Evaluating the Effects of Perinatal Exposures to BPSIP on Hepatic Cholesterol Metabolism in Female and Male Offspring ICR Mice.

BACKGROUND: A broad suite of bisphenol S (BPS) derivatives as alternatives for BPS have been identified in various human biological samples, including 4-hydroxyphenyl 4-isopropoxyphenylsulfone (BPSIP) detected in human umbilical cord plasma and breast milk. However, very little is known about the health outcomes of prenatal BPS derivative exposure to offspring. OBJECTIVES: Our study aimed to investigate the response of hepatic cholesterol metabolism by sex in offspring of dams exposed to BPSIP. METHODS: Pregnant ICR mice were exposed to 5µg/kg body weight (BW)/day of BPSIP, BPS, or E2 through drinking water from gestational day one until the pups were weaned. The concentration of BPSIP, BPS, or E2 in the plasma and liver of pups was determined by liquid chromatography-tandem mass spectrometry. Metabolic phenotypes were recorded, and histopathology was examined for liver impairment. Transcriptome analysis was employed to characterize the distribution and expression patterns of differentially expressed genes across sexes. The metabolic regulation was validated by quantitative real-time PCR, immunohistochemistry, and immunoblotting. The role of estrogen receptors (ERs) in mediating sex-dependent effects was investigated using animal models and liver organoids. RESULTS: Pups of dams exposed to BPSIP showed a higher serum cholesterol level, and liver cholesterol levels were higher in females and lower in males than in the controls. BPSIP concentration in the male liver was 1.22±0.25 ng/g and 0.69±0.27 ng/g in the female liver. Histopathology analysis showed steatosis and lipid deposition in both male and female offspring. Transcriptome and gene expression analyses identified sex-specific differences in cholesterol biosynthesis, absorption, disposal, and efflux between pups of dams exposed to BPSIP and those in controls. In vivo, chromatin immunoprecipitation analysis revealed that the binding of ERα protein to key genes such as Hmgcr, Pcsk9, and Abcg5 was attenuated in BPSIP-exposed females compared to controls, while it was enhanced in males. In vitro, the liver organoid experiments demonstrated that restoration of differential expression induced by BPSIP in key genes, such as Hmgcr, Ldlr, and Cyp7a1, to levels comparable to the controls was only achieved when treated with a combination of ERα agonist and ERß agonist. DISCUSSION: Findings from this study suggest that perinatal exposure to BPSIP disrupted cholesterol metabolism in a sex-specific manner in a mouse model, in which ERα played a crucial role both in vivo and in vitro. Therefore, it is crucial to systematically evaluate BPS derivatives to protect maternal health during pregnancy and prevent the transmission of metabolic disorders across generations. https://doi.org/10.1289/EHP14643.

Dimeric-molecular beacon based intramolecular strand displacement amplification enables robust analysis of miRNA.

Given the critical role of miRNAs in regulating gene expression and their potential as biomarkers for various diseases, accurate and sensitive miRNA detection is essential for early diagnosis and monitoring of conditions such as cancer. In this study, we introduce a dimeric molecular beacon (Di-MB) based isothermal strand displacement amplification (ISDA) system (Di-MB-ISDA) for enhanced miRNA detection. The Di-MB system is composed of two monomeric MBs (Mono-MBs) connected by a double-stranded DNA linker with single-stranded sequences in the middle, facilitating binding with the flexible arms of the Mono-MBs. This design forms a compact, high-density structure, significantly improving biostability against nuclease degradation. In the absence of target miRNA, the Di-MB maintains its stable structure. When target miRNA is present, it binds to the stem-loop regions, causing the hairpin structure to unfold and expose the stem sequences. These sequences serve as templates for the built-in primers, triggering DNA replication through an intramolecular recognition mechanism. This spatial confinement effect accelerates the strand displacement reaction, allowing the target miRNA to initiate additional reaction cycles and amplify the detection signal. The Di-MB-ISDA system addresses key challenges such as poor biostability and limited sensitivity seen in traditional methods. By enhancing biostability and optimizing reaction conditions, this system demonstrates robust performance for miRNA detection with a detection limit of 100 pM. The findings highlight the potential of Di-MB-ISDA for sensitive and accurate miRNA analysis, paving the way for its application in biomedical study and disease diagnosis in complex biological samples.

Cross-Priming-Linked Hierarchical Isothermal Amplification Programming Progressive Activating Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a in miRNA Signaling.

Cascade isothermal nucleic acid amplification, which integrates several different amplification protocols to enhance the assay performance, is widely utilized in biosensing, particularly for detecting microRNAs (miRNAs), crucial biomarkers associated with tumor initiation and progression. However, striking a balance between a high amplification efficiency and simplicity in design remains a challenge. Therefore, methods achieving high amplification efficiency without significantly increasing complexity are highly favored. In this study, we propose a novel approach for miRNA detection, employing cross-priming-linked hierarchical isothermal amplification (CP-HIA) to progressively activate the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. The CP-HIA method strategically combines nicking-rolling circle amplification (n-RCA) and palindrome-aided circular strand displacement amplification (p-CSDA) for miRNA detection. Remarkably, this method utilizes only two main probes. Its key innovation lies in the interactive cross-priming strategy, wherein the amplification product from n-RCA is recycled to further drive p-CSDA, and vice versa. This interactive process establishes a hierarchical amplification, significantly enriching the activation probes for progressive CRISPR/Cas12a activation and subsequent target signal amplification. Consequently, the method exhibits greatly enhanced analytical performance, including high sensitivity and specificity in detecting low concentrations of miRNA. As low as 1.06 fM miRNA can thus be quantitatively detected, and the linear response of the miRNA is from 10 fM to 10 nM. These features demonstrate its potential for early disease diagnosis and monitoring. We anticipate that the CP-HIA method will serve as a promising platform for developing advanced molecular diagnostic tools for biomedical research.

Intramolecular Accelerated Assembly of Molecular Beacons: A DNA Nanoarchitecture-based Spatial Confinement Strategy toward Terminal Deoxynucleotidyl Transferase Biosensing.

Physiological function analysis of terminal deoxynucleotidyl transferase (TdT) in clinical medicine and hematopathology highlights its significance to be extensively utilized as a diagnostic biomarker for leukemia diagnosis. Herein, taking advantage of the spatial-confinement effect on a three-dimensional (3D) DNA nanoarchitecture, we reported a target-triggered intramolecular accelerated molecular beacon (MB) assembly for rapid and real-time analysis of TdT activity. In this strategy, the 3D DNA nanoarchitecture is first engineered via a cross-linking network hybridization chain reaction (HCR). A number of MBs, which were designed with a polythymine (poly-T) loop, were then conjugated on the scaffold DNA nanoarchitecture, allowing the obtained MB-DNA nanoarchitecture to contain lots of free 3'-hydroxyl (OH) termini inside or outside the super DNA nanostructure. Moreover, the distance between different MBs is closed, and the local concentration of MB is significantly improved owing to the confinement of MBs on this DNA nanoarchitecture. Once encountered with target TdT, the free -OH groups can be recognized by TdT immediately to catalyze the template-independent incorporation of adenine nucleotides, which results in the generation of multiple poly-A chains that rapidly react with many MBs via an intramolecular accelerated assembly process. The time-dependent substantial enhancement of the fluorescence from MBs can thus be applied for robustly analyzing TdT. Our observations suggest that the DNA nanostructure-based spatial confinement effect enables a high molecular collision frequency to accelerate the reaction kinetics, and the super DNA nanoarchitecture exhibits a better nuclease resistance to maintain signal stability. With these advantages, TdT can be rapidly detected with high sensitivity, specificity, and biostability.

NFAT and NF-κB dynamically co-regulate TCR and CAR signaling responses in human T cells.

While it has been established that the responses of T cells to antigens are combinatorially regulated by multiple signaling pathways, it remains elusive what mechanisms cells utilize to quantitatively modulate T cell responses during pathway integration. Here, we show that two key pathways in T cell signaling, calcium/nuclear factor of activated T cells (NFAT) and protein kinase C (PKC)/nuclear factor κB (NF-κB), integrate through a dynamic and combinatorial strategy to fine-tune T cell response genes. At the cis-regulatory level, the two pathways integrate through co-binding of NFAT and NF-κB to immune response genes. Pathway integration is further regulated temporally, where T cell receptor (TCR) and chimeric antigen receptor (CAR) activation signals modulate the temporal relationships between the nuclear localization dynamics of NFAT and NF-κB. Such physical and temporal integrations together contribute to distinct modes of expression modulation for genes. Thus, the temporal relationships between regulators can be modulated to affect their co-targets during immune responses, underscoring the importance of dynamic combinatorial regulation in cellular signaling.

Modulating gene regulation function by chemically controlled transcription factor clustering.

Recent studies have suggested that transcriptional protein condensates (or clusters) may play key roles in gene regulation and cell fate determination. However, it remains largely unclear how the gene regulation function is quantitatively tuned by transcription factor (TF) clustering and whether TF clustering may confer emergent behaviors as in cell fate control systems. Here, to address this, we construct synthetic TFs whose clustering behavior can be chemically controlled. Through single-parameter tuning of the system (i.e., TF clustering propensity), we provide lines of evidence supporting the direct transcriptional activation and amplification of target genes by TF clustering. Single-gene imaging suggests that such amplification results from the modulation of transcriptional dynamics. Importantly, TF clustering propensity modulates the gene regulation function by significantly tuning the effective TF binding affinity and to a lesser extent the ultrasensitivity, contributing to bimodality and sustained response behavior that are reminiscent of canonical cell fate control systems. Collectively, these results demonstrate that TF clustering can modulate the gene regulation function to enable emergent behaviors, and highlight the potential applications of chemically controlled protein clustering.

Realgar transforming solution suppresses angiogenesis and tumor growth by inhibiting VEGF receptor 2 signaling in vein endothelial cells.

Realgar (As4S4), as an arsenic sulfide mineral drug, has a good therapeutic reputation for anticancer in Traditional Chinese Medicine, and has recently been reported to inhibit angiogenesis in tumor growth. However, considering the poor solubility and low bioavailability of realgar, large dose of realgar and long period of treatment are necessary for achieving the effective blood medicine concentration. In present study, we resolved the crucial problem of poor solubility of realgar by using intrinsic biotransformation in microorganism, and investigated underlying mechanisms of realgar transforming solution (RTS) for antiangiogenesis. Our results demonstrated that RTS had a strong activity to inhibit HUVECs proliferation, migration, invasion, and tube formation. Moreover, RTS inhibited VEGF/bFGF-induced phosphorylation of VEGFR2 and the downstream protein kinases including ERK, FAK, and Src. In vivo zebrafish and chicken chorioallantoic membrane model experiments showed that RTS remarkably blocked angiogenesis. Finally, compared with the control, administration of 2.50 mg/kg RTS reached more than 50% inhibition against H22 tumor allografts in KM mice, but caused few toxic effects in the host. The antiangiogenic effect was indicated by CD31 immunohistochemical staining and alginate-encapsulated tumor cell assay. In summary, our findings suggest that RTS inhibits angiogenesis and may be a potential drug candidate in anticancer therapy.

Autophagy enhanced antitumor effect in K562 and K562/ADM cells using realgar transforming solution.

Realgar transforming solution (RTS) can be produced from a biotransformation process by using microorganisms cultured with realgar in our lab. RTS has been demonstrated as a novel arsenic anti-leukemia agent in K562 and K562/ADM. However, its underlying mechanism is unclear. In this study, we showed that RTS could strongly induce apoptosis in K562 and K562/ADM cells. After the cells were treated by RTS, apoptotic population were increased compared to control and clearly distinguishable by DAPI nuclei staining. With increasing the dose of RTS, more cells arrested in S phase and G2/M phase. Secondly, we also showed that RTS could induce autophagy via up-regulation of LC3, p62/SQSTM1 and inhibition of mTOR in a much lower arsenic dosage in contrast to ATO and realgar. In addition, autophagy induced by RTS partially due to the degradation of fusion oncoprotein Bcr-Abl, which is associated with multidrug resistant in (MDR)-CML. Our results also showed that the apoptotic rate decreased when autophagic flux was attenuated by CQ via inhibiting cleaved-caspase-3 and alleviating Bcl-2 level. These suggested that RTS triggered autophagy is a pro-death process in CML and MDR-CML cells. In conclusion, our findings demonstrated that RTS could serve as a promising arsenic candidate for anti-CML/MDR-CML by inducing apoptosis and autophagy and is more potent than ATO and realgar.

p53 dynamics orchestrates with binding affinity to target genes for cell fate decision.

Emerging evidence support that temporal dynamics is pivotal for signaling molecules in orchestrating smart responses to diverse stimuli. p53 is such a signaling molecule that employs temporal dynamics for the selective activation of downstream target genes and ultimately for cell fate decision. Yet how this fine-tuned p53 machinery is quantitatively decoded remains largely unclear. Here we report a quantitative mechanism defining how p53 dynamics orchestrates with binding affinity to target genes for cell fate decision. Treating cells with a genotoxic drug doxorubicin at various doses and durations, we found that a mild and prolonged challenge triggered sequential p53 pulses and ultimately resulted in a terminal pulse enacting apoptosis in a comparable rate with that induced by an acute and high-dose treatment. To transactivate proapoptotic genes and thereafter executing apoptosis, p53 must exceed a certain threshold and accumulate for sufficient time at levels above it. Effective cumulative levels above the threshold, defined as E∫p53, but not the total accumulation levels of p53, precisely discriminate survival and apoptotic cells. p53 accumulation below this threshold, even with prolonging time to reach a total level comparable to that from the accumulation over the threshold, could not transactivate proapoptotic genes to which the binding affinity of p53 is lower than that of proarrest genes, and this property is independent of dynamic features. Our findings indicate that the dynamic feature per se does not directly control cell fate, but rather it orchestrates with the binding affinity to target genes to confer an appropriate time window for cell fate choice. Our study provides a quantitative mechanism unifying p53 dynamics and binding affinity to target genes, providing novel insights to understand how p53 can respond quantitatively to chemotherapeutic drugs, and guiding the design of metronomic regimens for chemotherapeutic drugs.

Metabolomics-Proteomics Combined Approach Identifies Differential Metabolism-Associated Molecular Events between Senescence and Apoptosis.

Apoptosis and senescence are two types of cell fates in response to chemotherapy. Besides canonical pathways that mediate cell fates, cancer cell metabolism has been revealed as a crucial factor affecting cell fate decisions and thus represents a new target for antitumor therapy. Therefore, a comprehensive description of metabolic pathways underlying cell senescence and apoptosis in response to chemotherapy is highly demanded for therapeutic exploitation of both processes. Herein we employed a metabolomics-proteomics combined approach to identify metabolism-associated molecular events that mediate cellular responses to senescence and apoptosis using doxorubicin-treated human breast cancer cells MCF7 as models. Such biomics approach revealed that tricarboxylic acid cycle, pentose phosphate pathway, and nucleotide synthesis pathways were significantly upregulated in the senescent model, whereas fatty acid synthesis was reduced. In apoptotic cells, an overall reduced activity of major metabolic pathways was observed except for the arginine and proline pathway. Combinatorially, these data show the utility of biomics in exploring biochemical mechanism-based differences between apoptosis and senescence and reveal an unprecedented finding of the metabolic events that were induced for survival by facilitating ROS elimination and DNA damage repair in senescent cells, while they were downregulated in apoptotic cells when DNA damage was irreparable.

Metabolic Pathway Extension Approach for Metabolomic Biomarker Identification.

Discovery of metabolomic biomarkers represents an important task in disease diagnosis and therapy. Although the development of various analytical tools and online libraries facilitates the identification of biomarkers, the fast and reliable identification of new biomarkers that are not included in databases still represents a major bottleneck in the field of metabolomics. Here, we developed a metabolic pathway extension (MPE) approach to the fast characterization of metabolomic biomarkers. This approach was proposed based on a core concept that the whole metabolome is built from a limited number of initial metabolites via various kinds and multiple steps of metabolic reactions, and thus, theoretically, the whole metabolome might be mapped from the initial metabolites and metabolic reactions. Carnitine was used as an example of initial metabolites to validate this concept and the usefulness of MPE approach. The intragastric dosing of carnitine to mice induced a significant alternation of a total of 97 metabolites. Mass differences between each pair of metabolites were calculated and then matched with those of typical metabolic pathways automatically by an in-house developed program. Diagnostic ions and neutral losses were used for validating the matches. With this approach, 93 out of a total of 97 metabolites were putatively identified, while only half of them could be traced from the currently available online database. The MPE approach was further validated by applying to the identification of carnitine-associated biomarkers in a typical mice model of fasting, and extended to the development of bile acids submetabolome. Our study indicates that the MPE approach is highly useful for rapid and reliable identification of metabolically and structurally associated biomarkers.