Signaling Processes Research Articles

Temporal multiscale modeling of biochemical regulatory networks: Calcium-regulated hepatocyte lipid and glucose metabolism

Hepatocyte lipid and glucose metabolism is regulated not only by major hormones like insulin and glucagon but also by many other factors, including calcium ions. Recently, mitochondria-associated membrane (MAM) dysfunction combined with incorrect IP3-receptor regulation has been shown to result in abnormal calcium signaling in hepatocytes. This dysfunction could further lead to hepatic metabolism pathology. However, the exact contribution of MAM dysfunction, incorrect IP3-receptor regulation and insulin resistance to the calcium-insulin-glucagon interplay is not understood yet. In this work, we analyze the role of abnormal calcium signaling and insulin dysfunction in hepatocytes by proposing a model of hepatocyte metabolic regulatory network with a detailed focus on the model construction details besides the biological aspect. In this work, we analyze the role of abnormal calcium signaling and insulin dysfunction in hepatocytes by proposing a model of hepatocyte metabolic regulatory network. We focus on the model construction details, model validation, and predictions. We describe the dynamic regulation of signaling processes by sigmoid Hill function. In particular, we study the effect of both the Hill function slope and the distance between Hill function extremes on metabolic processes in hepatocytes as a model of nonspecific insulin dysfunction. We also address the significant time difference between characteristic time of glucose hepatic processing and a typical calcium oscillation period in hepatocytes. Our modeling results show that calcium signaling dysfunction results in an abnormal increase in postprandial glucose levels, an abnormal glucose decrease in fasting, and a decreased amount of stored glycogen. An insulin dysfunction of glucose phosphorylation, glucose dephosphorylation, and glycogen breakdown also cause a noticeable effect. We also get some insight into the so-called hepatic insulin resistance paradox, confirming the hypothesis regarding indirect insulin action on hepatocytes via dysfunctional adipocyte lipolysis.

Abstract PO2-25-07: PEGylated Functional Upstream Domain (PEG-FUD): an anti-cancer therapy for breast Cancer

Abstract Background: Breast cancer (BC) is the most common cancer diagnosed in women. In breast cancers, the extracellular matrix (ECM) is known to play a key role in disease progression by mediating cell signaling processes that drive cancer cell proliferation, migration and invasion. Work by our group identified that aligned collagen fibers perpendicular to the tumor boundary are prognostic of poor patient prognosis. Further, through matrix targeted proteomics we determined that the ECM protein, Fibronectin (FN), organizes with aligned collagen fibers in BC tissues. FN is known as the master regulator of ECM assembly because FN deposition precedes and regulates the deposition of several other ECM proteins. Clinical evidence shows that high expression of FN levels in BC patients correlates with an increased mortality risk. Due to the abundant expression of FN in the tumor microenvironment, FN has been a useful biomarker for cancer imaging and therapy. Despite multiple efforts in developing therapies that target the ECM, currently there are no effective ECM treatments available due to toxicity and lack of specificity. Methods: To target abnormal FN deposition, we used a FN binding peptide, Functional Upstream Domain (FUD), derived from the F1 adhesin from Streptococcus pyogenes. PEGylated-FUD (PEG-FUD) is a potent inhibitor of FN assembly which binds the 70 kDa N-terminal region of FN with high affinity. We hypothesize that PEG-FUD will localize to the tumor site and that targeted disruption of FN assembly with PEG-FUD will reduce ECM deposition, thus block tumor progression in-vivo. In this study, we used an in vivo imaging system to assess the biodistribution of 20-kDa PEG-FUD following subcutaneous injection of Cy5 labeled peptide in 4T1 mammary tumor bearing mice. Additionally, in a second therapeutic experiment, we treated 4T1 mammary tumor bearing mice with 20-kDa PEG-FUD every 48 hr for a total of 10 treatments and measured tumor volume as an indicator of primary tumor burden. Results: As anticipated, we observed PEG-FUD’s accumulation and localization in 4T1 tumors. Additionally, PEG-FUD treatment significantly reduced tumor growth compared to saline treatment. There were no observed changes in standard toxicity assessments such as body weight and spleen weight among treatment groups. After PEG-FUD’s therapeutic treatment, we observed a significant reduction in the deposition of FN matrix and an increase in cleaved caspase-3, a known marker for apoptosis by western blotting providing a potential mechanism of action of PEG-FUD inducing changes in tumor growth. Conclusions: PEG-FUD’s localization in tumors suggests its utility as a solo cancer imaging agent while providing indications that PEG-FUD can be used to deliver other therapeutics to the tumor site as a conjugate in the future. In addition, therapeutic treatment of PEG-FUD inhibited tumor growth providing preliminary evidence for PEG-FUD’s use as a tumor targeting anti-cancer agent. Citation Format: Metti Gari. PEGylated Functional Upstream Domain (PEG-FUD): an anti-cancer therapy for breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-25-07.

Emerging Concepts of Mechanisms Controlling Cardiac Tension: Focus on Familial Dilated Cardiomyopathy (DCM) and Sarcomere-Directed Therapies.

Novel therapies for the treatment of familial dilated cardiomyopathy (DCM) are lacking. Shaping research directions to clinical needs is critical. Triggers for the progression of the disorder commonly occur due to specific gene variants that affect the production of sarcomeric/cytoskeletal proteins. Generally, these variants cause a decrease in tension by the myofilaments, resulting in signaling abnormalities within the micro-environment, which over time result in structural and functional maladaptations, leading to heart failure (HF). Current concepts support the hypothesis that the mutant sarcomere proteins induce a causal depression in the tension-time integral (TTI) of linear preparations of cardiac muscle. However, molecular mechanisms underlying tension generation particularly concerning mutant proteins and their impact on sarcomere molecular signaling are currently controversial. Thus, there is a need for clarification as to how mutant proteins affect sarcomere molecular signaling in the etiology and progression of DCM. A main topic in this controversy is the control of the number of tension-generating myosin heads reacting with the thin filament. One line of investigation proposes that this number is determined by changes in the ratio of myosin heads in a sequestered super-relaxed state (SRX) or in a disordered relaxed state (DRX) poised for force generation upon the Ca2+ activation of the thin filament. Contrasting evidence from nanometer-micrometer-scale X-ray diffraction in intact trabeculae indicates that the SRX/DRX states may have a lesser role. Instead, the proposal is that myosin heads are in a basal OFF state in relaxation then transfer to an ON state through a mechano-sensing mechanism induced during early thin filament activation and increasing thick filament strain. Recent evidence about the modulation of these mechanisms by protein phosphorylation has also introduced a need for reconsidering the control of tension. We discuss these mechanisms that lead to different ideas related to how tension is disturbed by levels of mutant sarcomere proteins linked to the expression of gene variants in the complex landscape of DCM. Resolving the various mechanisms and incorporating them into a unified concept is crucial for gaining a comprehensive understanding of DCM. This deeper understanding is not only important for diagnosis and treatment strategies with small molecules, but also for understanding the reciprocal signaling processes that occur between cardiac myocytes and their micro-environment. By unraveling these complexities, we can pave the way for improved therapeutic interventions for managing DCM.

The differential effects of acute and prolonged L-NAME exposure on A23187-induced NO-dependent dilation in human arterioles

The Ca2+ ionophore A23187 induces endothelium-dependent and non-receptor-mediated vasodilation in human adipose arterioles (HAA). Preliminary data from our group suggest that while A23187 dilation mainly depends on NO as a vasodilator factor in healthy HAAs, there is a component of non-NO and likely hydrogen peroxide (H2O2)-mediated dilation, especially in arterioles from patients with coronary artery disease (CAD). The mechanisms underlying the release of two different vasodilator factors remain largely unclear. There is evidence that NO-mediated S-nitrosylation inhibits the catalytic activity of mitochondrial electron transport chain (ETC) complexes or other reactive oxygen species (ROS)-producing enzymes such as NADPH oxidases (NOX). The objective of this study was to compare the effects of acute (30 minutes) and prolonged (90 minutes) nitric oxide (NO) synthase inhibitor nitro-L-arginine methyl ester (L-NAME) exposure on A23187 dilation to elucidate whether prolonged incubation with L-NAME results in transition from NO-dependent to H2O2-dependent vasodilation in HAAs. HAAs (~150 μm ID), isolated from fat tissues obtained from subjects with and without CAD, were cannulated and pressurized at 60 mmHg for videomicroscopic measurement of vessel diameters. In non-CAD HAAs constricted with endothelin-1, A23187 (10−11 to 10−7 M) elicited concentration-dependent dilation (maximal dilation at 10−7 M: 68±5%, n=5), and this dilation was largely abolished after acute exposure to L-NAME (maximal dilation at 10−7 M: 21±7%, n=5). Prolonged L-NAME incubation restored A23187-dependent dilation (maximal dilation at 10−7 M: 74±2%, n=5), which was subsequently inhibited by hydrogen peroxide scavenger catalase (500 U/ml; maximal dilation at 10−7 M: 14±4%, n=5). HAAs obtained from CAD patients, responded to A23187 in a similar dose-dependent fashion (maximal dilation at 10−7 M: 68±3%, n=5), and this dilation was not affected by acute L-NAME exposure (maximal dilation at 10−7 M: 65±3%, n=5), but was reduced by catalase (maximal dilation at 10−7 M: 31±6%, n=6). To further determine the role of mitochondria-derived ROS in A23187-induced dilation, we treated arterioles with ETC inhibitor rotenone (1 mM). In non-CAD HAAs not pretreated with L-NAME, rotenone had no effect on A23187-induced dilation (maximal dilation at 10−7 M: 68±6%, n=5). In the presence of rotenone, however, prolonged L-NAME incubation no longer restored A23187-dependent dilation (maximal dilation at 10−7 M: 20±3%, n=5). Together, these data provide the first evidence that in healthy HAAs, prolonged L-NAME exposure changes the mechanism of vasodilation from NO to H2O2-mediated CAD-like vasodilation in response to A23187. In addition, the signaling process likely involves mitochondrial ROS. To elucidate the precise mechanisms underlying this process, future studies will be required to evaluate whether in healthy vasculature, basal NO inhibits mitochondrial ETC or other proteins such as NOXs through NO-mediated protein modification, which can be reversed by prolonged L-NAME incubation thereby promoting a switch from NO to H2O2 -mediated dilation. This work was supported by the National Heart, Lung, and Blood Institute [R01HL096647]. 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.

Lighting ATR/Chk1 by mesoscale TopBP1 condensates

Biomolecular condensation has gained considerable attention as a fundamental mechanism in cell signaling and various biological processes. A recent study by Egger et al. provides valuable insights into the constituents of topoisomerase IIβ binding protein 1 (TopBP1) condensates and sheds light on the mechanism of Chk1 activation by ataxia telangiectasia-mutated and Rad3-related (ATR) at the interface of these condensates.

Time of day does not impact spinal serotonin levels in humans.

Spinal serotonin enables neuro-motor recovery (i.e., plasticity) in patients with debilitating paralysis. While there exists time of day fluctuations in serotonin-dependent spinal plasticity, it is unknown, in humans, whether this is due to dynamic changes in spinal serotonin levels or downstream signaling processes. The primary objective of this study was to determine if time of day variations in spinal serotonin levels exists in humans. To assess this, intrathecal drains were placed in seven adults with cerebrospinal fluid (CSF) collected at diurnal (05:00 to 07:00) and nocturnal (17:00 to 19:00) intervals. High performance liquid chromatography with mass spectrometry was used to quantify CSF serotonin levels with comparisons being made using univariate analysis. From the 7 adult patients, 21 distinct CSF samples were collected: 9 during the diurnal interval and 12 during nocturnal. Diurnal CSF samples demonstrated an average serotonin level of 216.6 67.7nM. Nocturnal CSF samples demonstrated an average serotonin level of 206.7 75.8nM. There was no significant difference between diurnal and nocturnal CSF serotonin levels (p =.762). Within this small cohort of spine healthy adults, there were no differences in diurnal versus nocturnal spinal serotonin levels. These observations exclude spinal serotonin levels as the etiology for time of day fluctuations in serotonin-dependent spinal plasticity expression.

Abstract 2082: Neutrophil Programming In Atherosclerosis

Emerging evidence suggest the involvement of neutrophils during the progression of human atherosclerosis through secreting inflammatory mediators and causing damages to endothelial vasculature. However, compared to other well-recognized immune cells such as monocytes/macrophages, mechanisms underlying the regulation of neutrophils during atherosclerosis are relatively less studied and understood. This presents a major impediment in our thorough understanding of the pathogenesis of atherosclerosis. In this study, we reported that both murine and human neutrophil population possesses a minor homeostatic resolving subset characterized by reduced expression of inflammatory mediators (LTB4, elastase), elevated expression of anti-inflammatory resolving mediators (RvD1, CD200R), correlated with reduced expression of inflammatory signaling adaptor TRAM. We observed that TRAM deficient neutrophils are effectively reprogrammed into the resolving state, and can potently improve vascular integrity and suppress atherosclerosis pathogenesis when adoptively transfused into recipient atherosclerotic animals. Mechanistically, TRAM deficiency reduces the expression of LOX5AP, dislodges nuclear localization of LOX5, and favors the generation of RvD1 instead of LTB4. We also observed that human TRAM-low neutrophils are less inflammatory, as reflected in reduced swarming and reduced induction of LTB4/elastase. TRAM serves as a stress sensor of oxLDL and/or free cholesterol, triggers inflammatory signaling processes that facilitate elastase release. Our data defines a novel resolving neutrophil subset due to reduced TRAM expression which may hold therapeutic potential for reducing inflammation and treating atherosclerosis.

The disruptive role of LRG1 on the vasculature and perivascular microenvironment.

The establishment of new blood vessels, and their subsequent stabilization, is a critical process that facilitates tissue growth and organ development. Once established, vessels need to diversify to meet the specific needs of the local tissue and to maintain homeostasis. These processes are tightly regulated and fundamental to normal vessel and tissue function. The mechanisms that orchestrate angiogenesis and vessel maturation have been widely studied, with signaling crosstalk between endothelium and perivascular cells being identified as an essential component. In disease, however, new vessels develop abnormally, and existing vessels lose their specialization and function, which invariably contributes to disease progression. Despite considerable research into the vasculopathic mechanisms in disease, our knowledge remains incomplete. Accordingly, the identification of angiocrine and angiopathic molecules secreted by cells within the vascular microenvironment, and their effect on vessel behaviour, remains a major research objective. Over the last decade the secreted glycoprotein leucine-rich α-2 glycoprotein 1 (LRG1), has emerged as a significant vasculopathic molecule, stimulating defective angiogenesis, and destabilizing the existing vasculature mainly, but not uniquely, by altering both canonical and non-canonical TGF-β signaling in a highly cell and context dependent manner. Whilst LRG1 does not possess any overt homeostatic role in vessel development and maintenance, growing evidence provides a compelling case for LRG1 playing a pleiotropic role in disrupting the vasculature in many disease settings. Thus, LRG1 has now been reported to damage vessels in various disorders including cancer, diabetes, chronic kidney disease, ocular disease, and lung disease and the signaling processes that drive this dysfunction are being defined. Moreover, therapeutic targeting of LRG1 has been widely proposed to re-establish a quiescent endothelium and normalized vasculature. In this review, we consider the current status of our understanding of the role of LRG1 in vascular pathology, and its potential as a therapeutic target.

Structural basis of closed groove scrambling by a TMEM16 protein.

Activation of Ca2+-dependent TMEM16 scramblases induces phosphatidylserine externalization, a key step in multiple signaling processes. Current models suggest that the TMEM16s scramble lipids by deforming the membrane near a hydrophilic groove and that Ca2+ dependence arises from the different association of lipids with an open or closed groove. However, the molecular rearrangements underlying groove opening and how lipids reorganize outside the closed groove remain unknown. Here we directly visualize how lipids associate at the closed groove of Ca2+-bound fungal nhTMEM16 in nanodiscs using cryo-EM. Functional experiments pinpoint lipid-protein interaction sites critical for closed groove scrambling. Structural and functional analyses suggest groove opening entails the sequential appearance of two π-helical turns in the groove-lining TM6 helix and identify critical rearrangements. Finally, we show that the choice of scaffold protein and lipids affects the conformations of nhTMEM16 and their distribution, highlighting a key role of these factors in cryo-EM structure determination.

Cell-type and sex-specific rhythmic gene expression in the nucleus accumbens.

Circadian rhythms are critical for human health and are highly conserved across species. Disruptions in these rhythms contribute to many diseases, including psychiatric disorders. Previous results suggest that circadian genes modulate behavior through specific cell types in the nucleus accumbens (NAc), particularly dopamine D1-expressing medium spiny neurons (MSNs). However, diurnal rhythms in transcript expression have not been investigated in NAc MSNs. In this study we identified and characterized rhythmic transcripts in D1- and D2-expressing neurons and compared rhythmicity results to homogenate as well as astrocyte samples taken from the NAc of male and female mice. We find that all cell types have transcripts with diurnal rhythms and that top rhythmic transcripts are largely core clock genes, which peak at approximately the same time of day in each cell type and sex. While clock-controlled rhythmic transcripts are enriched for protein regulation pathways across cell type, cell signaling and signal transduction related processes are most commonly enriched in MSNs. In contrast to core clock genes, these clock-controlled rhythmic transcripts tend to reach their peak in expression about 2-h later in females than males, suggesting diurnal rhythms in reward may be delayed in females. We also find sex differences in pathway enrichment for rhythmic transcripts peaking at different times of day. Protein folding and immune responses are enriched in transcripts that peak in the dark phase, while metabolic processes are primarily enriched in transcripts that peak in the light phase. Importantly, we also find that several classic markers used to categorize MSNs are rhythmic in the NAc. This is critical since the use of rhythmic markers could lead to over- or under-enrichment of targeted cell types depending on the time at which they are sampled. This study greatly expands our knowledge of how individual cell types contribute to rhythms in the NAc.

Triptolide, a Cancer Cell Proliferation Inhibitor, Causes Zebrafish Muscle Defects by Regulating Notch and STAT3 Signaling Pathways.

Triptolide is a natural compound in herbal remedies with anti-inflammatory and anti-proliferative properties. We studied its effects on critical signaling processes within the cell, including Notch1 and STAT3 signaling. Our research showed that triptolide reduces cancer cell proliferation by decreasing the expression of downstream targets of these signals. The levels of each signal-related protein and mRNA were analyzed using Western blot and qPCR methods. Interestingly, inhibiting one signal with a single inhibitor alone did not significantly reduce cancer cell proliferation. Instead, MTT assays showed that the simultaneous inhibition of Notch1 and STAT3 signaling reduced cell proliferation. The effect of triptolide was similar to a combination treatment with inhibitors for both signals. When we conducted a study on the impact of triptolide on zebrafish larvae, we found that it inhibited muscle development and interfered with muscle cell proliferation, as evidenced by differences in the staining of myosin heavy chain and F-actin proteins in confocal fluorescence microscopy. Additionally, we noticed that inhibiting a single type of signaling did not lead to any significant muscle defects. This implies that triptolide obstructs multiple signals simultaneously, including Notch1 and STAT3, during muscle development. Chemotherapy is commonly used to treat cancer, but it may cause muscle loss due to drug-related adverse reactions or other complex mechanisms. Our study suggests that anticancer agents like triptolide, inhibiting essential signaling pathways including Notch1 and STAT3 signaling, may cause muscle atrophy through anti-proliferative activity.

Personalized identification and characterization of genome-wide gene expression differences between patient-matched intracranial and extracranial melanoma metastasis pairs

Melanoma is the most serious type of skin cancer that frequently spreads to other organs of the human body. Especially melanoma metastases to the brain (intracranial metastases) are hard to treat and a major cause of death of melanoma patients. Little is known about molecular alterations and altered mechanisms that distinguish intra- from extracranial melanoma metastases. So far, almost all existing studies compared intracranial metastases from one set of patients to extracranial metastases of an another set of melanoma patients. This neglects the important facts that each melanoma is highly individual and that intra- and extracranial melanoma metastases from the same patient are more similar to each other than to melanoma metastases from other patients in the same organ. To overcome this, we compared the gene expression profiles of 16 intracranial metastases to their corresponding 21 patient-matched extracranial metastases in a personalized way using a three-state Hidden Markov Model (HMM) to identify altered genes for each individual metastasis pair. This enabled three major findings by considering the predicted gene expression alterations across all patients: (i) most frequently altered pathways include cytokine-receptor interaction, calcium signaling, ECM-receptor interaction, cAMP signaling, Jak-STAT and PI3K/Akt signaling, (ii) immune-relevant signaling pathway genes were downregulated in intracranial metastases, and (iii) intracranial metastases were associated with a brain-like phenotype gene expression program. Further, the integration of all differentially expressed genes across the patient-matched melanoma metastasis pairs led to a set of 103 genes that were consistently down- or up-regulated in at least 11 of the 16 of the patients. This set of genes contained many genes involved in the regulation of immune responses, cell growth, cellular signaling and transport processes. An analysis of these genes in the TCGA melanoma cohort showed that the expression behavior of 11 genes was significantly associated with survival. Moreover, a comparison of the 103 genes to three closely related melanoma metastasis studies revealed a core set of eight genes that were consistently down- or upregulated in intra- compared to extracranial metastases in at least two of the three related studies (down: CILP, DPT, FGF7, LAMP3, MEOX2, TMEM119; up: GLDN, PMP2) including FGF7 that was also significantly associated with survival. Our findings contribute to a better characterization of genes and pathways that distinguish intra- from extracranial melanoma metastasis and provide important hints for future experimental studies to identify potential targets for new therapeutic approaches.

Correlating transcription and protein expression profiles of immune biomarkers following lipopolysaccharide exposure in lung epithelial cells.

Universal and early recognition of pathogens occurs through recognition of evolutionarily conserved pathogen associated molecular patterns (PAMPs) by innate immune receptors and the consequent secretion of cytokines and chemokines. The intrinsic complexity of innate immune signaling and associated signal transduction challenges our ability to obtain physiologically relevant, reproducible and accurate data from experimental systems. One of the reasons for the discrepancy in observed data is the choice of measurement strategy. Immune signaling is regulated by the interplay between pathogen-derived molecules with host cells resulting in cellular expression changes. However, these cellular processes are often studied by the independent assessment of either the transcriptome or the proteome. Correlation between transcription and protein analysis is lacking in a variety of studies. In order to methodically evaluate the correlation between transcription and protein expression profiles associated with innate immune signaling, we measured cytokine and chemokine levels following exposure of human cells to the PAMP lipopolysaccharide (LPS) from the Gram-negative pathogen Pseudomonas aeruginosa. Expression of 84 messenger RNA (mRNA) transcripts and 69 proteins, including 35 overlapping targets, were measured in human lung epithelial cells. We evaluated 50 biological replicates to determine reproducibility of outcomes. Following pairwise normalization, 16 mRNA transcripts and 6 proteins were significantly upregulated following LPS exposure, while only five (CCL2, CSF3, CXCL5, CXCL8/IL8, and IL6) were upregulated in both transcriptomic and proteomic analysis. This lack of correlation between transcription and protein expression data may contribute to the discrepancy in the immune profiles reported in various studies. The use of multiomic assessments to achieve a systems-level understanding of immune signaling processes can result in the identification of host biomarker profiles for a variety of infectious diseases and facilitate countermeasure design and development.

Resolving candidate genes of duck ovarian tissue transplantation via RNA-Seq and expression network analyses

This study aims to identify candidate genes related to ovarian development after ovarian tissue transplantation through transcriptome sequencing (RNA-seq) and expression network analyses, as well as to provide a reference for determining the molecular mechanism of improving ovarian development following ovarian tissue transplantation. We collected ovarian tissues from 15 thirty-day-old ducks and split each ovary into 4 equal portions of comparable sizes before orthotopically transplanting them into 2-day-old ducks. Samples were collected on days 0 (untransplanted), 3, 6, and 9. The samples were paraffin sectioned and then subjected to Hematoxylin-Eosin (HE) staining and follicular counting. We extracted RNA from ovarian samples via the Trizol method to construct a transcriptome library, which was then sequenced by the Illumina Novaseq 6000 sequencing platform. The sequencing results were examined for differentially expressed genes (DEG) through gene ontology (GO) function and the Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses, gene set enrichment analysis (GSEA), weighted correlation network analysis (WGCNA), and protein-protein interaction (PPI) networks. Some of the candidate genes were selected for verification using real-time fluorescence quantitative PCR (qRT-PCR). Histological analysis revealed a significant reduction in the number of morphologically normal follicles at 3, 6, and 9 d after ovarian transplantation, along with significantly higher abnormality rates (P < 0.05). The transcriptome analysis results revealed 2,114, 2,224, and 2,257 upregulated DEGs and 2,647, 2,883, and 2,665 downregulated DEGs at 3, 6, and 9 d after ovarian transplantation, respectively. Enrichment analysis revealed the involvement multiple pathways in inflammatory signaling, signal transduction, and cellular processes. Furthermore, WGCNA yielded 13 modules, with 10, 4, and 6 candidate genes mined at 3, 6 and 9 d after ovarian transplantation, respectively. Transcription factor (TF) prediction showed that STAT1 was the most important TF. Finally, the qRT-PCR verification results revealed that 12 candidate genes exhibited an expression trend consistent with sequencing data. In summary, significant differences were observed in the number of follicles in duck ovaries following ovarian transplantation. Candidate genes involved in ovarian vascular remodeling and proliferation were screened using RNA-Seq and WGCNA.

Upregulated Matrisomal Proteins and Extracellular Matrix Mechanosignaling Underlie Obesity-Associated Promotion of Pancreatic Ductal Adenocarcinoma.

Diet-induced obesity (DIO) promotes pancreatic ductal adenocarcinoma (PDAC) in mice expressing KRasG12D in the pancreas (KC mice), but the precise mechanisms remain unclear. Here, we performed multiplex quantitative proteomic and phosphoproteomic analysis by liquid chromatography-tandem mass spectrometry and further bioinformatic and spatial analysis of pancreas tissues from control-fed versus DIO KC mice after 3, 6, and 9 months. Normal pancreatic parenchyma and associated proteins were steadily eliminated and the novel proteins, phosphoproteins, and signaling pathways associated with PDAC tumorigenesis increased until 6 months, when most males exhibited cancer, but females did not. Differentially expressed proteins and phosphoproteins induced by DIO revealed the crucial functional role of matrisomal proteins, which implies the roles of upstream regulation by TGFβ, extracellular matrix-receptor signaling to downstream PI3K-Akt-mTOR-, MAPK-, and Yap/Taz activation, and crucial effects in the tumor microenvironment such as metabolic alterations and signaling crosstalk between immune cells, cancer-associated fibroblasts (CAFs), and tumor cells. Staining tissues from KC mice localized the expression of several prognostic PDAC biomarkers and elucidated tumorigenic features, such as robust macrophage infiltration, acinar-ductal metaplasia, mucinous PanIN, distinct nonmucinous atypical flat lesions (AFLs) surrounded by smooth muscle actin-positive CAFs, invasive tumors with epithelial-mesenchymal transition arising close to AFLs, and expanding deserted areas by 9 months. We next used Nanostring GeoMX to characterize the early spatial distribution of specific immune cell subtypes in distinct normal, stromal, and PanIN areas. Taken together, these data richly contextualize DIO promotion of Kras-driven PDAC tumorigenesis and provide many novel insights into the signaling pathways and processes involved.

CXXC5 drove inflammation and ovarian cancer proliferation via transcriptional activation of ZNF143 and EGR1

CXXC5, a zinc-finger protein, is known for its role in epigenetic regulation via binding to unmethylated CpG islands in gene promoters. As a transcription factor and epigenetic regulator, CXXC5 modulates various signaling processes and acts as a key coordinator. Altered expression or activity of CXXC5 has been linked to various pathological conditions, including tumorigenesis. Despite its known role in cancer, CXXC5's function and mechanism in ovarian cancer are unclear. We analyzed multiple public databases and found that CXXC5 is highly expressed in ovarian cancer, with high expression correlating with poor patient prognosis. We show that CXXC5 expression is regulated by oxygen concentration and is a direct target of HIF1A. CXXC5 is critical for maintaining the proliferative potential of ovarian cancer cells, with knockdown decreasing and overexpression increasing cell proliferation. Loss of CXXC5 led to inactivation of multiple inflammatory signaling pathways, while overexpression activated these pathways. Through in vitro and in vivo experiments, we confirmed ZNF143 and EGR1 as downstream transcription factors of CXXC5, mediating its proliferative potential in ovarian cancer. Our findings suggest that the CXXC5-ZNF143/EGR1 axis forms a network driving ovarian cell proliferation and tumorigenesis, and highlight CXXC5 as a potential therapeutic target for ovarian cancer treatment.

Exploring MiR-484 Regulation by Polyalthia longifolia: A Promising Biomarker and Therapeutic Target in Cervical Cancer through Integrated Bioinformatics and an In Vitro Analysis.

MiR-484, implicated in various carcinomas, holds promise as a prognostic marker, yet its relevance to cervical cancer (CC) remains unclear. Our prior study demonstrated the Polyalthia longifolia downregulation of miR-484, inhibiting HeLa cells. This study investigates miR-484's potential as a biomarker and therapeutic target in CC through integrated bioinformatics and an in vitro analysis. MiR-484 levels were analyzed across cancers, including CC, from The Cancer Genome Atlas. The limma R package identified differentially expressed genes (DEGs) between high- and low-miR-484 CC cohorts. We assessed biological functions, tumor microenvironment (TME), immunotherapy, stemness, hypoxia, RNA methylation, and chemosensitivity differences. Prognostic genes relevant to miR-484 were identified through Cox regression and Kaplan-Meier analyses, and a prognostic model was captured via multivariate Cox regression. Single-cell RNA sequencing determined cell populations related to prognostic genes. qRT-PCR validated key genes, and the miR-484 effect on CC proliferation was assessed via an MTT assay. MiR-484 was upregulated in most tumors, including CC, with DEGs enriched in skin development, PI3K signaling, and immune processes. High miR-484 expression correlated with specific immune cell infiltration, hypoxia, and drug sensitivity. Prognostic genes identified were predominantly epidermal and stratified patients with CC into risk groups, with the low-risk group showing enhanced survival and immunotherapeutic responses. qRT-PCR confirmed FGFR3 upregulation in CC cells, and an miR-484 mimic reversed the P. longifolia inhibitory effect on HeLa proliferation. MiR-484 plays a crucial role in the CC progression and prognosis, suggesting its potential as a biomarker for targeted therapy.

PhuEGO: A Network-Based Method to Reconstruct Active Signaling Pathways From Phosphoproteomics Datasets

Signaling networks are critical for virtually all cell functions. Our current knowledge of cell signaling has been summarized in signaling pathway databases, which, while useful, are highly biased toward well-studied processes, and do not capture context specific network wiring or pathway cross-talk. Mass spectrometry-based phosphoproteomics data can provide a more unbiased view of active cell signaling processes in a given context, however, it suffers from low signal-to-noise ratio and poor reproducibility across experiments. While progress in methods to extract active signaling signatures from such data has been made, there are still limitations with respect to balancing bias and interpretability. Here we present phuEGO, which combines up-to-three-layer network propagation with ego network decomposition to provide small networks comprising active functional signaling modules. PhuEGO boosts the signal-to-noise ratio from global phosphoproteomics datasets, enriches the resulting networks for functional phosphosites and allows the improved comparison and integration across datasets. We applied phuEGO to five phosphoproteomics data sets from cell lines collected upon infection with SARS CoV2. PhuEGO was better able to identify common active functions across datasets and to point to a subnetwork enriched for known COVID-19 targets. Overall, phuEGO provides a flexible tool to the community for the improved functional interpretation of global phosphoproteomics datasets.

Metabolic Diaschisis in Mild Traumatic Brain Injury.

Neurophysiological diaschisis presents in traumatic brain injury (TBI) as functional impairment distant to the lesion site caused by axonal neuroexcitation and deafferentation. Diaschisis studies in TBI models have evaluated acute phase functional and microstructural changes. Here, in vivo biochemical changes and cerebral blood flow (CBF) dynamics following TBI are studied with magnetic resonance. Behavioral assessments, magnetic resonance spectroscopy (MRS), and CBF measurements on rats followed cortical impact TBI. Data were acquired pre-TBI and 1-3 h, 2-days, 7-days, and 14-days post-TBI. MRS was performed on the ipsilateral and contralateral sides in the cortex, striatum, and thalamus. Metabolites measured by MRS included N-acetyl aspartate (NAA), aspartate (Asp), lactate (Lac), glutathione (GSH), and glutamate (Glu). Lesion volume expanded for 2 days post-TBI and then decreased. Ipsilateral CBF dropped acutely versus baseline on both sides (-62% ipsilateral, -48% contralateral, p < 0.05) but then recovered in cortex, with similar changes in ipsilateral striatum. Metabolic changes versus baseline included increased Asp (+640% by Day 7 post-TBI, p < 0.05) and Lac (+140% on Day 2 post-TBI, p < 0.05) in ipsilateral cortex, while GSH (-67% acutely, p < 0.05) and NAA decreased (-50% on Day 2, p < 0.05). In contralateral cortex Lac decreased (-73% acutely, p < 0.05). Analysis of variance showed significance for Side (p < 0.05), Time after TBI (p < 0.05), and interactions (p < 0.005) for Asp, GSH, Lac, and NAA. Transient decreases of GSH (-30%, p < 0.05, acutely) and NAA (-23% on Day 2, p < 0.05) occurred in ipsilateral striatum with reduced GSH (-42%, p < 0.005, acutely) in the contralateral striatum. GSH was decreased in ipsilateral thalamus (-59% ipsilateral on Day 2, p < 0.05). Delayed increases of total choline were seen in the contralateral thalamus were noted as well (+21% on Day 7 post-TBI, p < 0.05). Both CBF and neurometabolite concentration changes occurred remotely from the TBI site, both ipsilaterally and contralaterally. Decreased Lac levels on the contralateral cortex following TBI may be indicative of reduced anaerobic metabolism during the acute phase. The timing and locations of the changes suggest excitatory and inhibitory signaling processes are affecting post-TBI metabolic fluctuations.

Pushing the Frontiers of Cancer Research: Highlights from the Frontiers in Cancer Science Conference 2023.

The 15th annual Frontiers in Cancer Science (FCS) conference gathered scientific experts who shared the latest research converging upon several themes of cancer biology. These themes included the dysregulation of metabolism, cell death, and other signaling processes in cancer cells; using patient "omics" datasets and single-cell and spatial approaches to investigate heterogeneity, understand therapy resistance, and identify targets; innovative strategies for inhibiting tumors, including rational drug combinations and improved drug delivery mechanisms; and advances in models that can facilitate screening for cancer vulnerabilities and drug testing. We hope the insights from this meeting will stimulate further progress in the field.