Metabolic reprogramming of terminally exhausted CD8+ T cells by IL-10 enhances anti-tumor immunity.

T cell exhaustion presents one of the major hurdles to cancer immunotherapy. Among exhausted CD8+ tumor-infiltrating lymphocytes, the terminally exhausted subset contributes directly to tumor cell killing owing to its cytotoxic effector function. However, this subset does not respond to immune checkpoint blockades and is difficult to be reinvigorated with restored proliferative capacity. Here, we show that a half-life-extended interleukin-10-Fc fusion protein directly and potently enhanced expansion and effector function of terminally exhausted CD8+ tumor-infiltrating lymphocytes by promoting oxidative phosphorylation, a process that was independent of the progenitor exhausted T cells. Interleukin-10-Fc was a safe and highly efficient metabolic intervention that synergized with adoptive T cell transfer immunotherapy, leading to eradication of established solid tumors and durable cures in the majority of treated mice. These findings show that metabolic reprogramming by upregulating mitochondrial pyruvate carrier-dependent oxidative phosphorylation can revitalize terminally exhausted T cells and enhance the response to cancer immunotherapy.

Structure of the platelet glycoprotein Ib receptor in complex with a novel antithrombotic agent.

Agkisacucetin, a snake C-type lectin-like protein isolated from the venom of Deinagkistrodon acutus (formerly Agkistrodon acutus), is a novel antithrombotic drug candidate in phase 2 clinical trials. Agkisacucetin specifically recognizes the platelet surface receptor glycoprotein Ib α chain (GPIbα) to block GPIb and von Willebrand factor (VWF). In this study, we solved the crystal structure of the GPIbα N-terminal domain (residues 1-305) in complex with agkisacucetin to understand their molecular recognition mechanism. The crystal structure showed that agkisacucetin primarily contacts GPIbα at the C-terminal part of the conserved leucine-rich repeat (LRR) domain (LRR-6 to LRR-8) and the previously described "ß-switch" region through the ß chain. In addition, we found that agkisacucetin α chain contacts part of the GPIbα C-terminal peptide after the LRR domain through complementary charge interactions. This C-terminal peptide plays a key role in GPIbα and thrombin recognition. Therefore, our structure revealed that agkisacucetin can sterically block the interaction between the GPIb receptor and VWF and thrombin proteins to inhibit platelet function. Our structural work provides key molecular insights into how an antithrombotic drug candidate recognizes the GPIb receptor to modulate platelet function to inhibit thrombosis.

Development of IL-15/IL-15Rα sushi domain-IgG4 Fc complexes in Pichia pastoris with potent activities and prolonged half-lives.

BACKGROUND: Interleukin-15 (IL-15) is a critical cytokine for the development, proliferation, and function of natural killer (NK) cells, NKT cells, and CD8+ memory T cells and has become one of the most promising protein molecules for the treatment of cancer and viral diseases. However, there are several limitations in applying IL-15 in therapy, such as its low yield in vitro, limited potency, and short half-life in vivo. To date, there are several recombinant IL-15 agonists based on configurational modifications that are being pursued in the treatment of cancer, such as ALT-803, which are mainly produced from mammalian cells. RESULTS: In this study, we designed two different forms of the IL-15 complex, which were formed by the noncovalent assembly of IL-15 with dimeric or monomeric sushi domain of IL-15 receptor α (SuIL-15Rα)-IgG4 Fc fusion protein and designated IL-15/SuIL-15Rα-dFc and IL-15/SuIL-15Rα-mFc, respectively. The two IL-15 complexes were expressed in Pichia pastoris (P. pastoris), and their activities and half-lives were evaluated and compared. Pharmacokinetic analysis showed that IL-15/SuIL-15Rα-dFc had a half-life of 14.26 h while IL-15/SuIL-15Rα-mFc had a half-life of 9.16 h in mice, which were much longer than the 0.7-h half-life of commercial recombinant human IL-15 (rhIL-15). Treatment of mice with intravenous injection of the two IL-15 complexes resulted in significant increases in NK cells, NKT cells, and memory CD8+ T cells, which were not observed after rhIL-15 treatment. Treatment of human peripheral blood mononuclear cells (PBMCs) from healthy donors with the two IL-15 complexes yielded enhanced NK and CD8+ T cell activation and proliferation, which was comparable to the effect of rhIL-15. CONCLUSIONS: These findings indicate that the IL-15/SuIL-15Rα-dFc and IL-15/SuIL-15Rα-mFc produced in P. pastoris exhibit potent activities and prolonged half-lives and may serve as superagonists for immunotherapy in further research and applications.

Improved the expression level of active transglutaminase by directional increasing copy of mtg gene in Pichia pastoris.

BACKGROUND: The microbial transglutaminase (MTG) is inactive when only the mature sequence is expressed in Pichia pastoris. Although co-expression of MTG and its N-terminal pro-peptide can obtain the active MTG, the enzyme activity was still low. One of the basic steps for strain improvement is to ensure a sufficient level of transcription of the heterologous gene, based on promoter strength and gene copy number. To date, high-copy-number recombinants of P. pastoris are achievable only by cloning of gene concatemers, so methods for rapid and reliable multicopy strains are therefore desirable. RESULTS: The coexpression strains harboring different copies mtg were obtained successfully by stepwise increasing Zeocin concentration based on the rDNA sequence of P. pastoris. The genome of coexpression strains with the highest enzyme activity was analyzed by real-time fluorescence quantitative PCR, and three copies of mtg gene (mtg-3c) was calculated according to the standard curve of gap and mtg genes (gap is regarded as the single-copy reference gene). The maximum enzyme activity of mtg-3c was up to 1.41 U/mL after being inducted for 72 h in 1 L flask under optimal culture conditions, and two protein bands were observed at the expected molecular weights (40 kDa and 5 kDa) by Western blot. Furthermore, among the strains detected, compared with mtg-2c, mtg-6c or mtg-8c, mtg-3c is the highest expression level and enzyme activity, implying that mtg-3c is the most suitable for co-expression pro-peptide and MTG. CONCLUSIONS: This study provides an effective strategy for improving the expression level of active MTG by directional increasing of mtg copies in P. pastoris.

PAX5 interacts with RIP2 to promote NF-κB activation and drug-resistance in B-lymphoproliferative disorders.

Paired box protein 5 (PAX5) plays a lineage determination role in B-cell development. However, high expression of PAX5 has been also found in various malignant diseases, including B-lymphoproliferative disorders (B-LPDs), but its functions and mechanisms in these diseases are still unclear. Here, we show that PAX5 induces drug resistance through association and activation of receptor-interacting serine/threonine-protein kinase 2 (RIP2; also known as RIPK2), and subsequent activation of NF-κB signaling and anti-apoptosis gene expression in B-lymphoproliferative cells. Furthermore, PAX5 is able to interact with RIP1 and RIP3, modulating both RIP1-mediated TNFR and RIP2-mediated NOD1 and NOD2 pathways. Our findings describe a new function of PAX5 in regulating RIP1 and RIP2 activation, which is at least involved in chemotherapeutic drug resistance in B-LPDs.

Biotransformation of menadione to its prenylated derivative MK-3 using recombinant Pichia pastoris.

Prenylated quinones, especially menaquinones, have significant physiological activities, but are arduous to synthesize efficiently. Due to the relaxed aromatic substrate specificity and prenylation regiospecificity at the ortho- site of the phenolic hydroxyl group, the aromatic prenyltransferase NovQ from Streptomyces may be useful in menaquinone synthesis from menadione. In this study, NovQ was overexpressed in Pichia pastoris. After fermentation optimization, NovQ production increased by 1617%. Then the different effects of metal ions, detergents and pH on the activity of purified NovQ were investigated to optimize the prenylation reaction. Finally, purified NovQ and cells containing NovQ were used for menadione prenylation in vitro and in vivo, respectively. Menaquinone-1 (MK-1) was detected as the only product in vitro with γ,γ-dimethylallyl pyrophosphate and menadione hydroquinol substrates. MK-3 at a concentration of 90.53 mg/L was detected as the major product of whole cell catalysis with 3-methyl-2-buten-1-ol and menadione hydroquinol substrates. This study realized whole cell catalysis converting menadione to menaquinones.

Elimination of N-glycosylation by site mutation further prolongs the half-life of IFN-α/Fc fusion proteins expressed in Pichia pastoris.

BACKGROUND: Interferon (IFN)-α has been commonly used as an antiviral drug worldwide; however, its short half-life in circulation due to its low molecular weight and sensitivity to proteases impacts its efficacy and patient compliance. RESULTS: In this study, we present an IgG1 Fc fusion strategy to improve the circulation half-life of IFN-α. Three different forms of IgG1 Fc fragments, including the wild type, aglycosylated homodimer and aglycosylated single chain, were each fused with IFN-α and designated as IFN-α/Fc-WT, IFN-α/Fc-MD, and IFN-α/Fc-SC, respectively. The recombinant proteins were expressed in Pichia pastoris and tested using antiviral and pharmacokinetic assays in comparison with the commercial pegylated-IFN-α (PEG-IFN-α). The in vitro study demonstrated that IFN-α/Fc-SC has the highest antiviral activity, while IFN-α/Fc-WT and IFN-α/Fc-MD exhibited antiviral activities comparable to that of PEG-IFN-α. The in vivo pharmacokinetic assay showed that both IFN-α/Fc-WT and IFN-α/Fc-MD have a longer half-life than PEG-IFN-α in SD rats, but IFN-α/Fc-SC has the shortest half-life among them. Importantly, the circulating half-life of 68.3 h for IFN-α/Fc-MD was significantly longer than those of 38.2 h for IFN-α/Fc-WT and 22.2 h for PEG-IFN-α. CONCLUSIONS: The results demonstrate that the elimination of N-glycosylation by mutation of putative N-glycosylation site further prolongs the half-life of the IFN-α/Fc fusion protein and could present an alternative strategy for extending the half-life of low-molecular-weight proteins expressed by P. pastoris for in vivo studies as well as for future clinical applications.

High-temperature cultivation of recombinant Pichia pastoris increases endoplasmic reticulum stress and decreases production of human interleukin-10.

BACKGROUND: The yeast Pichia pastoris (P. pastoris) has become a popular 'cell factory' for producing heterologous proteins, but production widely varies among proteins. Cultivation temperature is frequently reported to significantly affect protein production; however, the underlying mechanisms of this effect remain unclear. RESULTS: A P. pastoris strain expressing recombinant human interleukin-10 (rhIL-10) under the control of the AOX1 promoter was used as the model in this study. This system shows high-yield rhIL-10 production with prolonged methanol-induction times when cultured at 20°C but low-yield rhIL-10 production and higher cell death rates when cultured at 30°C. Further investigation showed that G3-pro-rhIL10, an immature form of rhIL-10 that contains the glycosylation-modified signal peptide, remained in the ER for a prolonged period at 30°C. The retention resulted in higher ER stress levels that were accompanied by increased ROS production, Ca(2+) leakage, ER-containing autophagosomes, shortened cortical ER length and compromised induction of the unfolded protein response (UPR). In contrast, G3-pro-rhIL10 was quickly processed and eliminated from the ER at 20°C, resulting in a lower level of ER stress and improved rhIL-10 production. CONCLUSIONS: High-temperature cultivation of an rhIL-10 expression strain leads to prolonged retention of immature G3-pro-rhIL10 in ER, causing higher ER stress levels and thus greater yeast cell death rates and lower production of rhIL-10.

IL-10-expressing CAR T cells resist dysfunction and mediate durable clearance of solid tumors and metastases.

The success of chimeric antigen receptor (CAR) T cell therapy in treating several hematopoietic malignancies has been difficult to replicate in solid tumors, in part because of T cell exhaustion and eventually dysfunction. To counter T cell dysfunction in the tumor microenvironment, we metabolically armored CAR T cells by engineering them to secrete interleukin-10 (IL-10). We show that IL-10 CAR T cells preserve intact mitochondrial structure and function in the tumor microenvironment and increase oxidative phosphorylation in a mitochondrial pyruvate carrier-dependent manner. IL-10 secretion promoted proliferation and effector function of CAR T cells, leading to complete regression of established solid tumors and metastatic cancers across several cancer types in syngeneic and xenograft mouse models, including colon cancer, breast cancer, melanoma and pancreatic cancer. IL-10 CAR T cells also induced stem cell-like memory responses in lymphoid organs that imparted durable protection against tumor rechallenge. Our results establish a generalizable approach to counter CAR T cell dysfunction through metabolic armoring, leading to solid tumor eradication and long-lasting immune protection.

Grooved Microneedle Patch Augments Adoptive T Cell Therapy Against Solid Tumors via Diverting Regulatory T Cells.

The efficacy of adoptive T cell therapy (ACT) for the treatment of solid tumors remains challenging. In addition to the poor infiltration of effector T (Teff) cells limited by the physical barrier surrounding the solid tumor, another major obstacle is the extensive infiltration of regulatory T (Treg) cells, a major immunosuppressive immune cell subset, in the tumor microenvironment. Here, this work develops a grooved microneedle patch for augmenting ACT, aiming to simultaneously overcome physical and immunosuppressive barriers. The microneedles are engineered through an ice-templated method to generate the grooved structure for sufficient T-cell loading. In addition, with the surface modification of chemokine CCL22, the MNs could not only directly deliver tumor-specific T cells into solid tumors through physical penetration, but also specifically divert Treg cells from the tumor microenvironment to the surface of the microneedles via a cytokine concentration gradient, leading to an increase in the ratio of Teff cells/Treg cells in a mouse melanoma model. Consequently, this local delivery strategy of both T cell receptor T cells and chimeric antigen receptor T cells via the CCL22-modified grooved microneedles as a local niche could significantly enhance the antitumor efficacy and reduce the on-target off-tumor toxicity of ACT.

Production and characterization of recombinant 9 and 15 kDa granulysin by fed-batch fermentation in Pichia pastoris.

Granulysin is a cytolytic, proinflammatory protein produced by human cytolytic T-lymphocytes and natural killer cells. Granulysin has two stable isoforms with molecular weight of 9 and 15 kDa; the 9-kDa form is a result of proteolytic maturation of the 15-kDa precursor. Recombinant 9-kDa granulysin exhibits cytolytic activity against a variety of microbes, such as bacteria, parasites, fungi, yeast and a variety of tumor cell lines. However, it is difficult to produce granulysin in large quantities by traditional methods. In this study, we developed a simple and robust fed-batch fermentation process for production and purification of recombinant 9- and 15-kDa granulysin using Pichia pastoris in a basal salt medium at high cell density. The granulysin yield reaches at least 100 mg/l in fermentation, and over 95 % purity was achieved with common His-select affinity and ion exchange chromatography. Functional analysis revealed that the yeast-expressed granulysin displayed dose-dependent target cytotoxicity. These results suggest that fermentation in P. pastoris provides a sound strategy for large-scale recombinant granulysin production that may be used in clinical applications and basic research.

Nicotine dependence affects cardiopulmonary endurance and physical activity in college students in Henan, China.

INTRODUCTION: This study aimed to investigate the impact of smoking on physical activity level, emotional status, and cardiopulmonary endurance in healthy young Chinese college students in order to develop future nicotine dependence management solutions. METHODS: This survey-based study was conducted in college students aged 19-26 years who were currently smoking. Cardio-respiratory endurance was assessed by estimating VO2max. Participants were given a questionnaire containing five factors from the Cigarette Dependence Scale-5 (CDS-5), also assessed were variables for physical activity level, using the Global Physical Activity Questionnaire (GPAQ), and emotional status. The sports training behavior was assessed using the Coaching Behavior Scale for Sport (CBS-S). RESULTS: A total of 400 participants were randomly selected and included in the study. All of them were current smokers. The highest percentage of participants had a score of 4 on the CDS-5 (n=93, 23.2%), scored 3-5 on each module of sports training, and experienced negative emotions, particularly depression (n=172; 43.0%) and anger (n=162; 40.5%). VO2max levels were significantly lower in participants with high nicotine dependence (CDS-5 score 4-5), and they correlated negatively with CDS-5 scores (r= -0.883, p<0.001). Nicotine dependence scores were negatively correlated with physical activity levels (r= -0.830, p<0.001), and high nicotine dependence scores were independently related to low physical activity (adjusted odds ratio, AOR=14.66; 95% CI: 4.98-43.19 , p <0.001). CONCLUSIONS: Tobacco smoking has a negative impact on emotional status. It also reduces cardiopulmonary endurance by reducing VO2max levels and negatively affects physical activity. Accordingly, it is critical to implement effective tobacco prevention programs for college students, such as a smoking warning system and physical exercise training, as well as to educate them on how to quit smoking.

Effect of exercise intervention on smoking cessation: a meta-analysis.

Background: Exercise has emerged as an effective approach to promote individual health and has shown potential in aiding smoking cessation. However, the specific benefits of exercise in smoking cessation remain unclear, and conflicting findings across studies may be attributed to variations in study populations and intervention characteristics. This study aims to conduct a meta-analysis to evaluate the impact of exercise interventions on tobacco dependence in smokers and assess the effectiveness of exercise in facilitating smoking cessation. Methods: A comprehensive search was performed in databases including PubMed, Web of Science, Embase, The Cochrane Library, and Scopus to identify relevant randomized controlled trials published before 30 October 2022. The Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines were followed during the review process. The quality of evidence (QoE) was assessed with GRADE (grading of recommendations, assessment, development and evaluations) methodology. Results: Acute exercise was found to significantly reduce smoking cravings [MD = -1.84, 95% CI (-2.92, -0.76), p < 0.001; SMD = -1.64, 95% CI (-2.22, -1.05), p < 0.001] and alleviate most withdrawal symptoms in smokers. However, there was no significant difference in the smoking cessation rate between the exercise group and the control group (p > 0.05). Exercise was associated with increased positive mood [SMD = 0.36, 95% CI (0.14, 0.58), p = 0.001] and reduced negative mood in smokers [SMD = -0.26, 95% CI (-0.39, -0.12), p < 0.001]. Conclusion: Acute exercise interventions effectively reduce cravings and withdrawal symptoms in smokers. However, long-term exercise interventions do not significantly improve the smoking cessation rate. Exercise can help reduce negative mood and enhance positive mood in smokers. Smokers with high levels of tobacco dependence may derive less benefit from exercise. Factors such as literature quality, exercise intervention characteristics, and exercise adherence may influence the effectiveness of interventions. Trial registration: This research protocol was registered in the International Prospective Register for Systematic Reviews (PROSPERO https://www.crd.york.ac.uk/PROSPERO/). Registration number: CRD42022326109.

Comprehensive Analysis of RNA-Binding Protein-Related lncRNA in Breast Invasive Carcinoma.

BACKGROUND: RNA-binding proteins (RBPs), which form complexes or single/multiple RNA-binding domains, have a functional role in regulating and determining the function or stability of the bound RNAs in various cancers, including breast invasive carcinoma (BRCA). However, the biological functions and clinical implications of RBP-related long noncoding RNAs (lncRNAs) in BRCA remain largely unknown. METHODS: Herein, we first identified and characterized RBP-related lncRNAs in BRCA. Then we built an RBP-related lncRNA signature (RBPLSig) and explored the clinical evaluation and prediction performance of the RBPLSig by bioinformatic analysis. In addition, to optimize treatment plans, prediction online tools was developed to predict the patient survival rate. Lastly, to verify the function of lncRNA WAC antisense RNA 1 (WAC-AS1), the experiments such as Quantitative real-time PCR (qRT-PCR), lncRNA knockdown, CCK-8, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining were performed. We also gained the potential mechanisms of the druggable compounds of the WAC-AS1 related RBP gene, putative NSUN6, using molecular docking. RESULTS: The results showed that RBPLSig, as an independent prognostic factor for BRCA patients, was involved in numerous malignancy-associated immunoregulatory pathways. We found different immune statuses and responses to immunotherapy, chemotherapy, and targeted therapy between the high- and low-risk groups stratified by RBPLSig. CONCLUSIONS: Our data broaden the comprehensive understanding of the biological functions of RBP-related lncRNAs, and demonstrate a novel and independent RBPLSig to assess prognosis and the immune microenvironment, thus helping to guide treatment decisions for BRCA.

The role of APOBEC3C in modulating the tumor microenvironment and stemness properties of glioma: evidence from pancancer analysis.

Background: It is now understood that APOBEC3 family proteins (A3s) are essential in tumor progression, yet their involvement in tumor immunity and stemness across diverse cancer types remains poorly understood. Methods: In the present study, comprehensive genome-wide statistical and bioinformatic analyses were conducted to elucidate A3 family expression patterns, establishing clinically relevant correlations with prognosis, the tumor microenvironment(TME), immune infiltration, checkpoint blockade, and stemness across cancers. Different experimental techniques were applied, including RT-qPCR, immunohistochemistry, sphere formation assays, Transwell migration assays, and wound-healing assays, to investigate the impact of A3C on low-grade glioma (LGG) and glioblastoma multiforme (GBM), as well as its function in glioma stem cells(GSCs). Results: Dysregulated expression of A3s was observed in various human cancer tissues. The prognostic value of A3 expression differed across cancer types, with a link to particularly unfavorable outcomes in gliomas. A3s are associated with the the TME and stemness in multiple cancers. Additionally, we developed an independent prognostic model based on A3s expression, which may be an independent prognostic factor for OS in patients with glioma. Subsequent validation underscored a strong association between elevated A3C expression and adverse prognostic outcomes, higher tumor grades, and unfavorable histology in glioma. A potential connection between A3C and glioma progression was established. Notably, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses implicated A3C in immune system-related diseases, with heightened A3C levels contributing to an immunosuppressive tumor microenvironment (TME) in glioma. Furthermore, in vitro experiments substantiated the role of A3C in sustaining and renewing glioma stem cells, as A3C deletion led to diminished proliferation, invasion, and migration of glioma cells. Conclusion: The A3 family exhibits heterogeneous expression across various cancer types, with its expression profile serving as a predictive marker for overall survival in glioma patients. A3C emerges as a regulator of glioma progression, exerting its influence through modulation of the tumor microenvironment and regulation of stemness.

Purification and characterization of human IL-10/Fc fusion protein expressed in Pichia pastoris.

Interleukin (IL)-10 is an anti-inflammatory cytokine that could be potentially applied for clinical therapy. However, its short circulating half-life in the serum limits its clinical applications. In this study, we designed a fusion protein containing human IL-10 and an IgG Fc fragment (hIL-10/Fc), and expressed it in Pichia pastoris. This hIL-10/Fc fusion protein was purified from the culture supernatant using MabSelect affinity chromatography and size-exclusion chromatography. The hIL-10/Fc yield was about 5mg/L in shake flasks, with purity exceeding 95%. In addition, the hIL-10/Fc fusion protein suppressed the phytohemagglutinin-induced IFN-γ production in human peripheral blood mononuclear cells. Pharmacokinetic study also revealed that hIL-10/Fc has a prolonged circulating half-life of about 30h in rats. More importantly, the hIL-10/Fc fusion protein displayed highly specific biological activity, which was slightly higher than that of the commercial recombinant human IL-10 (rhIL-10). Therefore, P. pastoris is useful in the large-scale production of hIL-10/Fc fusion protein for both research and therapeutic applications.

[Correlation of homocysteine in plasma with NOS and endogenous CO in the penile corpus cavernosum of type 2 diabetic rats].

OBJECTIVE: To study the correlation of homocysteine (Hcy) in plasma with nitric oxide synthetase (NOS) and endogenous carbon monoxide (CO) in the penile corpus cavernosum of type 2 diabetic rats. METHODS: This study included 40 male Wistar rats, 10 as controls (Group A) and the other 30 as diabetes mellitus (DM) models. Four weeks after the model establishment, the model rats were divided into a DM group (Group B, n = 10), an insulin treated group (Group C, n = 10), and a folic acid and vitamin B12 treated group (Group D, n = 10). All the rats were injected with apomorphine and observed for penile erection at 8 and 12 weeks, and the levels of total plasma Hcy (tHcy), NOS and CO in the penile corpus cavernosum were measured at 12 weeks. RESULTS: Compared with Group A, the level of tHcy was significantly increased, while NOS and CO activities in the penile cavernous tis-sue and erectile function remarkably decreased in Group B (P < 0.01). The incidence rate of high Hcy was 55% in the DM rats. In comparison, the level of tHcy was obviously decreased, and the NOS activity and erectile function markedly increased in Groups C and D (P < 0.01). The Hcy level showed a significant negative correlation with NOS activity (rA = -0.89, rB = -0.76, rc = -0.91, rD = -0.91) and CO content (TA = -0.82, r, = -0.77, rc = -0.93, rD = -0.81). CONCLUSION: High plasma Hcy can decrease NOS and CO activities in the penile corpus cavernosum, and consequently induce erectile dysfunction in DM rats, while insulin, folic acid and vitamin B12 can improve their penile erectile function by increasing NOS and CO activities.

Deoxyschizandrin Inhibits the Proliferation, Migration, and Invasion of Bladder Cancer Cells through ALOX5 Regulating PI3K-AKT Signaling Pathway.

Objective: Deoxyschizandrin has a significant inhibitory effect on a variety of tumor cells. However, the effect of Deoxyschizandrin on bladder cancer cells and its mechanism are still unclear. Methods: Bladder cancer cells were treated with different concentrations of Deoxyschizandrin for 24 h, 48 h, and 72 h. The inhibition rate of cell proliferation was detected by CCK-8 assay. The changes of cell migration and invasion were detected by wound healing and Transwell assay. Based on the structure of Deoxyschizandrin, the protein targets of Deoxyschizandrin were predicted by bioinformatics database and verified by RNA and protein. Then, the expressions of ALOX5 and PI3K-AKT signaling pathway proteins were detected by Western blot in bladder cancer cells treated with Deoxyschizandrin. Result: Deoxyschizandrin inhibited the proliferation, migration, and invasion of bladder cancer cells in a time- and concentration-dependent manner. Bioinformatics analysis showed that Deoxyschizandrin had 100 protein targets; among them, the score of ALOX5 was the highest, and the mRNA and protein levels of ALOX5 decreased after treatment with different concentrations of Deoxyschizandrin. Western blot results showed that compared with the control group, Deoxyschizandrin could significantly reduce the expression of p-PI3K and p-AKT, and overexpression of ALOX5 could significantly enhance the expression of p-PI3K and p-AKT. Compared with Deoxyschizandrin or overexpression of ALOX5, the expression of p-PI3K and p-AKT of Deoxyschizandrin combined with overexpression of ALOX5 recovered. Conclusion: Deoxyschizandrin inhibits the proliferation, migration, and invasion of bladder cancer cells through ALOX5 regulating PI3K-AKT signaling pathway.

A pan-cancer landscape of IGF2BPs and their association with prognosis, stemness and tumor immune microenvironment.

Background: The human insulin-like growth factor 2 mRNA binding proteins 1-3 (IGF2BP1-3, also called IMP1-3) play essential roles in mRNA regulation, including its splicing, translocation, stability, and translation. However, knowledge regarding the involvement of IGF2BPs in tumor immunity and stemness across cancer types is still lacking. Methods: In this study, we comprehensively analyzed pan-cancer multi-omic data to determine the correlation of IGF2BPs mRNA and protein expression with various cancer parameters such as mutation frequency, prognostic value, the tumor microenvironment (TME), checkpoint blockade, tumor immune infiltration, stemness and drug sensitivity. Validation of the expression of IGF2BPs in cancer samples and glioma cells were performed by quantitative real-time (qRT)-PCR, and immunofluorescence staining. Investigation of the functional role of IGF2BP3 in glioma stem cells(GSCs) were performed by sphere formation, cytotoxicity, transwell, and wound healing assays. Results: We found that IGF2BP1 and 3 are either absent or expressed at very low levels in most normal tissues. However, IGF2BP1-3 can be re-expressed in a broad range of cancer types and diverse cancer cell lines, where their expression often correlates with poor prognosis. Immunofluorescence staining and qRT-PCR analyses also showed that the expression of IGF2BP2 and IGF2BP3 were higher in cancer tissues than that in adjacent normal tissues. Moreover, IGF2BPs are associated with TME and stemness in human pan-cancer. Remarkably, IGF2BP3 participated in the maintenance and self-renewal of glioma stem cell (GSCs). Knockdown of IGF2BP3 attenuated GSC and glioma cell proliferation, invasion, and migration. Conclusions: Our systematic pan-cancer study confirmed the identification of IGF2BPs as therapeutic targets and highlighted the need to study their association with stemness, and the TME, which contribute to the cancer drug-discovery research. Especially, preliminary studies demonstrate the IGF2BP3 as a potential negative regulator of glioma tumorigenesis by modulating stemness.

lncRNA C2dat2 facilitates autophagy and apoptosis via the miR-30d-5p/DDIT4/mTOR axis in cerebral ischemia-reperfusion injury.

Cerebral ischemia-reperfusion injury (CIRI) is an important pathophysiological process of ischemic stroke associated with various physiological and pathological processes, including autophagy and apoptosis. In this study, we examined the role and mechanism of long noncoding RNA CAMK2D-associated transcript 2 (C2dat2) in regulating CIRI in vivo and in vitro. C2dat2 up-regulation facilitated neuronal autophagy and apoptosis induced by CIRI. Mechanistically, C2dat2 acts as a competing endogenous RNA (ceRNA) to negatively regulate miR-30d-5p expression. More specifically, miR-30d-5p targeted the 3'-untranslated region of DNA damage-inducible transcript 4 (DDIT4) and silenced its target mRNA DDIT4. Additionally, C2dat2 binding with heat shock cognate 70/heat shock protein 90 blocked RNA-induced silencing complex assembly to abolish the miR-30d-5p targeting of DDIT4 and inhibited miR-30d-5p to silence its target mRNA DDIT4. Further analysis showed that C2dat2 knockdown conspicuously inhibited the up-regulation of DDIT4 and Beclin-1 levels and LC3B II/I ratio and the down-regulation of P62 and phosphorylated mammalian target of rapamycin (mTOR)/mTOR and phosphorylated-P70S6K/P70S6K ratio in Neuro-2a cells after oxygen-glucose deprivation/reoxygenation. This study first revealed that C2dat2/miR-30d-5p/DDIT4/mTOR forms a novel signaling pathway to facilitate autophagy and apoptosis induced by CIRI, contributing to the better understanding of the mechanisms of CIRI and enriching the ceRNA hypothesis in CIRI.