Expression Ratio Research Articles

A machine learning–based radiomics approach to predict MUC4 mutation and response to immunotherapy in cholangiocarcinoma.

e16203 Background: The immunogenicity of MUC4 made it a tumor antigen for immunotherapeutic interventions in cholangiocarcinoma (CCA). Identifying CCA patients with elevated MUC4 expression would be crucial for MUC4-targeted immunotherapies and the prediction response to immunotherapy. Methods: In this retrospective study, 55 CCA patients who received first-line treatment with pembrolizumab in combination with gemcitabine plus cisplatin (GP) chemotherapy were enrolled. Enrolled patients underwent surgery before, and their postoperative specimens were subjected to whole exome sequencing and proteomic sequencing, and matched magnetic resonance imaging (MRI) data were collected. We employed a random forest classifier based on radiomic features to predict the status of key genes mutation including MUC4 and tested it on an internal and an external validation CCA cohort. Odds ratio of MUC4 expression for objective response to pembrolizumab plus GP was tested with logistic regression analysis. Association of MUC4 expression with progression-free survival (PFS) and overall survival (OS) was analyzed with a Cox proportional hazard regression. Results: A radiomics nomogram that incorporates radiomics signature and MUC4 mutation showed good calibration and discrimination in the training cohort (AUC = 0.762), internal validation cohort (AUC = 0.812) and external validation cohort (AUC = 0.801). The radiomics nomogram was an independent preoperative predictor of overall and recurrence-free survival. High expression of MUC4 portended significantly lower overall and recurrence-free survival than decreased MUC4 expression levels (P < 0.001). Findings from cell experiments revealed targeting MUC4 with bosutinib showed inhibitory effects on cell proliferation and promoted apoptosis. Additionally, the combination of bosutinib with a PD-1 inhibitor increased the inhibitory effect. Investigating the feasibility of translating these findings into clinical trials for patients with CCA is an important next step. Conclusions: The integration of MUC4-based immunotherapy and incorporating radiomics-based biomarkers could significantly advance personalized medicine and contribute to more effective treatment strategies for CCA patients.

Baseline heterogeneity of myeloid and stroma cells to predict pCR, baseline lymph node (LN) metastasis, and 1-year EFS in locally advanced ESCC with neoadjuvant immunotherapy: Final results from a single-arm, phase II clinical trial (SEEK-01).

e14661 Background: Response of neoadjuvant immunotherapy can vary among patients in locally advanced ESCC, with the lack of a promising biomarker which need fully capture the heterogeneity of TME. Now, based on final results of the SEEK-01 study (2023 ASCO, P#4047), we have, for the first time, established a precise correlation from baseline TME to pathological response, baseline LN status, and 1-year EFS. Methods: In SEEK-01 study, 22 locally advanced ESCC patients (cT2-4NxM0) were enrolled. Following neoadjuvant chemotherapy (paclitaxel-albumin+carboplatin) combined with tislelizumab, patients underwent minimal invasive esophagectomy (MIE). Fresh tumor tissues were obtained via endoscopy prior to neoadjuvant therapy for scRNAseq analysis. Pathological response was evaluated using the irRVT score, leading to classification into pCR (irRVT=0%), MPR (0<irRVT<10%), and non-responder (irRVT>60%). Baseline LN status was determined based on pathological diagnosis after surgery. We identified subclusters of tumor-associated myeloid cells and stroma cells that significantly predicted pathological response, baseline LN metastasis and recurrence within 1 year. Prediction model of efficacy for neoadjuvant immunotherapy was developed using baseline expression ratios of differential subclusters. Results: Among 22 enrolled patients, 18 completed neoadjuvant therapy and MIE. 4 achieved pCR, 2 got MPR and 6 were non-responder. 12 of 18 proved baseline LN metastasis. 5 of 18 suffered recurrence within 1 year. Myeloid cells, cancer-associated fibroblast (Caf) and endothelium (Endo) shew heterogeneity that can predict pathological response, baseline LN metastasis or recurrence within 1 year. Upgrade of CXCL9+ tumor-associated-macrophage (TAM), LAMP3+mregDC3 was a strong predictor of pCR, while the upgrade of STAT1+TAM, C1q+TAM, IL1b+TAM, IL1b+ Neutrophil, C1q+ Neutrophil was a solid biomarker of non-responder. Besides, presence of PTGS1+Caf, RGS5+Caf, S100B+Caf, RND1+Endo and S100+Endo highly correlated with baseline LN metastasis and recurrence within 1 year, which was better than PET-CT. On this basis, we constructed predictive models for pathological response. With further validated by public scRNAseq and TCGA database, it showed excellent predictive ability and robustness. Conclusions: The final results of the SEEK-01 study demonstrate the differential baseline infiltration of myeloid cells that can predict pathological response of neoadjuvant immunotherapy. The characteristic changes in Caf and Endo in TME can predict occult LN metastasis and 1-year EFS. A predictive model for efficacy of neoadjuvant immunotherapy in locally advanced ESCC was developed to facilitate individualized and precise neoadjuvant therapy before treatment. Clinical trial information: NCT05807542 .

DCLK1 Expression and its Functional Significance in Intrahepatic Cholangiocarcinoma and Bil-IN Stage.

Although several studies in some neoplasms have reported correlation between the expression levels of Doublecortin-like kinase1(DCLK1) and carcinogenesis, its role in cholangiocarcinoma remains unknown. DCLK1 expression in normal epithelium (NE), biliary intraepithelial neoplasia (BilIN)1∼3, and intrahepatic cholangiocarcinoma (ICC) were investigated immuno-histochemically. The molecular effects of DCLK1 were investigated by gene silencing using RNAi [DCLK1-tagrgeting (siDCLK1)]. The human ICC cell lines HuCCT1 and HuH28 were transfected with these siRNAs, and used for assays in the presence or absence of DCLK1 inhibitors. The positive ratio of DCLK1 expression in ICC was higher than that in NE, and equally distributed among BilIN1∼3 (NE: BilIN1: BilIN2: BilIN3: ICC=62%: 91%: 97%: 100%: 95%, p<0.05). In the wound healing assay, the migration of the siDCLK1-treated cells was significantly inhibited compared to the NT-treated cells (p<0.05). In the cell invasion assay, the invasion of the siDCLK1-treated cells was significantly inhibited compared to the NT-treated cells (p<0.05). In the presence of the DCLK1 inhibitor, cell proliferative capacity at 24 hours was decreased in a concentration-dependent manner. DCLK1 was highly expressed in the early stage of ICC carcinogenesis. Human ICC cell growth was suppressed in vitro by siRNA silencing of DCLK1 or after treatment with the DCLK1 inhibitor, indicating DCLK1 may be molecular target for ICC therapy.

CAFs-Associated Genes (CAFGs) in Pancreatic Ductal Adenocarcinoma (PDAC) and Novel Therapeutic Strategy.

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive cancer with striking fibrosis, and its mortality rate is ranked second across human cancers. Cancer-associated fibroblasts (CAFs) play a critical role in PDAC progression, and we reviewed the molecular understanding of PDAC CAFs and novel therapeutic potential at present. CAFs-associated genes (CAFGs) were tentatively classified into three categories by stroma specificity representing stroma/epithelia expression ratios (SE ratios). The recent classification using single cell transcriptome technology clarified that CAFs were composed of myofibroblasts (myCAFs), inflammatory CAFs (iCAFs), and other minor ones (e.g., POSTN-CAFs and antigen presenting CAFs, apCAFs). LRRC15 is a myCAFs marker, and myCAFs depletion by diphtheria toxin induces the rapid accumulation of cytotoxic T lymphocytes (CTLs) and therefore augment PDL1 antibody treatments. This finding proposes that myCAFs may be a critical regulator of tumor immunity in terms of PDAC progression. myCAFs are located in CAFs adjacent to tumor cells, while iCAFs marked by PDPN and/or COL14A1 are distant from tumor cells, where hypoxic and acidic environments being located in iCAFs putatively due to poor blood supply is consistent with HIF1A and GPR68 expressions. iCAFs may be shared with SASP (secretion-associated phenotypes) in senescent CAFs. myCAFs are classically characterized by CAFGs induced by TGFB1, while chemoresistant CAFs with SASP may dependent on IL6 expression and accompanied by STAT3 activation. Recently, it was found that the unique metabolism of CAFs can be targeted to prevent PDAC progression, where PDAC cells utilize glucose, whereas CAFs in turn utilize lactate, which may be epigenetically regulated, mediated by its target genes including CXCR4. In summary, CAFs have unique molecular characteristics, which have been rigorously clarified as novel therapeutic targets of PDAC progression.

Utility of microRNA analysis in exhaled breath condensate of sarcoidosis and mediastinal tuberculosis patients: a pilot study.

Sarcoidosis and tuberculosis (TB) are the two most common causes of granulomatous mediastinal lymphadenopathy. These often exhibit overlapping clinical and radiological characteristics, rendering accurate diagnosis difficult. MicroRNA (miRNA) analysis is increasingly utilised as a potential biomarker for various diseases. Exhaled breath condensate (EBC) is a noninvasive technique for biomarker evaluation in different respiratory conditions. We attempted to identify differentially expressed miRNAs in the EBC of sarcoidosis and mediastinal TB patients. EBC was obtained from subjects with a definitive diagnosis of sarcoidosis and mediastinal TB. EBC was also obtained from age- and sex-matched control subjects. From EBC, miRNA isolation, cDNA preparation and qPCR array were performed. Differentially expressed miRNAs were shortlisted. Further validation was conducted in the EBC of a new subset. Subjects with a definitive diagnosis of sarcoidosis (50) and TB (50), and control subjects (50) were included. qPCR array from EBC (20 subjects from each group) shortlisted eight differentially expressed miRNAs (miR-126, miR-132, miR-139-3p, miR-139-5p, miR-181c, miR-454, miR-512-3p and miR-362-5p). In the validation set (EBC of 30 subjects from each group), miR-126 and miR-132 were differentially expressed significantly. The miR-126 and miR-132 expression ratio could differentiate sarcoidosis from mediastinal TB with an AUC of 0.618 (82% specificity and 41% sensitivity). While EBC miRNA expression is significantly and differently altered in sarcoidosis and mediastinal TB, a simple ratiometric approach failed to provide clinically useful signatures for differentiating between the two in patients with mediastinal lymphadenopathy.

Reduced histone H3K4 trimethylation in oral mucosa of patients with DYT-KMT2B

BackgroundDYT-KMT2B, also known as DYT28, is a childhood-onset hereditary dystonia caused by KMT2B mutation. The pathogenesis of DYT-KMT2B involves haploinsufficiency of KMT2B, an enzyme that catalyzes specific histone methylation (H3K4me3). Dysmorphic features in patients with DYT-KMT2B suggest that KMT2B dysfunction may extend beyond the neuronal system. Therefore, valuable diagnostic insights may be obtained from readily available tissue samples. ObjectivesTo explore the altered H3K4me3 levels in non-neural tissue of DYT-KMT2B patients. MethodsA database analysis was performed to determine in which parts of the body and in which cells KMT2B is highly expressed. Twelve clinically and genetically diagnosed patients with DYT-KMT2B and 12 control subjects participated in this study. Oral mucosa–derived purified histone proteins were analyzed using Western blotting with anti-H3K4me3 and anti-H4 antibodies. ResultsHigher expression of KMT2B was observed in oral keratinocytes and gingival fibroblasts, constituting the oral mucosa. In oral mucosa analyses, DYT-KMT2B cases exhibited markedly reduced H3K4me3 levels compared with the controls. Using a cutoff window of 0.90–0.98, the H3K4me3/H4 expression ratio was able to distinguish patient groups. ConclusionsOral mucosa H3K4me3 analysis is currently not sufficient as a diagnostic tool for DYT-KMT2B, but has the advantage for screening test since it is a non-invasive means.

Impact of Ebola virus nucleoprotein on VP40 virus-like particle production: a computational approach

Ebola virus (EBOV) matrix protein VP40 can assemble and bud as virus-like particles (VLPs) when expressed alone in mammalian cells. Nucleoprotein (NP) could be recruited to VLPs as inclusion body (IB) when co-expressed, and increase VLP production. However, the mechanism behind it remains unclear. Here, we use a computational approach to study NP-VP40 interactions. Our simulations indicate that NP may enhance VLP production through stabilizing VP40 filaments and accelerating the VLP budding step. Further, both the relative timing and amount of NP expression compared to VP40 are important for the effective production of IB-containing VLPs. We predict that relative NP/VP40 expression ratio and time are important for efficient production of IB-containing VLPs. We conclude that disrupting the expression timing and amount of NP and VP40 could provide new avenues to treat EBOV infection. This work provides quantitative insights into EBOV proteins interactions and how virion generation and drug efficacy could be influenced.

MEASUREMENT OF PLATELET ACTIVATION MARKERS CD62P AND CD63 TO DETERMINE THE EFFICIENCY OF DUAL THERAPY WITH ASPIRIN AND CLOPIDOGREL: A PRELIMINARY STUDY

Background: A dual antiplatelet therapy with aspirin and clopidogrel is advised to prevent stent thrombosis. However, the most appropriate assay for evaluating the antiplatelet effects remains undefined. Objective: This study aimed to assess the efficacy of measuring platelet surface activation markers, CD62P and CD63, using flow cytometry to determine the effectiveness of dual antiplatelet therapy. Methods: Thirty patients who received aspirin plus clopidogrel before cerebral artery stent implantation and thirty unrelated healthy controls were enrolled. The expression of CD62P and CD63 was measured using flow cytometry. The diagnostic performances were evaluated and compared with the conventional light-transmitted aggregometry (LTA) using 5.0 and 20.0 µM of adenosine di-phosphate (ADP). Results: The expression ratios of both markers were significantly lower in the patients receiving dual antiplatelet therapy than controls (p &lt; 0.001). The area under the receiver operating characteristic (ROC) curve (AUC) of CD62P using flow cytometry and the LTA using 5.0 and 20.0 µM of ADP were comparable (0.97 vs. 0.95 and 0.97 vs. 0.96, respectively), whereas those of CD63 was lower than the LTA (0.88 vs. 0.96). The sensitivity of CD62P and CD63 were 71.4% and 60.0%, and the specificity of CD62P and CD63 were 100.0% and 96.7%, respectively. Conclusion: Flow cytometry measurements of CD62P could be utilized to identify the efficacy of dual antiplatelet therapy. Additional studies are suggested to support this issue and its effects on clinical outcomes.

#1864 Phosphorilation of SHP-1 switch does not indicate lupus nephritis activity

Abstract Background and Aims Protein tyrosine kinases (PTKs) are enzymes responsible for the phosphorylation of tyrosine residues in lymphocytes. In 2017, Mkaddem et al. suggested that unique PTK signatures in peripheral leukocytes (PBMC) are associated with lupus nephritis (LN) activity [1]. Lymphocytes express two important PTKs, Lyn and Fyn, that modulate inhibitory or activatory signals depending on ligand avidity or phosphorylation status. SHP-1 tyrosine 536 phosphorylation (pSHP1-Y536) by Lyn activates inhibitory signaling and Fyn-dependent phosphorylation of SHP-1 serine 591 (pSHP1-S591) inactivates the phosphatase, enabling activatory immunoreceptor signaling. We hypothesized that active and refractory patients with proliferative LN have a different PTK profile in PBMC, with lower expression of pSHP1-Y536 and greater expression of pSHP1-S591, in comparison with those who have responded to treatment. Method In a cohort from the Royal Free Hospital, UCL (London, UK) we performed western blotting of PBMC lysates for SHP1-phospho-S591 and-Y536 from healthy individuals (HC; n = 19), LN patients in remission (n = 11) and with ongoing disease activity (n = 4). Densitometry of bands was carried out and a ratio of S591:Y536 expression was calculated (both normalized to actin). For the flow cytometry analysis, we followed up a Brazilian cohort of LN (n = 24) from Hospital das Clinicas, UFPE (Recife, Brazil). All patients had ongoing active LN with initial renal biopsy (class III, IV or/and V). PBMC samples were collected at 0, 3, 6, 9, 12, 18, and 24 months after biopsy. Isolated PBMC samples were stored at −80°C. PBMC samples were stained with antibodies against pSHP1 S591 and p-SHP1 Y536 and cell viability was checked using zombie red dye. Samples were acquired on the Accuri C6 cytometer (BD) and analyzed using the FLOJO software (BD). Results The western blotting relative expression of pSHP-1 S591:Y536 in PBMC was not significantly different when we compared HC with active LN (p = 0.098) and active with remission LN (p = 0.10), Fig. 1. The results obtained by flow cytometry expression ratio of pSHP1-S591:Y536 did not show a significant correlation with the median of proteinuria (Fig. 2), C3, C4, or SLEDAI from time of active kidney biopsy during LN flare throughout induction treatment. Conclusion There was no expression pattern of pSHP-1 Y536 and S591 in PBMC of LN patients, regardless of their renal activity or inflammatory status. Therefore, we cannot state those markers as a potential “liquid biopsy” for LN.

Galectin-receptor interaction: a key player in liver fibrosis induced by Schistosoma japonicum infection

BackgroundSchistosoma japonicum eggs lodge in the liver and induce a fibrotic granulomatous immune response in the liver of host. Galectin 3 (Gal-3) is a protein implicated in fibrosis in multiple organs. However, the pathology and molecular mechanisms promoting hepatic granuloma formation remain poorly understood.MethodsTo investigate the effect of blocking galectin-receptor interactions by α-lactose on liver immunopathology in mice with S. japonicum infection, C57BL/6 mice were infected with S. japonicum and alpha (α)-lactose was intraperitoneally injected to block the interactions of galectins and their receptors.ResultsCompared with S. japonicum-infected mice, there were significantly decreased Gal-3 mRNA and protein expression levels, decreased intensity of Gal-3 fluorescence in the liver, decreased serum ALT and AST levels, decreased egg numbers of S. japonicum in the liver section, attenuated hepatic and spleen pathology, and alleviated liver fibrosis accompanied with decreased protein expression levels of fibrosis markers [α-smooth muscle actin (α-SMA), collagen I, and collagen IV] in the liver of S. japonicum-infected mice blocked galectin-receptor interactions with hematoxylin-eosin staining, Masson’s trichrome staining, immunohistochemistry, or Western blot analysis. Compared with S. japonicum-infected mice, blocking galectin-receptor interactions led to increased eosinophil infiltration and higher eosinophil cationic protein (ECP) expression in the liver, accompanied by increased mRNA levels of eosinophil granule proteins [ECP and eosinophil peroxidase (EPO)], IL-5, CCL11, and CCR3 in the liver and decreased mRNA levels of Gal-3 and M2 macrophage cytokines (TGF-β, IL-10, and IL-4) in the liver and spleen by using quantitative real-time reverse transcription-polymerase chain reaction. In addition, there were increased Beclin1 protein expression and protein expression ratio of LC3B-II/LC3B-I and decreased p62 protein expression and protein expression ratios of phospho-mTOR/mTOR and phospho-AKT/AKT by Western blot; increased double-labeled F4/80+/LC3B+ cells by immunofluorescence staining; increased M1 macrophage polarization in the liver of S. japonicum-infected mice blocked galectin-receptor interactions by flow cytometric analysis and immunofluorescence staining.ConclusionsOur data found that blockage of galectin-receptor interactions downregulated Gal-3, which in turn led to reduced liver functional damage, elevated liver eosinophil recruitment, promoted macrophage autophagy through the Akt/mTOR signaling pathway, and alleviated liver pathology and fibrosis. Therefore, Gal-3 plays a pivotal role during S. japonicum infection and could be a target of pharmacologic potential for liver fibrosis induced by S. japonicum infection.Graphical

Identification and characterization of whole blood gene expression and splicing quantitative trait loci during early to mid-lactation of dairy cattle

BackgroundCharacterization of regulatory variants (e.g., gene expression quantitative trait loci, eQTL; gene splicing QTL, sQTL) is crucial for biologically interpreting molecular mechanisms underlying loci associated with complex traits. However, regulatory variants in dairy cattle, particularly in specific biological contexts (e.g., distinct lactation stages), remain largely unknown. In this study, we explored regulatory variants in whole blood samples collected during early to mid-lactation (22–150 days after calving) of 101 Holstein cows and analyzed them to decipher the regulatory mechanisms underlying complex traits in dairy cattle.ResultsWe identified 14,303 genes and 227,705 intron clusters expressed in the white blood cells of 101 cattle. The average heritability of gene expression and intron excision ratio explained by cis-SNPs is 0.28 ± 0.13 and 0.25 ± 0.13, respectively. We identified 23,485 SNP-gene expression pairs and 18,166 SNP-intron cluster pairs in dairy cattle during early to mid-lactation. Compared with the 2,380,457 cis-eQTLs reported to be present in blood in the Cattle Genotype-Tissue Expression atlas (CattleGTEx), only 6,114 cis-eQTLs (P < 0.05) were detected in the present study. By conducting colocalization analysis between cis-e/sQTL and the results of genome-wide association studies (GWAS) from four traits, we identified a cis-e/sQTL (rs109421300) of the DGAT1 gene that might be a key marker in early to mid-lactation for milk yield, fat yield, protein yield, and somatic cell score (PP4 > 0.6). Finally, transcriptome-wide association studies (TWAS) revealed certain genes (e.g., FAM83H and TBC1D17) whose expression in white blood cells was significantly (P < 0.05) associated with complex traits.ConclusionsThis study investigated the genetic regulation of gene expression and alternative splicing in dairy cows during early to mid-lactation and provided new insights into the regulatory mechanisms underlying complex traits of economic importance.

Role of small nucleolar RNAs in alternative splicing of the doublesex gene in the silkworm, Bombyx mori.

More and more evidence shows that small noncoding RNAs (ncRNAs) play diverse roles in development, stress response and other cellular processes, but functional study of intermediate-size ncRNAs is still rare. Here, the expression profile of 16 intermediate-size ncRNAs in ovary and testis of silkworm Bombyx mori were analyzed. Twelve ncRNAs, including 5 small nucleolar RNAs (snoRNAs) and 7 unclassified ncRNAs, accumulated more in the testis than in the ovary of silkworm, especially Bm-163, Bm-51 and Bm-68. Four ncRNAs (including three orphan snoRNAs and one unclassified ncRNA) had higher expression level in the ovary than in the testis, especially Bm-86. Overexpression of the testis-enriched snoRNA Bm-68 in the female led to the accumulation of male-specific isoform of doublesex (BmdsxM) and increased the expression ratio of BmdsxM: BmdsxF. While overexpression of ovary-enriched snoRNA Bm-86 in the male decreased the expression ratio of BmdsxM: BmdsxF, indicating the roles of the two snoRNAs played in the alternative splicing of Bmdsx of silkworm, which will provide new clues for the functional study of snoRNAs in insects.

Role of local vasoconstrictors in diving performance of two pinniped species

Diving mammals extend submergence time through bradycardia and reduced peripheral perfusion to certain tissues. Thus, local blood flow regulation under hypoxia may be an essential physiological strategy that fine-tunes metabolic expenditures during breath-hold. We focused on a potent local vasoconstrictor, Endothelin-1 (ET1) and its receptors ETR-A and ETR-B, to determine their potential role in mammalian diving. We hypothesized that less perfused organs (kidney) would have higher signatures of local vasoconstriction (i.e. higher ET1 and receptors), compared to organs known to have higher relative perfusion underwater (heart/left ventricle). Additionally, we compared pinniped species with higher (Weddell seal) and lower (California sea lion) relative dive capabilities. Differences in mRNA expression of vasoconstrictive elements were assessed by 2-way ANOVA to compare tissue and species. In adult animals (n=7 both species) there were significant differences in expression of ET1 between species (F1,16=9.2, p=0.08) and tissues (F1,16=18.4, p=0.0006), with sea lions having consistently higher levels of ET1. ET1 expression was highest in the kidney, with sea lion kidney expressing 25x more ET1 mRNA than heart (posthoc p=0.0007). The expression ratio of ET A and B receptors also differed across tissue (F1,16=19.83 p=0.0004) with B/A highest in the kidney in both species. This is counter to expectations that submergence capability would relate to vasoconstriction potential across species, but supports the hypothesis that within a species, the less-perfused kidney should demonstrate higher potential for local vasoconstriction compared to the heart. Relative expression of ET receptors between tissues also runs counter to our hypothesis. In both divers, the heart expresses relatively more ETR-A, which has a critical role in vasoconstrictive signaling. On the other hand, ETR-B can signal for either vasoconstriction or vasodilation depending on its location within the endothelium or smooth muscle. Kidney in both species expresses relatively more ETR-B, indicating potential for nuanced vasoregulatory control through the endothelin pathway in this tissue. Receptor localization will be determined by immuno-staining to investigate functional differences between tissues. We also investigated the development of diving by comparing the same biomarkers in adults versus pups (n=10 seals, n=3 sea lions) using a separate 2-way ANOVA for each species. We expected that signatures of vasoregulatory potential would be less evident in the novice diving pups. Concordantly, ET1 is lower in sea lion pups than adults (F1,12=5.097, p=0.043); ET1 mRNA is 23x lower in the pup kidney compared to adult (posthoc p=0.02). Receptor expression followed the same patterns in both species, with the highest expression of ETR-A noted in the pup heart, indicating high vasoconstrictive potential in pups, but through tissue specific mechanisms. These results point to differences in the control of local vasoconstriction that occur with development and highlight the physiological components essential to the development of diving. Funding by NSF #2020706. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex Is Not a Transcriptomic Binary: Towards a Broad Physiologic Omics Insight Into Sex Chromosome Genes

Background: As social definitions of sex and gender expand, there is a greater demand for understanding the clinical challenges of intersex individuals, transgender individuals, and those with hormonal and transcriptomic profiles that do not align with traditional binary standards. Larger population studies are elucidating comorbidities associated with Klinefelter’s Syndrome, Turner’s Syndrome, 47XXX, 47XYY, and other sex chromosome syndromes even though the sex chromosomes are underpowered in traditional GWAS. Though research has focused on how estrogen and testosterone supplementation can influence comorbidity risk, we identify a need for greater understanding of isoform and tissue-specific expression and functions of X and Y genes to understand their potential interactions with hormones and inform physiological outcomes. Methods: Analysis of RNA-Sequencing data quantified in transcripts per million (TPM) from GTex (extracted June 2022, n = 17,382), NCBI SRA Blood PAXgene tube analysis (extracted June 2021, n = 12,559), and NCBI SRA “skin” RNA sequencing (extracted June 2021, n = 8,274). Results: We display an expression panel of 572 isoforms of 73 sex-biased genes across 54 male (n = 11,588) and female (n=5,798) tissue samples and highlight isoform-specific expression differences within the brain, arteries, heart, skeletal muscle, and gastrointestinal tract. Six X/Y gene pairs show broad ratios over tissues highlighting the role of X-chromosome dosage and X-chromosome inactivation escape differences in various tissues. Several genome-wide eQTLs across multiple tissues have sex differences and are found within X/Y chromosome transcription factor binding sites such as ZFX/ZFY. Stratifying blood and skin RNA-Sequencing data by XIST/KDM5D ratios to determine transcriptomic “sex”, we conduct an exploratory analysis of bioprojects with five or more outliers of X:Y chromosome expression ratios (Males: PRJNA683803 (HIV), PRJNA768419 (Sepsis), PRJNA492829 (Vaccine trial), PRJNA496323 (Psoriasis and atopic dermatitis), PRJNA699562 (COVID-19). Females: PRJNA683803 (HIV), PRJNA794277 (Sepsis), PRJNA492965 (Vaccine trial), PRJNA526259 (Myalgic encephalomyelitis)).Conclusion: Transcriptomics enables broader insights to sex chromosomes and their contributions to physiology outside of the binary male/female knowledge. This work was supported by the NIH NIAID and OD grant R01AI171984. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Expression analysis of necroptosis related genes and lncRNAs in patients with pituitary neuroendocrine tumors

Necroptosis can either be the cause of tumorigenesis or it can impede its process. Recently, it has been proved that long non-coding RNAs (lncRNAs) have different crucial roles at cellular level, especially on cell death. Regarding the important role of necroptosis and lncRNAs in the pathogenesis of different cancers, especially pituitary adenomas (PAs), we assessed expression levels of two necroptosis related genes, namely TRADD and BIRC2, in addition to three related lncRNAs, namely FLVCR1-DT, MAGI2-AS3, and NEAT1 in PAs compared with adjacent normal tissues (ANTs). TRADD had no significant difference between two groups; however, BIRC2, FLVCR1-DT, MAGI2-AS3, and NEAT1 were upregulated in PAs compared to ANTs (Expression ratios [95% CI] = 2.3 [1.47–3.6], 2.13 [1.02–4.44], 3.01 [1.76–5.16] and 2.47 [1.37–4.45], respectively). When taking into account different types of PAs, significant upregulation of BIRC2, MAGI2-AS3 and NEAT1 was recorded in non-functioning PAs compared with corresponding ANTs (Expression ratios [95% CI] =1.9 [1.04–3.43], 2.69 [1.26–5.72] and 2.22 [0.98–5.01], respectively). Additionally, higher levels of BIRC2 were associated with higher flow of CSF (P value=0.048). Moreover, higher Knosp classified tumors had lower levels of BIRC2 (P value=0.001). Finally, lower levels of MAGI2-AS3 were associated with larger tumor size (P value=0.006). NEAT1 expression was correlated with FLVCR1-DT and TRADD. TRADD expression was correlated with FLVCR1-DT. Additional correlation was observed between expression of BIRC2 and MAGI2-AS3. In sum, this study provides evidence that dysregulated levels of studied genes could contribute to the pathogenesis of pituitary tumors.

In search of the ratio of miRNA expression as robust biomarkers for constructing stable diagnostic models among multi-center data.

MicroRNAs (miRNAs) are promising biomarkers for the early detection of disease, and many miRNA-based diagnostic models have been constructed to distinguish patients and healthy individuals. To thoroughly utilize the miRNA-profiling data across different sequencing platforms or multiple centers, the models accounting the batch effects were demanded for the generalization of medical application. We conducted transcription factor (TF)-mediated miRNA-miRNA interaction network analysis and adopted the within-sample expression ratios of miRNA pairs as predictive markers. The ratio of the expression values between each miRNA pair turned out to be stable across multiple data sources. A genetic algorithm-based classifier was constructed to quantify risk scores of the probability of disease and discriminate disease states from normal states in discovery, with a validation dataset for COVID-19, renal cell carcinoma, and lung adenocarcinoma. The predictive models based on the expression ratio of interacting miRNA pairs demonstrated good performances in the discovery and validation datasets, and the classifier may be used accurately for the early detection of disease.

The endonuclease FEN1 mediates activation of STAT3 and facilitates proliferation and metastasis in breast cancer.

The metastasis accounts for most deaths from breast cancer (BRCA). Understanding the molecular mechanisms of BRCA metastasis is urgently demanded. Flap Endonuclease 1 (FEN1), a pivotal factor in DNA metabolic pathways, contributes to tumor growth and drug resistance, however, little is known about the role of FEN1 in BRCA metastasis. In this study, FEN1 expression and its clinical correlation in BRCA were investigated using bioinformatics, showing being upregulated in BRCA samples and significant relationships with tumor stage, node metastasis, and prognosis. Immunohistochemistry (IHC) staining of local BRCA cohort indicated that the ratio of high FEN1 expression in metastatic BRCA tissues rose over that in non-metastatic tissues. The assays of loss-of-function and gain-of-function showed that FEN1 enhanced BRCA cell proliferation, migration, invasion, xenograft growth as well as lung metastasis. It was further found that FEN1 promoted the aggressive behaviors of BRCA cells via Signal Transducer and Activator of Transcription 3 (STAT3) activation. Specifically, the STAT3 inhibitor Stattic thwarted the FEN1-induced enhancement of migration and invasion, while the activator IL-6 rescued the decreased migration and invasion caused by FEN1 knockdown. Additionally, overexpression of FEN1 rescued the inhibitory effect of nuclear factor-κB (NF-κB) inhibitor BAY117082 on phosphorylated STAT3. Simultaneously, the knockdown of FEN1 attenuated the phosphorylation of STAT3 promoted by the NF-κB activator tumor necrosis factor α (TNF-α). These results indicate a novel mechanism that NF-κB-driven FEN1 contributes to promoting BRCA growth and metastasis by STAT3 activation.

Preventive effect of free radical scavenger edaravone lotion on cyclophosphamide chemotherapy-induced alopecia.

We investigated the inhibitory effect of edaravone (EDR) lotion on chemotherapy-induced alopecia (CIA) to improve the quality of life for patients with cancer. Wistar rats were intraperitoneally injected with cyclophosphamide (CPA, 75mg/kg) to induce CIA and divided into six groups: (1) Control; (2) EDR 0%; (3) EDR 0.3%; (4) EDR 3%. The TUNEL-positive area was examined histologically, and mRNA expression levels of the apoptosis-related factors, such as B-cell/CLL lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax), were determined. In the three CPA-treated groups, a decrease in the coverage score (percentage of hairs covered) was observed from days 16 to 18. In addition, coverage scores on day 21, the last day of observation, showed a tendency for the suppression of hair loss to increase, though hair loss was observed in all groups. The coverage scores of the EDR 0.3% and 3% groups after day 17 were significantly higher than those of the EDR 0% group. The TUNEL-positive area of skin tissue on day 16 was extensive in the EDR 0% group and decreased in the EDR 0.3% and 3% groups. The mRNA expression ratio of Bcl-2/Bax on day 21 was maintained at the same level as that of the control group only in the EDR 3% group. This study confirmed the use of EDR lotion to inhibit hair loss, indicating that the clinical application of EDR lotion may improve the quality of life for patients with cancer and their willingness to undergo treatment.

Utilizing 5' UTR Engineering Enables Fine-Tuning of Multiple Genes within Operons to Balance Metabolic Flux in Bacillus subtilis.

The application of synthetic biology tools to modulate gene expression to increase yield has been thoroughly demonstrated as an effective and convenient approach in industrial production. In this study, we employed a high-throughput screening strategy to identify a 5' UTR sequence from the genome of B. subtilis 168. This sequence resulted in a 5.8-fold increase in the expression level of EGFP. By utilizing the 5' UTR sequence to overexpress individual genes within the rib operon, it was determined that the genes ribD and ribAB serve as rate-limiting enzymes in the riboflavin synthesis pathway. Constructing a 5' UTR library to regulate EGFP expression resulted in a variation range in gene expression levels exceeding 100-fold. Employing the same 5' UTR library to regulate the expression of EGFP and mCherry within the operon led to a change in the expression ratio of these two genes by over 10,000-fold. So, employing a 5' UTR library to modulate the expression of the rib operon gene and construct a synthetic rib operon resulted in a 2.09-fold increase in riboflavin production. These results indicate that the 5' UTR sequence identified and characterized in this study can serve as a versatile synthetic biology toolkit for achieving complex metabolic network reconstruction. This toolkit can facilitate the fine-tuning of gene expression to produce target products.

The Effects of Artificial Diets on the Expression of Molecular Marker Genes Related to Honey Bee Health.

Honey bees are commonly used to study metabolic processes, yet the molecular mechanisms underlying nutrient transformation, particularly proteins and their effects on development, health, and diseases, still evoke varying opinions among researchers. To address this gap, we investigated the digestibility and transformation of water-soluble proteins from four artificial diets in long-lived honey bee populations (Apis mellifera ligustica), alongside their impact on metabolism and DWV relative expression ratio, using transcriptomic and protein quantification methods. Diet 2, characterized by its high protein content and digestibility, was selected for further analysis from the other studied diets. Subsequently, machine learning was employed to identify six diet-related molecular markers: SOD1, Trxr1, defensin2, JHAMT, TOR1, and vg. The expression levels of these markers were found to resemble those of honey bees who were fed with Diet 2 and bee bread, renowned as the best natural food. Notably, honey bees exhibiting chalkbrood symptoms (Control-N) responded differently to the diet, underscoring the unique nutritional effects on health-deficient bees. Additionally, we proposed a molecular model to elucidate the transition of long-lived honey bees from diapause to development, induced by nutrition. These findings carry implications for nutritional research and beekeeping, underscoring the vital role of honey bees in agriculture.