Positive roles of the Ca2+ sensors GbCML45 and GbCML50 in improving cotton Verticillium wilt resistance.

As a universal second messenger, cytosolic calcium (Ca2+) functions in multifaceted intracellular processes, including growth, development and responses to biotic/abiotic stresses in plant. The plant-specific Ca2+ sensors, calmodulin and calmodulin-like (CML) proteins, function as members of the second-messenger system to transfer Ca2+ signal into downstream responses. However, the functions of CMLs in the responses of cotton (Gossypium spp.) after Verticillium dahliae infection, which causes the serious vascular disease Verticillium wilt, remain elusive. Here, we discovered that the expression level of GbCML45 was promoted after V. dahliae infection in roots of cotton, suggesting its potential role in Verticillium wilt resistance. We found that knockdown of GbCML45 in cotton plants decreased resistance while overexpression of GbCML45 in Arabidopsis thaliana plants enhanced resistance to V. dahliae infection. Furthermore, there was physiological interaction between GbCML45 and its close homologue GbCML50 by using yeast two-hybrid and bimolecular fluorescence assays, and both proteins enhanced cotton resistance to V. dahliae infection in a Ca2+-dependent way in a knockdown study. Detailed investigations indicated that several defence-related pathways, including salicylic acid, ethylene, reactive oxygen species and nitric oxide signalling pathways, as well as accumulations of lignin and callose, are responsible for GbCML45- and GbCML50-modulated V. dahliae resistance in cotton. These results collectively indicated that GbCML45 and GbCML50 act as positive regulators to improve cotton Verticillium wilt resistance, providing potential targets for exploitation of improved Verticillium wilt-tolerant cotton cultivars by genetic engineering and molecular breeding.

Molecular mechanism of Saikosaponin-d in the treatment of gastric cancer based on network pharmacology and in vitro experimental verification.

The study aimed to utilize network pharmacology combined with cell experiments to research the mechanism of action of Saikosaponin-d in the treatment of gastric cancer. Drug target genes were obtained from the PubChem database and the Swiss Target Prediction database. Additionally, target genes for gastric cancer were obtained from the GEO database and the Gene Cards database. The core targets were then identified and further analyzed through gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and GESA enrichment. The clinical relevance of the core targets was assessed using the GEPIA and HPA databases. Molecular docking of drug monomers and core target proteins was performed using Auto Duck Tools and Pymol software. Finally, in vitro cellular experiments including cell viability, apoptosis, cell scratch, transwell invasion, transwell migration, qRT-PCR, and Western blot were conducted to verify these findings of network pharmacology. The network pharmacology analysis predicted that the drug monomers interacted with 54 disease targets. Based on clinical relevance analysis, six core targets were selected: VEGFA, IL2, CASP3, BCL2L1, MMP2, and MMP1. Molecular docking results showed binding activity between the Saikosaponin-d monomer and these core targets. Saikosaponin-d could inhibit gastric cancer cell proliferation, induce apoptosis, and inhibit cell migration and invasion.

Hawthorn with "homology of medicine and food": a review of anticancer effects and mechanisms.

Over the past few years, there has been a gradual increase in the incidence of cancer, affecting individuals at younger ages. With its refractory nature and substantial fatality rate, cancer presents a notable peril to human existence and wellbeing. Hawthorn, a medicinal food homology plant belonging to the Crataegus genus in the Rosaceae family, holds great value in various applications. Due to its long history of medicinal use, notable effects, and high safety profile, hawthorn has garnered considerable attention and plays a crucial role in cancer treatment. Through the integration of modern network pharmacology technology and traditional Chinese medicine (TCM), a range of anticancer active ingredients in hawthorn have been predicted, identified, and analyzed. Studies have shown that ingredients such as vitexin, isoorientin, ursolic acid, and maslinic acid, along with hawthorn extracts, can effectively modulate cancer-related signaling pathways and manifest anticancer properties via diverse mechanisms. This review employs network pharmacology to excavate the potential anticancer properties of hawthorn. By systematically integrating literature across databases such as PubMed and CNKI, the review explores the bioactive ingredients with anticancer effects, underlying mechanisms and pathways, the synergistic effects of drug combinations, advancements in novel drug delivery systems, and ongoing clinical trials concerning hawthorn's anticancer properties. Furthermore, the review highlights the preventive health benefits of hawthorn in cancer prevention, offering valuable insights for clinical cancer treatment and the development of TCM with anticancer properties that can be used for both medicinal and edible purposes.

Overexpression of Abscisic Acid Biosynthesis Gene OsNCED3 Enhances Survival Rate and Tolerance to Alkaline Stress in Rice Seedlings.

Alkaline stress with high pH levels could significantly influence plant growth and survival. The enzyme 9-cis-epoxycarotenoid dioxygenase (NCED) serves as a critical bottleneck in the biosynthesis of abscisic acid (ABA), making it essential for regulating stress tolerance. Here, we show that OsNCED3-overexpressing rice lines have increased ABA content by up to 50.90% and improved transcription levels of numerous genes involved in stress responses that significantly enhance seedling survival rates. Overexpression of OsNCED3 increased the dry weight contents of the total chlorophyll, proline, soluble sugar, starch, and the activities of antioxidant enzymes of rice seedlings, while reducing the contents of O2·-, H2O2, and malondialdehyde under hydroponic alkaline stress conditions simulated by 10, 15, and 20 mmol L-1 of Na2CO3. Additionally, the OsNCED3-overexpressing rice lines exhibited a notable increase in the expression of OsNCED3; ABA response-related genes OsSalT and OsWsi18; ion homeostasis-related genes OsAKT1, OsHKT1;5, OsSOS1, and OsNHX5; and ROS scavenging-related genes OsCu/Zn-SOD, OsFe-SOD, OsPOX1, OsCATA, OsCATB, and OsAPX1 in rice seedling leaves. The results of these findings suggest that overexpression of OsNCED3 upregulates endogenous ABA levels and the expression of stress response genes, which represents an innovative molecular approach for enhancing the alkaline tolerance of rice seedlings.

RNF216 Inhibits the Replication of H5N1 Avian Influenza Virus and Regulates the RIG-I Signaling Pathway in Ducks.

The RING finger (RNF) family, a group of E3 ubiquitin ligases, plays multiple essential roles in the regulation of innate immunity and resistance to viral infection in mammals. However, it is still unclear whether RNF proteins affect the production of IFN-I and the replication of avian influenza virus (AIV) in ducks. In this article, we found that duck RNF216 (duRNF216) inhibited the duRIG-I signaling pathway. Conversely, duRNF216 deficiency enhanced innate immune responses in duck embryonic fibroblasts. duRNF216 did not interacted with duRIG-I, duMDA5, duMAVS, duSTING, duTBK1, or duIRF7 in the duck RIG-I pathway. However, duRNF216 targeted duTRAF3 and inhibited duMAVS in the recruitment of duTRAF3 in a dose-dependent manner. duRNF216 catalyzed K48-linked polyubiquitination of duck TRAF3, which was degraded by the proteasome pathway. Additionally, AIV PB1 protein competed with duTRAF3 for binding to duRNF216 to reduce degradation of TRAF3 by proteasomes in the cytoplasm, thereby slightly weakening duRNF216-mediated downregulation of IFN-I. Moreover, although duRNF216 downregulated the IFN-ß expression during virus infection, the expression level of IFN-ß in AIV-infected duck embryonic fibroblasts overexpressing duRNF216 was still higher than that in uninfected cells, which would hinder the viral replication. During AIV infection, duRNF216 protein targeted the core protein PB1 of viral polymerase to hinder viral polymerase activity and viral RNA synthesis in the nucleus, ultimately strongly restricting viral replication. Thus, our study reveals a new mechanism by which duRNF216 downregulates innate immunity and inhibits AIV replication in ducks. These findings broaden our understanding of the mechanisms by which the duRNF216 protein affects AIV replication in ducks.

The adipose-neural axis is involved in epicardial adipose tissue-related cardiac arrhythmias.

Dysfunction of the sympathetic nervous system and increased epicardial adipose tissue (EAT) have been independently associated with the occurrence of cardiac arrhythmia. However, their exact roles in triggering arrhythmia remain elusive. Here, using an in vitro coculture system with sympathetic neurons, cardiomyocytes, and adipocytes, we show that adipocyte-derived leptin activates sympathetic neurons and increases the release of neuropeptide Y (NPY), which in turn triggers arrhythmia in cardiomyocytes by interacting with the Y1 receptor (Y1R) and subsequently enhancing the activity of the Na+/Ca2+ exchanger (NCX) and calcium/calmodulin-dependent protein kinase II (CaMKII). The arrhythmic phenotype can be partially blocked by a leptin neutralizing antibody or an inhibitor of Y1R, NCX, or CaMKII. Moreover, increased EAT thickness and leptin/NPY blood levels are detected in atrial fibrillation patients compared with the control group. Our study provides robust evidence that the adipose-neural axis contributes to arrhythmogenesis and represents a potential target for treating arrhythmia.

Global dynamics and cytokinin participation of salt gland development trajectory in recretohalophyte Limonium bicolor.

Salt gland is an epidermal Na+ secretory structure that enhances salt resistance in the recretohalophyte sea lavender (Limonium bicolor). To elucidate the salt gland development trajectory and related molecular mechanisms, we performed single-cell RNA sequencing of L. bicolor protoplasts from young leaves at salt gland initiation and differentiation stages. Dimensionality reduction analyses defined 19 transcriptionally distinct cell clusters, which were assigned into 4 broad populations-promeristem, epidermis, mesophyll, and vascular tissue-verified by in situ hybridization. Cytokinin was further proposed to participate in salt gland development by the expression patterns of related genes and cytological evidence. By comparison analyses of Single-cell RNA sequencing with exogenous application of 6-benzylaminopurine, we delineated 5 salt gland development-associated subclusters and defined salt gland-specific differentiation trajectories from Subclusters 8, 4, and 6 to Subcluster 3 and 1. Additionally, we validated the participation of TRIPTYCHON and the interacting protein Lb7G34824 in salt gland development, which regulated the expression of cytokinin metabolism and signaling-related genes such as GLABROUS INFLORESCENCE STEMS 2 to maintain cytokinin homeostasis during salt gland development. Our results generated a gene expression map of young leaves at single-cell resolution for the comprehensive investigation of salt gland determinants and cytokinin participation that helps elucidate cell fate determination during epidermis formation and evolution in recretohalophytes.

Novel Photocatalyst Based on Through-Space Charge Transfer Induced Intersystem Crossing Enables Rapid and Efficient Polymerization Under Low-Power Excitation Light.

Currently, most photoredox catalysis polymerization systems are limited by high excitation power, long polymerization time, or the requirement of electron donors due to the precise design of efficient photocatalysts still poses a great challenge. Herein, we propose a new approach: the creation of efficient photocatalysts having low ground state oxidation potentials and high excited state energy levels, along with through-space charge transfer (TSCT) induced intersystem crossing (ISC) properties. A cabazole-naphthalimide (NI) dyad (NI-1) characterized by long triplet excited state lifetime (τT=62 µs), satisfactory ISC efficiency (ΦΔ=54.3 %) and powerful reduction capacity [Singlet: E1/2 (PC+1/*PC)=-1.93 eV, Triplet: E1/2 (PC+1/*PC)=-0.84 eV] was obtained. An efficient and rapid polymerization (83 % conversion of 1 mM monomer in 30 s) was observed under the conditions of without electron donor, low excitation power (10 mW cm-2) and low catalyst (NI-1) loading (<50 µM). In contrast, the conversion rate was lower at 29 % when the reference catalyst (NI-4) was used for photopolymerization under the same conditions, demonstrating the advantage of the TSCT photocatalyst. Finally, the TSCT material was used as a photocatalyst in practical lithography for the first time, achieving pattern resolutions of up to 10 µm.

The Role and Mechanism of Paeoniae Radix Alba in Tumor Therapy.

Tumors have a huge impact on human life and are now the main cause of disease-related deaths. The main means of treatment are surgery and radiotherapy, but they are more damaging to the organism and have a poor postoperative prognosis. Therefore, we urgently need safe and effective drugs to treat tumors. In recent years, Chinese herbal medicines have been widely used in tumor therapy as complementary and alternative therapies. Medicinal and edible herbs are popular and have become a hot topic of research, which not only have excellent pharmacological effects and activities, but also have almost no side effects. Therefore, as a typical medicine and food homology, some components of Paeoniae Radix Alba (PRA, called Baishao in China) have been shown to have good efficacy and safety against cancer. Numerous studies have also shown that Paeoniae Radix Alba and its active ingredients treat cancer through various pathways and are also one of the important components of many antitumor herbal compound formulas. In this paper, we reviewed the literature on the intervention of Paeoniae Radix Alba in tumors and its mechanism of action in recent years and found that there is a large amount of literature on its effect on total glucosides of paeony (TGP) and paeoniflorin (PF), as well as an in-depth discussion of the mechanism of action of Paeoniae Radix Alba and its main constituents, with a view to promote the clinical development and application of Paeoniae Radix Alba in the field of antitumor management.

The evolution of Earth's surficial Mg cycle over the past 2 billion years.

The surficial cycling of Mg is coupled with the global carbon cycle, a predominant control of Earth's climate. However, how Earth's surficial Mg cycle evolved with time has been elusive. Magnesium isotope signatures of seawater (δ26Mgsw) track the surficial Mg cycle, which could provide crucial information on the carbon cycle in Earth's history. Here, we present a reconstruction of δ26Mgsw evolution over the past 2 billion years using marine halite fluid inclusions and sedimentary dolostones. The data show that δ26Mgsw decreased, with fluctuations, by about 1.4‰ from the Paleoproterozoic to the present time. Mass balance calculations based on this δ26Mgsw record reveal a long-term decline in net dolostone burial (NDB) over the past 2 billion years, due to the decrease in dolomitization in the oceans and the increase in dolostone weathering on the continents. This underlines a previously underappreciated connection between the weathering-burial cycle of dolostone and the Earth's climate on geologic timescales.

Bacterial detection based on Förster resonance energy transfer.

The huge economic loss and threat to human health caused by bacterial infection have attracted the public's concern, and there is an urgent need to relieve and improve the tough problem. Therefore, it is significant to establish a facile, rapid, and sensitive method for bacterial detection considering the shortcomings of existing methods. Förster resonance energy transfer (FRET)-based sensors have exhibited immense potential and applicability for bacterial detection given their high signal-to-noise ratio and high sensitivity. This review focuses on the development of FRET-based fluorescence assays for bacterial detection. We summarize the principle of FRET-based assays, discuss the commonly used recognition molecules and further introduce three frequent construction strategies. Based on the strategies and materials, relevant applications are presented. Moreover, some restrictions of FRET fluorescence sensors and development prospects are discussed. Suitable donor-acceptor pairs and stable recognition molecules are the essential conditions for sensors to play their roles, and there is still some room for development. Besides, applying FRET fluorescence sensors to point-of-care detection is still difficult. Future developments could focus on near-infrared fluorescent dyes and simultaneous detection of multiple analytes.

A validated LC-MS/MS method for the quantitation of daratumumab in rat serum using rapid tryptic digestion without IgG purification and reduction.

Daratumumab, a humanized monoclonal antibody utilized in treating immunoglobulin light-chain amyloidosis and relapsed/refractory multiple myeloma, was quantified in rat serum through a simple, economical and effective liquid chromatography tandem-mass spectrometry (LC-MS/MS) method. A surrogate peptide, LLIYDASNR, derived from trypsin hydrolysis, was quantitatively analyzed with LLIYDASN [13C6, 15N4] RAT as an internal standard. This corrected variations from sample pretreatment and mass spectrometry response, involving denaturation and trypsin hydrolysis in a two-step process lasting approximately 1 hour. Methodological validation demonstrated a linear range of 1 µg/mL to 1000 µg/mL in rat serum. Precision, accuracy, matrix effect, sensitivity, stability, selectivity, carryover, and interference met acceptance criteria. The validated LC-MS/MS approach was successfully applied to a pharmacokinetic study of daratumumab in rats at an intravenous dose of 15 mg/kg.

Chicken UFL1 Restricts Avian Influenza Virus Replication by Disrupting the Viral Polymerase Complex and Facilitating Type I IFN Production.

During avian influenza virus (AIV) infection, host defensive proteins promote antiviral innate immunity or antagonize viral components to limit viral replication. UFM1-specific ligase 1 (UFL1) is involved in regulating innate immunity and DNA virus replication in mammals, but the molecular mechanism by which chicken (ch)UFL1 regulates AIV replication is unclear. In this study, we first identified chUFL1 as a negative regulator of AIV replication by enhancing innate immunity and disrupting the assembly of the viral polymerase complex. Mechanistically, chUFL1 interacted with chicken stimulator of IFN genes (chSTING) and contributed to chSTING dimerization and the formation of the STING-TBK1-IRF7 complex. We further demonstrated that chUFL1 promoted K63-linked polyubiquitination of chSTING at K308 to facilitate chSTING-mediated type I IFN production independent of UFMylation. Additionally, chUFL1 expression was upregulated in response to AIV infection. Importantly, chUFL1 also interacted with the AIV PA protein to inhibit viral polymerase activity. Furthermore, chUFL1 impeded the nuclear import of the AIV PA protein and the assembly of the viral polymerase complex to suppress AIV replication. Collectively, these findings demonstrate that chUFL1 restricts AIV replication by disrupting the viral polymerase complex and facilitating type I IFN production, which provides new insights into the regulation of AIV replication in chickens.

Multi-objective ecological restoration priority in China: Cost-benefit optimization in different ecological performance regimes based on planetary boundaries.

In the context of the "United Nations Decade on Ecosystem Restoration", optimizing spatiotemporal arrangements for ecological restoration is an important approach to enhancing overall socioecological benefits for sustainable development. However, against the background of ecological degradation caused by the human use of most natural resources at levels that have approached or exceeded the safe and sustainable boundaries of ecosystems, it is key to explain how to optimize ecological restoration by classified management and optimal total benefits. In response to these issues, we combined spatial heterogeneity and temporal dynamics at the national scale in China to construct five ecological performance regimes defined by indicators that use planetary boundaries and ecological pressures which served as the basis for prioritizing ecological restoration areas and implementing zoning control. By integrating habitat conservation, biodiversity, water supply, and restoration cost constraints, seven ecological restoration scenarios were simulated to optimize the spatial layout of ecological restoration projects (ERPs). The results indicated that the provinces with unsustainable freshwater use, climate change, and land use accounted for more than 25%, 66.7%, and 25%, respectively, of the total area. Only 30% of the provinces experienced a decrease in environmental pressure. Based on the ecological performance regimes, ERP sites spanning the past 20 years were identified, and more than 50% of the priority areas were clustered in regime areas with increased ecological stress. As the restoration area targets doubled (40%) from the baseline (20%), a multi-objective scenario presents a trade-off between expanded ERPs in areas with highly beneficial effects and minimal restoration costs. In conclusion, a reasonable classification and management regime is the basis for targeted restoration. Coordinating multiple objectives and costs in ecological restoration is the key to maximizing socio-ecological benefits. Our study offered new perspectives on systematic and sustainable planning for ecological restoration.

Precise molecular engineering for the preparation of pyridinium photosensitizers with efficient ROS generation and photothermal conversion.

Organic photosensitizers (PSs) with aggregation-induced emission properties have great development potential in the integrated application of multi-mode diagnosis and treatment of photodynamic therapy (PDT) and photothermal therapy (PTT). However, preparing high-quality PSs with both optical and biological properties, high reactive oxygen species (ROS) and photothermal conversion ability are undoubtedly a great challenge. In this work, a series of pyridinium AIE PSs modified with benzophenone have been synthesized. A wide wavelength range of fluorescent materials was obtained by changing the conjugation and donor-acceptor strength. TPAPs5 has a significant advantage over similar compounds, and we have also identified the causes of high ROS generation and high photothermal conversion in terms of natural transition orbitals, excited state energy levels, ground-excited state configuration differences and recombination energy. Interestingly, migration of target sites was also found in biological imaging experiments, which also provided ideas for the design of double-targeted fluorescent probes. Therefore, the present work proposed an effective molecular design strategy for synergistic PDT and PTT therapy.

Identification of bZIP Transcription Factors That Regulate the Development of Leaf Epidermal Cells in Arabidopsis thaliana by Single-Cell RNA Sequencing.

Epidermal cells are the main avenue for signal and material exchange between plants and the environment. Leaf epidermal cells primarily include pavement cells, guard cells, and trichome cells. The development and distribution of different epidermal cells are tightly regulated by a complex transcriptional regulatory network mediated by phytohormones, including jasmonic acid, and transcription factors. How the fate of leaf epidermal cells is determined, however, is still largely unknown due to the diversity of cell types and the complexity of their regulation. Here, we characterized the transcriptional profiles of epidermal cells in 3-day-old true leaves of Arabidopsis thaliana using single-cell RNA sequencing. We identified two genes encoding BASIC LEUCINE-ZIPPER (bZIP) transcription factors, namely bZIP25 and bZIP53, which are highly expressed in pavement cells and early-stage meristemoid cells. Densities of pavement cells and trichome cells were found to increase and decrease, respectively, in bzip25 and bzip53 mutants, compared with wild-type plants. This trend was more pronounced in the presence of jasmonic acid, suggesting that these transcription factors regulate the development of trichome cells and pavement cells in response to jasmonic acid.

Investigation on the mechanism of the combination of eremias multiocellata and cisplatin in reducing chemoresistance of gastric cancer based on in vitro and in vivo experiments.

BACKGROUND: Cisplatin (DDP) is one of the important chemotherapy drugs for patients with advanced gastric cancer and metastasis, but its resistance is a bottleneck problem that affects clinical efficacy and patient survival. Eremias multiocellata (EM) is a traditional Chinese herbal medicine, which has been used in the treatment of precancerous lesions, gastric cancer, liver fibrosis, and other digestive diseases. However, the mechanism of reducing chemotherapy resistance to gastric cancer is still unclear. METHODS: We used the MTT assay to evaluate the proliferative viability of gastric cancer parental cell line MKN45 and its drug-resistant cell line MKN45/DDP, and compared their drug-resistance indices. The migration and invasion abilities of MKN45/DDP drug-resistant cells were evaluated using the Transwell assay. Apoptosis in MKN45/DDP drug-resistant cells was detected using flow cytometry. The effect of a combination of EM and cisplatin on the levels of reactive oxygen species (ROS) and lipid peroxides (LPO) in cisplatin-resistant gastric cancer cells was detected using ROS fluorescent probes and a lipid peroxidation assay kit in conjunction with flow cytometry. The effect of EM combined with cisplatin on the level of iron ions was detected by fluorescence probe and confocal laser technique. Hematoxylin-eosin staining (HE staining) was used to detect the histopathologic morphology of drug-resistant gastric cancer in nude mice. Ferroptosis-related proteins were measured using immunohistochemistry. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect tumor drug resistance-related genes. The NF-κB/Snail pathway-related proteins, PI3K/AKT/mTOR pathway-related proteins, and drug resistance-related proteins were detected by Western blot. RESULTS AND CONCLUSIONS: The results of in vitro and in vivo experiments showed that EM combined with DDP could effectively inhibit the migration and invasive ability of MKN45/DDP cells, as well as induce apoptosis of MKN45/DDP cells; the combination of the two drugs could significantly increase the levels of ROS, lipid peroxidation and divalent ferric ions in MKN45/DDP cells, at the same time reducing the levels of Ferroptosis-related proteins, which could induce Ferroptosis. In addition, EM combined with DDP can also exert the effect of reversing DDP resistance and increasing the sensitivity of gastric cancer drug-resistant cells to DDP by regulating the NF-κB/Snail signaling pathway, PI3K/AKT/mTOR signaling pathway, and the expression of drug resistance-related proteins and genes.

Generation of Functional and Mature Sympathetic Neurons from Human Pluripotent Stem Cells via a Neuroepithelial Route.

The sympathetic nervous system (SNS) is a crucial branch of the autonomic nervous system (ANS) that is responsible for regulating visceral function and various physiological processes. Dysfunction of the SNS can lead to various diseases, such as hypertension and metabolic disorders. However, obtaining sympathetic neurons from human tissues for research is challenging. The current research aimed at recapitulating the process of human sympathetic neuron development and achieved the successful establishment of a stepwise, highly efficient in vitro differentiation protocol. This protocol facilitated the generation of functional and mature sympathetic neurons from human pluripotent stem cells (hPSCs) using a chemical-defined induction medium. Initially, each differentiation stage was refined to derive sympathoadrenal progenitors (SAPs) from hPSCs through neural epithelial cells (NECs) and trunk neural crest stem cells (NCSCs). hPSC-derived SAPs could be expanded in vitro for at least 12 passages while maintaining the expression of SAP-specific transcription factors and neuronal differentiation potency. SAPs readily generated functional sympathetic neurons (SymNs) when cultured in the neuronal maturation medium for 3-4 weeks. These SymNs expressed sympathetic markers, exhibited electrophysiological properties, and secreted sympathetic neurotransmitters. More importantly, we further demonstrated that hPSC-derived SymNs can efficiently regulate the adipogenesis of human adipose-derived stem cells (ADSCs) and lipid metabolism in vitro. In conclusion, our study provided a simple and robust protocol for generating functional sympathetic neurons from hPSCs, which may be an invaluable tool in unraveling the mechanisms of SNS-related diseases.

Discovery of Pyrido[2,3-d]pyrimidin-7-one Derivatives as Highly Potent and Efficacious Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) Inhibitors for Cancer Treatment.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is an extracellular enzyme responsible for hydrolyzing cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), the endogenous agonist for the stimulator of interferon genes (STING) pathway. Inhibition of ENPP1 can trigger STING and promote antitumor immunity, offering an attractive therapeutic target for cancer immunotherapy. Despite progress in the discovery of ENPP1 inhibitors, the diversity in chemical structures and the efficacy of the agents are far from desirable, emphasizing the demand for novel inhibitors. Herein, we describe the design, synthesis, and biological evaluation of a series of ENPP1 inhibitors based on the pyrido[2,3-d]pyrimidin-7-one scaffold. Optimization efforts led to compound 31 with significant potency in both ENPP1 inhibition and STING pathway stimulation in vitro. Notably, 31 demonstrated in vivo efficacy in a syngeneic 4T1 mouse triple negative breast cancer model. These findings provide a promising lead compound with a novel scaffold for further drug development in cancer immunotherapy.

A robust and validated LC-MS/MS method for the quantification of ramucirumab in rat and human serum using direct enzymatic digestion without immunoassay.

A new liquid chromatography tandem mass spectrometry (LC-MS/MS) method was established to quantify the anti-gastric cancer fully human monoclonal antibody (ramucirumab) in rat and human serum. The surrogate peptide (GPSVLPLAPSSK) for ramucirumab was generated by trypsin hydrolysis and quantified using the isotopically labeled peptide GPSVLPLAPSSK[13C6, 15N2]ST containing two more amino acids at the carboxyl end as an internal standard to correct for variations introduced during the enzymatic hydrolysis process and any mass spectrometry changes. Additionally, the oxidation and deamidation of unstable peptides (VVSVLTVLHQDWLNGK and NSLYLQMNSLR) were detected. The quantitative range of the proposed method was 1-1000 µg/mL, and complete methodological validation was performed. The precision, accuracy, matrix effect, sensitivity, stability, selectivity, carryover, and interference of the measurements met the required standards. The validated LC-MS/MS method was applied to pharmacokinetic studies in rats administered ramucirumab at 15 mg/kg intravenously. Overall, a robust, efficient, and cost-effective LC-MS/MS method was successfully developed for quantifying ramucirumab in rat and human serum.