Fold Change Research Articles

Abstract 1017: Characterizing immune cell infiltration in colorectal cancer tumor microenvironment among Native Hawaiians

Abstract Background Colorectal cancer (CRC) is recognized as a heterogeneous disease, ranking third in new cancer diagnoses and second in cancer-related deaths globally and nationally. CRC incidence is higher in the state of Hawaii compared to the U.S. overall. This study focuses on profiling immune cell infiltration in Native Hawaiian(NH)-specific CRC tumor microenvironments. Identifying the race-specific tumor infiltrating microenvironment may uncover potential mechanisms for CRC development in the NH population. Methods Deidentified, archival formalin-fixed paraffin-embedded tumor samples from Native Hawaiian patients diagnosed with primary CRC between 1990 and 2006 were obtained from the Hawaii Tumor Registry. RNA was extracted from tumor and adjacent normal tissues. RNA-seq data from Caucasian American (CA) CRC samples were obtained from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). Differentially expressed genes for each cohort were identified via DESeq2 with Benjamini-Hochberg multiple testing q-value<0.05 and a cutoff of 2-fold change. From the gene expression profiles, we applied the ESTIMATE algorithm to calculate the overall immune cell infiltration score for each sample. Based on a deconvolution algorithm, known as CIBERSORT to estimate the abundance of 22 immune cell types, we profiled the tumor-infiltrating immune cells present in each cohort’s paired samples, respectively. To improve the accuracy, p-value and root mean squared error were counted for each sample. Data with p<0.05 were filtered and selected for the next analysis. The fraction of immune cell subpopulations was evaluated and compared between paired tumor and adjacent normal tissues in NH and CA cohorts. Results In the NH cohort, we found the overall immune cell infiltration score was significantly lower in cancer tissues than the adjacent normal tissues (p<0.05), and there were significantly higher fractions of activated dendritic cells, resting natural killer (NK), and follicular helper T cells in tumor tissues compared to adjacent non-tumor tissues (p<0.05). Furthermore, the fractions of activated NK cells and plasma cells were significantly lower in tumor tissues than in adjacent non-tumor tissues in NH (p<0.05). Resting NK cells are upregulated in both the NH and the CA cohorts, while plasma cells are the only immune cell type downregulated in either cohort. In the NH cohort, activated dendritic cells and follicular helper T cells are exclusively upregulated, while activated NK cells are downregulated relative to the CA cohort. Conclusion This study demonstrates that the NH cohort displays distinctive immunological variances between tumor and adjacent non-tumor tissues which distinguishes them from the CA cohort. This emphasizes the significance of acknowledging population-specific genetic variations which may help understand race specific difference in CRC. Citation Format: Yuanyuan Fu, Gino Quintal, Masaki Nasu, Mayumi Jijiwa, Sudhir K. Rai, Isam Mohd-Ibrahim, Yu Chen, Hua Yang, Zhanwei Wang, Christina Wai, Owen Chan, Brenda Y. Hernandez, Youping Deng. Characterizing immune cell infiltration in colorectal cancer tumor microenvironment among Native Hawaiians [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1017.

Detection of hepatocellular carcinoma methylation markers in salivary DNA.

Alterations to DNA methylation have been identified in both hepatocellular carcinoma (HCC) tumor and circulating DNA from affected individuals. These markers have potential utility in HCC screening. Adherence to HCC screening is poor and acceptable HCC screening tests are needed. A feasibility study was performed on a subset of case patients and control subjects from a prior study of risk factors for HCC. Case patients (n=12) included adults aged 47-85 years with a first diagnosis of HCC between 2011 and 2016 and without viral hepatitis. Control subjects (n=12) were matched on age, sex, and state of residence. Participants provided saliva samples for DNA genotyping. Log fold change in salivary DNA methylation at 1359 CpG sites representing 25 candidate genes previously associated with HCC was compared across case patients and control subjects. The quantity of DNA ranged from 9.65 to 257.79 μg. The purity of DNA isolates was good, with mean OD260/280 ratio of 1.78 (SD: 0.14). Of 25 candidate genes, 16 had at ≥1 CpG site with detectable differences in methylation across HCC case patients and control subjects. Sites differentially methylated in HCC case patients included genes encoding tumor suppressors (PRDM2, RUNX3, p15/16, and RASSF1/5), regulators of cell cycle progression (DAPK1 and TP73), and DNA repair (MGMT and GSTP1). No associations met the significance threshold 3.7 × 10-5 required for multiple comparisons. Salivary DNA may be a feasible alternative to blood samples in the era of novel DNA-based screening tests for HCC. The ease of saliva-based testing supports further investigation of its potential.

MicroRNAs as biomarkers of brain injury in neonatal encephalopathy: an observational cohort study

Neonatal Encephalopathy (NE) is a major cause of lifelong disability and neurological complications in affected infants. Identifying novel diagnostic biomarkers in this population may assist in predicting MRI injury and differentiate neonates with NE from those with low-cord pH or healthy neonates and may help clinicians make real-time decisions. To compare the microRNA (miRNA) profiles between neonates with NE, healthy controls, and neonates with low cord pH. Moreover, miRNA concentrations were compared to brain injury severity in neonates with NE. This is a retrospective analysis of miRNA profiles from select samples in the biorepository and data registry at the University of Florida Health Gainesville. The Firefly miRNA assay was used to screen a total of 65 neurological miRNA targets in neonates with NE (n = 36), low cord pH (n = 18) and healthy controls (n = 37). Multivariate statistical techniques, including principal component analysis and orthogonal partial least squares discriminant analysis, and miRNA Enrichment Analysis and Annotation were used to identify miRNA markers and their pathobiological relevance. A set of 10 highly influential miRNAs were identified, which were significantly upregulated in the NE group compared to healthy controls. Of these, miR-323a-3p and mir-30e-5p displayed the highest fold change in expression levels. Moreover, miR-34c-5p, miR-491-5p, and miR-346 were significantly higher in the NE group compared to the low cord pH group. Furthermore, several miRNAs were identified that can differentiate between no/mild and moderate/severe injury in the NE group as measured by MRI. MiRNAs represent promising diagnostic and prognostic tools for improving the management of NE.

PSCA gene expression in bladder, colorectal and prostate cancer patients from Basrah governorate southern of Iraq

Cancer causes death in all countries of the world, with approximately (9.6) million deaths in 2018. Developing countries have high cancer rates. Furthermore, there are risk factors that lead to cancer, including economic and political instability and bad lifestyles. This is the first study of PSCA gene expression with BC, CRC and PC in Iraq, especially in Basrah governorate. This paper involved one hundred and one blood samples being collected from bladder, colorectal and prostate cancer. On the other hand, one hundred and one blood samples with no cancer were collected as a control group. Two ml of peripheral blood was drawn for RNA extraction, then total RNA was reverse transcribed to cDNA, and PSCA mRNA was measured using qPCR. The results showed PSCA gene was over expressed in bladder cancer (BC) with an expression level at ± 8.63 for patients and controls estimated with ±4.16. The gene expression was a 2-fold change in patients compared to healthy control. While the PSCA gene was over-expressed in colorectal cancer (CRC) with an expression level ± 8.16 for patients and controls estimated at ±3.30. That means the gene expression was a 2.47-fold change in patients compared to healthy control. The PSCA gene was over expressed in prostate cancer (PC) with an expression level ± 9.47 for patients and controls estimated at ±4.22. The gene expression was a 2.24-fold change in patients compared to healthy control. In the present study, PSCA gene was expressed at a significantly higher level in the BC, CRC and PC patients compared in the controls group.

Cellular energy regulates mRNA degradation in a codon-specific manner

Codon optimality is a major determinant of mRNA translation and degradation rates. However, whether and through which mechanisms its effects are regulated remains poorly understood. Here we show that codon optimality associates with up to 2-fold change in mRNA stability variations between human tissues, and that its effect is attenuated in tissues with high energy metabolism and amplifies with age. Mathematical modeling and perturbation data through oxygen deprivation and ATP synthesis inhibition reveal that cellular energy variations non-uniformly alter the effect of codon usage. This new mode of codon effect regulation, independent of tRNA regulation, provides a fundamental mechanistic link between cellular energy metabolism and eukaryotic gene expression.

Validating a Four-gene Set for H-ARS Severity Prediction in Peripheral Blood Samples of Irradiated Rhesus Macaques.

Increased radiological and nuclear threats require preparedness. Our earlier work identified a set of four genes (DDB2, FDXR, POU2AF1 and WNT3), which predicts severity of the hematological acute radiation syndrome (H-ARS) within the first three days postirradiation In this study of 41 Rhesus macaques (Macaca mulatta, 27 males, 14 females) irradiated with 5.8-7.2 Gy (LD29-50/60), including some treated with gamma-tocotrienol (GT3, a radiation countermeasure) we independently validated these genes as predictors in both sexes and examined them after three days. At the Armed Forces Radiobiology Research Institute/Uniformed Services University of the Health Sciences, peripheral whole blood (1 ml) of Rhesus macaques was collected into PAXgene® Blood RNA tubes pre-irradiation after 1, 2, 3, 35 and 60 days postirradiation, stored at -80°C for internal experimental analyses. Leftover tubes from these already ongoing studies were kindly provided to Bundeswehr Institute of Radiobiology. RNA was isolated (QIAsymphony), converted into cDNA, and for further gene expression (GE) studies quantitative RT-PCR was performed. Differential gene expression (DGE) was measured relative to the pre-irradiation Rhesus macaques samples. Within the first three days postirradiation, we found similar results to human data: 1. FDXR and DDB2 were up-regulated, FDXR up to 3.5-fold, and DDB2 up to 13.5-fold in the median; 2. POU2AF1 appeared down regulated around tenfold in nearly all Rhesus macaques; 3. Contrary to human data, DDB2 was more up-regulated than FDXR, and the difference of the fold change (FC) ranged between 2.4 and 10, while the median fold changes of WNT3, except days 1 and 35, were close to 1. Nevertheless, 46% of the Rhesus macaques showed down-regulated WNT3 on day one postirradiation, which decreased to 12.2% on day 3 postirradiation. Considering the extended phase, there was a trend towards decreased fold changes at day 35, with median-fold changes ranging from 0.7 for DDB2 to 0.1 for POU2AF1, and on day 60 postirradiation, DGE in surviving animals was close to pre-exposure values for all four genes. In conclusion, the diagnostic significance for radiation-induced H-ARS severity prediction of FDXR, DDB2, and POU2AF1 was confirmed in this Rhesus macaques model. However, DDB2 showed higher GE values than FDXR. As shown in previous studies, the diagnostic significance of WNT3 could not be reproduced in Rhesus macaques; this could be due to the choice of animal model and methodological challenges.

Transkingdom Network Analysis (TkNA): a systems framework for inferring causal factors underlying host-microbiota and other multi-omic interactions.

We present Transkingdom Network Analysis (TkNA), a unique causal-inference analytical framework that offers a holistic view of biological systems by integrating data from multiple cohorts and diverse omics types. TkNA helps to decipher key players and mechanisms governing host-microbiota (or any multi-omic data) interactions in specific conditions or diseases. TkNA reconstructs a network that represents a statistical model capturing the complex relationships between different omics in the biological system. It identifies robust and reproducible patterns of fold change direction and correlation sign across several cohorts to select differential features and their per-group correlations. The framework then uses causality-sensitive metrics, statistical thresholds and topological criteria to determine the final edges forming the transkingdom network. With the subsequent network's topological features, TkNA identifies nodes controlling a given subnetwork or governing communication between kingdoms and/or subnetworks. The computational time for the millions of correlations necessary for network reconstruction in TkNA typically takes only a few minutes, varying with the study design. Unlike most other multi-omics approaches that find only associations, TkNA focuses on establishing causality while accounting for the complex structure of multi-omic data. It achieves this without requiring huge sample sizes. Moreover, the TkNA protocol is user friendly, requiring minimal installation and basic familiarity with Unix. Researchers can access the TkNA software at https://github.com/CAnBioNet/TkNA/ .

Molecular basis of distinct responses to drought between rice and wheat genotypes

The water-intensive cultivation of rice in flooded conditions poses a great challenge to global water resources, particularly in the face of climate change. Wheat requires significantly less water than rice with fairly high yields. A main research question is whether there are any drought adaptive mechanisms in rice similar to those existing in wheat. We report a study, where precise moisture stress was imposed at the vegetative stage using a high-throughput drought simulator phenomics facility. Variability in stress response was captured through untargeted metabolome analysis. In the first experiment, using two rice cultivars with contrasting drought responses (IR64 and Apo) and a wheat cultivar, Weebill, we found distinct variations in regulation of metabolite levels under well-watered or water-limited conditions, both between and within species. Several biosynthetic pathways, including those of polyamines, flavonoids, flavones, and specific metabolites like sphinganine-1-phosphate and anthranilate were enhanced under water-limited conditions. These metabolites were more abundant in wheat than in rice. Among the rice cultivars, Apo had higher abundance of metabolites than IR64. Regulatory patterns governing these metabolic pathways provided basis for stress responses of the tolerant rice cultivar and wheat. A second experiment was conducted to screen rice germplasm lines for acquired tolerance traits contrasting for drought responsiveness. Eight genotypes (four sensitive and four tolerant ones) were selected to validate the candidate metabolites from the first experiment. In the third experiment, an RT-PCR analysis of the key regulatory genes governing the metabolic pathways was conducted, using plant materials from both experiments. The results of this third experiment provided a strong validation for the relevance of these metabolic pathways under stress in rice. We identified promising rice genotypes that exhibited fold change levels of stress resistance metabolites comparable to those of the wheat cultivar Weebill. This study helped identify key candidate metabolites that can be either developed as markers or as elicitors, and genotypes that can be used as trait donor lines for rice improvement.

Loss of ERβ Disrupts Gene Regulation in Primordial and Primary Follicles.

Loss of ERβ increases primordial follicle growth activation (PFGA), leading to premature ovarian follicle reserve depletion. We determined the expression and gene regulatory functions of ERβ in dormant primordial follicles (PdFs) and activated primary follicles (PrFs) using mouse models. PdFs and PrFs were isolated from 3-week-old Erβ knockout (Erβnull) mouse ovaries, and their transcriptomes were compared with those of control Erβfl/fl mice. We observed a significant (≥2-fold change; FDR p-value ≤ 0.05) deregulation of approximately 5% of genes (866 out of 16,940 genes, TPM ≥ 5) in Erβnull PdFs; ~60% (521 out of 866) of the differentially expressed genes (DEGs) were upregulated, and 40% were downregulated, indicating that ERβ has both transcriptional enhancing as well as repressing roles in dormant PdFs. Such deregulation of genes may make the Erβnull PdFs more susceptible to increased PFGA. When the PdFs undergo PFGA and form PrFs, many new genes are activated. During PFGA of Erβfl/fl follicles, we detected a differential expression of ~24% genes (4909 out of 20,743; ≥2-fold change; FDR p-value ≤ 0.05; TPM ≥ 5); 56% upregulated and 44% downregulated, indicating the gene enhancing and repressing roles of Erβ-activated PrFs. In contrast, we detected a differential expression of only 824 genes in Erβnull follicles during PFGA (≥2-fold change; FDR p-value ≤ 0.05; TPM ≥ 5). Moreover, most (~93%; 770 out of 824) of these DEGs in activated Erβnull PrFs were downregulated. Such deregulation of genes in Erβnull activated follicles may impair their inhibitory role on PFGA. Notably, in both Erβnull PdFs and PrFs, we detected a significant number of epigenetic regulators and transcription factors to be differentially expressed, which suggests that lack of ERβ either directly or indirectly deregulates the gene expression in PdFs and PrFs, leading to increased PFGA.

Screening by Q Exactive liquid chromatography/tandem mass spectrometry identified Choline, 25-hydroxyvitamin D2, and SM(d18:0/16:1(9Z) (OH)) as biomarkers for high-grade serous ovarian cancer

High-grade serous ovarian cancer (HGSOC) has a high death rate and poor prognosis. The main causes of poor prognosis are asymptomatic early disease, no effective screening method at present, and advanced disease. Changes in cellular metabolism are characteristic of cancer, and plasma metabolome analysis can be used to identify biomarkers. In this study, we used Q Exactive liquid chromatography tandem mass spectrometry (LC–MS/MS, QE) to compare the differentiation between plasma samples (22 HGSOC samples and 22 normal samples). In total, we detected 124 metabolites, and an orthogonal partial least-squares-discriminant analysis (OPLS-DA) model was useful to distinguish HGSOC patients from healthy controls. Choline, 25-hydroxyvitamin D2, and sphingomyelin (d18:0/16:1(9Z) (OH))/SM(d18:0/16:1(9Z) (OH)) showed significantly differential plasma levels in HGSOC patients under the conditions of variable importance in projection (VIP) > 1, p < 0.05 using Student's t-test, and fold change (FC) ≥ 1.5 or ≤ 0.667. Metabolic pathway analysis can provide valuable information to enhance the understanding of the underlying pathophysiology of HGSOC. In conclusion, the Q Exactive LC/MS/MS method validation-based plasma metabolomics approach may have potential as a convenient screening method for HGSOC and may be a method to monitor tumor recurrence in patients with HGSOC after surgery SignificanceHigh-grade serous ovarian cancer (HGSOC) has a high death rate and poor prognosis. The main causes of poor prognosis are asymptomatic early disease, no effective screening method at present, and advanced disease. Changes in cellular metabolism are characteristic of cancer, and plasma metabolome analysis can be used to identify biomarkers. In this study, we used Q Exactive liquid chromatography tandem mass spectrometry (LC–MS/MS, QE) to compare the differentiation between plasma samples (20 HGSOC samples and 20 normal samples). In total, we detected 124 metabolites, and an orthogonal partial least-squares-discriminant analysis (OPLS-DA) model was useful to distinguish HGSOC patients from healthy controls. Choline, 25-hydroxyvitamin D2, and sphingomyelin (d18:0/16:1(9Z) (OH))/SM(d18:0/16:1(9Z) (OH)) showed significantly differential plasma levels in HGSOC patients under the conditions of variable importance in projection (VIP) > 1, p < 0.05 using Student's t-test, and fold change (FC) ≥ 1.5 or ≤ 0.667. Metabolic pathway analysis can provide valuable information to enhance the understanding of the underlying pathophysiology of HGSOC. In conclusion, the Q Exactive LC/MS/MS method validation-based plasma metabolomics approach may have potential as a convenient screening method for HGSOC and may be a method to monitor tumor recurrence in patients with HGSOC after surgery.

Expression of Rejection-Associated Transcripts in Early Protocol Renal Transplant Biopsies Is Associated with Tacrolimus Exposure and Graft Outcome.

Subclinical inflammation in protocol biopsies relates to tacrolimus exposure and human leukocyte antigen (HLA) matching. We aimed to characterize transcripts associated with rejection and tacrolimus exposure and the latter's association with transplant outcomes. We tested whether gene expression is associated with rejection using strictly normal protocol biopsies (n = 17) and biopsies with T cell-mediated rejection (TCMR) or antibody-mediated rejection (ABMR) according to Banff criteria (n = 12). Subsequently, we analyzed these transcripts in a set of 4-month protocol biopsies (n = 137) to assess their association with donor and recipient characteristics, the intensity of immunosuppression, and the graft outcome. Differential expression (false discovery rate (FDR) < 0.01, fold (change (FC) > 3) between normal and rejection biopsies yielded a set of 111 genes. In the protocol biopsy cohort (n = 137), 19 out of these 111 genes correlated with tacrolimus trough levels at the time of biopsy (TAC-C0), and unsupervised analysis split this cohort into two clusters. The two clusters differed in donor age and tacrolimus trough levels. Subclinical rejection, including borderline lesions, tended to occur in the same cluster. Logistic regression analysis indicated that TAC-C0 at the time of biopsy (OR: 0.83, 95%CI:0.72-0.06, p = 0.0117) was associated with cluster 2. In a follow-up averaging 70 ± 30 months, this patient group displayed a significant decline in renal function (p = 0.0135). The expression of rejection-associated transcripts in early protocol biopsies is associated with tacrolimus exposure and a faster decline in renal function.

Comparison of the gene expression profile of testicular tissue before and after sexual maturity in Qianbei Ma goats

BackgroundWith long-term research on the reproductive ability of Qianbei Ma goat, we found that the puberty of the male goats comes at the age of 3 months and reaches sexual maturity at 4 months,the male goats are identified as physically mature at 9 months and able to mate. Compared with other kinds of breeds of goats, Qianbei Ma goat is featured with more faster growth and earlier sexual maturity.Therefore, in order to explore the laws of growth of Qianbei Ma goat before sexual maturity(3-month-old)and after sexual maturity (9-month-old). The testicular tissue was collected to explore their changes in morphology through HE staining, the serum was collected to detect the hormone content, and the mRNA expression profile of the testis was analyzed by transcriptomics. In this way, the effect of testicular development on the reproduction of Qianbei ma goats was further analyzed.ResultsThe results showed that the area and diameter of spermatogenic tubules were larger at 9 months than 3 months, and the number of spermatocytes, interstitial cells, spermatogonia and secondary spermatocytes in the lumen of the tubules showed a similar trend. The appearance of spermatozoa at age 3 months indicated that puberty had begun in Qianbei Ma goats. The Elasa test for testosterone, luteinizing hormone, follicle stimulating hormone and anti-Müllerian hormone showed that the levels of these hormones in the serum at age 9 months were all highly significantly different than those at age 3 months (P < 0.01). There were 490 differentially expressed genes (DEGs) between the (|log2(fold change)| > 1 and p value < 0.05) 3-month-old and 9-month-old groups, of which 233 genes were upregulated and 257 genes were downregulated (3 months of age was used as the control group and 9 months of age was used as the experimental group). According to the GO and KEGG enrichment analyses of DEGs, PRSS58, ECM1, WFDC8 and LHCGR are involved in testicular development and androgen secretion, which contribute to the sexual maturation of Qianbei Ma goats.ConclusionsPotential biomarker genes and relevant pathways involved in the regulation of testicular development and spermatogenesis in Qianbei Ma goats were identified, providing a theoretical basis and data support for later studies on the influence of testicular development and spermatogenesis before and after sexual maturity in Qianbei Ma goats.

High throughput biological sequence analysis using machine learning-based integrative pipeline for extracting functional annotation and visualization

The Differential Gene Expression (DGE) approach to find out the expressed genes relies on measures such as log-fold change and adjusted p-values. Although fold change is commonly employed in gene expression studies, especially in microarray and RNA sequencing experiments to quantify alterations in a gene’s expression level, a limitation and potential hazard of relying on fold change in this context is its inherent bias. As a consequence, it might incorrectly categorize genes that have significant differences but minor ratios, resulting in poor detection of mutations in genes with high expression levels. In contrast, machine learning offers a more comprehensive view, adept at capturing the non-linear complexities of gene expression data and providing robustness against noise that inspired us to utilize machine learning models to explore differential gene expression based on feature importance in Type 2 Diabetes (T2D), a significant global health concern, in this study. Moreover, we validated biomarkers based on our findings expressed genes with previous studies to ensure the effectiveness of our ML models in this work which led us to go through to analysis pathways, gene ontologies, protein-protein interactions, transcription factors, miRNAs, and drug predictions to deal with T2D. This study aims to consider the machine learning technique as a good way to know about expressed genes profoundly not relying on the DGE approach, and to control or reduce the risk of T2D patients by helping drug developer researchers.

PLA2G4A and ACHE modulate lipid profiles via glycerophospholipid metabolism in platinum-resistant gastric cancer

BackgroundBioactive lipids involved in the progression of various diseases. Nevertheless, there is still a lack of biomarkers and relative regulatory targets. The lipidomic analysis of the samples from platinum-resistant in gastric cancer patients is expected to help us further improve our understanding of it.MethodsWe employed LC–MS based untargeted lipidomic analysis to search for potential candidate biomarkers for platinum resistance in GC patients. Partial least squares discriminant analysis (PLS-DA) and variable importance in projection (VIP) analysis were used to identify differential lipids. The possible molecular mechanisms and targets were obtained by metabolite set enrichment analysis and potential gene network screened. Finally, verified them by immunohistochemical of a tissue microarray.ResultsThere were 71 differential lipid metabolites identified in GC samples between the chemotherapy-sensitivity group and the chemotherapy resistance group. According to Foldchange (FC) value, VIP value, P values (FC > 2, VIP > 1.5, p < 0.05), a total of 15 potential biomarkers were obtained, including MGDG(43:11)-H, Cer(d18:1/24:0) + HCOO, PI(18:0/18:1)-H, PE(16:1/18:1)-H, PE(36:2) + H, PE(34:2p)-H, Cer(d18:1 + hO/24:0) + HCOO, Cer(d18:1/23:0) + HCOO, PC(34:2e) + H, SM(d34:0) + H, LPC(18:2) + HCOO, PI(18:1/22:5)-H, PG(18:1/18:1)-H, Cer(d18:1/24:0) + H and PC(35:2) + H. Furthermore, we obtained five potential key targets (PLA2G4A, PLA2G3, DGKA, ACHE, and CHKA), and a metabolite-reaction-enzyme-gene interaction network was built to reveal the biological process of how they could disorder the endogenous lipid profile of platinum resistance in GC patients through the glycerophospholipid metabolism pathway. Finally, we further identified PLA2G4A and ACHE as core targets of the process by correlation analysis and tissue microarray immunohistochemical verification.ConclusionPLA2G4A and ACHE regulated endogenous lipid profile in the platinum resistance in GC patients through the glycerophospholipid metabolism pathway. The screening of lipid biomarkers will facilitate earlier precision medicine interventions for chemotherapy-resistant gastric cancer. The development of therapies targeting PLA2G4A and ACHE could enhance platinum chemotherapy effectiveness.

Unraveling the Dual-Stretch-Mode Impact on Tension Gauge Tethers' Mechanical Stability.

Tension gauge tethers (TGTs), short DNA segments serving as extracellular tension sensors, are instrumental in assessing the tension dynamics in mechanotransduction. These TGTs feature an initial shear-stretch region and an unzip-stretch region. Despite their utility, no theoretical model has been available to estimate their tension-dependent lifetimes [τ(f)], restricting insights from cellular mechanotransduction experiments. We have now formulated a concise expression for τ(f) of TGTs, accommodating contributions from both stretch regions. Our model uncovers a tension-dependent energy barrier shift occurring when tension surpasses a switching force of approximately 13 pN for the recently developed TGTs, greatly influencing τ(f) profiles. Experimental data from several TGTs validated our model. The calibrated expression can predict τ(f) of TGTs at 37 °C based on their sequences with minor fold changes, supporting future applications of TGTs.

Pulmonary Effects of Traumatic Brain Injury in Mice: A Gene Set Enrichment Analysis.

Acute lung injury occurs in 20-25% of cases following traumatic brain injury (TBI). We investigated changes in lung transcriptome expression post-TBI using animal models and bioinformatics. Employing unilateral controlled cortical impact for TBI, we conducted microarray analysis after lung acquisition, followed by gene set enrichment analysis of differentially expressed genes. Our findings indicate significant upregulation of inflammation-related genes and downregulation of nervous system genes. There was enhanced infiltration of adaptive immune cells, evidenced by positive enrichment in Lung-Th1, CD4, and CD8 T cells. Analysis using the Tabula Sapiens database revealed enrichment in lung-adventitial cells, pericytes, myofibroblasts, and fibroblasts, indicating potential effects on lung vasculature and fibrosis. Gene set enrichment analysis linked TBI to lung diseases, notably idiopathic pulmonary hypertension. A Venn diagram overlap analysis identified a common set of 20 genes, with FOSL2 showing the most significant fold change. Additionally, we observed a significant increase in ADRA1A→IL6 production post-TBI using the L1000 library. Our study highlights the impact of brain trauma on lung injury, revealing crucial gene expression changes related to immune cell infiltration, cytokine production, and potential alterations in lung vasculature and fibrosis, along with a specific spectrum of disease influence.

Plasma Circulating mRNA Profile for the Non-Invasive Diagnosis of Colorectal Cancer Using NanoString Technologies.

Colorectal cancer (CRC) is one of the most prevalent cancers and the second leading cause of cancer deaths in developed countries. Early CRC may have no symptoms and symptoms usually appear with more advanced diseases. Regular screening can identify people who are at increased risk of CRC in order to offer earlier treatment. A cost-effective non-invasive platform for the screening and monitoring of CRC patients allows early detection and appropriate treatment of the disease, and the timely application of adjuvant therapy after surgical operation is needed. In this study, a cohort of 71 plasma samples that include 48 colonoscopy- and histopathology-confirmed CRC patients with TNM stages I to IV were recruited between 2017 and 2019. Plasma mRNA profiling was performed in CRC patients using NanoString nCounter. Normalized data were analyzed using a Mann-Whitney U test to determine statistically significant differences between samples from CRC patients and healthy subjects. A multiple-group comparison of clinical phenotypes was performed using the Kruskal-Wallis H test for statistically significant differences between multiple groups. Among the 27 selected circulating mRNA markers, all of them were found to be overexpressed (gene expression fold change > 2) in the plasma of patients from two or more CRC stages. In conclusion, NanoString-based targeted plasma CRC-associated mRNAs circulating the marker panel that can significantly distinguish CRC patients from a healthy population were developed for the non-invasive diagnosis of CRC using peripheral blood samples.

Investigating antibiotic free feed additives for growth promotion in poultry: effects on performance and microbiota

The poultry industry is evolving towards antibiotic-free production to meet market demands and decelerate the increasing spread of the antimicrobial resistance. The growing need for antibiotic free products has challenged producers to decrease or completely stop using antimicrobials as feed supplements in broiler diet to improve feed efficiency, growth rate, and intestinal health. Natural feed additives (e.g., probiotics and phytobiotics) are promising alternatives to substitute antimicrobial growth promoters. The goal of our study was to characterize the effects of a Probiotic and an Essential Oils blend on broilers' performance and perform a time-series analysis to describe their excreta microbiome. A total of 320 Cobb 500 (1-day-old) chicks were raised for 21 d in 32 randomly allocated cages. Treatments consisted of 4 experimental diets: a basal diet, and a basal diet mixed with an Antibiotic (bacitracin methylene disalicylate), an essential oils blend (oregano oil, rosemary, and red pepper), or a Probiotic (Bacillus subtilis). Body weight (on 1, 10, and 21d), and feed intake (10d and 21d) were recorded and feed conversion ratio was calculated. Droppings were collected daily (1-21d) to characterize broilers' excreta microbiota by targeted sequencing of the bacterial 16S rRNA gene. The Probiotic significantly improved feed conversion ratio for starter phase 1 to 10d (P = 0.03), grower phase 10 to 21d (P = 0.05), and total period 1 to 21d (P = 0.01) compared to the Antibiotic. Feed supplements did not affect alpha diversity but did impact microbial beta diversity (P < 0.01). Age also impacted microbiome turnover as differences in alpha and beta diversity were detected. Furthermore, when compared to the basal diet, the probiotic and antibiotic significantly impacted relative abundance of Bifidobacterium (log2 fold change -1.44, P = 0.03), Intestinimonas (log2 fold change 0.560, P < 0.01) and Ligilactobacillus (log2 fold change -1.600, P < 0.01). Overall, Probiotic supplementation but not essential oils supplementation positively impacted broilers' growth performance by directly causing directional shifts in broilers' excreta microbiota structure.

Abstract P06: Characterization of the Immune Tumor Microenvironment in a Diverse Cohort of Patients with Multiple Myeloma

Abstract Multiple myeloma (MM) is the most common blood cancer in African American (AA) individuals. AAs have an increased risk of developing Monoclonal Gammopathy of Undetermined Significance (MGUS) and MM compared to non-Hispanic White (NHW) individuals. While several studies have shown a pivotal role for the immune tumor microenvironment (iTME) in MM pathogenesis, the composition of the iTME in AAs with MM is under investigated. Viably frozen whole bone marrow (BM) aspirates from 128 individuals (53 AA and 75 NHW) with either MM (n=65), MGUS (n=28), smoldering MM (SMM, n=13), or healthy controls (n=22) were evaluated by single cell RNA-sequencing (scRNA-seq) or mass cytometry (CyTOF). We characterized 34 (14 AA and 20 NHW) by scRNA-seq and 94 (39 AA and 55 NHW) by CyTOF. CyTOF samples were stained with Maxpar Direct Immune Profiling Assay Kit (Fluidigm), acquired on a Helios mass cytometer and analyzed in Cytobank. Data were analyzed by logistic equivalent regression model with p&amp;lt;0.05 for significance. ScRNA-seq samples were processed as in Yao, 2022. Data were normalized using scvi-tools and clustered by Uniform manifold approximation and projection (UMAP). Differentially expressed markers between clusters were identified using Model-based Analysis of Single-cell Transcriptomics (MAST) in Seurat R package at FDR &amp;lt; 0.05 and log2 fold change &amp;gt; 0.55. ScRNA-seq of the CD45+ non-tumor lymphocytes revealed 17 immune populations (HSC, erythroblast, MDSC, GMP, CD1c+ DC, CD4+ and CD8+ T cell, CD14+ M1 macrophage, CD16+monocyte, monocyte, pDC, naïve B, pre-B, pro-B, memory B, NK/MAIT). CD3E/D+cells were clustered into naïve/central memory (CCR7+, LEF1+, SELL+), memory (GZMK+), effector (GZMB+, GZMH+, FGFBP2+), g/d (TRDC+), MAIT (SLC4A10+, KLRB1+) and IFN CD8+ T cells (IFIT1+, IFIT2+,IFIT3+). Disease progression was associated with increases in median CD4+ and CD8+ GZMK+ T cells, memory B cells and a reduction in all monocyte clusters. Within the diseased cohort in comparison to NHW patients, AA patients had increased CD3+ T cells (CD4 and CD8+) (AA 57.5% vs. NHW 44.3%) and a reduction in overall monocytes (AA 10.5% vs. 26.5%). These results were validated by CyTOF, which confirmed an increase in CD3+ T cells (AA 53.7% vs. NHW 35.7%, p&amp;lt;0.001) including increases in CD8+ (AA 7.0% vs. NHW 3.2%, p&amp;lt;0.05) and CD4+ terminal effector T cells (AA 5.7% vs. NHW 2.7%, p&amp;lt;0.05) and a reduction in monocytes (AA 18.3% vs. NHW 26.6%) among AAs. After adjusting for age and sex, AAs retained an increase in T cells (OR 1.59 (95%CI 1.2-2.01, p=0.00023)) and a reduction in monocytes (OR 0.88 (95%CI 0.78-1, p=0.045)). Despite the increase in CD8+ T cells in AA patients, we identified reduced GZMK+ T cells and reductions in exhaustion markers (TIGIT, LAG3, TOX) in CD8+ T cells in comparison to NHWs. Nevertheless, the distribution of the immune populations was similar between healthy AA and NHW individuals. Our multiomics approach to characterize the iTME identified significant differences among AA compared to NHW patients with MM. Citation Format: Suganti Shivaram, Hongwei Tang, Huihuang Yan, Shulan Tian, Guyu Qin, Emilie Anderson, Neeraj Sharma, Abiola Bolarinwa, Cinthya Zepeda-Mendoza, Stacey Lehman, Felicia Gomez, Amit Mitra, Surendra Dasari, Taxiarchis Kourelis, Shaji Kumar, Linda Baughn. Characterization of the Immune Tumor Microenvironment in a Diverse Cohort of Patients with Multiple Myeloma [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P06.

CRISPR/Cas9-edited ROS1 + non-small cell lung cancer cell lines highlight differential drug sensitivity in 2D vs 3D cultures while reflecting established resistance profiles

IntroductionThe study of resistance-causing mutations in oncogene-driven tumors is fundamental to guide clinical decisions. Several point mutations affecting the ROS1 kinase domain have been identified in the clinical setting, but their impact requires further exploration, particularly in improved pre-clinical models. Given the scarcity of solid pre-clinical models to approach rare cancer subtypes like ROS1 + NSCLC, CRISPR/Cas9 technology allows the introduction of mutations in patient-derived cell lines for which resistant variants are difficult to obtain due to the low prevalence of cases within the clinical setting.MethodsIn the SLC34A2-ROS1 rearranged NSCLC cell line HCC78, we knocked-in through CRISPR/Cas9 technology three ROS1 drug resistance-causing mutations: G2032R, L2026M and S1986Y. Such variants are located in different functional regions of the ROS1 kinase domain, thus conferring TKI resistance through distinct mechanisms. We then performed pharmacological assays in 2D and 3D to assess the cellular response of the mutant lines to crizotinib, entrectinib, lorlatinib, repotrectinib and ceritinib. In addition, immunoblotting assays were performed in 2D-treated cell lines to determine ROS1 phosphorylation and MAP kinase pathway activity. The area over the curve (AOC) defined by the normalized growth rate (NGR_fit) dose–response curves was the variable used to quantify the cellular response towards TKIs.ResultsSpheroids derived from ROS1G2032R cells were significantly more resistant to repotrectinib (AOC fold change = − 7.33), lorlatinib (AOC fold change = − 6.17), ceritinib (AOC fold change = − 2.8) and entrectinib (AOC fold change = − 2.02) than wild type cells. The same cells cultured as a monolayer reflected the inefficacy of crizotinib (AOC fold change = − 2.35), entrectinib (AOC fold change = − 2.44) and ceritinib (AOC fold change = − 2.12) in targeting the ROS1 G2032R mutation. ROS1L2026M cells showed also remarkable resistance both in monolayer and spheroid culture compared to wild type cells, particularly against repotrectinib (spheroid AOC fold change = − 2.19) and entrectinib (spheroid AOC fold change = − 1.98). ROS1S1986Y cells were resistant only towards crizotinib in 2D (AOC fold change = − 1.86). Overall, spheroids showed an increased TKI sensitivity compared to 2D cultures, where the impact of each mutation that confers TKI resistance could be clearly distinguished. Western blotting assays qualitatively reflected the patterns of response towards TKI observed in 2D culture through the levels of phosphorylated-ROS1. However, we observed a dose–response increase of phosphorylated-Erk1/2, suggesting the involvement of the MAPK pathway in the mediation of apoptosis in HCC78 cells.ConclusionIn this study we knock-in for the first time in a ROS1 + patient-derived cell line, three different known resistance-causing mutations using CRISPR/Cas9 in the endogenous translocated ROS1 alleles. Pharmacological assays performed in 2D and 3D cell culture revealed that spheroids are more sensitive to TKIs than cells cultured as a monolayer. This direct comparison between two culture systems could be done thanks to the implementation of normalized growth rates (NGR) to uniformly quantify drug response between 2D and 3D cell culture. Overall, this study presents the added value of using spheroids and positions lorlatinib and repotrectinib as the most effective TKIs against the studied ROS1 resistance point mutations.