Amonafide-based H2O2-responsive theranostic prodrugs: Exploring the correlation between H2O2 level and anticancer efficacy.

Leveraging the elevated hydrogen peroxide (H2O2) levels in cancer cells, H2O2-activated prodrugs have emerged as promising candidates for anticancer therapy. Notably, the efficacy of these prodrugs is influenced by the varying H2O2 levels across different cancer cell types. In this context, we have developed a novel H2O2-activated prodrug, PBE-AMF, which incorporates a phenylboronic ester (PBE) motif. Upon H2O2 exposure, PBE-AMF liberates the fluorescent and cytotoxic molecule amonafide (AMF), functioning as a theranostic agent. Our studies with PBE-AMF have demonstrated a positive correlation between intracellular H2O2 concentration and anticancer activity. The breast cancer cell line MDA-MB-231, characterized by high H2O2 content, showed the greatest susceptibility to this prodrug. Subsequently, we replaced the PBE structure with phenylboronic acid (PBA) to obtain the prodrug PBA-AMF, which exhibited enhanced stability, aqueous solubility, and tumor cell selectivity. This selectivity is attributed to its affinity for sialic acid, which is overexpressed on the surfaces of cancer cells. In vitro assays confirmed that PBA-AMF potently and selectively inhibited the proliferation of MDA-MB-231 cells, while sparing non-cancerous MCF-10A cells. Mechanistic investigations indicated that PBA-AMF impedes tumor proliferation by inhibiting DNA synthesis, reducing ATP levels, inducing apoptosis, and arresting the cell cycle. Our work broadens the range of small molecule H2O2-activated anticancer theranostic prodrugs, which are currently limited in number. We anticipate that the applications of PBA-AMF will extend to a wider spectrum of tumors and other diseases associated with increased H2O2 levels, thereby offering new horizons in cancer diagnostics and treatment.

Cdc42 Regulates the Expression of Cytoskeleton and Microtubule Network Proteins to Promote Invasion and Metastasis of Progeny Cells Derived from CoCl2-induced Polyploid Giant Cancer Cells.

Purpose: Our previous studies have shown that CoCl2 can induce the formation of polyploid giant cancer cells (PGCCs) and PGCCs could produce progeny cells via asymmetric division. In this study, the molecular mechanism by which PGCCs generate progeny cells with high invasion and migration abilities was explored. Methods: In this study, PGCCs induced by CoCl2 produced progeny cells via asymmetric division, which was observed dynamically using laser scanning confocal microscopy. Cell cycle in LoVo and Hct116 before and after CoCl2 treatment was analyzed by flow cytometry. Cell function experiments, co-immunoprecipitation, mass spectrometry analysis, ML141 treatment, western blotting, and siRNA transfection experiments were used to demonstrate that Cdc42/PAK1 was involved in the regulation of cytoskeleton expression. The proliferation, migration, and invasion abilities of PGCCs and progeny cells were compared in PGCCs and progeny cells with and without inhibiting the expression of Cdc42 and PAK1. Results: G2/M phase arrest appeared in CoCl2-treated LoVo and Hct116 cells. After CoCl2 treatment, an increased expression of Cdc42 and PAK1 led to a decrease in the expression of stathmin and an increase in the expression of phosphorylated stathmin, which is located in the nucleus of PGCCs and progeny cells. PTPN14 negatively regulates the expression of PAK1 and p38MAPK. Low levels of PTPN14 expression, a downstream regulatory protein of stathmin, endows progeny tumor cells generated by PGCCs with the ability to invade and metastasize. The expression of PKA1α, cathepsin B, and D increased in CoCl2-treated cells compared with that in the control cells, associated with the infiltration and migration of PGCCs with their progeny cells. Conclusion: CoCl2-induced overexpression of Cdc42 plays a critical role in increasing the infiltration and migration abilities of PGCCs and progeny cells by regulating cytoskeleton protein expression.

Correlation between inflammatory marker and lipid metabolism in patients with uterine leiomyomas.

Introduction: Obesity is a risk factor for the development of uterine leiomyoma (UL), and the inflammatory response plays a key role in the pathogenesis of UL. Our objective was to assess whether there was an independent relationship between inflammatory markers and triglycerides (TG) in patients with UL. Methods: 1,477 UL participants who were hospitalized at the Jining Medical University between January 2016 and December 2022 were included in this cross-sectional study. The independent and dependent variables measured at baseline were inflammatory markers and TG levels, respectively. The covariates were age, body mass index (BMI), UL and menstrual status. Based on the number of fibroids, the study population was divided into Single-group and Multiple-group. Results: Univariate and multiple regression analyses and stratified analyses revealed significant positive correlations between neutrophil-lymphocyte ratio and systemic immune inflammation index and TG, and significant negative correlations between monocyte-lymphocyte ratio and TG. Conclusion: The findings show a significant correlation between the inflammatory response and lipid metabolism levels in UL patients. This provides direction for further research into the pathophysiology of UL and also helps to formulate hypotheses for predictive models of UL.

Unique Probiotic Properties and Bioactive Metabolites of Saccharomyces boulardii.

Saccharomyces boulardii (S. boulardii) is a probiotic and is widely used to improve the nutritional and functional value of food. This study aimed to compare the probiotic properties of S. boulardii and Saccharomyces cerevisiae. A series of in vitro probiotic experiments was performed, including simulated gastrointestinal digestion, bile salt tolerance, hydrophobicity, self-aggregation, and antioxidant and antibacterial properties. Self-aggregation and hydrophobic properties of S. boulardii were relatively poor, but they showed high tolerance, antioxidant properties, and broad antibacterial properties. In addition, non-targeted metabolomics was used to comprehensively analyze the active metabolites of S. boulardii and the metabolic differences between S. boulardii and S. cerevisiae were compared. Saccharomyces boulardii produced many bioactive metabolites, which generally showed antioxidant, antibacterial, antitumor, anti-inflammatory, and other properties. In contrast to S. cerevisiae, S. boulardii produced phenyllactic acid and 2-hydroxyisocaproic acid. There were also significant differences in their metabolic pathways. These results may be of great significance in the medical and food industries and provide a basis for understanding the metabolism of S. boulardii. It also shows that metabolomics is an effective and novel method for screening microbial functional metabolites and identifying functional differences between similar microorganisms.

A nitric oxide and hydrogen sulfide dual-donating nanosystem for highly synergistic gas-radiotherapy against hepatocellular carcinoma.

A drug delivery system (DDS) based on gold-capped mesoporous silica nanoparticles (MSN) is fabricated for loading NOSH-aspirin, a nitric oxide (NO) and hydrogen sulfide (H2S) dual-donating cytotoxic molecule. The liver targeting and tumor microenvironment responsive properties of the nanosystem enable, for the first time, the concurrent delivery of NO and H2S from a DDS into hepatocellular carcinoma (HCC) cells. Combined gas-radiotherapy (GT-RT) from drug-loaded DDS (NOSH@MSN-Au-Gal) and X-ray irradiation shows highly synergistic anti-cancer activity against both normoxic and hypoxic HCC cells. Further studies revealed that the combined GT-RT not only retains the well-known anticancer mechanism of NO, H2S, and X-ray individually, but also alleviates HCC hypoxia via NO- and H2S- involved unique pathways. In mice, the GT-RT greatly slows the growth of both subcutaneous and orthotopic HCC tumors and shows high biocompatibility. The current work is expected to promote the clinical application of combined GT-RT as an effective cancer treatment.

Xenopus GLP-1-based glycopeptides as dual glucagon-like peptide 1 receptor/glucagon receptor agonists with improved in vivo stability for treating diabetes and obesity.

Peptide dual agonists toward both glucagon-like peptide 1 receptor (GLP-1R) and glucagon receptor (GCGR) are emerging as novel therapeutics for the treatment of type 2 diabetes mellitus (T2DM) patients with obesity. Our previous work identified a Xenopus GLP-1-based dual GLP-1R/GCGR agonist termed xGLP/GCG-13, which showed decent hypoglycemic and body weight lowering activity. However, the clinical utility of xGLP/GCG-13 is limited due to its short in vivo half-life. Inspired by the fact that O-GlcNAcylation of intracellular proteins leads to increased stability of secreted proteins, we rationally designed a panel of O-GlcNAcylated xGLP/GCG-13 analogs as potential long-acting GLP-1R/ GCGR dual agonists. One of the synthesized glycopeptides 1f was found to be equipotent to xGLP/GCG-13 in cell-based receptor activation assays. As expected, O-GlcNAcylation effectively improved the stability of xGLP/GCG-13 in vivo. Importantly, chronic administration of 1f potently induced body weight loss and hypoglycemic effects, improved glucose tolerance, and normalized lipid metabolism and adiposity in both db/db and diet induced obesity (DIO) mice models. These results supported the hypothesis that glycosylation is a useful strategy for improving the in vivo stability of GLP-1-based peptides and promoted the development of dual GLP-1R/GCGR agonists as antidiabetic/antiobesity drugs.

Duplication of SOX3 in an SRY-negative 46,XX male with prostatic utricle: case report and literature review.

BACKGROUND: 46,XX male disorders of sex development are rare. Approximately 80% of cases of testicular tissue differentiation may be due to translocation of SRY to the X chromosome or an autosome. SRY-negative 46,XX males show overexpression of pro-testis genes, such as SOX9 and SOX3, or failure of pro-ovarian genes, such as WNT4 and RSPO1, which induces testis differentiation, however, almost all testicles exhibit dysgenesis. Following inadequate exposure to androgens during the embryo stage, remnants of the Mullerian duct and incomplete closure of the urogenital sinus lead to enlargement of prostatic utricles. This condition is associated with proximal hypospadias and disorders of sex development. Many cases are asymptomatic, but show increased rates of postoperative complications and surgical failure. CASE PRESENTATION: A 5-year-old Chinese boy with scrotal hypospadias and bilateral cryptorchidism with prostatic utricles was presented. Gonadal histology showed ovo-testicular tissue on the right side and testicular tissue on the left side; all testicular tissue exhibited dysgenesis. Furthermore, chromosome karyotype analysis revealed 46,XX and, the presence of SRY was ruled out by polymerase chain reaction analysis. Whole-genome analysis showed the boy has a 1.4-Mb duplication in the Xq27.1q27.2 region (arr[hg19]Xq27.1q27.2:139585794-140996652) involving SOX3. No SOX3 duplication was observed in the parents, who had a normal phenotype. CONCLUSIONS: We report the first case of an SRY-negative 46 XX male with prostatic utricle caused by SOX3 duplication. SOX3 duplication may cause sex reversal, and all 46,XX SRY-negative males should be screened for SOX3 mutations. Gonadal biopsy is recommended to evaluate ovarian and testicular tissue development. Testicular dysgenesis and low exposure to male hormones during fetal development can lead to enlarged prostatic utricles. Thus endoscopic examination should be performed preoperatively to detect prostatic utricles in SRY-negative 46,XX males to determine the surgical plan and reduce postoperative complications.

N-acetyl-galactosamine modified metal-organic frameworks to inhibit the growth and pulmonary metastasis of liver cancer stem cells through targeted chemotherapy and starvation therapy.

Hepatocellular carcinoma (HCC) is a common high-mortality malignancy which still needs efficient treatments. HCC patients undergoing extrahepatic metastases are mostly with unsatisfactory prognosis. Therefore, specific attention has been paid to extrahepatic HCC metastasis. We integrated Sorafenib (Sor) and glucose oxidase (GOx) into a N-acetyl-galactosamine (GalNAc) modified zeolitic imidazolate framework (ZIF-8), designated as SG@GR-ZIF-8, for targeted bimodal therapies of chemotherapy and starvation therapy against HCC. The hepatic delivery of SG@GR-ZIF was mediated by the specific recognition of GalNAc residues with asialoglycoprotein (ASGPR) on the liver cell surface. Sor is a clinically approved anti-proliferation and anti-angiogenesis drug for advanced HCC treatment. GOx can efficiently induce cell death and disturb malignant progression by suppressing glucose supply of cancer cells, which is highly associated with metabolic rewiring in metastasis. The nano-formulation exhibit significant anti-metastatic HCC activity against C5WN1 cells, a liver cancer stem cell-like cell line with tumorigenicity and pulmonary metastasis activity. In a subcutaneous C5WN1-tumor carrying mouse model, SG@GR-ZIF exhibits potent synergistic anti-tumor activity with a tumor inhibition rate of 89% and a prolonged survival status. The growth and pulmonary metastasis of HCC in an orthotopic mouse model of HCC was remarkably suppressed in SG@GR-ZIF treated group. The therapeutic strategy targeting energy supply combined with first-line treatment holds great promise for the future treatment of metastatic HCC. STATEMENT OF SIGNIFICANCE: SG@GR-ZIF, a N-acetyl-galactosamine modified metal-organic framework carrying Sorafenib and glucose oxidase, was fabricated for treating metastatic hepatocellular carcinoma (HCC). Sorafenib is an anti-proliferation and anti-angiogenesis drug for advanced HCC treatment. Glucose oxidase blocks energy demand in HCC metastasis by depleting glucose. C5WN1 was used for therapeutic evaluations as a liver cancer stem cell-like cell line with tumorigenicity and pulmonary metastasis activity. In subcutaneous C5WN1-tumor bearing mice, SG@GR-ZIF exhibited a tumor inhibition rate of 89% and prolonged survival period. In orthotopic C5WN1-tumor carrying mice, the growth and pulmonary metastasis of hepatocarcinoma was remarkably suppressed by SG@GR-ZIF. Together, this study suggests the great potential of synergistic chemo/starvation therapy mediated by SG@GR-ZIF for treating metastatic HCC.

Bimodal Treatment of Hepatocellular Carcinoma by Targeted Minimally Interventional Photodynamic/Chemotherapy Using Glyco-Covalent-Organic Frameworks-Guided Porphyrin/Sorafenib.

Very limited treatment options are available to fight hepatocellular carcinoma (HCC), a serious global health concern with high morbidity and mortality. The integration of multiple therapies into one nanoplatform to exert synergistic therapeutic effects offers advantages over monotherapies. Here, we describe the construction of the nanoplatform Sor@GR-COF-366 for synergistic chemotherapy and photodynamic therapy (PDT) for HCC using a porphyrin-based covalent organic framework (COF-366) coated with N-acetyl-galactosamine (GalNAc) and rhodamine B (RhB), and loaded with the first-line agent, Sorafenib (Sor). The nanoplatform is targeted towards ASGPR-overexpressed HCC cells and liver tissues by GalNAc and observed by real-time imaging of RhB in vitro and in vivo. The nanoplatform Sor@GR-COF-366 exerts an enhanced synergistic tumor suppression effect in a subcutaneous HCC mouse model with a tumor inhibition rate (TGI) of 97% while significantly prolonging survival at very low toxicity. The potent synergistic therapeutic outcome is confirmed in an orthotopic mouse model of HCC with the TGI of 98% with a minimally invasive interventional PDT (IPDT). Sor@GR-COF-366 is a promising candidate to be combined with chemo-IPDT for the treatment of HCC. STATEMENT OF SIGNIFICANCE: This work describes the construction of covalent-organic frameworks (COFs) modified with glyco-moieties to serve as hepato-targeted multitherapy delivery systems. They combine minimally invasive interventional photodynamic therapy (IPDT) triggered synergism with chemotherapy treatment for hepatocellular carcinoma (HCC). With the aid of minimally invasive intervention, PDT can elicit potent anti-cancer activity for deep solid tumors. This platform shows strong therapeutic outcomes in both subcutaneous and orthotopic mouse models, which can significantly prolong survival. This work showed an effective combination of a biomedical nano-formulation with the clinical operational means in cancer treatment, which is greatly promising in clinical translation.

The role of cell division control protein 42 in tumor and non-tumor diseases: A systematic review.

Rho-GTPases control a variety of cellular functions mainly by regulating microtubule and actin dynamics, affecting the cytoskeleton, and are important regulators of the structural plasticity of dendrites and spines. Members of the Rho-GTPase family include Ras-related C3 botulinum toxin substrate 1 (Rac1), RhoA (Ras homologous), and cell division control protein 42 (Cdc42). Cdc42 is involved in the regulation of a variety of tumor and non-tumor diseases through a cascade of multiple signaling pathways. Active Cdc42 can regulate intercellular adhesion, cytoskeleton formation, and cell cycle, thus affecting cell proliferation, transformation, and dynamic balance as well as migration and invasion of tumor cells by regulating the expression of effector proteins. Here we discuss the role of Cdc42 in promoting metastasis, invasion, epithelial-mesenchymal transformation and angiogenesis in malignant tumors. The significant role of Cdc42 in non-tumor diseases is also discussed. Since Cdc42 plays a central role in the development of various diseases, small molecule inhibitors targeting Cdc42 have important clinical significance in the prevention and treatment of these diseases.

Arsenic Trioxide Promotes Tumor Progression by Inducing the Formation of PGCCs and Embryonic Hemoglobin in Colon Cancer Cells.

Arsenic trioxide (ATO) has been used to treat acute promyelocytic leukemia. However, it is not effective in treating solid tumors such as colorectal cancer. We have previously reported that polyploid giant cancer cells (PGCCs) exhibiting the characteristics of cancer stem cells can be generated by various inducers. In this study, ATO was used to induce the formation of PGCCs in LoVo and Hct116 colon cancer cell lines. The migration, invasion, and proliferation abilities of colon cancer cells with and without ATO treatment were assessed by wound-healing, transwell, and plate colony formation assays. The expression of epithelial to mesenchymal transition-related proteins and erythroid differentiation-related proteins in colon cancer cells was further evaluated by western blot and immunocytochemical assays. LoVo and Hct116 cells were transfected with a eukaryotic expression vector for green fluorescent protein (GFP), red fluorescent protein (RFP), H2B-GFP, and H2B-mCherry to study PGCCs formation via cell fusion. WB and ICC assays were performed to assess the expression of cell fusion-related proteins. MG132, small interfering RNA-glial cell missing 1 (GCM1), and chromatin immunoprecipitation-polymerase chain reaction assays were performed to study the role of GCM1/syncytin-1-mediated cell fusion. Clinically, the significance of cell fusion-related proteins and erythroid differentiation-related proteins expression in human colorectal cancer tissues was evaluated. Results of our study showed that ATO induced the formation of PGCCs, and the daughter cells derived from PGCCs gained a mesenchymal phenotype and exhibited strong migration, invasion, and proliferation abilities. PGCCs also produced embryonic hemoglobin-delta and -zeta with strong oxygen-binding ability and erythroid differentiation-related proteins after ATO treatment. In addition, cell fusion was observed during the formation of PGCCs, indicated by the presence of yellow fluorescence via the GCM1/syncytin-1 signaling pathway. Clinically, the expression of cell fusion-related and erythroid differentiation-related proteins gradually increased with the progression of human colorectal cancer tissues. In conclusion, ATO can promote tumor progression by inducing the formation of PGCCs via GCM1/syncytin-1-mediated cell fusion. PGCCs can produce daughter cells with high invasion and migration abilities and embryonic hemoglobin with strong oxygen binding ability, promoting survival of tumor cells in a hypoxic microenvironment.

High Migration and Invasion Ability of PGCCs and Their Daughter Cells Associated With the Nuclear Localization of S100A10 Modified by SUMOylation.

Our previous studies have confirmed that cobalt chloride (CoCl2) or chemoradiotherapy could induce the formation of polyploid tumor giant cells (PGCCs). Polyploid giant cancer cells are a special subpopulation of cancer cells that contribute to solid tumor heterogeneity. The size of PGCC was at least three times larger than regular diploid cancer cells. PGCCs have the properties of cancer stem cells (CSCs) and can express CSC markers CD44 and CD133. Daughter cells derived from PGCCs have strong proliferation, infiltration and migration abilities. However, the detailed molecular mechanism of daughter cells expressing mesenchymal phenotype and displaying strong abilities of proliferation and migration is unclear. As a plasminogen receptor, S100A10 which is closely associated with the invasion and metastasis of malignant tumors, was highly expressed in PGCCs with their daughter cells. In this study, CoCl2 was used to induce the formation of PGCCs in LoVo and HCT116 CRC cells. Cell functional experiments, co-immunoprecipitation, MG132 and ginkgolic acid treatment, western blot, and ChIP-Seq were used to identify the mechanism of S100A10 nuclear location. The proliferation and migration abilities of PGCCs and their daughter cells decreased significantly after S100A10 knockdown. In the control cells, S100A10 was mainly ubiquitinated, while in PGCCs and daughter cells, S100A10 was mainly SUMOylated, which was associated with S100A10 nuclear location. After SUMO1 was inhibited, the nuclear S100A10 in PGCCs and daughter cells decreased, and their proliferation and migration abilities significantly decreased. ChIP-Seq combined with real-time fluorescent quantitative PCR showed that S100A10 regulated the expression of neutrophil defensin 3 (DEFA3), receptor-type tyrosine-protein phosphatase N2 (PTPRN2), and rho guanine nucleotide exchange factor 18 (ARHGEF18), which were associated with actin dynamics and cytoskeleton remodeling. The expression of S100A10 in the nuclei and cytoplasm of rectal cancer after neoadjuvant chemoradiation (nCRT) and liver metastases increased compared with that in rectal cancer without nCRT. Taken together, the expression and nuclear localization of S100A10 modified by SUMOylation were associated with the high proliferation and migration of PGCCs and their daughter cells, and the differentiation, metastases, and relapse of CRCs by regulating the expression of ARHGEF18, PTPRN2, and DEFA3.

TPGS-Galactose-Modified Polydopamine Co-delivery Nanoparticles of Nitric Oxide Donor and Doxorubicin for Targeted Chemo-Photothermal Therapy against Drug-Resistant Hepatocellular Carcinoma.

The lack of cancer cell specificity and the occurrence of multidrug resistance (MDR) are two major obstacles in the treatment of hepatocellular carcinoma (HCC). To tackle these challenges, a novel nanoparticle (NP)-based drug delivery system (DDS) with a core/shell structure consisted of d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS)-galactose (Gal)/polydopamine (PDA) is fabricated. The NP is loaded with doxorubicin (DOX) and a nitric oxide (NO) donor N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine (BNN) sensitive to heat to afford NO-DOX@PDA-TPGS-Gal. The unique binding of Gal to asialoglycoprotein receptor (ASGPR) and the pH-sensitive degradation of NP ensure the targeted transportation of NP into liver cells and the release of DOX in HCC cells. The near-infrared (NIR) light further facilitates DOX release and initiates NO generation from BNN due to the photothermal property of PDA. In addition to the cytotoxicity contributed by DOX, NO, and heat, TPGS and NO act as MDR reversal agents to inhibit P-glycoprotein (P-gp)-related efflux of DOX by HepG2/ADR cells. The combined chemo-photothermal therapy (chemo-PTT) by NO-DOX@PDA-TPGS-Gal thus shows potent anti-cancer activity against drug-resistant HCC cells in vitro and in vivo and significantly prolongs the life span of drug-resistant tumor-bearing mice. The present work provides a useful strategy for highly targeted and MDR reversal treatment of HCC.

Nitric oxide-donating and reactive oxygen species-responsive prochelators based on 8-hydroxyquinoline as anticancer agents.

Metal ion chelators based on 8-hydroxyquinoline (8-HQ) have been widely explored for the treatment of many diseases. When aimed at being developed into potent anticancer agent, a largely unmet issue is how to avoid nonspecific chelation of metal ions by 8-HQ in normal cells or tissues. In the current work, a two-step strategy was employed to both enhance the anticancer activity of 8-HQ and improve its cancer cell specificity. Considering the well-known anticancer activity of nitric oxide (NO), NO donor furoxan was first connected to 8-HQ to construct HQ-NO conjugates. These conjugates were screened for their cytotoxicity, metal-binding ability, and NO-releasing efficiency. Selected conjugates were further modified with a ROS-responsive moiety to afford prochelators. Among all the target compounds, prodrug HQ-NO-11 was found to potently inhibit the proliferation of many cancer cells but not normal cells. The abilities of metal chelation and NO generation by HQ-NO-11 were confirmed by various methods and were demonstrated to be essential for the anticancer activity of HQ-NO-11. In vivo studies revealed that HQ-NO-11 inhibited the growth of SW1990 xenograft to a larger extent than 8-HQ. Our results showcase a general method for designing novel 8-HQ derivatives and shed light on obtaining more controllable metal chelators.

Glycoconjugates for glucose transporter-mediated cancer-specific targeting and treatment.

First observed in 1920s, the Warburg effects have inspired scientists to harness the unique glucose metabolism of cancer cells for targeted therapy for a century. Carbohydrate-drug conjugates are explicitly designed for selective uptake by cancer cells overexpressing glucose transporters. We summarize the progress in developing glycoconjugates for cancer-specific targeting and treatment over the past decade (2010-2020) and point to some future directions in this field.

Global profiling of N6 -methyladenosine methylation in maize callus induction.

Callus induction is a dedifferentiation process that accompanies a cell fate transition, and epigenetic regulation plays a crucial role in the process. N6 -methyladenosine (m6A) methylation is an important mechanism in post-transcriptional epigenetic regulation and functions in cell reprogramming. However, the function of m6A methylation during callus induction is still unknown. Here, we performed transcriptome-wide m6A-seq on immature maize embryos after culturing for 2, 4, or 8 days with or without the auxin analogue 2,4-D. A total of 26,794 unique m6A peaks were detected from 17,456 maize genes; and 2,338 specific, 2,4-D-induced m6A peaks (D-specific m6A) were detected only in embryos cultured with 2,4-D. Furthermore, a positive correlation between m6A methylation and mRNA abundance was discovered in the genes with D-specific m6A deposition, especially at the beginning of callus induction. Key genes involved in callus induction, i.e. BABY BOOM and LBD transcription factors, underwent m6A methylation, increasing their transcript levels, thus improving callus induction. These results revealed the importance of m6A methylation during the early stage of callus induction and provided new insights into the molecular mechanism of callus induction at an epitranscriptomic level.

Stapled and Xenopus Glucagon-Like Peptide 1 (GLP-1)-Based Dual GLP-1/Gastrin Receptor Agonists with Improved Metabolic Benefits in Rodent Models of Obesity and Diabetes.

Diabetes is characterized by pancreas dysfunction and is commonly associated with obesity. Hypoglycemic agents capable of improving ß-cell function and reducing body weight therefore are gaining increasing interest. Though glucagon-like peptide 1 receptor (GLP-1R)/cholecystokinin 2 receptor (CCK-2R) dual agonist ZP3022 potently increases ß-cell mass and improves glycemic control in diabetic db/db mice, the in vivo half-life (t1/2) is short, and its body weight reducing activity is limited. Here, we report the discovery of a series of novel GLP-1R/CCK-2R dual agonists. Starting from Xenopus GLP-1, dual cysteine mutation was conducted followed by covalent side chain stapling and albumin binder incorporation, resulting in a stabilized secondary structure, increased agonist potency, and improved stability. Further C-terminal conjugation of gastrin-6 generated GLP-1R/CCK-2R dual agonists, among which 6a and 6b showed higher stability and hypoglycemic activity than liraglutide and ZP3022. Desirably, 6a and 6b exhibited prominent metabolic benefits in diet-induced obesity mice without causing nausea responses and exerted considerable effects on ß-cell restoration in db/db mice. These preclinical studies suggest the potential role of GLP-1R/CCK-2R dual agonists as effective agents for treating diabetes and related metabolic disorders.

Rational design and biological evaluation of gemfibrozil modified Xenopus GLP-1 derivatives as long-acting hypoglycemic agents.

Novel methods for peptides structural modification and bioactivity optimization are highly needed in peptide-based drug discovery. Herein, we explored the use gemfibrozil (GFZ) as an albumin binder to enhance the stability and improve the bioactivity of peptides. Short-acting Xenopus glucagon-like peptide-1 (xGLP-1) analogues with anti-diabetic activity were selected as the starting point. Mono-GFZ conjugation, peptide sequence hybridization, and dimeric-GFZ derivatization were successively used to generate novel GFZ-xGLP-1 conjugates, biologically screened by various in vitro and in vivo models. Dimeric-GFZ modified conjugate 3b was finally identified as a promising anti-diabetic candidate with high albumin binding affinity, enhanced in vivo stability in SD rats, and long-acting hypoglycemic activity in db/db mice. Moreover, GFZ endowed 3b with strong lipid-regulating ability in DIO and db/db mice. In a twelve-week study, chronic administration of 3b in db/db mice resulted in sustained glycemic control, to a greater extent than liraglutide and semaglutide. In addition, 3b showed comparable therapeutic efficacies to liraglutide and semaglutide on HbA1c and pancreas islets protection. Our studies reveal 3b as a potential candidate for the treatment of metabolic diseases and indicate dimeric-GFZ modification as a novel method for peptide optimization.

Label-Free Comparative Proteomic Analysis Combined with Laser-Capture Microdissection Suggests Important Roles of Stress Responses in the Black Layer of Maize Kernels.

The black layer (BL) is traditionally used as an indicator for kernel harvesting in maize, as it turns visibly dark when the kernel reaches physiological maturity. However, the molecular roles of BL in kernel development have not been fully elucidated. In this work, microscopy images showed that BL began to appear at a growth stage earlier than 10 days after pollination (DAP), and its color gradually deepened to become dark as the development period progressed. Scanning electron microscopy observations revealed that BL is a tissue structure composed of several layers of cells that are gradually squeezed and compressed during kernel development. Laser-capture microdissection (LCM) was used to sample BL and its neighboring inner tissue, basal endosperm transfer layer (BETL), and outer tissue, inner epidermis (IEP), from 20 DAP of kernels. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling (MALDI-TOF MS profiling) detected 41, 104, and 120 proteins from LCM-sampled BL, BETL, and IEP, respectively. Gene ontology (GO) analysis indicated that the 41 BL proteins were primarily involved in the response to stress and stimuli. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis found that the BL proteins were enriched in several defense pathways, such as the ascorbate and aldarate metabolic pathways. Among the 41 BL proteins, six were BL-specific proteins that were only detected from BL. Annotations of five BL-specific proteins were related to stress responses. During kernel development, transcriptional expression of most BL proteins showed an increase, followed by a decrease, and reached a maximum zero to 20 DAP. These results suggest a role for BL in stress responses for protecting filial tissue against threats from maternal sides, which helps to elucidate the biological functions of BL.

Clinicopathological Significances of Cancer Stem Cell-Associated HHEX Expression in Breast Cancer.

Aberrant expression of the transcription factor hematopoietic ally expressed homeobox/proline-rich homeodomain (HHEX/PRH) is implicated in numerous cancers. However, the association of HHEX with breast cancer (BC) remains unclear. In this study, HHEX mRNA and protein expression were analyzed using the Oncomine, UALCAN, GEPIA, TCGAportal, and HPA databases. We evaluated the effect of HHEX on clinicopathological parameters using Kaplan-Meier plotter, OncoLnc, TCGAportal, PROGgeneV2, and BC-GenExMiner. Western blotting was performed to compare the level of HHEX in breast samples of Tientsin Albino 2 mice, human breast precancerous lesions, benign breast tumors, and BC. The correlation between HHEX and cancer stem cells was investigated using the GEO (GSE52327 and GSE94865) and GEPIA datasets. Networks between HHEX and survival-related gene marker sets and microRNAs were analyzed using GEPIA, StarBase, and Cytoscape. Results of this study showed that HHEX expression in BC was significantly lower than those in breast precancerous lesions and benign breast tumors at both mRNA and protein levels. BC patients with lower HHEX expression had significantly worse overall survival and disease-free survival. Moreover, HHEX significantly affected the clinicopathology of BC. Specifically, low HHEX expression was correlated with the following groups of patients: age ≤51 years, ER-negative or PR-negative patients, HER-2 positive, triple-negative breast cancer, and basal-like BC. Immunohistochemical analysis of the breast samples showed significant differences of HHEX staining index (P < 0.001) among the three groups. To further investigate the mechanism, we determined the intersection of differentially expressed genes related to BC stem cells and those genes after HHEX expression was altered. This led to the identification of four potentially regulated genes-CXL12, BLNK, PAG1, and LPXN. Using StarBase and km-plotter, the negative regulation of HHEX expression and survival trends, including miR-130b, miR-30e, and miR-301b were joined into miRNA-HHEX-mRNA potential regulatory network. The abilities of proliferation, migration and invasion increased in MDA-MB-231 and BT-549 breast cancer cell lines after HHEX down expression and decreased after HHEX overexpression compared them in the control cells. In conclusion, these data suggest that HHEX expression is downregulated in BC and HHEX may regulate the development of BC through the stem cell-related genes.