Iberiotoxin and clofilium regulate hyperactivation, acrosome reaction, and ion homeostasis synergistically during human sperm capacitation.

Potassium channels play essential roles in the regulation of male fertility. However, potassium channels mediating K+ currents in human sperm (IKSper ) remain controversial. Besides SLO3, the SLO1 potassium channel is a potential candidate for human sperm KSper. This study intends to elucidate the function of SLO1 potassium channel during human sperm capacitation. Human sperm were treated with iberiotoxin (IbTX, a SLO1 specific inhibitor) and clofilium (SLO3 inhibitor) separately or simultaneously during in vitro capacitation. A computer-assisted sperm analyzer was used to assess sperm motility. The sperm acrosome reaction (AR) was analyzed using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin staining. Sperm protein tyrosine phosphorylation was studied using western blotting. Intracellular Ca2+ , K+ , Cl- , and pH were analyzed using ion fluorescence probes. Independent inhibition with IbTX or clofilium decreased the sperm hyperactivation, AR, and protein tyrosine phosphorylation, and was accompanied by an increase in [K+ ]i , [Cl- ]i , and pHi , but a decrease in [Ca2+ ]i . Simultaneously inhibition with IbTX and clofilium lower sperm hyperactivation and AR more than independent inhibition. The increase in [K+ ]i , [Cl- ]i , and pHi , and the decrease in [Ca2+ ]i were more pronounced. This study suggested that the SLO1 potassium channel may have synergic roles with SLO3 during human sperm capacitation.

Circ_0058608 contributes to the progression and taxol resistance of non-small cell lung cancer by sponging miR-1299 to upregulate GBP1.

Circular RNAs (circRNAs) act as key regulators in human cancers and chemoresistance. Here, we aimed to explore the role and mechanism of circ_0058608 in nonsmall cell lung cancer (NSCLC) and taxol resistance. The expression of circ_0058608, microRNA-1299 (miR-1299) and guanylate binding protein 1 (GBP1) mRNA was determined by quantitative real-time PCR. In-vitro and in-vivo assays were conducted using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), colony formation, transwell assays, flow cytometry and animal xenograft experiments. The interaction between miR-1299 and circ_0058608 or GBP1 was confirmed by the dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Circ_0058608 was overexpressed in NSCLC tissues/cells and taxol-resistant NSCLC tissues/cells. Circ_0058608 knockdown inhibited NSCLC cell proliferation and metastasis and also suppressed tumor growth in vivo. Moreover, circ_0058608 knockdown increased taxol sensitivity by increasing taxol-induced apoptosis in taxol-resistant NSCLC cells. Moreover, circ_0058608 silencing enhanced taxol-induced tumor growth of NSCLC in vivo. MiR-1299 was a target of circ_0058608, and the effects of circ_0058608 knockdown on NSCLC cell progression and taxol resistance were reversed by miR-1299 inhibition. Additionally, miR-1299 could interact with GBP1, and miR-1299 suppressed NSCLC cell progression and taxol resistance by targeting GBP1. Furthermore, circ_0058608 could regulate GBP1 expression by sponging miR-1299. Circ_0058608 promoted the progression and taxol resistance of NSCLC by regulating the miR-1299/GBP1 axis.

Hsp90 modulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways.

BACKGROUND: Heat shock protein 90 (Hsp90) is a highly abundant eukaryotic molecular chaperone that plays important roles in client protein maturation, protein folding and degradation, and signal transduction. Previously, we found that both Hsp90 and its co-chaperone cell division cycle protein 37 (Cdc37) were expressed in human sperm. Hsp90 is known to be involved in human sperm capacitation via unknown underlying mechanism(s). As Cdc37 was a kinase-specific co-chaperone of Hsp90, Hsp90 may regulate human sperm capacitation via other kinases. It has been reported that two major mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (Erk1/2) and p38, are expressed in human sperm in the same locations as Hsp90 and Cdc37. Phosphorylated Erk1/2 has been shown to promote sperm hyperactivated motility and acrosome reaction, while phosphorylated p38 inhibits sperm motility. Therefore, in this study we explored whether Hsp90 modulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways. METHODS: Human sperm was treated with the Hsp90-specific inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) during capacitation. Computer-assisted sperm analyzer (CASA) was used to detect sperm motility and hyperactivation. The sperm acrosome reaction was analyzed by using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (PSA-FITC) staining. The interactions between Hsp90, Cdc37, Erk1/2 and p38 were assessed using co-immunoprecipitation (Co-IP) experiments. Western blotting analysis was used to evaluate the levels of protein expression and phosphorylation. RESULTS: Human sperm hyperactivation and acrosome reaction were inhibited by 17-AAG, suggesting that Hsp90 is involved in human sperm capacitation. In addition, Co-IP experiments revealed that 17-AAG reduced the interaction between Hsp90 and Cdc37, leading to the dissociation of Erk1/2 from the Hsp90-Cdc37 protein complex. Western blotting analysis revealed that levels of Erk1/2 and its phosphorylated form were subsequently decreased. Decreasing of Hsp90-Cdc37 complex also affected the interaction between Hsp90 and p38. Nevertheless, p38 dissociated from the Hsp90 protein complex and was activated by autophosphorylation. CONCLUSIONS: Taken together, our findings indicate that Hsp90 is involved in human sperm hyperactivation and acrosome reaction. In particular, Hsp90 and its co-chaperone Cdc37 form a protein complex with Erk1/2 and p38 to regulate their kinase activity. These results suggest that Hsp90 regulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways.

Reduced CENPU expression inhibits lung adenocarcinoma cell proliferation and migration through PI3K/AKT signaling.

CENPU (centromere protein U), a centromere component essential for mitosis, relates with some cancers progression. However, it is not well illustrated in lung adenocarcinoma (LAC). Here, we aimed to investigate the potential effect of CENPU on LAC progression and prognosis. In this experiment, expression level of CENPU and association between its expression and LAC patients' clinicopathological characteristics and prognosis were analyzed. The proliferation, migration and invasive abilities of LAC cells were determined by CCK-8, colony formation, transwell assays. Western blot was used to detect PI3K/AKT signaling key proteins. We found CENPU level was overexpressed in LAC tissues on comparing normal tissues. Moreover, CENPU overexpression correlated with clinicopathological variables and predicted an independent prognostic indicator in LAC patients. Functionally, CENPU downregulation significantly inhibited LAC cell proliferation, migration and invasion in, which was possibly mediated by PI3K/AKT pathway inactivation. Our findings insinuate targeting CENPU may be a potential therapeutic strategy for LAC.

[Effects of resveratrol on aging of mesenchymal stem cells and its mechanism].

OBJECTIVE: To investigate the effects of resveratrol (Res) on aging of marrow mesenchymal stem cells (MSCs), and to explore its mechanism. METHODS: MSCs were isolated from young SD rats and cultured in vitro. The optimal D-gal concentration for induction of MSCs senescence was determined. Then MSCs were randomly divided into four groups, namely the control group, 10µmol/L, 50µmol/L and 100µmol/L Res groups. After the cells were treated with different concentration of Res for 48 h, the senescence-associated changes were examined with senescence-associated-ß-galactosidase (SA-ß-gal) staining; the expression of p53, p16 and γ-H2AX was evaluated by Western blot. The total active oxygen species (ROS) level was determined by flow cytometry with DCFH-DA staining. In order to assess the effect of Res on the mitochondrial function, MitoSox Red staining was used to detect mitochondrial ROS levels in each group, mitochondrial membrane potential was detected by JC-1 assay, mPTP method was used to detect mitochondrial membrane channel opening level, and Western blot was used to detect the expression level of cytoplasmic cytochrome C (Cyt-C). RESULTS: D-gal 10 and 50 g/L significantly increased the number of SA-ß-gal positive cells and the level of mitochondrial ROS (all P<0.01). Therefore, 10 g/L D-gal was used to induce the senescence of MSCs in subsequent experiment. Compared with the control group, the number of SA-ß-gal positive cells in Res groups significantly decreased (all P<0.01), the expression of p53, p16 and γ-H2AX decreased, and the total and mitochondrial ROS level also decreased (all P<0.01). Moreover, mitochondrial membrane potential, open level of mitochondrial membrane channels and the levels of cytoplasm Cyt-C in the Res treatment groups decreased compared with the control group (P<0.05 or P<0.01). CONCLUSIONS: Resveratrol can protect the mitochondrial function of MSCs, and effectively delay the MSC senescence.

LncRNAs regulate the cyclic growth and development of hair follicles in Dorper sheep.

Introduction: Hair follicles in Dorper sheep are characterized by seasonal cyclic growth and development, consequently resulting in hair shedding during spring. The cyclic growth and development of hair follicles are regulated by several influencing factors such as photoperiods, hormones, age of the animal, genes, long non-coding RNAs (lncRNAs), and signaling pathways. Methods: In the present study, skin samples of five shedding sheep (S), used as experimental animals, and three non-shedding sheep (N), used as controls, were collected at three time points (September 27, 2019; January 3, 2020; and March 17, 2020) for RNA sequencing (RNA-seq) technology. Nine different groups (S1-vs-S2, S1-vs-S3, S2-vs-S3, N1- vs-N2, N1-vs-N3, N2-vs-N3, S1-vs-N1, S2-vs-N2, and S3-vs-N3) were compared using FDR < 0.05 and log 21 FC >as thresholds to assess the differences in the expression of lncRNAs. Results and discussion: In total, 395 differentially expressed (DE) lncRNAs were screened. Cluster heatmap analysis identified two types of expression patterns, namely, high expression during the anagen phase (A pattern) and high expression during the telogen phase (T pattern). Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the target genes were largely enriched in the Estrogen signaling pathway, PI3K-Akt signaling pathway, Fc gamma R-mediated phagocytosis, and cell adhesion molecules (CAMs), which are associated with hair follicle cyclic growth and development-related pathways. In addition, 17 pairs of lncRNAs-target genes related to hair follicle cyclic growth and development were screened, and a regulatory network was constructed. Altogether, candidate lncRNAs and their regulated target genes were screened that contributed to sheep hair follicle cyclic growth and development. We believe these findings will provide useful insights into the underlying regulatory mechanisms.

Characterization of human anti-EpCAM antibodies for developing an antibody-drug conjugate.

We previously generated fully human antibody-producing TC-mAb mice for obtaining potential therapeutic monoclonal antibodies (mAbs). In this study, we investigated 377 clones of fully human mAbs against a tumor antigen, epithelial cell adhesion molecule (EpCAM), to determine their antigen binding properties. We revealed that a wide variety of mAbs against EpCAM can be obtained from TC-mAb mice by the combination of epitope mapping analysis of mAbs to EpCAM and native conformational recognition analysis. Analysis of 72 mAbs reacting with the native form of EpCAM indicated that the EpCL region (amino acids 24-80) is more antigenic than the EpRE region (81-265), consistent with numerous previous studies. To evaluate the potential of mAbs against antibody-drug conjugates, mAbs were directly labeled with DM1, a maytansine derivative, using an affinity peptide-based chemical conjugation (CCAP) method. The cytotoxicity of the conjugates against a human colon cancer cell line could be clearly detected with high-affinity as well as low-affinity mAbs by the CCAP method, suggesting the advantage of this method. Thus, this study demonstrated that TC-mAb mice can provide a wide variety of antibodies and revealed an effective way of identifying candidates for fully human ADC therapeutics.

Hsp90 interacts with Cdc37, is phosphorylated by PKA/PKC, and regulates Src phosphorylation in human sperm capacitation.

BACKGROUND: Heat shock protein 90 (Hsp90) signaling pathways participate in protein phosphorylation during sperm capacitation. However, the underlying mechanism is largely unknown. OBJECTIVE: The aim of this study was to explore the interaction between Hsp90 and its co-chaperone protein, cell division cycle protein Cdc37 (Cdc37), in human spermatozoa. MATERIALS AND METHODS: We examined the effects of H-89 (a protein kinase A [PKA] inhibitor) and Go6983 (a protein kinase C [PKC] inhibitor) on the phosphorylation of serine, threonine, and tyrosine residues in Hsp90; the effect of 17-allylamino-17-demethoxygeldanamycin (17-AAG, Hsp90 inhibitor) on Y416-Src phosphorylation; and the effects of 17-AAG and geldanamycin on threonine phosphorylation during human sperm capacitation. RESULTS: Hsp90 co-localized and interacted with Cdc37. During human sperm capacitation, Hsp90 phosphorylation at serine, threonine, and tyrosine residues was inhibited by H-89 and Go6983. In addition, phosphorylation of residue Y416 in the tyrosine kinase Src (its active site) was inhibited by 17-AAG, and the threonine phosphorylation levels of some proteins were decreased by 17-AAG and geldanamycin. DISCUSSION AND CONCLUSION: Taken together, our data showed that the interaction of Hsp90 with Cdc37 regulates total protein threonine phosphorylation and Src phosphorylation via its serine, threonine, and tyrosine phosphorylation, which are controlled by PKA and PKC during human sperm capacitation. The results of this study help understand the mechanism underlying Hsp90 regulation of sperm function.

CPEB3-mediated MTDH mRNA translational suppression restrains hepatocellular carcinoma progression.

Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is a sequence-specific RNA-binding protein. We had reported that CPEB3 is involved in hepatocellular carcinoma (HCC) progression. However, the underlying mechanisms of CPEB3 in HCC remain unclear. In this study, we firstly performed RNA immunoprecipitation to uncover the transcriptome-wide CPEB3-bound mRNAs (CPEB3 binder) in HCC. Bioinformatic analysis indicates that CPEB3 binders are closely related to cancer progression, especially HCC metastasis. Further studies confirmed that metadherin (MTDH) is a direct target of CPEB3. CPEB3 can suppress the translation of MTDH mRNA in vivo and in vitro. Besides, luciferase assay demonstrated that CPEB3 interacted with 3'-untranslated region of MTDH mRNA and inhibited its translation. Subsequently, CPEB3 inhibited the epithelial-mesenchymal transition and metastasis of HCC cells through post-transcriptional regulation of MTDH. In addition, cpeb3 knockout mice are more susceptible to carcinogen-induced hepatocarcinogenesis and subsequent lung metastasis. Our results also indicated that CPEB3 was a good prognosis marker, which is downregulated in HCC tissue. In conclusion, our results demonstrated that CPEB3 played an important role in HCC progression and targeting CPEB3-mediated mRNA translation might be a favorable therapeutic approach.

Performance evaluation of activated carbon with different pore sizes and functional groups for VOC adsorption by molecular simulation.

Volatile organic compounds (VOCs) are growing pollutants now that cause air pollution and threaten human health. In this paper, the Grand Canonical Monte Carlo was used to simulate the adsorption performance of activated carbon on VOCs (benzene, toluene, acetone and methanol). After simulating different pore sizes (0.902 nm,1.997 nm,3 nm and 4 nm) adsorption performances of activated carbon, activated carbon with a pore size of 1.997 nm was selected to further study the influence of functional groups (carboxyl, amino, hydroxyl and hydrogen), and the capillary condensation was explained by the Kelvin equation. Furthermore, effects of functional groups under saturated vapor pressure (P0) of VOCs that range from 0 to 0.1 P0 were explained by the accessible volume and intermolecular interaction potential, respectively. Under pressure range of 0-0.1 P0, at the beginning of adsorption of acetone and methanol, carboxyl and amino groups could reduce the threshold pressure while hydroxyl and hydrogen have the opposite effect. For benzene and toluene, all functional groups have little effect on the threshold pressure, and they reduce the adsorption capacity instead. It could be concluded that the activated carbon could achieve the best adsorption effect on acetone and methanol, on the contrary, the addition of functional groups on benzene and toluene will weaken their adsorption performance.

Telmisartan inhibits NSCLC A549 cell proliferation and migration by regulating the PI3K/AKT signaling pathway.

Expression of angiotensin II (Ang II), a key biological peptide in the renin-angiotensin system, is closely associated with the occurrence and development of cancer. Ang II binds two receptor subtypes, the Ang II type 1 receptor (AT1R) and the AT2R, to mediate a series of biological effects. Telmisartan, a specific AT1R blocker, has been reported to have potential as an anticancer drug for treating renal cancer. In the present study, whether telmisartan had an effect on non-small cell lung cancer (NSCLC) cell proliferation and migration was investigated. The Cell Counting kit-8 assay revealed that telmisartan significantly inhibited the growth of the NSCLC A549 cell line in a time- and dose-dependent manner. In a transwell assay, telmisartan significantly inhibited cellular invasion and migration. Furthermore, it was determined that the expression of the anti-apoptotic protein B-cell lymphoma was decreased, and that of the pro-apoptotic proteins caspase-3 and Bcl-associated X increased in the A549 cells treated with telmisartan. Additionally, levels of phosphorylated RAC serine/threonine-protein kinase (p-AKT), p-mechanistic target of rapamycin, p70-S6 kinase and cyclin D1 was decreased in the telmisartan-treated group. Therefore, the current study reveals that telmisartan-induced NSCLC apoptosis may be regulated via the phosphoinositide 3-kinase/AKT signaling pathway, which indicates that it may be a potential novel drug for clinical NSCLC treatment.

In-situ gelation of sodium alginate supported on melamine sponge for efficient removal of copper ions.

Sodium alginate-melamine sponge composites were fabricated by in-situ gelation of sodium alginate supported on the commercial melamine sponge (MS). MS serves as the three-dimensional skeleton for alginate coating and can effectively avoid the shrink of alginate, and thus makes the alginate-MS composites user-friendly and facile to recover. In the adsorption test, the adsorption process is pseudo-second order and matches the Langmuir model with the maximum adsorption capacity of 90.1mg/g for copper ions. The alginate-MS is recyclable and presents enhanced mechanical properties compared with those of pristine MS. All these properties make such alginate-MS a promising candidate as an adsorbent for heavy metal removal.

Simple fabrication of easy handling millimeter-sized porous attapulgite/polymer beads for heavy metal removal.

A simple method was offered to synthesize porous attapulgite (ATP)/polymer beads. The polymeric network acts as both holder to support ATP inside the inner pore structure and as the obstacle to avoid the agglomeration of ATP during the adsorption process. Moreover, such beads are millimeter-size and can float on water surface, making such beads easy-to-handle and facile-to-recover without weight loss. The maximum adsorption capacity of such beads for Cu(II) and Cd(II) were 25.3 and 32.7mg/g, respectively, and pH value plays a key role in determining the adsorption capacities. The ATP/polymer beads also show good recycling ability and chemical stability. All these properties make such beads promising candidates as adsorbents for heavy metal removal.

High glucose induces the aging of mesenchymal stem cells via Akt/mTOR signaling.

It has previously been demonstrated that glucose is important in the process of stem cell aging. However, the mechanisms of cell senescence induced by high glucose (HG) remain to be elucidated. The preliminary study indicated that D­galactose induced mesenchymal stem cell (MSCs) aging. The present study demonstrated, following treatment with 11.0 or 22.0 mM HG for 14 days, that HG significantly promoted MSCs aging and the expression levels of phosphorylated (p-)phosphatidylinositol 3-kinase/protein kinase B (Akt) and p­mammalian target of rapamycin signaling (mTOR) in the HG groups were increased compared with the control group. However, following Akt inhibition with 1.0 or 10.0 nM MK­2206, which is an Akt­specific small molecule inhibitor, the senescence­cell value in the HG group was significantly decreased compared with the control group. These results indicated that HG induced MSCs senescence and this effect was primarily mediated via the Akt/mTOR signaling pathway.

Coenzyme Q10 inhibits the aging of mesenchymal stem cells induced by D-galactose through Akt/mTOR signaling.

Increasing evidences indicate that reactive oxygen species are the main factor promoting stem cell aging. Recent studies have demonstrated that coenzyme Q10 (CoQ10) plays a positive role in organ and cellular aging. However, the potential for CoQ10 to protect stem cell aging has not been fully evaluated, and the mechanisms of cell senescence inhibited by CoQ10 are still poorly understood. Our previous study had indicated that D-galactose (D-gal) can remarkably induce mesenchymal stem cell (MSC) aging through promoting intracellular ROS generation. In this study, we showed that CoQ10 could significantly inhibit MSC aging induced by D-gal. Moreover, in the CoQ10 group, the expression of p-Akt and p-mTOR was clearly reduced compared with that in the D-gal group. However, after Akt activating by CA-Akt plasmid, the senescence-cell number in the CoQ10 group was significantly higher than that in the control group. These results indicated that CoQ10 could inhibit D-gal-induced MSC aging through the Akt/mTOR signaling.