the case group was statistically significantly higher than in healthy individuals. The ratio of GAS5 gene expression in people with Parkinson’s disease was lower, with a statistically significant difference. The ratio of HULC gene expression was higher in the case group, but it did not show a statistically significant difference with the control group.ConclusionThe involvement of long lncRNAs that increase apoptosis may play a role in the pathogenesis of the disease, which may be used for identification and therapeutic purposes.