[Baicalin induces ferroptosis in HepG2 cells by inhibiting ROS-mediated PI3K/Akt/FoxO3a signaling pathway].

This study aims to investigate whether baicalin induces ferroptosis in HepG2 cells and decipher the underlying mechanisms based on network pharmacology and cell experiments. HepG2 cells were cultured in vitro and the cell viability was detected by the cell counting kit-8(CCK-8). The transcriptome data of hepatocellular carcinoma were obtained from the Cancer Genome Atlas(TCGA), and the ferroptosis gene data from FerrDb V2. The DEG2 package was used to screen the differentially expressed genes(DEGs), and the common genes between DEGs and ferroptosis genes were selected as the target genes that mediate ferroptosis to regulate hepatocellular carcinoma progression. The functions and structures of the target genes were analyzed by Gene Ontology(GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment with the thresholds of P<0.05 and |log_2(fold change)|>0.5. DCFH-DA probe was used to detect the changes in the levels of cellular reactive oxygen species(ROS) in each group. The reduced glutathione(GSH) assay kit was used to measure the cellular GSH level, and Fe~(2+) assay kit to determine the Fe~(2+) level. Real-time quantitative PCR(RT-PCR) was employed to measure the mRNA levels of glutathione peroxidase 4(GPX4) and solute carrier family 7 member 11(SLC7A11) in each group. Western blot was employed to determine the protein levels of GPX4, SLC7A11, phosphatidylinositol 3-kinase(PI3K), p-PI3K, protein kinase B(Akt), p-Akt, forkhead box protein O3a(FoxO3a), and p-FoxO3a in each group. The results showed that treatment with 200 µmol·L~(-1) baicalin for 48 h significantly inhibited the viability of HepG2 cells. Ferroptosis in hepatocellular carcinoma could be regulated via the PI3K/Akt signaling pathway. The cell experiments showed that baicalin down-regulated the expression of SLC7A11 and GPX4, lowered the GSH level, and increased ROS accumulation and Fe~(2+) production in HepG2 cells. However, ferrostatin-1, an ferroptosis inhibitor, reduced baicalin-induced ROS accumulation, up-regulated the expression of SLC7A11 and GPX4, elevated the GSH level, and decreased PI3K, Akt, and FoxO3a phosphorylation. In summary, baicalin can induce ferroptosis in HepG2 cells by inhibiting the ROS-mediated PI3K/Akt/FoxO3a pathway.

Inhibition of ACSS2-mediated histone crotonylation alleviates kidney fibrosis via IL-1ß-dependent macrophage activation and tubular cell senescence.

Histone lysine crotonylation (Kcr), as a posttranslational modification, is widespread as acetylation (Kac); however, its roles are largely unknown in kidney fibrosis. In this study, we report that histone Kcr of tubular epithelial cells is abnormally elevated in fibrotic kidneys. By screening these crotonylated/acetylated factors, a crotonyl-CoA-producing enzyme ACSS2 (acyl-CoA synthetase short chain family member 2) is found to remarkably increase histone 3 lysine 9 crotonylation (H3K9cr) level without influencing H3K9ac in kidneys and tubular epithelial cells. The integrated analysis of ChIP-seq and RNA-seq of fibrotic kidneys reveal that the hub proinflammatory cytokine IL-1ß, which is regulated by H3K9cr, play crucial roles in fibrogenesis. Furthermore, genetic and pharmacologic inhibition of ACSS2 both suppress H3K9cr-mediated IL-1ß expression, which thereby alleviate IL-1ß-dependent macrophage activation and tubular cell senescence to delay renal fibrosis. Collectively, our findings uncover that H3K9cr exerts a critical, previously unrecognized role in kidney fibrosis, where ACSS2 represents an attractive drug target to slow fibrotic kidney disease progression.

Intravenous immunoglobulin protects the integrity of the intestinal epithelial barrier and inhibits ferroptosis induced by radiation exposure by activating the mTOR pathway.

Radiation exposure often leads to serious health problems in humans. The intestinal epithelium is sensitive to radiation damage, and radiation causes destruction of the intestinal epithelial barrier, which leads to radiation enteritis (RE), the loss of fluids, and the translocation of intestinal bacteria and toxins; radiation can even threaten survival. In this study, we aimed to explore the influence of IVIg on the integrity of the intestinal epithelial barrier after RE. Using a RE mouse model, we investigated the protective effects of intravenous immunoglobulin (IVIg) on the epithelial junctions of RE mice and validated these findings with intestinal organoids cultured in vitro. In addition, transmission electron microscopy (TEM), western blotting (WB) and immunostaining were used to further investigate changes in intestinal epithelial ferroptosis and related signaling pathways. When RE occurs, the intestinal epithelial barrier is severely damaged. IVIg treatment significantly ameliorated this damage to epithelial tight junctions both in vivo and in vitro. Notably, IVIg alleviated RE by inhibiting intestinal epithelial ferroptosis in RE mice. Mechanistically, IVIg promoted activation of the mTOR pathway and inhibited ferroptosis in the intestinal epithelium of mice. Rapamycin, which is a potent inhibitor of the mTOR protein, significantly abolished the protective effect of IVIg against radiation-induced damage to intestinal epithelial tight junctions. Overall, IVIg can prevent RE-induced damage to the intestinal epithelial barrier and inhibit ferroptosis by activating the mTOR pathway; this study provides a new treatment strategy for patients with RE caused by radiotherapy or accidental nuclear exposure.

Case report: remedial microdissection testicular sperm extraction after onco-microdissection testicular sperm extraction failure.

BACKGROUND: Testicular cancer (TC) mostly occurs in men aged 14 to 44. Studies have shown that TC seriously damages male fertility, and 6% to 24% of patients with TC were even found to suffer from azoospermia when they are diagnosed. At present, some studies have pointed out that onco-microdissection testicular sperm extraction (mTESE) can extract sperm from tumor testicles. However, there are almost no reports on remedial measures after onco-mTESE failure. Given the valuable opportunity for fertility preservation in patients with TC and azoospermia, it is necessary to provide effective remedial methods for patients with failed onco-mTESE. METHODS: Two young men, who were diagnosed with TC and also found to have azoospermia, tried onco-mTESE while undergoing radical orchiectomy for fertility preservation. However, sperm extraction failed in both patients. Subsequently, the isolated testicular tissue of the patient in case 1 suffered from TC again, and the patient in case 2 was scheduled to receive multiple cycles of gonadotoxic chemotherapy. Because both had a plan to have a birth in the future, we performed remedial mTESE. RESULTS: Sperm was successfully extracted from both patients. The patient recovered well, without complications. The patient couple in case 1 underwent 1 intracytoplasmic sperm injection (ICSI) cycle but did not achieve clinical pregnancy. CONCLUSIONS: There is still an opportunity to extract sperm successfully using onco-mTESE, despite the difficulty of fertility preservation in TC patients with azoospermia. If sperm extraction from the tumor testis fails, implementing remedial mTESE as early as possible would likely preserve the last chance of fertility for these patients.

Passivation protein-adhesion platform promoting stent reendothelialization using two-electron-assisted oxidation of polyphenols.

Superhydrophilic surfaces play an important role in nature. Inspired by this, scientists have designed various superhydrophilic materials that are widely used in the field of biomaterials, such as PEG molecular brushes and zwitterionic materials. However, superhydrophilic coatings with only anti-fouling properties do not satisfy the requirements for rapid reendothelialization of cardiovascular stent surfaces. Herein, a novel polyphenol superhydrophilic surface with passivated protein-adsorption properties was developed using two-electron oxidation of dopamine and polyphenols. This coating has a multiscale effects: 1) macroscopically: anti-fouling properties of superhydrophilic; 2) microscopically: protein adhesion properties of active groups (quinone-, amino-, hydroxyphenyl groups and aromatic ring). Polyphenols not only enhance the ability of coating to passivate protein-adsorption, but also make the coating have polyphenol-related biological functions. Therefore, the polyphenol and passivated protein-adsorption platform together maintain the stability of the scaffold microenvironment. This, in turn, provides favorable conditions for the growth of endothelial cells on the scaffold surface. In vivo implantation of the coated stents into the abdominal aorta resulted in uniform and dense endothelial cells covering the surface of the neointima. Moreover, new endothelial cells secreted large amounts of functional endothelial nitric oxide synthase like healthy endothelial cells. These results indicate that the polyphenol superhydrophilic coating potentially resists intra-stent restenosis and promotes surface reendothelialization. Hence, polyphenol superhydrophilic coatings with passivated protein-adsorption properties constructed by two-electron-assisted oxidation are a highly effective and versatile surface-modification strategy for implantable cardiovascular devices.

VIGA: a one-stop tool for eukaryotic virus identification and genome assembly from next-generation-sequencing data.

Identification of viruses and further assembly of viral genomes from the next-generation-sequencing data are essential steps in virome studies. This study presented a one-stop tool named VIGA (available at https://github.com/viralInformatics/VIGA) for eukaryotic virus identification and genome assembly from NGS data. It was composed of four modules, namely, identification, taxonomic annotation, assembly and novel virus discovery, which integrated several third-party tools such as BLAST, Trinity, MetaCompass and RagTag. Evaluation on multiple simulated and real virome datasets showed that VIGA assembled more complete virus genomes than its competitors on both the metatranscriptomic and metagenomic data and performed well in assembling virus genomes at the strain level. Finally, VIGA was used to investigate the virome in metatranscriptomic data from the Human Microbiome Project and revealed different composition and positive rate of viromes in diseases of prediabetes, Crohn's disease and ulcerative colitis. Overall, VIGA would help much in identification and characterization of viromes, especially the known viruses, in future studies.

Hairpin/DNA Ring Ternary Complex Initiated Rolling Circle Amplification for an Elevated Accuracy and Its Application in Analyzing Let-7a.

Rolling circle amplification (RCA) is an attractive isothermal nucleic acid amplification approach and has been widely applied in constructing a variety of biosensors. However, the inevitable drawbacks of lacking enough selectivity greatly hindered further applications of RCA-based approaches. Here, we develop a novel RCA-based approach by integrating the specific target recognition capability of the hairpin/DNA ring ternary complex and multiple signal amplification and successfully applied it for let-7a detection. In this method, let-7a specifically unfolds the hairpin probe (Hp probe) in the ternary complex to induce target recycle and RCA- and DNAzyme-based signal generation. Based on this, the established approach exhibits a high selectivity to let-7a, and the response of the approach to one base pair mismatched sequences was 24.9%, indicating a significantly improved specificity. Meanwhile, the limit of detection is as low as 342 aM, which can meet the high requirement for a trace amount of miRNA detection. In all, we believe that the established approach can offer a new avenue for miRNA detection and post-tumor care.

[Association of Body Mass Index and Weight Gain With Obesity-Related Breast Cancer Risk Biomarkers in Adult Chinese Women].

Objective: To investigate the associatiojn of body mass index (BMI) at different stages of life and weight gain in adulthood with obesity-related breast cancer risk biomarkers and to provide evidence for formulating policies concerning the prevention and control of breast cancer. Methods: A cross-sectional study was designed based on the follow-up cohort of southwest China community-based breast cancer screening of women. Using sequential sampling, eligible participants were enrolled from the cohort as the subjects of the study. Information on the basic risk factors was collected and the height, weight, and plasma biomarker levels were measured. Multiple linear regression model was applied to analyze the associations of early adulthood BMI (defined as the BMI of the participant at age 20), adulthood BMI (defined as the BMI measured at the time of enrollment), and weight gain in adulthood with the biomarkers. The concentrations of the biomarkers were incorporated in the model after log transformation. Results: The average age of the 442 participants was 49 (45, 54) years old, the average early adulthood BMI and adulthood BMI were 21.47 (19.56, 23.11) and 24.10 (22.59, 25.97) kg/m 2, respectively, and the average weight gain in adulthood was 6.60 (2.00, 11.00) kg. Adulthood BMI was negatively associated with adiponectin level ( ß=-0.026, 95% CI: -0.045--0.008, P=0.006), and positively associated with C-reactive protein level ( ß=0.095, 95% CI: 0.054-0.137, P<0.001) and leptin receptor level ( ß=0.090, 95% CI: 0.063-0.117, P<0.001). No association was found between adulthood BMI and resistin levels or between adulthood BMI and insulin-like growth factor-binding protein-3 levels. BMI in early adulthood was found to be negatively associated with only insulin-like growth factor-binding protein-3 levels ( ß=-0.039, 95% CI: -0.068--0.010, P=0.009). Further analysis of adulthood weight gain after the age of 20 revealed that average annual weight gain in adulthood was negatively associated with adiponectin levels and positively associated with 4 other biomarkers. Furthermore, compared with those of women whose weight remained stable, the adiponectin level of women whose weight gain in adulthood exceeded 5.00 kg was much lower ( ß=-0.185, 95% CI: -0.320--0.049, P=0.008), while their insulin-like growth factor-binding protein-3 ( ß=0.389, 95% CI: 0.183-0.594, P<0.001) and leptin receptor ( ß=0.245, 95% CI: 0.048-0.442, P=0.015) levels were higher. Conclusion: Weight gain in adulthood is strongly associated with the changes in obesity-related breast cancer risk biomarkers. Women should maintain a stable weight throughout adulthood and it is preferred that their weight gain should not exceed 5.00 kg.

Stepwise formation of a chemodynamic therapy agent of {Cu8} macrocyclic complex recognized by iodide ions.

An atomically precise Cu(I) macrocyclic complex Cu8I was developed for chemodynamic therapy (CDT) research. The {Cu8} macrocyclic skeleton gradually forms with the selective recognition of iodide ions, and the monitoring of intermediate fragments during Cu8I formation using time-dependent electrospray ionization mass spectrometry indicates the following possible formation process: [Cu1] → [Cu2] → [Cu3] → [Cu4] → [Cu5I] → [Cu6I] → [Cu7I] → [Cu8I] when recognized by iodide ions. Furthermore, the Cu(I)-mediated Fenton-like reaction in Cu8I catalyzes the production of toxic ˙OH from H2O2, which results in efficient tumor suppression.

Increases in BMI contribute to worsening inflammatory biomarkers related to breast cancer risk in women: a longitudinal study.

BACKGROUND: Inflammatory adipokines and cytokines play a pivotal role in linking obesity and breast cancer (BC) risk in women. We investigated the longitudinal associations between BMI change and trajectories of inflammatory biomarkers related to BC risk. METHODS: A longitudinal study was conducted among 442 Chinese women with 3-year repeated measures from 2019 to 2021. Plasma circulating inflammatory biomarkers related to BC risk, including adiponectin (ADP), resistin (RETN), soluble leptin receptor (sOB-R), insulin-like growth factor-binding protein-3 (IGFBP-3), and C-reactive protein (CRP), were examined annually. Linear mixed-effect models (LMM) were applied to investigate associations of time-varying BMI with trajectories of biomarkers. We additionally examined the modification effect of baseline BMI groups, menopausal status, and metabolic syndrome. RESULTS: BMI was associated with increased levels of RETN, CRP, sOB-R, and decreased levels of ADP at baseline. An increasing BMI rate was significantly associated with an average 3-year increase in RETN (ß = 0.019, 95% CI 0.004 to 0.034) and sOB-R (ß = 0.022, 95% CI 0.009 to 0.035), as well as a decrease in ADP (ß = - 0.006, 95% CI - 0.012 to 0.001). These associations persisted across different baseline BMI groups. An increasing BMI rate was significantly associated with an average 3-year increase in CRP levels among normal weight (ß = 0.045, 95% CI 0.001 to 0.088) and overweight (ß = 0.060, 95% CI 0.014 to 0.107) women. As BMI increased over time, a more remarkable decrease in ADP was observed among women with metabolic syndrome (ß = - 0.016, 95% CI - 0.029 to - 0.004) than those without metabolic syndrome at baseline. CONCLUSIONS: A higher increase rate of BMI was associated with poorer trajectories of inflammatory biomarkers related to BC risk. Recommendations for BMI reduction may benefit BC prevention in women, particularly for those with metabolic syndrome.

Associations between changes of smartphone pedometer-assessed step counts and levels of obesity-related breast cancer biomarkers in non-cancer women: A population-based observational study.

While a higher level of physical activity (PA) is inversely associated with a higher breast cancer (BC) risk, the health benefits of daily steps on obesity-related BC biomarkers remain unclear. We aimed to understand the associations of changes in step counts with levels of five obesity-related BC biomarkers during a two-year follow-up. In total, 144 non-cancer women (47.96 ± 5.72) were observed on both 2019 and 2021. A structured questionnaire, daily steps and fasting blood samples were collected before (t0, 2019) and after (t1, 2021). Levels of biomarkers (IGF-binding proteins 3, adiponectin, soluble leptin receptor, C-reactive protein, and resistin) were assayed by ELISA. Participants were divided into persistent low steps, decreasing steps, increasing steps, and persistent high steps. Associations of categories on proposed biomarkers were estimated using linear regression models, with persistent low steps as reference. Associations between time-varying step counts with biomarkers were quantified using mixed linear models. Compared with persistent low steps, increasing steps is associated with a reduction in C-reactive protein level (ß=-0.74, 95%CI=-1.23--0.26, P-value = 2.98 × 10-3). An inverse association between time-varying step counts with C-reactive protein level was identified, consistent across different obesity types and baseline step level categories. No association with daily step counts was observed for other proteins.

Organic carbon preservation in wetlands: Iron oxide protection vs. thermodynamic limitation.

The sequestration of organic carbon (OC) in wetland sediments is influenced by the presence of oxygen or lack thereof. The mechanisms of OC sequestration under redox fluctuations, particularly by the co-mediation of reactive iron (Fe) protection and thermodynamic limitation by the energetics of the OC itself, remain unclear. Over the past 26 years, a combination of field surveys and remote sensing images had revealed a strong decline in both natural and constructed wetland areas in Tianjin. This decline could be attributed to anthropogenic landfill practices and agricultural reclamation efforts, which may have significant impacts on the oxidation-reduction conditions for sedimentary OC. The Fe-bound OC (CBD extraction) decreased by 2 to 10-fold (from 8.3 to 10% to 0.7-4.5%) with increasing sediment depth at three sites with varying water depths (WD). The high-resolution spectro-microscopy analysis demonstrated that Fe (oxyhydr)oxides were colocalized with sedimentary OC. Corresponding to lower redox potential, the nominal oxidation state of C (NOSC), which corresponds to the energy content in OC, became more negative (energy content increased) with increasing sediment depth. Taken together, the preservation of sedimentary OC is contingent on the prevailing redox conditions: In environments where oxygen availability is high, reactive Fe provides protection for OC, while in anoxic environments, thermodynamic constraints (i.e., energetic constraints) limit the oxidation of OC.

Incidence and risk factors of acute kidney injury in cancer patients treated with immune checkpoint inhibitors: a systematic review and meta-analysis.

Background: The incidence and risk factors of acute kidney injury (AKI) in patients with malignancies receiving immune checkpoint inhibitors (ICIs) are being extensively reported with their widespread application. Objective: This study aimed to quantify the incidence and identify risk factors of AKI in cancer patients treated with ICIs. Methods: We searched the electronic databases of PubMed/Medline, Web of Science, Cochrane and Embase before 1 February 2023 on the incidence and risk factors of AKI in patients receiving ICIs and registered the protocol in PROSPERO (CRD42023391939). A random-effect meta-analysis was performed to quantify the pooled incidence estimate of AKI, identify risk factors with pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) and investigate the median latency period of ICI-AKI in patients treated with ICIs. Assessment of study quality, meta-regression, and sensitivity and publication bias analyses were conducted. Results: In total, 27 studies consisting of 24048 participants were included in this systematic review and meta-analysis. The overall pooled incidence of AKI secondary to ICIs was 5.7% (95% CI: 3.7%-8.2%). Significant risk factors were older age (OR: 1.01, 95% CI: 1.00-1.03), preexisting chronic kidney disease (CKD) (OR: 2.90, 95% CI: 1.65-5.11), ipilimumab (OR: 2.66, 95% CI: 1.42-4.98), combination of ICIs (OR: 2.45, 95% CI: 1.40-4.31), extrarenal immune-related adverse events (irAEs) (OR: 2.34, 95% CI: 1.53-3.59), and proton pump inhibitor (PPI) (OR: 2.23, 95% CI: 1.88-2.64), nonsteroidal anti-inflammatory drug (NSAID) (OR: 2.61, 95% CI: 1.90-3.57), fluindione (OR: 6.48, 95% CI: 2.72-15.46), diuretic (OR: 1.78, 95% CI: 1.32-2.40) and angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin-receptor blockers (ARBs) (pooled OR: 1.76, 95% CI: 1.15-2.68) use. Median time from ICIs initiation to AKI was 108.07 days. Sensitivity and publication bias analyses indicated robust results for this study. Conclusion: The occurrence of AKI following ICIs was not uncommon, with an incidence of 5.7% and a median time interval of 108.07 days after ICIs initiation. Older age, preexisting chronic kidney disease (CKD), ipilimumab, combined use of ICIs, extrarenal irAEs, and PPI, NSAID, fluindione, diuretics and ACEI/ARB use are risk factors for AKI in patients receiving ICIs. Systematic review registration: https://www.crd.york.ac.uk/prospero/, identifier CRD42023391939.

Influence of liver function after laparoscopy-assisted vs totally laparoscopic gastrectomy.

BACKGROUND: Previously, some studies have proposed that total laparoscopic gastrectomy (TLG) is superior to laparoscopic-assisted gastrectomy (LAG) in terms of safety and feasibility based on the related intraoperative operative parameters and incidence of postoperative complications. However, there are still few studies on the changes in postoperative liver function in patients undergoing LG. The present study compared the postoperative liver function of patients with TLG and LAG, aiming to explore whether there is a difference in the influence of TLG and LAG on the liver function of patients. AIM: To investigate whether there is a difference in the influence of TLG and LAG on the liver function of patients. METHODS: The present study collected 80 patients who underwent LG from 2020 to 2021 at the Digestive Center (including the Department of Gastrointestinal Surgery and the Department of General Surgery) of Zhongshan Hospital affiliated with Xiamen University, including 40 patients who underwent TLG and 40 patients who underwent LAG. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyltransferase (GGLT), total bilirubin (TBIL), direct bilirubin (DBIL) and indirect bilirubin (IBIL), and other liver function-related test indices were compared between the 2 groups before surgery and on the 1st, 3rd, and 5th d after surgery. RESULTS: The levels of ALT and AST in the 2 groups were significantly increased on the 1st to 2nd postoperative days compared with those before the operation. The levels of ALT and AST in the TLG group were within the normal range, while the levels of ALT and AST in the LAG group were twice as high as those in the TLG group (P < 0.05). The levels of ALT and AST in the 2 groups showed a downward trend at 3-4 d and 5-7 d after the operation and gradually decreased to the normal range (P < 0.05). The GGLT level in the LAG group was higher than that in the TLG group on postoperative days 1-2, the ALP level in the TLG group was higher than that in the LAG group on postoperative days 3-4, and the TBIL, DBIL and IBIL levels in the TLG group were higher than those in the LAG group on postoperative days 5-7 (P < 0.05). No significant difference was observed at other time points (P > 0.05). CONCLUSION: Both TLG and LAG can affect liver function, but the effect of LAG is more serious. The influence of both surgical approaches on liver function is transient and reversible. Although TLG is more difficult to perform, it may be a better choice for patients with gastric cancer combined with liver insufficiency.

Molecular Characterization of Two Wamide Neuropeptide Signaling Systems in Mollusk Aplysia.

Neuropeptides with the C-terminal Wamide (Trp-NH2) are one of the last common ancestors of peptide families of eumetazoans and play various physiological roles. In this study, we sought to characterize the ancient Wamide peptides signaling systems in the marine mollusk Aplysia californica, i.e., APGWamide (APGWa) and myoinhibitory peptide (MIP)/Allatostatin B (AST-B) signaling systems. A common feature of protostome APGWa and MIP/AST-B peptides is the presence of a conserved Wamide motif in the C-terminus. Although orthologs of the APGWa and MIP signaling systems have been studied to various extents in annelids or other protostomes, no complete signaling systems have yet been characterized in mollusks. Here, through bioinformatics, molecular and cellular biology, we identified three receptors for APGWa, namely, APGWa-R1, APGWa-R2, and APGWa-R3. The EC50 values for APGWa-R1, APGWa-R2, and APGWa-R3 are 45, 2100, and 2600 nM, respectively. For the MIP signaling system, we predicted 13 forms of peptides, i.e., MIP1-13 that could be generated from the precursor identified in our study, with MIP5 (WKQMAVWa) having the largest number of copies (4 copies). Then, a complete MIP receptor (MIPR) was identified and the MIP1-13 peptides activated the MIPR in a dose-dependent manner, with EC50 values ranging from 40 to 3000 nM. Peptide analogs with alanine substitution experiments demonstrated that the Wamide motif at the C-terminus is necessary for receptor activity in both the APGWa and MIP systems. Moreover, cross-activity between the two signaling systems showed that MIP1, 4, 7, and 8 ligands could activate APGWa-R1 with a low potency (EC50 values: 2800-22,000 nM), which further supported that the APGWa and MIP signaling systems are somewhat related. In summary, our successful characterization of Aplysia APGWa and MIP signaling systems represents the first example in mollusks and provides an important basis for further functional studies in this and other protostome species. Moreover, this study may be useful for elucidating and clarifying the evolutionary relationship between the two Wamide signaling systems (i.e., APGWa and MIP systems) and their other extended neuropeptide signaling systems.

Identification of three elevenin receptors and roles of elevenin disulfide bond and residues in receptor activation in Aplysia californica.

Neuropeptides are ubiquitous intercellular signaling molecules in the CNS and play diverse roles in modulating physiological functions by acting on specific G-protein coupled receptors (GPCRs). Among them, the elevenin signaling system is now believed to be present primarily in protostomes. Although elevenin was first identified from the L11 neuron of the abdominal ganglion in mollusc Aplysia californica, no receptors have been described in Aplysia, nor in any other molluscs. Here, using two elevenin receptors in annelid Platynereis dumerilii, we found three putative elevenin GPCRs in Aplysia. We cloned the three receptors and tentatively named them apElevR1, apElevR2, and apElevR3. Using an inositol monophosphate (IP1) accumulation assay, we demonstrated that Aplysia elevenin with the disulfide bond activated the three putative receptors with low EC50 values (ranging from 1.2 to 25 nM), supporting that they are true receptors for elevenin. In contrast, elevenin without the disulfide bond could not activate the receptors, indicating that the disulfide bond is required for receptor activity. Using alanine substitution of individual conserved residues other than the two cysteines, we showed that these residues appear to be critical to receptor activity, and the three different receptors had different sensitivities to the single residue substitution. Finally, we examined the roles of those residues outside the disulfide bond ring by removing these residues and found that they also appeared to be important to receptor activity. Thus, our study provides an important basis for further study of the functions of elevenin and its receptors in Aplysia and other molluscs.

A Cu(I)-based Fenton-like agent inducing mitochondrial damage for photo-assisted enhanced chemodynamic therapy.

Increasing the reactive oxygen species (ROS) content at the tumor site is one of the effective strategies to promote intracellular oxidative stress and improve therapeutic efficiency. Herein, an atomically precise cinnamaldehyde-derived metal-organic Cu(I) complex (denoted as DC-OD-Cu) was rationally constructed. DC-OD-Cu could preferentially accumulate in the mitochondria of HeLa cells due to the mitochondria-targeting ability of triphenylphosphine, which was accompanied by the generation of large amounts of highly toxic hydroxyl radicals (˙OH) via Cu(I)-mediated Fenton-like reactions. Meanwhile, greater ROS generation jointly results in mitochondrial damage under white LED light irradiation. Furthermore, the in vitro and in vivo results suggested that DC-OD-Cu possesses favorable cytotoxicity and inhibits tumor growth. We believe that this research might provide a controllable strategy to construct multifunctional metal organic complexes for ROS-involved CDT.

Characterization of an Aplysia vasotocin signaling system and actions of posttranslational modifications and individual residues of the ligand on receptor activity.

The vasopressin/oxytocin signaling system is present in both protostomes and deuterostomes and plays various physiological roles. Although there were reports for both vasopressin-like peptides and receptors in mollusc Lymnaea and Octopus, no precursor or receptors have been described in mollusc Aplysia. Here, through bioinformatics, molecular and cellular biology, we identified both the precursor and two receptors for Aplysia vasopressin-like peptide, which we named Aplysia vasotocin (apVT). The precursor provides evidence for the exact sequence of apVT, which is identical to conopressin G from cone snail venom, and contains 9 amino acids, with two cysteines at position 1 and 6, similar to nearly all vasopressin-like peptides. Through inositol monophosphate (IP1) accumulation assay, we demonstrated that two of the three putative receptors we cloned from Aplysia cDNA are true receptors for apVT. We named the two receptors as apVTR1 and apVTR2. We then determined the roles of post-translational modifications (PTMs) of apVT, i.e., the disulfide bond between two cysteines and the C-terminal amidation on receptor activity. Both the disulfide bond and amidation were critical for the activation of the two receptors. Cross-activity with conopressin S, annetocin from an annelid, and vertebrate oxytocin showed that although all three ligands can activate both receptors, the potency of these peptides differed depending on their residue variations from apVT. We, therefore, tested the roles of each residue through alanine substitution and found that each substitution could reduce the potency of the peptide analog, and substitution of the residues within the disulfide bond tended to have a larger impact on receptor activity than the substitution of those outside the bond. Moreover, the two receptors had different sensitivities to the PTMs and single residue substitutions. Thus, we have characterized the Aplysia vasotocin signaling system and showed how the PTMs and individual residues in the ligand contributed to receptor activity.