Investigation of Signaling in Primary Populations of Human Senescent T Cells.

Mitogen-activated protein kinases, a family of three stress-related kinases, the Erks and Jnks and p38s, are activated by three-layer transphosphorylation cascades and are important for the activation, differentiation, and effector functions of lymphocytes. Recent studies on the aged immune systems from both humans and mice have uncovered a different mode of MAPK signaling that is independent of canonical activation cascades and instead occurs through simultaneous self-phosphorylation reactions within the sestrin-MAPK activation complex (sMAC), an immune-inhibitory complex not previously observed. In this chapter, we discuss methodologies to study these pathways at the population and single cell level, which allows rejuvenating immune cell differentiation and fate.

Targeting LINC00665/miR-199b-5p/SERPINE1 axis to inhibit trastuzumab resistance and tumorigenesis of gastric cancer via PI3K/AKt pathway.

Long noncoding RNAs (lncRNAs) serve as critical mediators of tumor progression and drug resistance in cancer. Herein, we identified a lncRNA, LINC00665, associated with trastuzumab resistance and development in gastric cancer (GC). LINC00665 was highly expressed in GC tissues and high expression of LINC00665 was correlated with poor prognosis. LINC00665 knockdown was verified to suppress migration, invasion, and resistance to trastuzumab in GC. Furthermore, we found that LINC00665 participates in the infiltration of naive B cells, mast cells, and T follicular helper (Tfh) cells. Mechanistically, LINC00665 was confirmed to regulate tumorigenesis and trastuzumab resistance by activating PI3K/AKt pathway. LINC00665 sponged miR-199b-5p to interact with SERPINE1 expression, resulting in the increase of phosphorylation of AKt, thus participating in the PI3K/AKt pathway. To summarize, LINC00665 facilitated the tumorigenesis and trastuzumab resistance of GC by sponging miR-199b-5p and promoting SERPINE1 expression, which further activated PI3K/AKt signaling; this finding reveals a new mechanism by which LINC00665 modulates tumor development and drug resistance in GC.

Measuring Calcium Signaling at the Primary Cilia.

Cellular signaling is nature's ingenious way for cells to perceive their surroundings and transmit external cues to internal compartments. Due to its critical role in cellular functions, the intricate machinery of molecular signaling has been intensively studied. A diverse arsenal of techniques exists to quantify the molecules involved in these processes. Among them, calcium stands out as a ubiquitous signaling molecule with roles in countless biological pathways. To elucidate its function as a second messenger, methods for measuring intracellular calcium have steadily evolved. This chapter introduces various methods for investigating calcium signaling cascades in cells as well as in cilia (thin hairlike projections) specifically, where calcium signaling is triggered by different cilial manipulation techniques.

T-2 toxin induces cardiac fibrosis by causing metabolic disorders and up-regulating Sirt3/FoxO3α/MnSOD signaling pathway-mediated oxidative stress.

T-2 toxin, an omnipresent environmental contaminant, poses a serious risk to the health of humans and animals due to its pronounced cardiotoxicity. This study aimed to elucidate the molecular mechanism of cardiac tissue damage by T-2 toxin. Twenty-four male Sprague-Dawley rats were orally administered T-2 toxin through gavage for 12 weeks at the dose of 0, 10, and 100 nanograms per gram body weight per day (ng/(g·day)), respectively. Morphological, pathological, and ultrastructural alterations in cardiac tissue were meticulously examined. Non-targeted metabolomics analysis was employed to analyze alterations in cardiac metabolites. The expression of the Sirt3/FoxO3α/MnSOD signaling pathway and the level of oxidative stress markers were detected. The results showed that exposure to T-2 toxin elicited myocardial tissue disorders, interstitial hemorrhage, capillary dilation, and fibrotic damage. Mitochondria were markedly impaired, including swelling, fusion, matrix degradation, and membrane damage. Metabonomics analysis unveiled that T-2 toxin could cause alterations in cardiac metabolic profiles as well as in the Sirt3/FoxO3α/MnSOD signaling pathway. T-2 toxin could inhibit the expressions of the signaling pathway and elevate the level of oxidative stress. In conclusion, the T-2 toxin probably induces cardiac fibrotic impairment by affecting amino acid and choline metabolism as well as up-regulating oxidative stress mediated by the Sirt3/FoxO3α/MnSOD signaling pathway. This study is expected to provide targets for preventing and treating T-2 toxin-induced cardiac fibrotic injury.

Measuring IP3 Generation in Real-Time Using a NanoBiT Luminescence Biosensor.

G protein-coupled receptors that activate Gq/11 regulate a range of physiological processes including neurotransmission, energy homeostasis, blood pressure regulation, and calcium homeostasis. Activation of Gq/11-coupled receptors stimulates the generation of inositol 1,4,5-trisphosphate (IP3), which mobilizes intracellular calcium release from the endoplasmic reticulum. This chapter describes an assay that uses a NanoBiT-IP3 luminescent biosensor to detect increases in IP3 in live cells. It describes how to perform these assays to assess signaling by the ghrelin receptor and the calcium-sensing receptor in HEK293 cells.

Quantifying Gq Signaling Using the IP1 Homogenous Time-Resolved Fluorescence (HTRF) Assay.

G protein-coupled receptors (GPCRs) play pivotal roles in cellular signaling and can regulate several cellular functions such as proliferation, secretion, protein expression, and cellular metabolism. Coupling of GPCRs to members of the Gq/11 protein family results in activation of inositol trisphosphate (IP3) and accumulation of calcium intracellularly. This protocol chapter outlines a step-by-step guide for utilizing the inositol phosphate-1 (IP1) accumulation assay, a time-resolved fluorescence resonance energy transfer (TR-FRET) method, to investigate Gq-IP3 signaling. The assay serves as a valuable tool for those conducting pharmacological investigations and compound screening targeting this critical cellular pathway. This protocol chapter covers experimental setup, sample preparation, and data analysis, providing researchers with an in-depth guide to explore the pharmacology of Gq-coupled receptors.

Analyzing Mitochondrial Calcium Influx in Isolated Mitochondria.

Mitochondria play a crucial role in Ca2+ signaling and homeostasis and can contribute to shaping the cytosolic Ca2+ landscape as well as regulate a variety of pathways including energy production and cell death. Dysregulation of mitochondrial Ca2+ homeostasis promotes pathologies including neurodegenerative diseases, cardiovascular disorders, and metabolic syndromes. The significance of mitochondria to Ca2+ signaling and regulation underscores the value of methods to assess mitochondrial Ca2+ import. Here we present a plate reader-based method using the Ca2+-sensitive fluorescent probe calcium green-5 N to measure mitochondrial Ca2+ import in isolated cardiac mitochondria. This technique can be expanded to measure Ca2+ uptake in mitochondria isolated from other tissue types and from cultured cells.

Methods for Imaging Intracellular Calcium Signals in the Mouse Mammary Epithelium in Two and Three Dimensions.

The mammary gland has a central role in optimal mammalian development and survival. Contractions of smooth muscle-like basal (or myoepithelial) cells in the functionally mature mammary gland in response to oxytocin are essential for milk ejection and are tightly regulated by intracellular calcium (Ca2+). Using mice expressing a genetically encoded Ca2+ indicator (GCaMP6f), we present in this chapter a method to visualize at high spatiotemporal resolution changes in intracellular Ca2+ in mammary epithelial cells, both in vitro (2D) and ex vivo (3D). The procedure to optimally prepare mammary tissue and primary cells is presented in detail.

Real-Time Imaging of Calcium Dynamics in Human Sperm After Precise Single-Cell Stimulation.

Calcium signaling is a critical regulator of sperm activation and function during the processes of capacitation and fertilization. Here, we describe a combined method for calcium imaging of single, live human sperm in response to stimuli administered with a precisely targeted delivery technique. This protocol is an adaptation of techniques developed for studies of murine sperm [1, 2], and enables real-time monitoring of human sperm calcium dynamics with high spatiotemporal resolution and concurrent detection of acrosome exocytosis (AE), a functional endpoint of sperm capacitation and requirement for physiological fertilization.The described imaging technique provides a valuable tool for exploration of calcium regulation in human sperm, which is essential to answer important questions and knowledge gaps regarding the link between calcium dynamics, AE, and fertilization. The versatility of this technique can be amplified through use of various indicator dyes or integration with pharmacological strategies such as pre-treating sperm with inhibitors or activators targeting specific receptors, channels, or intracellular signaling pathways of interest. Beyond fundamental inquiries into sperm physiology, this method can also be applied to assess the impact of potential contraceptive compounds on calcium signaling, AE, and membrane integrity.

High-Throughput RPLC-MS/MS Quantification of Short- and Medium-Chain Fatty Acids.

Short- and medium-chain fatty acids (SMCFA) are monocarboxylic acids with a carbon chain length of 1-12 carbon atoms. They are mainly produced in humans by the gut microbiota, play crucial metabolic roles, are vital for intestinal health, and have multifaceted impact on immune and neurological functions. Accurate detection and quantification of SMCFA in different human biofluids is achieved using 3-nitro phenylhydrazine (3-NPH) derivatization of the free fatty acids followed by reverse phase liquid chromatography (RPLC) separation and detection by tandem mass spectrometry (MS/MS). Here, we describe the simultaneous measurement of 14 SMCFA and lactate in detail. All 3-NPH-SMCFA-hydrazones are separated in less than 5 min with an 8-min total run time (injection-to-injection). Linear dynamic range over 0.1-500 µM is achieved for most SCFAs, while it is 0.05-100 µM for MCFAs. Validation of the procedure depicts good linearity (R2 > 0.98) and repeatability (CV ≤ 20%). The lower limit of detection (LLOD) is 10-30 nM. The lower limit of quantification (LLOQ) is 50-100 nM for most analytes, while it is 0.5 µM for acetate. In conclusion, the method offers several benefits compared to alternative methods regarding throughput, selectivity, sensitivity, and robustness.

Targeting RhoA expression with formononetin and salvianolic acid B to mitigate pancreatic cancer-associated endothelial cells changes.

ETHNOPHARMACOLOGICAL RELEVANCE: According to the theory of Qi and blood in Traditional Chinese Medicine (TCM), the combination of Qi-reinforcing herbs and blood-activating herbs has a synergistic effect in improving blood stasis syndrome, especially in tumor treatment. The classic "Radix Astragali - Salvia miltiorrhiza" duo exemplifies this principle, renowned for invigorating Qi and activating blood flow, employed widely in tumor therapies. Our prior research underscores the potent inhibition of pancreatic tumor xenografts by the combination of Formononetin (from Radix Astragali) and Salvianolic acid B (from Salvia miltiorrhiza) in vitro. However, it remains unclear whether this combination can inhibit the abnormal vascularization of pancreatic tumors to achieve its anti-cancer effect. AIM OF THE STUDY: Abnormal vasculature, known to facilitate tumor growth and metastasis. Strategies to normalize tumor-associated blood vessels provide a promising avenue for anti-tumor therapy. This study aimed to unravel the therapeutic potential of Formononetin combined with Salvianolic acid B (FcS) in modulating pancreatic cancer's impact on endothelial cells, illuminate the underlying mechanisms that govern this therapeutic interaction, thereby advancing strategies to normalize tumor vasculature and combat cancer progression. MATERIALS AND METHODS: A co-culture system involving Human Umbilical Vein Endothelial Cells (HUVECs) and PANC-1 cells was established to investigate the potential of targeting abnormal vasculature as a novel anti-tumor therapeutic strategy. We systematically compared HUVEC proliferation, migration, invasion, and lumenogenesis in both mono- and co-culture conditions with PANC-1 (H-P). Subsequently, FcS treatment of the H-P system was evaluated for its anti-angiogenic properties. Molecular docking was utilized to predict the interactions between Formononetin and Salvianolic acid B with RhoA, and the post-treatment expression of RhoA in HUVECs was assessed. Furthermore, we utilized shRhoA lentivirus to elucidate the role of RhoA in FcS-mediated effects on HUVECs. In vivo, a zebrafish xenograft tumor model was employed to assess FcS's anti-tumor potential, focusing on cancer cell proliferation, migration, apoptosis, and vascular development. RESULTS: FcS treatment demonstrated a significant, dose-dependent inhibition of PANC-1-induced alterations in HUVECs, including proliferation, migration, invasion, and tube formation capabilities. Molecular docking analyses indicated potential interactions between FcS and RhoA. Further, FcS treatment was found to downregulate RhoA expression and modulated the PI3K/AKT signaling pathway in PANC-1-induced HUVECs. Notably, the phenotypic inhibitory effects of FcS on HUVECs were attenuated by RhoA knockdown. In vivo zebrafish studies validated FcS's anti-tumor activity, inhibiting cancer cell proliferation, metastasis, and vascular sprouting, while promoting tumor cell apoptosis. CONCLUSIONS: This study underscores the promising potential of FcS in countering pancreatic cancer-induced endothelial alterations. FcS exhibits pronounced anti-abnormal vasculature effects, potentially achieved through downregulation of RhoA and inhibition of the PI3K/Akt signaling pathway, thereby presenting a novel therapeutic avenue for pancreatic cancer management.

Luciferase Reporter Assay for Determining the Signaling Activity of Interferons.

The classic dual luciferase reporter assay has been widely used to rapidly and accurately determine the transcriptional activity of a given promoter induced by certain signal pathways in the cells. In particular, the sensitive characteristics of luciferase highlight its significance in many experiments, such as weak promoter analysis, transfection studies using small amounts of DNA, and detection in cell lines with low transfection efficiency. This chapter presents detailed information and experimental procedures for measuring interferon (IFN)-induced Interferon-Stimulated Response Element (ISRE) promoter activity using the dual luciferase reporter assay.

Identifying an Abnormal Phosphorylated Adaptor by Viral Kinase Using Mass Spectrometry.

Mass spectrometers are widely used to identify protein phosphorylation sites. The process usually involves selective isolation of phosphoproteins and subsequent fragmentation to identify both the peptide sequence and phosphorylation site. Immunoprecipitation could capture and purify the protein of interest, greatly reducing sample complexity before submitting it for mass spectrometry analysis. This chapter describes a method to identify an abnormal phosphorylated site of the adaptor protein by a viral kinase through immunoprecipitation followed by LC-MS/MS.

Engeletin attenuates the inflammatory response via inhibiting TLR4-NFκB signaling pathway in Crohn's disease-like colitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Smilax glabra rhizome has a long history been used for clinical purposes in traditional Chinese medicinal for treating various inflammatory conditions. Engeletin1 (ENG) is one of the most abundant bioactive compounds found in Smilax glabra rhizome, with anti-inflammatory, antioxidant, and ulcer-preventing activities. AIM OF THE STUDY: The purpose of this study was to investigate the ability of ENG to alleviate inflammatory symptoms and improve epithelial barrier integrity utilize a 2,4,6-trinitrobenzene sulfonic acid2 (TNBS)-induced murine model in Crohn's disease3 (CD)-like colitis, and to characterize the underlying anti-inflammatory mechanisms of action. MATERIALS AND METHODS: A colitis model was established in BALB/c mice and treated with ENG for 7 days. RAW264.7 macrophages were pre-treated with ENG and lipopolysaccharide4 (LPS) stimulation. The mice's weight and colon length were assessed. qPCR and Western blotting were used to analyze gene expression and TLR4-NFκB pathway. Flow cytometry was used to analyze the polarization states of the macrophages. RESULTS: Treatment with ENG was sufficient to significantly alleviate symptoms of inflammation and colonic epithelial barrier integrity in treated mice. Significant inhibition of TNF-α, IL-1ß, and IL-6 expression was observed following ENG treatment in vivo and in vitro. ENG was also determined to be capable of inhibiting the expression of iNOS and CD86, inhibited M1 macrophage polarization in vitro, as well as the TLR4-NFκB signaling pathway. Molecular docking showed a highly stable binding between ENG and TLR4. CONCLUSION: ENG has been proven to alleviate inflammation and ameliorate the damage of epithelial barrier in CD-like colitis. ENG also suppressed the M1 macrophages polarization and the inhibited inflammatory cytokines. TLR4-NFκB signaling pathway, especially TLR4, may be the target of ENG. These data offer a new insight into the therapeutic mechanisms of ENG.

A dual-channel fluorescent probe targeting lysosomes for differential detection of Cys/Hcy and GSH: Applications in food, pharmaceutical analysis and bioimaging.

Thiols function as antioxidants in food, prolonging shelf life and enhancing flavor. Moreover, thiols are vital biomolecules involved in enzyme activity, cellular signal transduction, and protein folding among critical biological processes. In this paper, the fluorescent probe PYL-NBD was designed and synthesized, which utilized the fluorescent molecule pyrazoline, the lysosome-targeted morpholine moiety, and the sensing moiety NBD. Probe PYL-NBD was tailored for the recognition of biothiols through single-wavelength excitation, yielding distinct fluorescence emission signals: blue for Cys, Hcy, and GSH; green for Cys, Hcy. Probe PYL-NBD exhibited rapid reaction kinetics (<10 min), distinct fluorescence response signals, and low detection limits (15.7 nM for Cys, 14.4 nM for Hcy, and 12.6 nM for GSH). Probe PYL-NBD enabled quantitative determination of Cys content in food samples and L-cysteine capsules. Furthermore, probe PYL-NBD had been successfully applied for confocal imaging with dual-channel detection of biothiols in various biological specimens, including HeLa cells, zebrafish, tumor sections, and Arabidopsis thaliana.

Identifying a Ubiquitinated Adaptor Protein by a Viral E3 Ligase Through Co-immunoprecipitation.

Co-immunoprecipitation is a technique widely utilized to isolate protein complexes and study protein-protein interactions. Ubiquitinated proteins could be identified by combining co-immunoprecipitation with SDS-PAGE followed by immunoblotting. In this chapter, we use Herpes Simplex Virus 1 immediate-early protein ICP0-mediated polyubiquitination of p50 as an example to describe the method to identify a ubiquitinated adaptor protein by a viral E3 ligase by co-immunoprecipitation.

Spatially restricted ecto-5'-nucleotidase expression promotes the growth of uterine leiomyomas by modulating Akt activity.

Found in as many as 80% of women, uterine leiomyomas are a frequent cause of abnormal uterine bleeding, pelvic pain, and infertility. Despite their significant clinical impact, the mechanisms responsible for driving leiomyoma growth remain poorly understood. After obtaining IRB permission, expression of ecto-5'-nucleotidase (NT5E, CD73) was assessed in matched specimens of myometrium and leiomyoma by real-time qPCR, Western blot, and immunohistochemistry (IHC). Adenosine concentrations were measured by enzyme-linked assay. Primary cultures were used to assess the impact of adenosine and/or adenosine receptor agonists on proliferation, apoptosis, and patterns of intracellular signaling in vitro. When compared to matched specimens of healthy myometrium, uterine leiomyomas were characterized by reduced CD73 expression. Largely limited to thin-walled vascular structures and the pseudocapsule of leiomyomas despite diffuse myometrial distribution. Restricted intra-tumoral CD73 expression was accompanied by decreased levels of intra-tumoral adenosine. In vitro, incubation of primary leiomyoma cultures with adenosine or its hydrolysis-resistant analog 2-chloro-adenosine (2-CL-AD) inhibited proliferation, induced apoptosis, and reduced proportion of myocytes in S- and G2-M phases of the cell cycle. Decreased proliferation was accompanied by reduced expression of phospho-Akt, phospho-Cdk2-Tyr15, and phospho-Histone H3. Enforced expression of the A2B adenosine receptor (ADORA2B) and ADORA2B-selective agonists similarly suppressed proliferation and inhibited Akt phosphorylation. Collectively, these observations broadly implicate CD73 and reduced extracellular concentrations of adenosine as key regulators of leiomyoma growth and potentially identify novel strategies for clinically managing these common tumors.

Bufalin Regulates STAT3 Signaling Pathway to Inhibit Corneal Neovascularization and Fibrosis After Alkali Burn in Rats.

PURPOSE: Bufalin (BU) is a bioactive ingredient extracted from the skin and parotid venom glands of Bufo raddei, which can effectively inhibit angiogenesis. The aim of this study was to investigate whether BU could affect corneal neovascularization (CoNV). METHODS: A rat CoNV model (right eye) was constructed by administration of NaOH, and the left eye served as a control. Corneal damage scores of rats were detected. Hematoxylin & eosin, TUNEL, and Masson staining examined pathological changes, apoptosis, and fibrosis of corneal tissues. Immunohistochemistry and western blotting assessed the expression of proteins. RESULTS: BU intervention resulted in a significant reduction in corneal inflammatory cells, repair of corneal epithelial hyperplasia, significant reduction in stromal edema, and reduction in vascular proliferation. BU can inhibit corneal neovascularization. CONCLUSION: This study demonstrated that BU inhibits CoNV, fibrosis, and inflammation by modulating the STAT3 signaling pathway, elucidating the intrinsic mechanism of its protective effect. BU has great potential in the treatment of CoNV caused by corneal alkali burns.

Real-world outcomes in patients with metastatic castration-resistant prostate cancer beyond progression after upfront androgen receptor signaling inhibitor.

BACKGROUND: Upfront androgen receptor signaling inhibitor (ARSI) along with androgen deprivation therapy is the current standard of care for metastatic castration-sensitive prostate cancer. However, evidence on second-line therapy after upfront ARSI is scarce. We aimed to evaluate the oncological outcome of ARSI versus docetaxel (DOC) after upfront ARSI therapy in a real-world clinical practice. METHODS: Subjects were metastatic castration-resistant prostate cancer (mCRPC) patients who had progressed within 2 years of upfront ARSI therapy and received ARSI (ARSI group) or DOC (DOC group) as a second-line therapy. Second-line progression-free survival (second-line PFS), and second-line overall survival (second-line OS) were assessed. Propensity score matching (PSM) was used to adjust the clinicopathological features and treatment patterns. RESULTS: A total of 101 mCRPC patients, 68 in the ARSI group, and 33 in the DOC group, were included in this analysis. Median second-line PFS was 6.3 months in the ARSI group and 4.9 months in the DOC group (p = 0.21). Median second-line OS was 25.0 months in the ARSI group and 14.2 months in the DOC group (p = 0.06). Prostate-specific antigen nadir ≤ 0.2 ng/ml during upfront ARSI therapy was significantly associated with improved second-line PFS. After PSM, no significant difference in second-line PFS and second-line OS were observed between the two groups. CONCLUSION: ARSI or DOC has comparable oncologic outcomes in terms of second-line PFS and second-line OS. Further prospective research with longer follow-ups will be needed to identify the optimal treatment after upfront ARSI therapy.

Signaling pathways in HPV-induced cervical cancer: Exploring the therapeutic promise of RNA modulation.

Cervical cancer, originating from the epithelial tissue of the uterine cervix, constitutes the most commonly diagnosed malignancy among women worldwide. The predominant etiological factor underpinning cervical carcinogenesis is persistent infection with high-risk human papillomavirus (HPV) genotypes, notably HPV-16 and HPV-18. Oncoproteins encoded by high-risk HPV interfere with multiple essential cellular signaling cascades. Specifically, E5, E6, and E7 proteins disrupt the signaling pathways like p53, retinoblastoma tumor suppressor protein (pRB), The phosphoinositide 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR), epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERK), and Wnt/ß-catenin, promoting HPV-mediated carcinogenesis. This dysregulation disrupts cell cycle control, apoptosis, and metastasis through modulation of microRNAs (miRNA) and key cellular processes. The novel therapeutic interventions for HPV prevention and detection are fundamental to patient management. RNA-based treatment modalities offer the potential for manipulating critical pathways involved in cervical carcinogenesis. RNA therapeutics offer novel approaches to drug development by targeting intracellular genetic elements inaccessible to conventional modalities. Additional advantages include rapid design, synthesis, and a reduced genotoxic profile compared to DNA-based therapies. Despite beneficial attributes, system stability and efficient delivery remain critical parameters. This study assessed the intricate relationship between HPV, cervical cancer, and various signaling pathways. The study explores miRNAs' diagnostic and therapeutic potential, mall interfering RNAs (siRNAs), and long non-coding RNAs (lncRNAs)in cervical cancer management. The review highlights the prospect of RNA-targeted therapies to modulate specific cancer signaling pathways. This approach offers a novel strategy for cervical cancer treatment through precise regulation of cancer signaling. Future research should concentrate on developing RNA-targeted interventions to improve cervical cancer treatment outcomes through increased therapeutic efficacy and specificity.