Significant Fold Change Research Articles

Triple and quadruple optimization for feature selection in cancer biomarker discovery

The proliferation of omics data has advanced cancer biomarker discovery but often falls short in external validation, mainly due to a narrow focus on prediction accuracy that neglects clinical utility and validation feasibility. We introduce three- and four-objective optimization strategies based on genetic algorithms to identify clinically actionable biomarkers in omics studies, addressing classification tasks aimed at distinguishing hard-to-differentiate cancer subtypes beyond histological analysis alone. Our hypothesis is that by optimizing more than one characteristic of cancer biomarkers, we may identify biomarkers that will enhance their success in external validation. Our objectives are to: (i) assess the biomarker panel’s accuracy using a machine learning (ML) framework; (ii) ensure the biomarkers exhibit significant fold-changes across subtypes, thereby boosting the success rate of PCR or immunohistochemistry validations; (iii) select a concise set of biomarkers to simplify the validation process and reduce clinical costs; and (iv) identify biomarkers crucial for predicting overall survival, which plays a significant role in determining the prognostic value of cancer subtypes. We implemented and applied triple and quadruple optimization algorithms to renal carcinoma gene expression data from TCGA. The study targets kidney cancer subtypes that are difficult to distinguish through histopathology methods. Selected RNA-seq biomarkers were assessed against the gold standard method, which relies solely on clinical information, and in external microarray-based validation datasets. Notably, these biomarkers achieved over 0.8 of accuracy in external validations and added significant value to survival predictions, outperforming the use of clinical data alone with a superior c-index. The provided tool also helps explore the trade-off between objectives, offering multiple solutions for clinical evaluation before proceeding to costly validation or clinical trials.

Differential abundance of microRNAs in seminal plasma extracellular vesicles (EVs) in Sahiwal cattle bull related to male fertility.

Sahiwal cattle, known for their high milk yield, are propagated through artificial insemination (AI) using male germplasm, largely contingent on semen quality. Spermatozoa, produced in the testes, carry genetic information and molecular signals essential for successful fertilization. Seminal plasma, in addition to sperm, contains nano-sized lipid-bound extracellular vesicles (SP-EVs) that carry key biomolecules, including fertility-related miRNAs, which are essential for bull fertility. The current study focused on miRNA profiling of SP-EVs from high-fertile (HF) and low-fertile (LF) Sahiwal bulls. SP-EVs were isolated using size exclusion chromatography (SEC) and characterized by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Western blotting detected the EV-specific protein markers TSG101 and CD63. The DLS analysis showed SP-EV sizes of 170-180nm in HF and 130-140nm in LF samples. The NTA revealed particle concentrations of 5.76 × 1010 to 5.86 × 1011 particles/mL in HF and 5.31 × 1010 to 2.70 × 1011 particles/mL in LF groups, with no significant differences in size and concentration between HF and LF. High-throughput miRNA sequencing identified 310 miRNAs in SP-EVs from both groups, with 61 upregulated and 119 downregulated in HF bull. Further analysis identified 41 miRNAs with significant fold changes and p-values, including bta-miR-1246, bta-miR-195, bta-miR-339b, and bta-miR-199b, which were analyzed for target gene prediction. Gene Ontology (GO) and KEGG pathway analyses indicated that these miRNAs target genes involved in transcription regulation, ubiquitin-dependent endoplasmic reticulum-associated degradation (ERAD) pathways, and signalling pathways. Functional exploration revealed that these genes play roles in spermatogenesis, motility, acrosome reactions, and inflammatory responses. qPCR analysis showed that bta-miR-195 had 80% higher expression in HF spermatozoa compared to LF, suggesting its association with fertility status (p < 0.05). In conclusion, this study elucidates the miRNA cargoes in SP-EVs as indicators of Sahiwal bull fertility, highlighting bta-miR-195 as a potential fertility factor among the various miRNAs identified.

Molecular Signature Associated With Acute Rejection in Vascularized Composite Allotransplantation.

A deeper understanding of acute rejection in vascularized composite allotransplantation is paramount for expanding its utility and longevity. There remains a need to develop more precise and accurate tools for diagnosis and prognosis of these allografts, as well as alternatives to traditional immunosuppressive regimens. Twenty-seven skin biopsies collected from 3 vascularized composite allotransplantation recipients, consisting of face and hand transplants, were evaluated by histology, immunohistochemistry staining, and gene expression profiling. Biopsies with clinical signs and symptoms of rejection, irrespective of histopathological grading, were significantly enriched for genes contributing to the adaptive immune response, innate immune response, and lymphocyte activation. Inflammation episodes exhibited significant fold change correlations between the face and hands, as well as across patients. Immune checkpoint genes were upregulated during periods of inflammation that necessitated treatment. A gene signature consisting of CCL5, CD8A, KLRK1, and IFNγ significantly predicted inflammation specific to vascularized composite allografts that required therapeutic intervention. The mechanism of vascularized composite allograft-specific inflammation and rejection appears to be conserved across different patients and skin on different anatomical sites. A concise gene signature can be utilized to ascertain graft status along with a continuous scale, providing valuable diagnostic and prognostic information to supplement current gold standards of graft evaluation.

Abstract C103: Exploring ethnicity-specific molecular mechanism of cholesterol and fatty acid synthesis pathway in Hispanic colorectal cancer

Abstract Colorectal cancer (CRC) is the most prevalent gastrointestinal cancer, the third most common malignancy and the second leading cause of cancer-related mortality worldwide. The American Cancer Society (ACS) estimates 152,810 new cases of colorectal cancer, with an estimated 53,010 deaths in 2024. Typically diagnosed in older adults, CRC incidence is rising among younger individuals, particularly in Hispanics (16.5%) compared to non-Hispanic whites (8.7%). Due to the lack of timely screenings in this ethnic group, further research is crucial to identify novel biomarkers and therapeutic targets for improved early detection and intervention. To accomplish this goal, we performed RNA-sequencing of Hispanic and non-Hispanic White (NHW) CRC tissues. The transcriptomic analysis revealed differential gene expression patterns in Hispanic and NHW populations when compared to respective normal adjacent tissues (NATs). We identified 3577 differentially expressed genes (DEGs) in Hispanic CRC samples and 2971 DEGs in NHW CRC samples, of which 1831 DEGs were unique to Hispanic. Based on the preliminary analysis we hypothesized that unique DEGs from the Hispanic population could help identify potential pathways contributing to CRC progression. Among these 1831 unique DEGs, many of the genes are associated with cholesterol and fatty acid synthesis pathway. The cholesterol biosynthesis pathway is a highly diverse and interconnected signaling pathway, playing essential roles in cellular function and systemic homeostasis. Its complexity and regulatory mechanisms make it a critical area of study in cancer. Studies indicate that Hispanics and African Americans with high cholesterol tend to have higher Body mass index (BMI) levels compared to Whites which has an increased risk of colorectal cancer. However, the role of cholesterol metabolism and fatty acid metabolism in CRC have not been studied from the Hispanic health disparities perspective. From the 1831 DEGs, we selected those in the cholesterol and fatty acid synthesis pathways with significant log2 fold changes (≤ -2 or ≥ +2) and adjusted p-values (FDR ≤ 0.05). Our bioinformatics analysis identified four key DEGs: LCN2, DHCR7, PCSK9 and SQLE. The expression levels of these genes were examined in Hispanic CRC vs. NHW CRC tissue samples using qRT-PCR. Elevated expression of LCN2 and SQLE was observed in late-stage Hispanic CRC tissues compared to NHW CRC tissues, while DHCR7 exhibited high expression in early-stage Hispanic CRC. On the other hand, PCSK9 showed low expression in both early and late stages of Hispanic CRC, suggesting potential ethnicity-specific molecular signatures in CRC progression. Moving forward, protein level studies and functional analysis of these DEGs in Hispanic CRC tissues and organoids will enhance our understanding of the role of cholesterol and fatty acid synthesis pathway proteins. Thus, elucidating novel molecular mechanisms underlying CRC progression could lead to identification of potential Hispanic specific biomarkers and therapeutic targets for early diagnosis and intervention. Citation Format: Somrita Roy, Soumya Nair, MD Zahirul Islam Khan, Sourav Roy. Exploring ethnicity-specific molecular mechanism of cholesterol and fatty acid synthesis pathway in Hispanic colorectal cancer [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 C103.

Identification of circadian clock-related immunological prognostic index and molecular subtypes in prostate cancer

BackgroundEvidence suggests that the circadian clock (CIC) is among the important factors for tumorigenesis. We aimed to provide new insights into CIC-mediated molecular subtypes and gene prognostic indexes for prostate cancer (PCa) patients undergoing radical prostatectomy (RP) or radical radiotherapy (RT).MethodsPCa data from TCGA was analyzed to identify differentially expressed genes (DEGs) with significant fold changes and p-values. A prognostic index called CIC-related gene prognostic index (CICGPI) was developed through clustering methods and survival analysis and validated on multiple data sets. The diagnostic accuracy of CICGPI for resistance to chemotherapy and radiotherapy was confirmed. Additionally, the interaction between tumor immune environment and CICGPI score was explored, along with their correlation with prognosis.ResultsTOP2A, APOE, and ALDH2 were used to classify the PCa patients into two subtypes. Cluster 2 had a higher risk of biochemical recurrence (BCR) than cluster 1 for PCa patients undergoing RP or RT. A CIC-related gene prognostic index (CICGPI) was constructed using the above three genes for PCa patents in the TCGA database. The CICGPI score showed good prognostic value in the TCGA database and was externally confirmed by PCa patients in GSE116918, MSKCC2010 and GSE46602. In addition, the CICGPI score had a certain and high diagnostic accuracy for tumor chemoresistance (AUC: 0.781) and radioresistance (AUC: 0.988). For gene set variation analysis, we observed that both beta alanine metabolism and limonene and pinene degradation were upregulated in cluster 1 for PCa patients undergoing RP or RT. For PCa patients undergoing RP, cell cycle, homologous recombination, mismatch repair, and DNA replication were upregulated in cluster 2. A strongly positive relationship between cancer-related fibroblasts and CICGPI score was observed in PCa patients undergoing RP or RT. Moreover, a high density of CAFs was highly closely associated with poorer BCR-free survival of PCa patients.ConclusionsIn this study, we established CIC-related immunological prognostic index and molecular subtypes, which might be useful for the clinical practice.

LncRNA PCIF1 promotes aerobic glycolysis in A549/DDP cells by competitively binding miR-326 to regulate PKM expression

ObjectiveUtilizing transcriptome analysis to investigate the mechanisms and therapeutic approaches for cisplatin resistance in non-small cell lung cancer (NSCLC). MethodsFirstly, the biological characters of A549 cells and A549/DDP cells were detected by RNA sequencing, CCK-8 and hippocampal energy analyzer. Then, the differential Genes were functionally enriched by GO and KEGG and the competitive endogenous RNA network map was constructed. Finally, the effects of the predicted biogenesis pathway on the biological functions of A549/DDP cells were verified by in vitro and in vivo experiments. ResultThe differentially transcribed genes of A549 and A549/DDP cells were analyzed by enrichment analysis and cell biological characteristics detection. The results showed that A549/DDP cells showed significantly increased resistance to cisplatin, glucose metabolism signaling pathway and glycolysis levels compared with A549 cells. Among glycolysis-related transcription genes, PKM had the most significant difference Fold Change is 8. LncRNA PCIF1 is a new marker of A549/DDP cells and can be used as a molecular sponge to regulate the expression of PKM. LncRNA PCIF1 targets miR-326 to induce PKM expression, promote glycolysis level, and enhance the resistance of A549/DDP cells to cisplatin. ConclusionLncRNA PCIF1 as biomarkers of A549/DDP cells, higher expression can induce the PKM, promote cell glycolysis, lead to the occurrence of cisplatin resistance. LncRNA PCIF1 can be considered as a potential target for treating cisplatin-resistant NSCLC.

Exploring the potential of Lactocaseibacillus rhamnosus PMC203 in inducing autophagy to reduce the burden of Mycobacterium tuberculosis

Mycobacterium tuberculosis, a lethal pathogen in human history, causes millions of deaths annually, which demands the development of new concepts of drugs. Considering this fact, earlier research has explored the anti-tuberculosis potential of a probiotic strain, Lactocaseibacillus rhamnosus PMC203, leading to a subsequent focus on the molecular mechanism involved in its effect, particularly on autophagy. In this current study, immunoblotting-based assay exhibited a remarkable expression of autophagy marker LC3-II in the PMC203 treated group compared to an untreated group. A remarkable degradation of p62 was also noticed within treated cells compared to control. Furthermore, the immunofluorescence-based assay showed significant fold change in fluorescence intensity for alexa-647-LC3 and alexa-488-LC3, whereas p62 was degraded noticeably. Moreover, lysosomal biogenesis generation was elevated significantly in terms of LAMP1 and acidic vesicular organelles. As a result, PMC203-induced autophagy played a vital role in reducing M. tuberculosis burden within the macrophages in treated groups compared to untreated group. A colony -forming unit assay also revealed a significant reduction in M. tuberculosis in the treated cells over time. Additionally, the candidate strain significantly upregulated the expression of autophagy induction and lysosomal biogenesis genes. Together, these results could enrich our current knowledge of probiotics-mediated autophagy in tuberculosis and suggest its implications for innovatively managing tuberculosis.

B Cell Lymphocytes as a Potential Source of Breast Carcinoma Marker Candidates.

Despite advances in the genomic classification of breast cancer, current clinical tests and treatment decisions are commonly based on protein-level information. Nowadays breast cancer clinical treatment selection is based on the immunohistochemical (IHC) determination of four protein biomarkers: Estrogen Receptor 1 (ESR1), Progesterone Receptor (PGR), Human Epidermal Growth Factor Receptor 2 (HER2), and proliferation marker Ki-67. The prognostic correlation of tumor-infiltrating T cells has been widely studied in breast cancer, but tumor-infiltrating B cells have not received so much attention. We aimed to find a correlation between immunohistochemical results and a proteomic approach in measuring the expression of proteins isolated from B-cell lymphocytes in peripheral blood samples. Shotgun proteomic analysis was chosen for its key advantage over other proteomic methods, which is its comprehensive and untargeted approach to analyzing proteins. This approach facilitates better characterization of disease-associated changes at the protein level. We identified 18 proteins in B cell lymphocytes with a significant fold change of more than 2, which have promising potential to serve as breast cancer biomarkers in the future.

Profiling several key milk miRNAs and analysing their signalling pathways in dairy sheep breeds during peak and late lactation.

The comprehensive understanding of microRNAs (miRNAs) in sheep milk during various lactation periods and their impact on milk yield and composition remains limited. This study aimed to investigate the expression patterns of four highly expressed miRNAs in sheep milk and their association with milk composition and yield parameters during peak and late lactation stages. A total of 40 healthy 4-year-old Akkaraman (n = 20) and Awassi (n = 20) ewes registered with the Ministry of Agriculture and Forestry of the Republic of Türkiye were used in the present study. For miRNA isolation from milk, the Qiagen miRNeasy Serum/Plasma Advanced Kit was utilised following the manufacturer's instructions. The expression levels of miRNAs were assessed using Qiagen miRNA PCR Assays. The significant fold changes in the expression levels of oar-miR-30a-5p, oar-miR-148a and oar-miR-181a were observed between peak and late lactation periods in the Awassi sheep breed. Conversely, only oar-miR-30a-5p and oar-miR-148a exhibited statistically significant changes in the Akkaraman sheep breed during the same lactation periods. Furthermore, oar-miR-21-5p demonstrated a significant fold change exclusively in peak lactation compared to Akkaraman and Awassi ewes. The findings suggest that the expression of the analysed miRNAs is influenced by both the lactation stage and different sheep breeds. This study offers valuable insights into the relationship between key miRNA expressions in sheep milk and milk composition and yield parameters during peak and late lactation, contributing to the existing knowledge in this field.

Cryptic Resilience: Decoding Molecular Networks in Pearl Millet for Enhanced Heat Stress

Pearl millet (&lt;em&gt;Pennisetum glaucum&lt;/em&gt;), a vital cereal crop renowned for its drought tolerance, is a cornerstone for smallholder farmers in arid and semi-arid regions, ranking as the fifth most significant cereal globally. Despite its resilience, the molecular mechanisms underlying its tolerance to heat stress remained elusive. To address this knowledge gap, we subjected ten-day-old pearl millet seedlings to an unprecedented temperature of 50°C for 60 seconds. Subsequent next-generation RNA sequencing aimed to unravel differential gene expression in heat-stressed seedlings compared to control conditions. Our analysis revealed a remarkable 29.8% differential expression in the genome sequence in response to heat stress. Heat-stressed pearl millet leaves exhibited differential expression in 11,483 genes, with fold changes ranging from 2 to 18.6 compared to the control group. Of these, 3,612 genes displayed upregulation, while 7,871 genes exhibited downregulation. These genes play roles in diverse biological processes involving crucial enzymes such as aminoacyl-tRNA synthetases, ligases, methyltransferases, oxidoreductases, and DNA-directed RNA polymerases. The Photosystem II Type I Chlorophyll-a/b-binding protein and heat shock proteins displayed the most significant fold changes in heat-stressed leaves. Moreover, various transcription factor families, including bHLH, ERF, NAC, WRKY, MYB-related, C2H2, bZIP, MYB, FAR1, and B3, vital in controlling pearl millet's response to heat stress, were linked to over 100 differentially expressed genes. The dataset generated through this research, shedding light on the molecular processes enabling pearl millet to withstand heat, holds immense value given the crop's role in food security and resilience to extreme weather. In the context of climate change and global warming, this knowledge lays the foundation for further studies on metabolic engineering and selecting crops resilient to high temperatures. Our transcriptomics approach provides comprehensive gene expression profiles of heat-stressed plants. It elucidates pearl millet's response to heat stress, offering a crucial resource for future investigations into crop adaptation strategies.

Comprehensive insights into the impact of bacterial indole-3-acetic acid on sensory preferences in Drosophila melanogaster

Several bacteria of environmental and clinical origins, including some human-associated strains secrete a cross-kingdom signaling molecule indole-3-acetic acid (IAA). IAA is a tryptophan (trp) derivative mainly known for regulating plant growth and development as a hormone. However, the nutritional sources that boost IAA secretion in bacteria and the impact of secreted IAA on non-plant eukaryotic hosts remained less explored. Here, we demonstrate significant trp-dependent IAA production in Pseudomonas juntendi NEEL19 when provided with ethanol as a carbon source in liquid cultures. IAA was further characterized to modulate the odor discrimination, motility and survivability in Drosophila melanogaster. A detailed analysis of IAA-fed fly brain proteome using high-resolution mass spectrometry showed significant (fold change, ± 2; p ≤ 0.05) alteration in the proteins governing neuromuscular features, audio-visual perception and energy metabolism as compared to IAA-unfed controls. Sex-wise variations in differentially regulated proteins were witnessed despite having similar visible changes in chemo perception and psychomotor responses in IAA-fed flies. This study not only revealed ethanol-specific enhancement in trp-dependent IAA production in P. juntendi, but also showed marked behavioral alterations in flies for which variations in an array of proteins governing odor discrimination, psychomotor responses, and energy metabolism are held responsible. Our study provided novel insights into disruptive attributes of bacterial IAA that can potentially influence the eukaryotic gut-brain axis having broad environmental and clinical implications.

Transcriptome exploration of ferroptosis-related genes in TGFβ- induced lens epithelial to mesenchymal transition during posterior capsular opacification development

BackgroundPosterior capsular opacification (PCO) is the main reason affecting the long-term postoperative result of cataract patient, and it is well accepted that fibrotic PCO is driven by transforming growth factor beta (TGFβ) signaling. Ferroptosis, closely related to various ocular diseases, but has not been explored in PCO.MethodsRNA sequencing (RNA-seq) was performed on both TGF-β2 treated and untreated primary lens epithelial cells (pLECs). Differentially expressed genes (DEGs) associated with ferroptosis were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to investigate their biological function. Additionally, protein-to-protein interactions among selected ferroptosis-related genes by PPI network and the top 10 genes with the highest score (MCC algorithm) were selected as the hub genes. The top 20 genes with significant fold change values were validated using quantitative real-time polymerase chain reaction (qRT-PCR).ResultsOur analysis revealed 1253 DEGs between TGF-β2 treated and untreated pLECs, uncovering 38 ferroptosis-related genes between two groups. Among these 38 ferroptosis-related genes,the most prominent GO enrichment analysis process involved in the response to oxidative stress (BPs), apical part of cell (CCs),antioxidant activity (MFs). KEGG were mainly concentrated in fluid shear stress and atherosclerosis, IL-17 and TNF signaling pathways, and validation of top 20 genes with significant fold change value were consistent with RNA-seq.ConclusionsOur RNA-Seq data identified 38 ferroptosis-related genes in TGF-β2 treated and untreated pLECs, which is the first observation of ferroptosis related genes in primary human lens epithelial cells under TGF-β2 stimulation.Graphical Our study initially observed ferroptosis related genes in primary human lens epithelial cells stimulated by TGF-β2. These findings may improve the understanding of the molecular mechanisms of PCO and provide a new direction for exploring the potential mechanisms of PCO

Phylogenetic analysis and antimony resistance of Leishmania major isolated from humans and rodents.

Cutaneous Leishmaniasis (CL) is one of the world's neglected diseases which is caused by Leishmania spp. The aim of this study was to assess molecular profile and antimony resistance of Leishmania isolated from human and rodent hosts. Samples were collected from suspected CL patients referred tohealth centres and wild rodent's traps in Gonbad-e-Qabus region, north-eastern Iran. Smears weresubjected to PCR-RFLP to identify Leishmania species. In addition, ITS1-PCR products weresequenced for phylogenetic analysis. Clinical isolates and rodent samples were subjected to MTTassay to determine IC50 values and in vitro susceptibilities. Expression levels of antimony resistance-related genes were determined in CL isolates. Out of 1,949 suspected patients with CL and 148rodents, 1,704 (87.4%) and 6 (4.05%) were positive with direct smear, respectively. Digestion patterns of BusRI (HaeIII) endonuclease enzyme were similar to what expected for Leishmania major. Phylogenetic analysis revealed that the highest interspecies similarity was found between current L.major sequences with L. major obtained from Russia and Uzbekistan. Out of 20 L. major samples tested, 13 (65%) were resistant to meglumine antimoniate (MA) treatment, with an activity index (AI) exceeding 4. The remaining 7 samples (35%) responded to MA treatment and were classified as sensitive isolates, with a confirmed sensitive phenotype based on their AI values. The comparison expression analysis of three major antimony resistance-associated genes in unresponsive clinical isolates demonstrated significant fold changes for TDR1 (4.78-fold), AQP1 (1.3-fold), and γ-GCS (1.17-fold) genes (P < 0.05). Herein, we demonstrate genetic diversity and antimony resistance of L.major isolated from human and reservoir hosts in north-eastern Iran, which could be the basis forplanning future control strategies.

The mouse retinal pigment epithelium mounts an innate immune defense response following retinal detachment

The retinal pigment epithelium (RPE) maintains photoreceptor viability and function, completes the visual cycle, and forms the outer blood-retinal barrier (oBRB). Loss of RPE function gives rise to several monogenic retinal dystrophies and contributes to age-related macular degeneration. Retinal detachment (RD) causes separation of the neurosensory retina from the underlying RPE, disrupting the functional and metabolic relationships between these layers. Although the retinal response to RD is highly studied, little is known about how the RPE responds to loss of this interaction. RNA sequencing (RNA-Seq) was used to compare normal and detached RPE in the C57BL6/J mouse. The naïve mouse RPE transcriptome was compared to previously published RPE signature gene lists and from the union of these 14 genes (Bmp4, Crim1, Degs1, Gja1, Itgav, Mfap3l, Pdpn, Ptgds, Rbp1, Rnf13, Rpe65, Slc4a2, Sulf1 and Ttr) representing a core signature gene set applicable across rodent and human RPE was derived. Gene ontology enrichment analysis (GOEA) of the mouse RPE transcriptome identified expected RPE features and functions, such as pigmentation, phagocytosis, lysosomal and proteasomal degradation of proteins, and barrier function. Differentially expressed genes (DEG) at 1 and 7 days post retinal detachment (dprd) were defined as mRNA with a significant (padj≤0.05) fold change (FC) of 0.67 ≥ FC ≥ 1.5 in detached versus naïve RPE. The RPE transcriptome exhibited dramatic changes at 1 dprd, with 2297 DEG identified. The KEGG pathways and biological process GO groups related to innate immune responses were significantly enriched. Lipocalin 2 (Lcn2) and several chemokines were upregulated, while numerous genes related to RPE functions, such as pigment synthesis, visual cycle, phagocytosis, and tight junctions were downregulated at 1 dprd. The response was largely transient, with only 18 significant DEG identified at 7 dprd, including upregulation of complement gene C4b. Validation studies confirmed RNA-Seq results. Thus, the RPE quickly downregulates cell-specific functions and mounts an innate immune defense response following RD. Our data demonstrate that the RPE contributes to the inflammatory response to RD and may play a role in attraction of immune cells to the subretinal space.

Abstract 4328: Erlotinib suppresses autophagy in colorectal cancer

Abstract Introduction: Oncogenic KRAS mutation is prevalent in approximately 45% of colorectal cancer (CRC) patients. Limited targeted therapies are available to inhibit the detrimental effects of KRAS mutation primarily pertaining to its unique structural intricacies. Erlotinib is an EGFR inhibitor which inducing autophagy in lung cancer cells. In this study, we followed the effects of Erlotinib treatment in mutant and wild-type colorectal cancer (CRC) cells. We report that Erlotinib exhibits selectivity between mutated KRAS and wild type, significantly modulating the expression of autophagy proteins ATG5, ULK1, Beclin1, and LC3A. Methodology: The 2 cell lines used in this study were HCT116 (KRAS mutant) and Hke3 (KRAS wild type). These cells were treated with Erlotinib for 24 hours and 48 hours, A cell viability assay was performed and subsequently followed by protein extraction. The expression of autophagy proteins ATG5, ULK1, Beclin1, and LC3A was determined. The TCGA COAD-READ patient dataset was utilized using GEPIA 2 software. A correlation was found between the autophagy proteins and the KRAS gene expression. Results: The cell viability assays showed that there was significantly more cell death in KRAS wild-type cells compared with KRAS mutant at time points ranging from 24 to 48 hours (p = 0.036). The experiment also showed that treatment of Erlotinib with concentrations of 20 uM, 35 uM, and 50 uM and significant down regulatory fold changes (p &amp;lt; 0.05). Both KRAS mutated and wild-type cells treated with Erlotinib expressed significant downregulation of ATG5, ULK1, Beclin1, and LC3A proteins at 48 hours (p &amp;lt; 0.05). The 20 uM treatment demonstrated a significance of p &amp;lt; 0.05 for all proteins. The 20 uM treatment resulted in fold changes of the KRAS mutated cells for ATG5, ULK1, Beclin1, and LC3A to be 1.42, 1.42, 1.81, and 2.09 respectively. A strong KRAS correlation was found with ATG5, ULK1, Beclin1, and LC3A using COAD &amp; READ tumor and COAD &amp; READ normal (R &amp;gt; 0.75, p &amp;lt; 0.05). Conclusion: This study highlights the effects of Erlotinib as a treatment for KRAS mutated CRC. Our results confirmed that Erlotinib had the opposite effect on CRC cells in relation to the expression of the autophagy proteins when compared to lung cancer cells. There were distinct effects of Erlotinib between KRAS wild-type and mutant cells in both cell viability and the expression of autophagy-related proteins. In both KRAS cell types, there was significant downregulation of autophagy proteins, however, the KRAS wild type had significantly more downregulation than the KRAS mutant. This data suggests that there is a relationship between KRAS mutation and the expression of the autophagy proteins. Our analysis of the patient datasets confirmed the strong correlation between KRAS mutation and the expression of autophagy proteins. These findings emphasize the need to explore further how Erlotinib can be augmented into the current treatment regimen as a benefit to CRC patients. Citation Format: Alexander Siegman, Aaron Shaykevich, Isaac Silverman, Sanjay Goel, Radhashree Maitra. Erlotinib suppresses autophagy in colorectal cancer [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 4328.

Integrated plasma metabolomic and cytokine analysis reveals a distinct immunometabolic signature in atopic dermatitis.

Disease models for atopic dermatitis (AD) have primarily focused on understanding underlying environmental, immunologic, and genetic etiologies. However, the role of metabolic mechanisms in AD remains understudied. To investigate the circulating blood metabolomic and cytokine profile of AD as compared to healthy control patients. This study collected plasma from 20 atopic dermatitis with moderate-to-severe itch (score of ≥5 on the itch Numeric Rating Scale and IGA score ≥3) and 24 healthy control patients. Mass-spectrometry based metabolite data were compared between AD and healthy controls. Unsupervised and supervised machine learning algorithms and univariate analysis analyzed metabolic concentrations. Metabolite enrichment and pathway analyses were performed on metabolites with significant fold change between AD and healthy control patients. To investigate the correlation between metabolites levels and cytokines, Spearman's rank correlation coefficients were calculated between metabolites and cytokines. Patients were recruited from the Johns Hopkins Itch Center and dermatology outpatient clinics in the Johns Hopkins Outpatient Center. The study included 20 atopic dermatitis patients and 24 healthy control patients. Fold changes of metabolites in AD vs healthy control plasma. In patients with AD, amino acids isoleucine, tyrosine, threonine, tryptophan, valine, methionine, and phenylalanine, the amino acid derivatives creatinine, indole-3-acrylic acid, acetyl-L-carnitine, L-carnitine, 2-hydroxycinnamic acid, N-acetylaspartic acid, and the fatty amide oleamide had greater than 2-fold decrease (all P-values<0.0001) compared to healthy controls. Enriched metabolites were involved in branched-chain amino acid (valine, leucine, and isoleucine) degradation, catecholamine biosynthesis, thyroid hormone synthesis, threonine metabolism, and branched and long-chain fatty acid metabolism. Dysregulated metabolites in AD were positively correlated cytokines TARC and MCP-4 and negatively correlated with IL-1a and CCL20. Our study characterized novel dysregulated circulating plasma metabolites and metabolic pathways that may be involved in the pathogenesis of AD. These metabolic pathways serve as potential future biomarkers and therapeutic targets in the treatment of AD.

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.

Biological insights from plasma proteomics of non-small cell lung cancer patients treated with immunotherapy.

Immune checkpoint inhibitors have made a paradigm shift in the treatment of non-small cell lung cancer (NSCLC). However, clinical response varies widely and robust predictive biomarkers for patient stratification are lacking. Here, we characterize early on-treatment proteomic changes in blood plasma to gain a better understanding of treatment response and resistance. Pre-treatment (T0) and on-treatment (T1) plasma samples were collected from 225 NSCLC patients receiving PD-1/PD-L1 inhibitor-based regimens. Plasma was profiled using aptamer-based technology to quantify approximately 7000 plasma proteins per sample. Proteins displaying significant fold changes (T1:T0) were analyzed further to identify associations with clinical outcomes using clinical benefit and overall survival as endpoints. Bioinformatic analyses of upregulated proteins were performed to determine potential cell origins and enriched biological processes. The levels of 142 proteins were significantly increased in the plasma of NSCLC patients following ICI-based treatments. Soluble PD-1 exhibited the highest increase, with a positive correlation to tumor PD-L1 status, and, in the ICI monotherapy dataset, an association with improved overall survival. Bioinformatic analysis of the ICI monotherapy dataset revealed a set of 30 upregulated proteins that formed a single, highly interconnected network, including CD8A connected to ten other proteins, suggestive of T cell activation during ICI treatment. Notably, the T cell-related network was detected regardless of clinical benefit. Lastly, circulating proteins of alveolar origin were identified as potential biomarkers of limited clinical benefit, possibly due to a link with cellular stress and lung damage. Our study provides insights into the biological processes activated during ICI-based therapy, highlighting the potential of plasma proteomics to identify mechanisms of therapy resistance and biomarkers for outcome.

Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria.

The RNA recognition motif (RRM) is the most common RNA-binding protein domain identified in nature. However, RRM-containing proteins are only prevalent in eukaryotic phyla, in which they play central regulatory roles. Here, we engineered an orthogonal post-transcriptional control system of gene expression in the bacterium Escherichia coli with the mammalian RNA-binding protein Musashi-1, which is a stem cell marker with neurodevelopmental role that contains two canonical RRMs. In the circuit, Musashi-1 is regulated transcriptionally and works as an allosteric translation repressor thanks to a specific interaction with the N-terminal coding region of a messenger RNA and its structural plasticity to respond to fatty acids. We fully characterized the genetic system at the population and single-cell levels showing a significant fold change in reporter expression, and the underlying molecular mechanism by assessing the in vitro binding kinetics and in vivo functionality of a series of RNA mutants. The dynamic response of the system was well recapitulated by a bottom-up mathematical model. Moreover, we applied the post-transcriptional mechanism engineered with Musashi-1 to specifically regulate a gene within an operon, implement combinatorial regulation, and reduce protein expression noise. This work illustrates how RRM-based regulation can be adapted to simple organisms, thereby adding a new regulatory layer in prokaryotes for translation control.

Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria

The RNA recognition motif (RRM) is the most common RNA-binding protein domain identified in nature. However, RRM-containing proteins are only prevalent in eukaryotic phyla, in which they play central regulatory roles. Here, we engineered an orthogonal post-transcriptional control system of gene expression in the bacterium Escherichia coli with the mammalian RNA-binding protein Musashi-1, which is a stem cell marker with neurodevelopmental role that contains two canonical RRMs. In the circuit, Musashi-1 is regulated transcriptionally and works as an allosteric translation repressor thanks to a specific interaction with the N-terminal coding region of a messenger RNA and its structural plasticity to respond to fatty acids. We fully characterized the genetic system at the population and single-cell levels showing a significant fold change in reporter expression, and the underlying molecular mechanism by assessing the in vitro binding kinetics and in vivo functionality of a series of RNA mutants. The dynamic response of the system was well recapitulated by a bottom-up mathematical model. Moreover, we applied the post-transcriptional mechanism engineered with Musashi-1 to specifically regulate a gene within an operon, implement combinatorial regulation, and reduce protein expression noise. This work illustrates how RRM-based regulation can be adapted to simple organisms, thereby adding a new regulatory layer in prokaryotes for translation control.