Log2 Fold Change Research Articles

Transcriptomic profiling highlights cell proliferation in the progression of experimental pulmonary hypertension in rats

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by pulmonary vascular remolding and occlusion, leading to the elevated pulmonary arterial pressures, right ventricular hypertrophy, and eventual heart failure if left untreated. Understanding the molecular mechanisms underlying the development and progression of pulmonary hypertension (PH) is crucial for devising efficient therapeutic approaches for the disease. Lung homogenates were collected weekly and underwent RNA-sequencing in the monocrotaline (MCT)-induced PH rat model to explore genes associated with PH progression. Statistical analyses revealed 1038, 1244, and 3125 significantly altered genes (P < 0.05, abs (log2fold change) > log21.5) between control and MCT-exposed rats during the first, second, and third week, respectively. Pathway enrichment analyses revealed involvement of cell cycle and innate immune system for the upregulated genes, GPCR and VEGF signaling for the downregulated genes. Furthermore, qRT-PCR validated upregulation of representative genes associated with cell cycle including Cdc25c (cell division cycle 25C), Cdc45, Top2a (topoisomerase IIα), Ccna2 (cyclin A2) and Ccnb1 (cyclin B1). Western blot and immunofluorescence analysis confirmed increases in PCNA, Ccna2, Top2a, along with other proliferation markers in the lung tissue of MCT-treated rats. In summary, RNA sequencing data highlights the significance of cell proliferation in progression of rodent PH.

Differential transcriptome study on the damage of testicular tissues caused by chronic infection of T. gondii in mice

BackgroundToxoplasma gondii is an intracellular protozoan parasite that is widely distributed in humans and warm-blooded animals. T. gondii chronic infections can cause toxoplasmic encephalopathy, adverse pregnancy, and male reproductive disorders. In male reproduction, the main function of the testis is to provide a stable place for spermatogenesis and immunological protection. The disorders affecting testis tissue encompass abnormalities in the germ cell cycle, spermatogenic retardation, or complete cessation of sperm development. However, the mechanisms of interaction between T. gondii and the reproductive system is unclear. The aims were to study the expression levels of genes related to spermatogenesis, following T. gondii infection, in mouse testicular tissue.MethodsRNA-seq sequencing was carried out on mouse testicular tissues from mice infected or uninfected with the T. gondii type II Prugniaud (PRU) strain and validated in combination with real-time quantitative PCR and immunofluorescence assays.ResultsThe results showed that there were 250 significant differentially expressed genes (DEGs) (P < 0.05, |log2fold change| ≧ 1). Bioinformatics analysis showed that 101 DEGs were annotated to the 1696 gene ontology (GO) term. While there was a higher number of DEGs in the biological process classification as a whole, the GO enrichment revealed a significant presence of DEGs in the cellular component classification. The Arhgap18 and Syne1 genes undergo regulatory changes following T. gondii infection, and both were involved in shaping the cytoskeleton of the blood–testis barrier (BTB). The number of DEGs enriched in the MAPK signaling pathway, the ERK1/2 signaling pathway, and the JNK signaling pathway were significant. The PTGDS gene is located in the Arachidonic acid metabolism pathway, which plays an important role in the formation and maintenance of BTB in the testis. The expression of PTGDS is downregulated subsequent to T. gondii infection, potentially exerting deleterious effects on the integrity of the BTB and the spermatogenic microenvironment within the testes.ConclusionsOverall, our research provides in-depth insights into how chronic T. gondii infection might affect testicular tissue and potentially impact male fertility. These findings offer a new perspective on the impact of T. gondii infection on the male reproductive system.Graphical

Sponging of five tumour suppressor miRNAs by lncRNA-KCNQ1OT1 activates BMPR1A/BMPR1B-ACVR2A/ACVR2B signalling and promotes chemoresistance in hepatocellular carcinoma

Diverse mechanisms have been established to understand the chemoresistance of hepatocellular carcinoma (HCC), but the contribution of non-coding RNAs is not surveyed well. Here, we aimed to explore the lncRNA-miRNA axis in Hepatitis C and B virus (HCV and HBV) infected HCC to investigate the molecular mechanism of chemoresistance and to identify a potential therapeutic target for HCC. The small RNA transcriptome analysis followed by qRT-PCR validation with the liver tissues of both HCV and HBV infected HCC patients revealed that miR-424-5p, miR-136-3p, miR-139-5p, miR-223-3p, and miR-375-3p were the most downregulated miRNAs in HCC compared to normal (log2 fold change ≤−1.5, Padj ≤ 0.05). In silico pathway analysis with the validated targets of each miRNA revealed that the signalling pathway regulating pluripotency of stem cells is commonly targeted by these five miRNAs. Subsequent validation by 3′UTR-luciferase assay and western blot analysis unveiled that these five miRNAs impeded either same or diverse genes, but all linked to BMP signalling pathway such as BMPR1A/BMPR1B by miR-139-5p, miR-136-3p, and miR-375-3p, and ACVR2A/ACVR2B by miR-424-5p and miR-223-3p. Furthermore, restoration of each miRNA in Huh7/SNU449 cells inhibited phosphorylation of downstream SMAD1/5 and ERK1/2, and attenuated Epithelial-mesenchymal transition, stemness, spheroid formation, chemoresistance, invasion and migration of cells. To investigate the mechanism of suppression of these miRNAs, “DIANA” tool was employed and lncRNA-KCNQ1OT1 was retrieved as interacting partner of all the five miRNAs. In vitro RNA pull-down assay revealed that lncRNA-KCNQ1OT1 physically interacted and sequestered these five miRNAs in the cytoplasm. Hence, KCNQ1OT1 was suppressed in Huh7/SNU449 cells using CRISPR technology and observed regression of oncogenic properties with enhanced chemosensitivity and reduced metastasis in cancer cells. Shrinkage of tumour size and volume in NOD-SCID mice injected with KCNQ1OT1-sgRNA cells further strengthened our observations. Thus, lncRNA-KCNQ1OT1 is the main regulator, which reduces the level of beneficiary miRNAs in the tumour milieu and modulates BMP signalling pathway to promote chemoresistance in HCC, suggesting lncRNA-KCNQ1OT1 might have robust potential to be a therapeutic target in HCC.

Effect of supplementing lysins and methionine to low-protein diets on growth performance, hepatic antioxidant capacity, immune status, and glycolytic activity of tibetan sheep

Reducing the levels of dietary protein is an effective nutritional approach in lowering feed cost and nitrogen emissions in ruminants. The purpose of this study was to evaluate the effects of dietary Lys/Met ratio in a low protein diet (10%, dry matter basis) on the growth performance and hepatic function (antioxidant capacity, immune status, and glycolytic activity) in Tibetan lambs. Ninety two-month-old rams with an average weight of 15.37 ± 0.92 kg were randomly assigned to LP-L (dietary Lys/Met = 1:1), LP-M (dietary Lys/Met = 2:1) and LP-H (dietary Lys/Met = 3:1) treatments. The trial was conducted over 100 d, including 10 d of adaption to the diets. Hepatic phenotypes, antioxidant capacity, immune status, glycolytic activity and gene expression profiling was detected after the conclusion of the feeding trials. The results showed that the body weight was higher in the LP-L group when compared to those on the LP-M group (P < 0.05). In addition, the activities of the catalase (CAT) and glutathione peroxidase (GSH-Px) in the LP-L group were significantly increased compared with the LP-M group (P < 0.05), while the malondialdehyde (MDA) levels in LP-H group were significantly decreased (P < 0.05). Compared with LP-H group, both hepatic glycogen (P < 0.01) and lactate dehydrogenase (LDH) (P < 0.05) were significantly elevated in LP-L group. For the LP-L group, the hepatocytes were arranged radially with the central vein in the center, and hepatic plates exhibited tight arrangement. Transcriptome analysis identified 29, 179, and 129 differentially expressed genes (DEGs) between the LP-M vs. LP-L, LP-H vs. LP-M, and LP-H vs. LP-L groups, respectively (Q-values < 0.05 and |log2Fold Change| > 1). Gene Ontology (GO) and correlation analyses showed that in the LP-L group, core genes (C1QA and JUNB) enriched in oxidoreductase activity were positively correlated with antioxidant indicators, while the MYO9A core gene enriched in the immune response was positively associated with immune indicators, and core genes enriched in molecular function (PDK3 and PDP2) were positively correlated with glycolysis indicators. In summary, low-protein diet with a low Lys/Met ratio (1:1) could reduce the hepatic oxidative stress and improve the glycolytic activity by regulating the expression of related genes of Tibetan sheep.

ADPORT-601: First-in-human study of adenosine 2A (A2A) and adenosine 2B (A2B) receptor antagonists in patients with select advanced solid tumors.

e14681 Background: Hypoxic tumors possess high levels of extracellular adenosine within the tumor microenvironment (TME) which suppresses both innate and adaptive immune responses by binding the adenosine receptors A2AR and A2BR. TT-10 (PORT-6) and TT-4 (PORT-7) are potent and selective antagonists of A2AR and A2BR, respectively. Both agents have shown monotherapy activity in both non-immunogenic (4T-1) and immunogenic (CT-26) tumor mouse models. Optimizing inhibition of A2AR and A2BR separately and in combination provides an opportunity to dissect the role of each signaling pathway within the TME. Methods: ADPORT-601 is a multi-center, open-label Phase 1 study, evaluating the safety, efficacy, PK and pharmacodynamics of PORT-6 in participants with mCRPC, RCC, or other selected solid tumors which are refractory to standard therapy. A subsequent arm will similarly test PORT-7 in participants with selected solid tumors. The Phase 1a cohorts utilize a standard 3+3 design. Phase 1b will be initiated once recommended doses have been established and will consist of monotherapy expansion cohorts for each drug, as well as both drugs combined at the optimal doses, and with other immunotherapy agents. A novel IHC-based assay will be implemented to select for patients with high A2AR expression. Metabolomic and gene expression profiling were performed on previously acquired bone biopsy and blood samples, respectively, to characterize the role of adenosine in CRPC. Results: 10 patients with mCRPC (n=6), RCC (n=3) or NSCLC (n=1) have been enrolled at escalating doses. Treatment-related AEs have been G1-2, with fatigue (29%), nausea (29%) and vomiting (14%) being the most reported. No related SAEs or dose limiting toxicities (DLTs) have been observed. Higher adenosine precursor metabolite (e.g., AMP, ADP, ATP, ADPR, inosine) levels were observed in image-guide bone metastatic lesions relative to non-tumor bearing bone controls (P&lt;0.05; n=10/group). A2AR and A2BR mRNA levels were enriched (1.3 and 1.4 log2 fold change, p&lt;0.05, respectively) in the plasma of mCRPC patients compared to those with localized disease. Updated safety, efficacy, PK and adenosine expression data will be reported at the meeting. Conclusions: PORT-6 is well tolerated to date. The TME within mCRPC bone metastases provides a unique target for A2AR and A2BR antagonism. Longitudinal blood and tissue samples, including bone biopsies, are being collected to enable biomarker analyses, allowing for elucidation of the relative contribution of A2AR and A2BR in suppressing anti-tumor immunity. Multi-omic translational analyses of tissue are planned to determine whether potent adenosine receptor antagonism will have an anti-tumor effect and convert the TME from immunosuppressive to proinflammatory. Clinical trial information: NCT04969315 .

Clinicopathologic and molecular landscape of invasive mucinous adenocarcinoma of the lung.

8037 Background: Invasive mucinous adenocarcinoma (IMA), accounting for 2–10% of lung adenocarcinomas, often mimics pneumonia with multicentric opacities in multiple lobes and both lungs. Notable for its microscopic skip lesions leading to tumor spread through air spaces (STAS), IMA is still under-researched in terms of its comprehensive clinical and genetic characteristics in large, multicenter studies. Methods: Primary lung cancer cases were obtained from the Tempus database, categorized into either IMA or non-IMA, and compared by patient, clinical, biopsy, and molecular characteristics. The normalization of RNA-seq data involved computing transcripts per million (TPM), performing log2 transformations, and adjusting for assay and batch effects. Significantly up- and down-regulated genes were defined as false discovery rate q-values &lt; 0.05 and |log2(fold change)| &gt; 0.5. Results: The study analyzed 18,857 cases, with 655 (3.5%) in the IMA group and 18,202 (96.5%) in the non-IMA group. The IMA group predominantly consisted of older individuals (median age 70 vs. 68; p&lt;0.001), had fewer smokers (63% vs. 73%; p&lt;0.001), and included more individuals of Hispanic or Latino ethnicity (4.3% vs. 2.7%; p=0.007). This group was more likely to be diagnosed with early-stage cancer and less likely to have stage 4 cancer at diagnosis (stage 1: 23% vs. 14%, stage 2: 18% vs. 7.3%, stage 3: 23% vs. 17%, stage 4: 36% vs. 62%; p&lt;0.001). They exhibited significantly lower tumor mutational and neoantigen burdens (median 3.1 vs. 4.6 mutations/Mb and 6 vs. 9 neoantigens/Mb, respectively; both p&lt;0.001), fewer positive PD-L1 statuses (27% vs. 58%; p&lt;0.001), and different immune cell infiltration patterns (Table). Gene expression analysis revealed 416 upregulated and 258 downregulated genes in the IMA group compared to the non-IMA group. Conclusions: This analysis underscores the unique clinicopathologic and molecular characteristics of IMAs. An improved understanding of IMA’s biological behavior could lead to the development of more effective, personalized treatment strategies. [Table: see text]

Identification and validation of immunity- and disulfidptosis-related genes signature for predicting prognosis in ovarian cancer

BackgroundOvarian cancer (OC) ranks as the fifth most prevalent neoplasm in women and exhibits an unfavorable prognosis. To improve the OC patient's prognosis, a pioneering risk signature was formulated by amalgamating disulfidptosis-related genes. MethodsA comparative analysis of OC tissues and normal tissues was carried out, and differentially expressed disulfidptosis-related genes (DRGs) were found using the criteria of |log2 (fold change) | > 0.585 and adjusted P-value <0.05. Subsequently, the TCGA training set was utilized to create a prognostic risk signature, which was validated by employing both the TCGA testing set and the GEO dataset. Moreover, the immune cell infiltration, mutational load, response to chemotherapy, and response to immunotherapy were analyzed. To further validate these findings, QRT-PCR analysis was conducted on ovarian tumor cell lines. ResultsA risk signature was created using fourteen differentially expressed genes (DEGs) associated with disulfidptosis, enabling the classification of ovarian cancer (OC) patients into high-risk group (HRG) and low-risk group (LRG). The HRG exhibited a lower overall survival (OS) compared to the LRG. In addition, the risk score remained an independent predictor even after incorporating clinical factors. Furthermore, the LRG displayed lower stromal, immune, and estimated scores compared to the HRG, suggesting a possible connection between the risk signature, immune cell infiltration, and mutational load. Finally, the QRT-PCR experiments revealed that eight genes were upregulated in the human OC cell line SKOV3 compared with the human normal OC line IOSE80, while six genes were down-regulated. ConclusionsA fourteen-biomarker signature composed of disulfidptosis-related genes could serve as a valuable risk stratification tool in OC, facilitating the identification of patients who may benefit from individualized treatment and follow-up management.

LINCATRA: Two-cycle method to amplify RNA for transcriptome analysis from formalin-fixed paraffin-embedded tissue

Whole transcriptome analysis (WTA) using RNA extracted from Formalin Fixed Paraffin Embedded (FFPE) tissue is an invaluable tool to understand the molecular pathology of disease. RNA extracted from FFPE tissue is either degraded and/or in very low quantities hampering gene expression analysis. Earlier studies described protocols applied for cellular RNA using poly-A primer-based linear amplification. The current study describes a method, LINCATRA (LINear amplifiCAtion of RNA for whole TRAnscriptome analysis). It employs random nonamer primer based method which can amplify short, fragmented RNA with high fidelity from as low as 5 ng to obtain enough material for WTA. The two-cycle method significantly amplified RNA at ∼1000 folds (p < 0.0001) improving the mean read lengths (p < 0.05) in WTA. Overall, increased mean read length positively correlated with on-target reads (Pearson's r = 0.71, p < 0.0001) in both amplified and unamplified RNA-seq analysis. Gene expression analysis compared between unamplified and amplified group displayed substantial overlap of the differentially expressed genes (DEGs) (log2 fold change cut-off < −2 and >2, p < 0.05) identified between lung cancer and asthma cohorts validating the method developed. This method is applicable in clinical molecular pathology field for both diagnostics and elucidation of disease mechanisms.

Use of integrated multi-omics analysis to determine antitumor efficacy of donafenib in combination with FADS2 inhibition in hepatocellular carcinoma.

e15083 Background: Pharmacotherapy remains a pivotal treatment strategy for patients with advanced hepatocellular carcinoma (HCC). Donafenib, globally the first targeted therapy proven to offer superior survival benefits and safety over sorafenib in a large-scale phase III clinical trial with head-to-head comparison, has been adopted for first-line treatment of advanced HCC. Given the limited basic research on Donafenib both domestically and internationally, and its constrained clinical efficacy, this study aims to explore combination drug therapy for HCC in an attempt to achieve further breakthroughs and enhance the anticancer activity of Donafenib. Methods: A xenograft tumor model in nude mice was established to observe the in vivo antitumor effects of Donafenib. Transcriptomics and proteomics were employed to characterize the gene expression changes in HCC cell lines after Donafenib intervention and to screen for key molecules. A series of assays, including the Cell Counting Kit-8 (CCK8), flow cytometry, scratch assays, Transwell assays, Western blotting, and in vivo tumorigenicity in nude mice, were conducted to determine the impact of the hub gene on the malignant behavior of HCC and to evaluate the combined antitumor effects of the molecular inhibition and Donafenib treatment in HCC. Results: Treatment with Donafenib significantly slowed tumor growth in a nude mouse xenograft model, resulting in a marked reduction in final tumor volumes compared to control subjects. Proteomic analyses post-Donafenib treatment identified FADS2 as the protein with the highest absolute log2 fold change among all differentially expressed proteins in HCC cells. This finding was corroborated by transcriptomic data, which indicated a concurrent downregulation of FADS2 at the mRNA level. Subsequent in vitro and in vivo assays validated the inhibitory effect of FADS2 blockade on the malignant biological behaviors of HCC cells. Importantly, the concomitant administration of Donafenib and the FADS2 inhibitor SC-26196 exhibited a synergistic antitumor action, significantly enhancing therapeutic efficacy in both HCC cell lines and xenografted tumors in nude mice. Conclusions: Combination therapy, a prevalent approach in clinical oncology, has the potential to augment treatment efficacy while simultaneously diminishing toxicity. This study employed an extensive multi-omics approach complemented by a range of both in vitro and in vivo experiments. It revealed that the synergistic use of Donafenib and SC-26196 markedly enhances Donafenib-induced the inhibition of cancer cell growth and metastasis, demonstrating a significant synergistic antitumor effect. These findings from our preclinical investigations may offer a promising new combinational drug regimen for the therapeutic management of HCC patients.

Using machine learning approach based on gene expression profiling to predict the risk of bone metastasis in lung cancer.

e20522 Background: Bone metastasis (BM) occurs in about 30% of patients with advanced lung cancer, and the prognosis of BM patients is very poor, with a median survival of 6-10 months. Therefore, we attempted to use machine learning methods based on gene expression profiles to construct a model to predict BM. Methods: Gene Expression Omnibus (GEO) database were searched, then these datasets (GSE10096, GSE29367, GSE29391, GSE76194, GSE175601) were included for analysis. Batch effects were removed using the ComBat function from the R package sva (v3.44.0). edgeR was used for differential expression analysis, and genes that had an absolute log2 fold change &gt; 1 and adjusted p-value &lt; 0.05, were selected as differentially expressed genes (DEGs) for modeling. Random forest (RF) modeling was performed using the R package random Forest (v4.7-1.1), and support vector machine (SVM) modeling and SVM Recursive Feature Elimination (SVM-RFE) were performed using the R package e1071 (v1.7-13). Survival curves were generated using the GEPIA2 R packages. Models performance was evaluated using the area under the receiver operating characteristics curve (AUC). Results: A total of 48 qualified cases were subjected to analysis, including 22 BM and 26 non-BM patients. Differential expression analysis revealed that a total of 327 DEGs were identified in the BM group. We divided the sample dataset into a training set and a test set. In the first approach, we attempted to use all 327 DEGs features to train the models. Among the evaluated models, the RF algorithm performed best with an AUC of 0.753, while the SVM algorithm had an AUC of 0.938. The second approach, we applied the SVM-RFE algorithm for feature selection, which ranked the features according to their importance, and the top 10 genes/features (TMC5, S100A4, EEF1A2, CLDN1, RALY, MYH10, MAGEC2, ALDH1A1, PI3, SALL1) were used to build a classification model (in the test set, sensitivity was100%, specificity: 81.8%, AUC:0.909). In addition, TCGA survival analysis of 10 genes was performed and found that the expression levels of five genes, including ALDH1A1, MYH10, RALY, CLDN1 and MAGEC2, were significantly associated with prognosis (p &lt; 0.1). Conclusions: Our study suggests that we have identified ten key genes associated with BM of lung cancer which could be potential therapeutic targets.

Highly prognostic signature for stage II colon cancer from the TOSCA trial: The PROSIT study.

3524 Background: The predictive and prognostic value of current biomarkers for guiding the treatment of stage II colon cancer (CC) is very limited. Here, we aimed to develop a prognostic transcriptional signature for high-risk stage II CC patients undergoing adjuvant treatment with either 3 or 6 months of FOLFOX4/CAPOX within the framework of the phase III TOSCA Italian trial. Methods: We conducted an observational retrospective study based on the TOSCA trial database, matching 62 patients with high-risk stage II CC who relapsed with 62 who did not. The matching criteria included the center of recruitment, pT stage, adjuvant treatment type and duration (3 or 6 months). Transcriptome sequencing was performed on material from centrally assessed FFPE blocks on an Illumina Novaseq 6000 instrument. Generated data were processed using the nf-core/rnaseq (v 3.14.0) pipeline, aligned to the human genome GRCh37/hg19, and analyzed for differential gene expression, requiring a minimum absolute log fold change of 1 and false discovery rate below 0.1. Enrichment analysis was performed using GSVA package with the z-score method. Results: Out of the 124 stage II CC cases considered for the present analysis, 60% received 3 months and 40% 6 months of adjuvant chemotherapy. The median age was 64 years (IQR 55.2-71.1), with 40% of the cancers being right-sided. Fifty-seven percent of the patients received FOLFOX, while the remaining were treated with XELOX. Of the 124 matched samples, 72 were of sufficient quality for RNA-seq.We identified a signature among the top 17 differentially expressed genes (absolute log fold-change &gt; 1, p-value &lt; 0.001, FDR &lt; 0.1, see Table). Patients exhibiting a lower prognostic signature (PROSIT-L) showed worse disease-free survival (DFS) compared to those with a high prognostic signature (PROSIT-H) (HR 3.5; 95% CI 1.53 – 7.9; p = 0.002), indicating its strong and independent prognostic value, regardless of the treatment received. Conclusions: The new transcriptomic signature may help in identifying stage II CC patients who are likely to relapse despite receiving adjuvant oxaliplatin-based treatment. [Table: see text]

Novel non-invasive molecular signatures for oral cavity cancer, by whole transcriptome and small non-coding RNA sequencing analyses: Predicted association with PI3K/AKT/mTOR pathway.

Identification of molecular biomarkers in the saliva and serum of oral cavity cancer patients represents a first step in the development of essential and efficient clinical tools for early detection and post-treatment monitoring. We hypothesized that molecular analyses of paired saliva and serum samples from an individual would likely yield better results than analyses of either serum or saliva alone. We performed whole-transcriptome and small non-coding RNA sequencing analyses on 32 samples of saliva and serum collected from the same patients with oral squamous cell carcinoma (OSCC) and healthy controls (HC). We identified 12 novel saliva and serum miRNAs and a panel of unique miRNA and mRNA signatures, significantly differentially expressed in OSCC patients relative to HC (log2 fold change: 2.6-26.8; DE: 0.02-0.000001). We utilized a combined panel of the 10 top-deregulated miRNAs and mRNAs and evaluated their putative diagnostic potential (>87% sensitivity; 100% specificity), recommending seven of them for further validation. We also identified unique saliva and serum miRNAs associated with OSCC and smoking history (OSCC smokers vs. never-smokers or HC: log2 fold change: 22-23; DE: 0.00003-0.000000001). Functional and pathway analyses indicated interactions between the discovered OSCC-related non-invasive miRNAs and mRNAs and their targets, through PI3K/AKT/mTOR signaling. Our data support our hypothesis that using paired saliva and serum from the same individuals and deep sequencing analyses can provide unique combined mRNA and miRNA signatures associated with canonical pathways that may have a diagnostic advantage relative to saliva or serum alone and may be useful for clinical testing. We believe this data will contribute to effective preventive care by post-treatment monitoring of patients, as well as suggesting potential targets for therapeutic approaches.

Integrated omics of Saccharomyces cerevisiae CENPK2-1C reveals pleiotropic drug resistance and lipidomic adaptations to cannabidiol

Yeast metabolism can be engineered to produce xenobiotic compounds, such as cannabinoids, the principal isoprenoids of the plant Cannabis sativa, through heterologous metabolic pathways. However, yeast cell factories continue to have low cannabinoid production. This study employed an integrated omics approach to investigate the physiological effects of cannabidiol on S. cerevisiae CENPK2-1C yeast cultures. We treated the experimental group with 0.5 mM CBD and monitored CENPK2-1C cultures. We observed a latent-stationary phase post-diauxic shift in the experimental group and harvested samples in the inflection point of this growth phase for transcriptomic and metabolomic analysis. We compared the transcriptomes of the CBD-treated yeast and the positive control, identifying eight significantly overexpressed genes with a log fold change of at least 1.5 and a significant adjusted p-value. Three notable genes were PDR5 (an ABC-steroid and cation transporter), CIS1, and YGR035C. These genes are all regulated by pleiotropic drug resistance linked promoters. Knockout and rescue of PDR5 showed that it is a causal factor in the post-diauxic shift phenotype. Metabolomic analysis revealed 48 significant spectra associated with CBD-fed cell pellets, 20 of which were identifiable as non-CBD compounds, including fatty acids, glycerophospholipids, and phosphate-salvage indicators. Our results suggest that mitochondrial regulation and lipidomic remodeling play a role in yeast’s response to CBD, which are employed in tandem with pleiotropic drug resistance (PDR). We conclude that bioengineers should account for off-target product C-flux, energy use from ABC-transport, and post-stationary phase cell growth when developing cannabinoid-biosynthetic yeast strains.

Circulating MicroRNAs as Predictors of Beta Cell Function in Youth-Onset Type 2 Diabetes: The TODAY study.

In the Treatment Options for Type 2 Diabetes in Adolescents and Youth (TODAY) study, an intervention trial followed by an observational phase, half the participants reached the primary outcome (HbA1c ≥ 8% for at least 6 months) within 4 years which was associated with a decrease in C-peptide oral disposition index (oDI). We aimed to identify circulating miRNA species associated with decline in beta cell function. Following a preliminary survey of select participants using nCounter Human v3 miRNA Panel (NanoString Technologies), polymerase chain reaction analyses were carried out for 17 miRNAs from 365 participants from samples at baseline, 24, 60, 96, and 120 months. Using a backward selection approach, four baseline miRNA log2 fold changes independently predicted treatment failure; however, baseline HbA1c was higher in those with treatment failure. Three baseline miRNA log2 fold changes remained significant predictors of this C-peptide oDI decline ≥20% (p < 0.05). Increased levels of miRNA-155 (OR:1.2, 95%CI:1.1-1.4) and miRNA-130b (OR:1.3, 95%CI: 1.0-1.7) were associated with oDI decline, while decreased levels of miRNA-126 (OR:0.6, 95%CI: 0.4-0.8) were associated with oDI decline. miRNA-122 was negatively correlated with C-peptide oDI at baseline and 24-months (R = 0.22, p < 0.01 and R = 0.19, p < 0.01, respectively), and positively correlated with proinsulin, at baseline, 24-, and 60- months (R = 0.26, p < 0.01, R = 0.26, p < 0.01, R = 0.18, p < 0.01, respectively). The miRNA species associated with beta cell function are associated with alterations in cellular metabolism and apoptosis, suggesting that differences in baseline abundance may serve as circulating markers of beta cell dysfunction and provide potential mechanistic insights into the aggressive nature of youth-onset type 2 diabetes.

Exploring the regulation of circulating factors involved in aortic valve sclerosis onset

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Ministero della Salute - Ricerca Finalizzata Background Aortic valve sclerosis (AVSc), the first stage of calcific aortic valve stenosis (CAVS), has a prevalence of 25-30% in subjects older than 65 years. Being a clinically silent phase, it is characterized only by non-uniform leaflet thickening due to fibrosis and microcalcifications, without the obstruction of the blood flow with an increased local oxidative stress, inflammation, and endothelial-to-mesenchymal transition (EndMT). However, to date, AVSc is not considered a pathology but 10% of cases evolve a full-blown CAVS. Aim Our aim is to investigate which systemic process(es) are activated in AVSc, in order to better define these subjects. These evaluations will allow us to identify potential druggable targets. Methods We consecutively enrolled 157 healthy subjects and detected AVSc presence by echocardiography. Plasma and serum samples were collected to evaluate circulating microRNA (RNA-sequencing) and cytokines (Luminex), respectively. Functional analysis was performed to reveal systemic pathway regulation. Healthy human valve endothelial cells were employed to evaluate viability (MTT assay) and EndMT (morphological analysis and quantitative PCR) Results AVSc was present in 77 patients (49%) and the differential expression analysis revealed that 59 microRNAs were down-regulated, while 93 were up-regulated in these subjects compared to healthy ones (adjusted-p&amp;lt;0.05). The functional analysis revealed that the major enriched pathways in AVSc subjects were related to inflammation. Indeed, serum GROα and IL1β levels were higher in AVSc vs healthy (p&amp;lt;0.05). In vitro, chronic GROα treatment resulted in lower cell viability (100.0±17.4 vs 61.8±16.5% of vital cells; p&amp;lt;0.0001), while IL1β treatment induced a decrease in cell roundness and an increase in total area (0.40±0.18 vs 0.47±0.19AU, p&amp;lt;0.0001; and 0.75±0.73 vs 0.45±0.48AU, p&amp;lt;0.0001; respectively). Quantitative PCR revealed EndMT activation: SNAI1 (1.87±0.57 log2(foldchange), p&amp;lt;0.0001) and SNAI2 (2.19±0.46 log2(foldchange), p&amp;lt;0.0001) only after IL1β treatment. Conclusions Inflammation is the most regulated systemic pathway in AVSc subjects and its master regulator, IL1β, seems to be involved in the EndMT progression of valve endothelial cells, while GROα contributes to their death. These effects could be related to the early triggers of AVSc onset, leading to fibrosis and eventually calcification of aortic leaflets.

MiRNAs as a tool for the diagnosis of suspected Brugada Syndrome

Abstract Background/Introduction Brugada syndrome (BrS) is a life-threatening arrhythmogenic disease that can lead to sudden unexplained death/cardiac arrest in otherwise healthy individuals. ECG represents the gold standard for the diagnosis. In 30% of cases, however, the syndrome is diagnosed only after a provocative test with sodium channel blockers [1]. Among the different approaches to support diagnosis, circulating biomarkers such as miRNAs may represent a tool that can provide a biological signature of the syndrome in patients with suspected BrS. Purpose Within the BrS and Artificial Intelligence Applications to Diagnosis system study, we collected and analyzed blood samples from patients with suspected BrS undergoing provocative tests aiming to assess whether miRNAs may be potential markers to identify the syndrome. Methods Patients undergoing provocative tests with sodium channel blockers (i.e., ajmaline, flecainide) were classified as positive (15 patients) and negative (13 patients) based on the test results. Blood samples were obtained when a Type-1 ECG pattern developed (for positive) or at the end of the provocative test without any electrographic changes (negative). miRNAs were extracted from circulating exosomes, obtained by blood samples, using a dedicated and innovative assay (exoRNeasy mini/midi kit, QIAGEN). They were quantified and quality tested by Agilent 2100 Bioanalyzer RNA assay; miRNA-seq was carried out with NovaSeq 6000 (Illumina, San Diego, CA). Data were analyzed through web-based platforms for network visual analytics (mirNET 2.0). Results The presence of 8 small RNAs up-regulated was reported in the positive group (hsa-miR-155-5p ,hsa-miR-10a-5p, hsa-miR-29a-3p, hsa-miR-142-5p, hsa-miR-30a-5p, hsa-miR-126-5p, hsa-miR-95-3p, hsa-miR-126-3p), with a log fold-change between 6.4 (as for hsa-miR-95-3p) and up to 615.0 (as for hsa-miR-126-3p) (Table 1). The network analysis results (Figure 1) on this group of miRNAs showed their involvement, among the others, in response to cytokine and muscle development (in terms of function analysis) and Ca2+pathway modulation (in terms of pathway analysis). Conclusion(s) In this work, a miRNA analysis of patients undergoing provocative tests for Type-1 BrS diagnosis was performed. Our results show a set of potential miRNAs that could be used to identify Type 1 BrS patients in whom the typical ECG pattern is basally lacking. Although further experiments and validation on a larger number of samples are necessary, our results could identify these miRNAs as potential early markers in patients with a suspected Type-1 BrS diagnosis.Upregulated miRNAs in BrS patientsMiRNA network analysis

A murine model of hypertensive heart disease in older women.

We propose a new mouse (C57Bl6/J) model combining several features of heart failure with preserved ejection fraction encountered in older women, including hypertension from Angiotensin II infusion (AngII), menopause, and advanced age. To mimic menopause, we delayed ovariectomy (Ovx) at 12 months of age. We also studied the effects of AngII infusion for 28 days in younger animals and the impact of losing gonadal steroids earlier in life. We observed that AngII effects on heart morphology were different in younger and adult mice (3- and 12-month-old; 20 and 19% increase in heart weight. P<0.01 for both) than in older animals (24-month-old; 6%; not significant). Ovariectomy at 12 months restored the hypertrophic response to AngII in elderly females (23%, p=0.0001). We performed a bulk RNA sequencing study of the left ventricle (LV) and left atrial gene expression in elderly animals, controls, and Ovx. AngII modulated (|Log2 fold change| ≥ 1) the LV expression of 170 genes in control females and 179 in Ovx ones, 64 being shared. In the left atrium, AngII modulated 235 genes in control females and 453 in Ovx, 140 shared. We observed many upregulated genes associated with the extracellular matrix regulation in both heart chambers. Many of these upregulated genes were shared between the ventricle and the atrium as well as in control and Ovx animals, namely for the most expressed Ankrd1, Nppb, Col3a1, Col1a1, Ctgf Col8a1, and Cilp. Several circadian clock LV genes were modulated differently by AngII between control and Ovx females (Clock, Arntl, Per2, Cry2, and Ciart). In conclusion, sex hormones, even in elderly female mice, modulate the heart's hypertrophic response to AngII. Our study identifies potential new markers of hypertensive disease in aging female mice and possible disturbances of their cardiac circadian clock.

Laser Capture Microscopy RNA Sequencing for Topological Mapping of Synovial Pathology During Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease in which the joint lining or synovium becomes highly inflamed and majorly contributes to disease progression. Understanding pathogenic processes in RA synovium is critical for identifying therapeutic targets. We performed laser capture microscopy (LCM) followed by RNA sequencing (LCM-RNAseq) to study regional transcriptomes throughout RA synovium. Synovial lining, sublining, and vessel samples were captured by LCM from seven patients with RA and seven patients with osteoarthritis (OA). RNAseq was performed on RNA extracted from captured tissue. Principal component analysis was performed on the sample set by disease state. Differential expression analysis was performed between disease states based on log2 fold change and q value parameters. Pathway analysis was performed using the Reactome Pathway Database on differentially expressed genes among disease states. Significantly enriched pathways in each synovial region were selected based on the false discovery rate. RA and OA transcriptomes were distinguishable by principal component analysis. Pairwise comparisons of synovial lining, sublining, and vessel samples between RA and OA revealed substantial differences in transcriptional patterns throughout the synovium. Hierarchical clustering of pathways based on significance revealed a pattern of association between biologic function and synovial topology. Analysis of pathways uniquely enriched in each region revealed distinct phenotypic abnormalities. As examples, RA lining samples were marked by anomalous immune cell signaling, RA sublining samples were marked by aberrant cell cycle, and RA vessel samples were marked by alterations in heme scavenging. LCM-RNAseq confirms reported transcriptional differences between the RA synovium and the OA synovium and provides evidence supporting a relationship between synovial topology and molecular anomalies in RA.

Association of Gene Expression and Tremor Network Structure.

Transcriptomic changes in the essential tremor (ET)-associated cerebello-thalamo-cortical "tremor network" and their association to brain structure have not been investigated. The aim was to characterize molecular changes associated with network-level imaging-derived phenotypes (IDP) found in ET. We performed an imaging-transcriptomic study in British adults using imaging-genome-wide association study summary statistics (UK Biobank "BIG40" cohort; n = 33,224, aged 40-69 years). We imputed imaging-transcriptomic associations for 184 IDPs and analyzed functional enrichment of gene modules and aggregate network-level phenotypes. Validation was performed in cerebellar-tissue RNA-sequencing data from ET patients and controls (n = 55). Among 237,896 individual predicted gene expression levels for 6063 unique genes/transcripts, we detected 2269 genome-wide significant associations (Bonferroni P < 2.102e-7, 0.95%). These were concentrated in intracellular volume fraction measures of white matter pathways and in genes with putative links to tremor (MAPT, ARL17A, KANSL1, SPPL2C, LRRC37A4P, PLEKHM1, and FMNL1). Whole-tremor-network cortical thickness was associated with a gene module linked to mitochondrial organization and protein quality control (r = 0.91, P = 2e-70), whereas white-gray T1-weighted magnetic resonance imaging (MRI) contrast in the tremor network was associated with a gene module linked to sphingolipid synthesis and ethanolamine metabolism (r = -0.90, P = 2e-68). Imputed association effect sizes and RNA-sequencing log-fold change in the validation dataset were significantly correlated for cerebellar peduncular diffusion MRI phenotypes, and there was a close overlap of significant associations between both datasets for gray matter phenotypes (χ2 = 6.40, P = 0.006). The identified genes and processes are potential treatment targets for ET, and our results help characterize molecular changes that could in future be used for patient treatment selection or prognosis prediction. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Exhaled breath analysis in patients with potentially curative lung cancer undergoing surgery: a longitudinal study

Exhaled breath analysis has emerged as a non-invasive and promising method for early detection of lung cancer, offering a novel approach for diagnosis through the identification of specific biomarkers present in a patient’s breath. For this longitudinal study, 29 treatment-naive patients with lung cancer were evaluated before and after surgery. Secondary electrospray ionization high-resolution mass spectrometry was used for exhaled breath analysis. Volatile organic compounds with absolute log2 fold change ⩾1 and q-values ⩾ 0.71 were selected as potentially relevant. Exhaled breath analysis resulted in a total of 3482 features. 515 features showed a substantial difference before and after surgery. The small sample size generated a false positive rate of 0.71, therefore, around 154 of these 515 features were expected to be true changes. Biological identification of the features with the highest consistency (m/z −242.18428 and m/z −117.0539) revealed to potentially be 3-Oxotetradecanoic acid and Indole, respectively. Principal component analysis revealed a primary cluster of patients with a recurrent lung cancer, which remained undetected in the initial diagnostic and surgical procedures. The change of exhaled breath patterns after surgery in lung cancer emphasizes the potential for lung cancer screening and detection.