RCDdb: A manually curated database and analysis platform for regulated cell death.

Regulated cell death is a pivotal regulatory mechanism governing the development and homeostasis of multicellular organisms. A comprehensive understanding of RCD's regulatory mechanisms is crucial for developing novel therapeutic strategies against diseases associated with cell death, such as cancer and neurodegenerative diseases. However, existing data repositories support limited types of cell death data and lack comprehensive annotation and analytical functionalities. Thus, establishing an extensive cell death database is an urgent imperative. To address this gap, we developed the Regulated Cell Death Database (RCDdb, chenyclab.com/RCDdb), the first comprehensively manually annotated database designed to support annotations and analytical capabilities across all RCD types. We compiled 3090 marker gene annotations associated with 15 RCD types from 2180 relevant articles. The RCDdb includes annotation data on these marker genes concerning diseases, drugs, pathways, proteins, and gene expressions. Furthermore, it provides 49 diverse visualization methods to present this information. More importantly, the RCDdb features three online analysis tools for identifying and analyzing RCD-related features within user-submitted data. Furthermore, the RCDdb offers a user-friendly interface for querying, browsing, analysis, and visualization of detailed information associated with each RCD category. This resource promises to significantly aid researchers in better understanding the mechanisms of cell death, thereby accelerating progress in research and therapeutic strategies aimed at combating RCD-related diseases.

Permeable Self-Association of Metal-Organic Framework 808/Ag-Based Fiber Membrane for Broad-Spectrum and Highly Efficient Degradation of Biological and Chemical War Agents.

The threat posed by biological and chemical warfare agents (BCWA) to national security, the environment, and personal health underscores the need for innovative chemical protective clothing. To address the limitations of conventional activated carbon materials, which are prone to falling off and adsorption saturation, an efficient self-association approach was introduced. In this study, we proposed the immobilization of metal-organic framework (MOF) 808 and Ag nanoparticles onto a polypropylene (PP) fiber membrane using a rapid self-association method facilitated by chitosan (CS). The MOF 808/Ag-based (PP-CS/808-Ag) fiber membrane demonstrated exceptional degradation efficiency, achieving a remarkable rate of t1/2 within 2 h for the mustard simulant 2-chloroethyl ethyl sulfide (2-CEES) and a rate of t1/2 = 4.12 min for the G-series simulant dimethyl 4-nitrophenylphosphate (DMNP). A theoretical computational model was developed to determine the overall reaction mechanism, and it was verified that MOF 808 and Ag nanoparticles were mainly involved in the hydrolysis process against 2-CEES and DMNP. The PP-CS/808-Ag composite fiber film was prepared as the core layer, and the fracture strength, bending resistance, and moisture permeability were better than those specified by many countries for biochemical protective clothing, showing that it has a broad application prospect in developing a generation of broad-spectrum bioprotective clothing.

A single excitation dual emission semi-salamo type multi-functional probe for sensitive pH and Cu2+ detection.

pH and Cu2+ ion concentration changes are linked to disorders like Alzheimer's and cancer. Rapid detection of pH and Cu2+ ions is critical for public health and environmental concerns. The semi-salamo-type probe (E)-2-hydroxy-1-naphthaldehyde O-(2-(aminooxy)ethyl) oxime (NSS) demonstrated substantial dual-functional performance, sensing pH change and Cu2+ ions. A single excitation and double emission characteristic on the probe NSS made it distinctive. Probe NSS exhibits pH-dependent excited state intramolecular proton transfer (ESIPT), and its optical properties vary based on the pH environment. Probe NSS detects pH changes from 2 to 11 by changing the "off-on-off" of the excited state intra-molecular proton transfer (ESIPT) mechanism, exhibiting rapid, reversible, and selective responses. In addition, the luminescent salamo-like naphthalene-based probe NSS can coordinate with Cu2+ ions, achieving great selectivity and sensitivity to identify Cu2+ ions with a detection limit of 0.84 ppb (13.2 nM) Probe NSS can detect Cu2+ ions in actual water samples such as tap water and yellow river water. The test strip loaded with probe NSS enabled quick and accurate detection of Cu2+ ions in water samples. Consequently, the versatile salamo-type probe NSS lays the foundation for developing high sensitivity and fast-response dual-mode pH meters as well as Cu2+ sensing.

Risk stratification for radioactive iodine refractoriness using molecular alterations in distant metastatic differentiated thyroid cancer.

Objective: Patients with radioactive iodine-refractory differentiated thyroid cancer (RAIR-DTC) are often diagnosed with delay and constrained to limited treatment options. The correlation between RAI refractoriness and the underlying genetic characteristics has not been extensively studied. Methods: Adult patients with distant metastatic DTC were enrolled and assigned to undergo next-generation sequencing of a customized 26-gene panel (ThyroLead). Patients were classified into RAIR-DTC or non-RAIR groups to determine the differences in clinicopathological and molecular characteristics. Molecular risk stratification (MRS) was constructed based on the association between molecular alterations identified and RAI refractoriness, and the results were classified as high, intermediate or low MRS. Results: A total of 220 patients with distant metastases were included, 63.2% of whom were identified as RAIR-DTC. Genetic alterations were identified in 90% of all the patients, with BRAF (59.7% vs. 17.3%), TERT promoter (43.9% vs. 7.4%), and TP53 mutations (11.5% vs. 3.7%) being more prevalent in the RAIR-DTC group than in the non-RAIR group, except for RET fusions (15.8% vs. 39.5%), which had the opposite pattern. BRAF and TERT promoter are independent predictors of RAIR-DTC, accounting for 67.6% of patients with RAIR-DTC. MRS was strongly associated with RAI refractoriness (P<0.001), with an odds ratio (OR) of high to low MRS of 7.52 [95% confidence interval (95% CI), 3.96-14.28; P<0.001] and an OR of intermediate to low MRS of 3.20 (95% CI, 1.01-10.14; P=0.041). Conclusions: Molecular alterations were associated with RAI refractoriness, with BRAF and TERT promoter mutations being the predominant contributors, followed by TP53 and DICER1 mutations. MRS might serve as a valuable tool for both prognosticating clinical outcomes and directing precision-based therapeutic interventions.

FUS unveiled in mitochondrial DNA repair and targeted ligase-1 expression rescues repair-defects in FUS-linked motor neuron disease.

This study establishes the physiological role of Fused in Sarcoma (FUS) in mitochondrial DNA (mtDNA) repair and highlights its implications to the pathogenesis of FUS-associated neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Endogenous FUS interacts with and recruits mtDNA Ligase IIIα (mtLig3) to DNA damage sites within mitochondria, a relationship essential for maintaining mtDNA repair and integrity in healthy cells. Using ALS patient-derived FUS mutant cell lines, a transgenic mouse model, and human autopsy samples, we discovered that compromised FUS functionality hinders mtLig3's repair role, resulting in increased mtDNA damage and mutations. These alterations cause various manifestations of mitochondrial dysfunction, particularly under stress conditions relevant to disease pathology. Importantly, rectifying FUS mutations in patient-derived induced pluripotent cells (iPSCs) preserves mtDNA integrity. Similarly, targeted introduction of human DNA Ligase 1 restores repair mechanisms and mitochondrial activity in FUS mutant cells, suggesting a potential therapeutic approach. Our findings unveil FUS's critical role in mitochondrial health and mtDNA repair, offering valuable insights into the mechanisms underlying mitochondrial dysfunction in FUS-associated motor neuron disease.

Night shift work was associated with functional outcomes in acute ischemic stroke patients treated with endovascular thrombectomy.

Objective: This study aimed to explore the impact of late night shift work on the functional outcomes of patients with acute ischemic stroke (AIS) treated with endovascular thrombectomy (EVT). Methods: Consecutive AIS patients who underwent EVT between June 2019 and June 2021 were enrolled and divided into non-night shift work and night shift work groups based on their occupational histories. The primary outcome was the modified Rankin Scale score defined 3-month functional outcome. The secondary outcomes were 3-month mortality, symptomatic intracerebral hemorrhage (sICH), ICH and early recanalization. Results: A total of 285 patients were enrolled, 35 patients (12.3%) were night shift workers, who were younger (P < 0.001) and had a significantly higher prevalence of smoking (P < 0.001), hyperlipidemia (P = 0.002), coronary heart disease (P = 0.031), and atrial fibrillation (P < 0.001). The 3-month favorable outcomes were achieved in 44.8% and 25.7% of patients in the non-night shift work and night shift work groups, respectively (adjusted odds ratio [OR]: 0.24, 95% CI: 0.10-0.57; adjusted P = 0.001). No difference was found in 3-month mortality (adjusted OR: 0.43; 95% CI: 0.14-1.25, adjusted P = 0.121), rates of ICH (adjusted OR: 0.73; 95% CI: 0.33-1.60; adjusted P = 0.430), sICH (adjusted OR: 0.75; 95% CI: 0.34-1.67; adjusted P = 0.487), or early successful recanalization (adjusted OR: 0.42; 95% CI: 0.12-1.56; adjusted P = 0.197). These results were consistent after PSM analysis. Conclusion: Our findings suggest that late night shift work is significantly associated with unfavorable outcomes in patients with AIS after EVT.

Dual-signal readout sensing of ATP content in single dental pulp stem cells during differentiation via functionalized glass nanopipettes.

Adenosine triphosphate (ATP) is regarded as the "energy currency" in living cells, so real-time quantification of content variation of intracellular ATP is highly desired for understanding some important physiological processes. Due to its single-molecule readout ability, nanopipette sensing has emerged as a powerful technique for molecular sensing. In this study, based on the effect of targeting-aptamer binding on ionic current, and fluorescence resonance energy transfer (FRET), we reported a dual-signal readout nanopipette sensing system for monitoring ATP content variation at the subcellular level. In the presence of ATP, the complementary DNA-modified gold nanoparticles (cDNAs-AuNPs) were released from the inner wall of the nanopipette, which leads to sensitive response variations in ionic current rectification and fluorescence intensity. The developed nanopipette sensor was capable of detecting ATP in single cells, and the fluctuation of ATP content in the differentiation of dental pulp stem cells (DPSCs) was further quantified with this method. The study provides a more reliable nanopipette sensing platform due to the introduction of fluorescence readout signals. Significantly, the study of energy fluctuation during cell differentiation from the perspective of energy metabolism is helpful for differentiation regulation and cell therapy.

Loss of SHROOM3 affects neuroepithelial cell shape through regulating cytoskeleton proteins in cynomolgus monkey organoids.

Neural tube defects (NTDs) are severe congenital neurodevelopmental disorders arising from incomplete neural tube closure. Although folate supplementation has been shown to mitigate the incidence of NTDs, some cases, often attributable to genetic factors, remain unpreventable. The SHROOM3 gene has been implicated in NTD cases that are unresponsive to folate supplementation; at present, however, the underlying mechanism remains unclear. Neural tube morphogenesis is a complex process involving the folding of the planar epithelium of the neural plate. To determine the role of SHROOM3 in early developmental morphogenesis, we established a neuroepithelial organoid culture system derived from cynomolgus monkeys to closely mimic the in vivo neural plate phase. Loss of SHROOM3 resulted in shorter neuroepithelial cells and smaller nuclei. These morphological changes were attributed to the insufficient recruitment of cytoskeletal proteins, namely fibrous actin (F-actin), myosin II, and phospho-myosin light chain (PMLC), to the apical side of the neuroepithelial cells. Notably, these defects were not rescued by folate supplementation. RNA sequencing revealed that differentially expressed genes were enriched in biological processes associated with cellular and organ morphogenesis. In summary, we established an authentic in vitro system to study NTDs and identified a novel mechanism for NTDs that are unresponsive to folate supplementation.

ESRP1 controls biogenesis and function of a large abundant multiexon circRNA.

While the majority of circRNAs are formed from infrequent back-splicing of exons from protein coding genes, some can be produced at quite high level and in a regulated manner. We describe the regulation, biogenesis and function of circDOCK1(2-27), a large, abundant circular RNA that is highly regulated during epithelial-mesenchymal transition (EMT) and whose formation depends on the epithelial splicing regulator ESRP1. CircDOCK1(2-27) synthesis in epithelial cells represses cell motility both by diverting transcripts from DOCK1 mRNA production to circRNA formation and by direct inhibition of migration by the circRNA. HITS-CLIP analysis and CRISPR-mediated deletions indicate ESRP1 controls circDOCK1(2-27) biosynthesis by binding a GGU-containing repeat region in intron 1 and detaining its splicing until Pol II completes its 157 kb journey to exon 27. Proximity-dependent biotinylation (BioID) assay suggests ESRP1 may modify the RNP landscape of intron 1 in a way that disfavours communication of exon 1 with exon 2, rather than physically bridging exon 2 to exon 27. The X-ray crystal structure of RNA-bound ESRP1 qRRM2 domain reveals it binds to GGU motifs, with the guanines embedded in clamp-like aromatic pockets in the protein.

lncRNA Sequencing Reveals Neurodegeneration-Associated FUS Mutations Alter Transcriptional Landscape of iPS Cells That Persists in Motor Neurons.

Fused-in sarcoma (FUS) gene mutations have been implicated in amyotrophic lateral sclerosis (ALS). This study aimed to investigate the impact of FUS mutations (R521H and P525L) on the transcriptome of induced pluripotent stem cells (iPSCs) and iPSC-derived motor neurons (iMNs). Using RNA sequencing (RNA Seq), we characterized differentially expressed genes (DEGs) and differentially expressed lncRNAs (DELs) and subsequently predicted lncRNA-mRNA target pairs (TAR pairs). Our results show that FUS mutations significantly altered the expression profiles of mRNAs and lncRNAs in iPSCs. Using this large dataset, we identified and verified six key differentially regulated TAR pairs in iPSCs that were also altered in iMNs. These target transcripts included: GPR149, NR4A, LMO3, SLC15A4, ZNF404, and CRACD. These findings indicated that selected mutant FUS-induced transcriptional alterations persist from iPSCs into differentiated iMNs. Functional enrichment analyses of DEGs indicated pathways associated with neuronal development and carcinogenesis as likely altered by these FUS mutations. Furthermore, ingenuity pathway analysis (IPA) and GO network analysis of lncRNA-targeted mRNAs indicated associations between RNA metabolism, lncRNA regulation, and DNA damage repair. Our findings provide insights into potential molecular mechanisms underlying the pathophysiology of ALS-associated FUS mutations and suggest potential therapeutic targets for the treatment of ALS.

Structural characterization of an isocytosine-specific deaminase VCZ reveals its application potential in the anti-cancer therapy.

Non-natural nucleobase isocytosine (IC) is the isomer of cytosine; its chemical derivate 5-fluoroisocytosine (5-FIC) together with the isocytosine-specific deaminase (ICD) VCZ was suggested to be potential practical enzyme/prodrug pair for cancer therapy through gene-directed enzyme-prodrug therapy (GDEPT) method. In this study, we have determined the crystal structures of apo-VCZ and its complex with 5-FU. We identified the critical residues for substrate binding and catalytic reaction. We also captured the substrate-induced conformational changes of VCZ, then proposed the conjectural reaction procedures of VCZ for converting the IC into the uracil. Moreover, we evaluated the therapeutic effect of wildtype or the mutated VCZ protein in the colorectal cancer cell lines. Our studies will shed light on optimizing the ICD/5-FIC pairs by modifying either the enzyme or the prodrug based on the structural observations, thereby improving the possibility of applying the ICD/5-FIC pair in clinical trials.

Malignant Middle Cerebral Artery Infarction during Early versus Late Endovascular Treatment in Acute Ischemic Stroke.

INTRODUCTION: Endovascular treatment (EVT) performed in the early time window has been shown to decrease the incidence of malignant middle cerebral artery infarction (MMI). However, the incidence of MMI in patients undergoing EVT during the late time window is unclear. This study aimed to investigate the prevalence of MMI in patients undergoing late EVT and compare it with that in patients undergoing early EVT. METHODS: We retrospectively analyzed consecutive patients with anterior large vessel occlusion stroke who underwent EVT at Xuanwu Hospital between January 2013 and June 2021. Eligible patients were divided into early EVT (within 6 h) and late EVT (6-24 h) groups according to the time from their stroke onset to puncture and compared. The occurrence of MMI post-EVT was the primary outcome. RESULTS: A total of 605 patients were recruited, of whom 300 (50.4%) underwent EVT within 6 h and 305 (49.6%) underwent EVT within 6-24 h. A total of 119 patients (19.7%) developed MMI. 68 patients (22.7%) in the early EVT group and 51 patients (16.7 %) in the late EVT group developed MMI (p = 0.066). After adjusting for covariate variables, late EVT was independently associated with a lower incidence of MMI (odds ratio, 0.404; 95% confidence interval, 0.242-0.675; p = 0.001). CONCLUSION: MMI is not an uncommon phenomenon in the modern thrombectomy era. Compared with the early time window, patients selected by stricter radiological criteria to undergo EVT in the late time window are independently associated with a lower incidence of MMI.

lncRNA Sequencing Reveals Neurodegeneration-associated FUS Mutations Alter Transcriptional Landscape of iPS Cells That Persists In Motor Neurons.

Fused-in Sarcoma (FUS) gene mutations have been implicated in amyotrophic lateral sclerosis (ALS). This study aimed to investigate the impact of FUS mutations (R521H and P525L) on the transcriptome of induced pluripotent stem cells (iPSCs) and iPSC-derived motor neurons (iMNs). Using RNA sequencing (RNA Seq), we characterized differentially expressed genes (DEGs), differentially expressed lncRNAs (DELs), and subsequently predicted lncRNA-mRNA target pairs (TAR pairs). Our results show that FUS mutations significantly altered expression profiles of mRNAs and lncRNAs in iPSCs. We identified key differentially regulated TAR pairs, including LMO3, TMEM132D, ERMN, GPR149, CRACD, and ZNF404 in mutant FUS iPSCs. We performed reverse transcription PCR (RT-PCR) validation in iPSCs and iMNs. Validation confirmed RNA-Seq findings and suggested that mutant FUS-induced transcriptional alterations persisted from iPSCs into differentiated iMNs. Functional enrichment analyses of DEGs indicated pathways associated with neuronal development and carcinogenesis that were likely altered by FUS mutations. Ingenuity Pathway Analysis (IPA) and GO network analysis of lncRNA-targeted mRNAs indicated associations related to RNA metabolism, lncRNA regulation, and DNA damage repair. Our findings provide insights into the molecular mechanisms underlying the pathophysiology of ALS-associated FUS mutations and suggest potential therapeutic targets for the treatment of ALS.

FUS Unveiled in Mitochondrial DNA Repair and Targeted Ligase-1 Expression Rescues Repair-Defects in FUS-Linked Neurodegeneration.

This study establishes the physiological role of Fused in Sarcoma (FUS) in mitochondrial DNA (mtDNA) repair and highlights its implications to the pathogenesis of FUS-associated neurodegenerative diseases such as Amyotrophic lateral sclerosis (ALS). Endogenous FUS interacts with and recruits mtDNA Ligase IIIα (mtLig3) to DNA damage sites within mitochondria, a relationship essential for maintaining mtDNA repair and integrity in healthy cells. Using ALS patient-derived FUS mutant cell lines, a transgenic mouse model, and human autopsy samples, we discovered that compromised FUS functionality hinders mtLig3's repair role, resulting in increased mtDNA damage and mutations. These alterations cause various manifestations of mitochondrial dysfunction, particularly under stress conditions relevant to disease pathology. Importantly, rectifying FUS mutations in patient-derived induced pluripotent cells (iPSCs) preserves mtDNA integrity. Similarly, targeted introduction of human DNA Ligase 1 restores repair mechanisms and mitochondrial activity in FUS mutant cells, suggesting a potential therapeutic approach. Our findings unveil FUS's critical role in mitochondrial health and mtDNA repair, offering valuable insights into the mechanisms underlying mitochondrial dysfunction in FUS-associated neurodegeneration.

Remote Ischemic Conditioning for Motor Recovery after Acute Ischemic Stroke.

BACKGROUND: Remote ischemic conditioning (RIC) has shown an impressive neuroprotective effect on acute ischemic stroke (AIS) in animal experiments. But whether chronic RIC improves long-term functional outcomes remains unclear. MATERIALS AND METHODS: We performed a non-randomized controlled trial. Eligible patients (aged 18 -80 y) with hemiplegia caused by AIS were allocated to the RIC group and the control group. All participants received normal protocol rehabilitation therapy. Patients in the RIC group underwent RIC twice daily for 90 days. The outcome included the 90-day Fugl-Meyer Assessment (FMA) scores and modified Rankin's scale (mRS) scores, as well as changes in angiogenesis-related factors in serum from baseline to 90 days. RESULTS: Twenty-seven patients were included in the analysis (13 in the RIC group and 14 in the control group). There was no significant difference in 90-day total FMA scores between the two groups. Lower limb FMA scores at day 90 were significantly higher in the RIC group (32.8±8.7 vs. 24.8±5.4, adjusted P =0.042). The proportion of favorable outcome (mRS<2) was higher in the RIC group than that in the control group, but no significant difference was detected (8 [61.5%] vs. 7 [50%], P =0.705). A significant increase has been found in the level of epidermal growth factor (EGF) in serum (9.4 [1.1 to 25.7] vs. -8.7 [-15.1 to 4.7], P =0.036) after chronic RIC procedure. CONCLUSION: This study investigated the role that RIC plays in AIS recovery, especially in motor function. RIC may have beneficial effects on lower limbs recovery by enhancing the EGF level. The effect of RIC on motor recovery should be further validated in future studies.

Efficient Modulation of Exon Skipping via Antisense Circular RNAs.

Splice-switching antisense oligonucleotides (ASOs) and engineered U7 small nuclear ribonucleoprotein (U7 Sm OPT) are the most commonly used methods for exon skipping. However, challenges remain, such as limited organ delivery and repeated dosing for ASOs and unknown risks of by-products produced by U7 Sm OPT. Here, we showed that antisense circular RNAs (AS-circRNAs) can effectively mediate exon skipping in both minigene and endogenous transcripts. We also showed a relatively higher exon skipping efficiency at the tested Dmd minigene than U7 Sm OPT. AS-circRNA specifically targets the precursor mRNA splicing without off-target effects. Moreover, AS-circRNAs with adeno-associated virus (AAV) delivery corrected the open reading frame and restored the dystrophin expression in a mouse model of Duchenne muscular dystrophy. In conclusion, we develop an alternative method for regulating RNA splicing, which might be served as a novel tool for genetic disease treatment.

CRISPR/Cas9 screen in human iPSC-derived cortical neurons identifies NEK6 as a novel disease modifier of C9orf72 poly(PR) toxicity.

INTRODUCTION: The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are hexanucleotide repeats in chromosome 9 open reading frame 72 (C9orf72). These repeats produce dipeptide repeat proteins with poly(PR) being the most toxic one. METHODS: We performed a kinome-wide CRISPR/Cas9 knock-out screen in human induced pluripotent stem cell (iPSC) -derived cortical neurons to identify modifiers of poly(PR) toxicity, and validated the role of candidate modifiers using in vitro, in vivo, and ex-vivo studies. RESULTS: Knock-down of NIMA-related kinase 6 (NEK6) prevented neuronal toxicity caused by poly(PR). Knock-down of nek6 also ameliorated the poly(PR)-induced axonopathy in zebrafish and NEK6 was aberrantly expressed in C9orf72 patients. Suppression of NEK6 expression and NEK6 activity inhibition rescued axonal transport defects in cortical neurons from C9orf72 patient iPSCs, at least partially by reversing p53-related DNA damage. DISCUSSION: We identified NEK6, which regulates poly(PR)-mediated p53-related DNA damage, as a novel therapeutic target for C9orf72 FTD/ALS.

RyR2 Serine-2030 PKA Site Governs Ca2+ Release Termination and Ca2+ Alternans.

BACKGROUND: PKA (protein kinase A)-mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of Ca2+ release and induction of cardiac Ca2+ alternans. METHODS: We performed single-cell endoplasmic reticulum Ca2+ imaging to assess the impact of S2030 mutations on Ca2+ release termination in human embryonic kidney 293 cells. Here we determined the role of the PKA site RyR2-S2030 in a physiological setting, we generated a novel mouse model harboring the S2030L mutation and carried out confocal Ca2+ imaging. RESULTS: We found that mutations, S2030D, S2030G, S2030L, S2030V, and S2030W reduced the endoplasmic reticulum luminal Ca2+ level at which Ca2+ release terminates (the termination threshold), whereas S2030P and S2030R increased the termination threshold. S2030A and S2030T had no significant impact on release termination. Furthermore, CaM-wild-type increased, whereas Ca2+ binding deficient CaM mutant (CaM-M [a loss-of-function CaM mutation with all 4 EF-hand motifs mutated]), PKA, and Ca2+/CaMKII (CaM-dependent protein kinase II) reduced the termination threshold. The S2030L mutation abolished the actions of CaM-wild-type, CaM-M, and PKA, but not CaMKII, in Ca2+ release termination. Moreover, we showed that isoproterenol and CaM-M suppressed pacing-induced Ca2+ alternans and accelerated Ca2+ transient recovery in intact working hearts, whereas CaM-wild-type exerted an opposite effect. The impact of isoproterenol was partially and fully reversed by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide and the CaMKII inhibitor N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide individually and together, respectively. S2030L abolished the impact of CaM-wild-type, CaM-M, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide-sensitive component, but not the N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide-sensitive component, of isoproterenol.

Pancreatic stellate cells exploit Wnt/ß-catenin/TCF7-mediated glutamine metabolism to promote pancreatic cancer cells growth.

Pancreatic stellate cells (PSCs) are crucial for metabolism and disease progression in pancreatic ductal adenocarcinoma (PDAC). However, detailed mechanisms of PSCs in glutamine (Gln) metabolism and tumor-stromal metabolic interactions have not been well clarified. Here we showed that tumor tissues displayed Gln deficiency in orthotopic PDAC models. Single-cell RNA sequencing analysis revealed metabolic heterogeneity in PDAC, with significantly higher expression of Gln catabolism pathway in stromal cells. Significantly higher glutamine synthetase (GS) protein expression was further validated in human tissues and cells. Elevated GS levels in tumor and stroma were independently prognostic of poorer prognosis in PDAC patients. Gln secreted by PSCs increased basal oxygen consumption rate in PCCs. Depletion of GS in PSCs significantly decreased PCCs proliferation in vitro and in vivo. Mechanistically, activation of Wnt signaling induced directly binding of ß-catenin/TCF7 complex to GS promoter region and upregulated GS expression. Rescue experiments testified that GS overexpression recovered ß-catenin knockdown-mediated function on Gln synthesis and tumor-promoting ability of PSCs. Overall, these findings identify the Wnt/ß-catenin/TCF7/GS-mediated growth-promoting effect of PSCs and provide new insights into stromal Gln metabolism, which may offer novel therapeutic strategies for PDAC.

Application of Indocyanine Green Fluorescence Imaging Combined with Laparoscopic Ultrasound in Laparoscopic Microwave Ablation of Liver Cancer.

BACKGROUND The aim of this study was to assess the effect of indocyanine green (ICG) fluorescence imaging combined with laparoscopic ultrasound in laparoscopic microwave ablation of liver cancer. MATERIAL AND METHODS This study retrospectively analyzed 61 patients who underwent laparoscopic microwave ablation of liver cancer, including laparoscopic microwave ablation with and without ICG fluoroscopy. RESULTS The operative times, ablation times, postoperative hospital stay, postoperative complication rate, hospitalization cost, postoperative liver function changes, and postoperative overall survival were similar between the 2 groups, but there was a statistically significant difference in recurrence-free survival (P<0.05). A total of 5 lesions were found in the fluorescence laparoscopy group that were not found by preoperative imaging, while no new lesions were found in the ordinary laparoscopy group. Fluorescence laparoscopy has obvious advantages over ordinary laparoscopy in finding small lesions that were not found before surgery. In terms of complete ablation rate, 3 patients in the ordinary laparoscopy group and 1 patient in the fluorescence laparoscopy group were judged to be incompletely ablated and were ablated again at 1 month after the operation. CONCLUSIONS For small hepatocellular carcinoma with severe liver cirrhosis and located on the liver surface, fluorescence laparoscopy can better reveal the location and boundary of the tumor, and fluorescence laparoscopy can detect tiny lesions that cannot be detected by preoperative imaging. The combination of fluorescence laparoscopy and microwave ablation has a good effect on the treatment of small hepatocellular carcinoma located on the surface of the liver that is difficult to distinguish.