Improvement in in?vitro fertilization outcome following in?vivo synchronization of oocyte maturation in mice

Synchronization of oocyte maturation in vitro has been shown to produce higher in vitro fertilization (IVF) rates than those observed in oocytes matured in vitro without synchronization. However, the increased IVF rates never exceeded those observed in oocytes matured in vivo without synchronization. This study was therefore designed to define the effect of in vivo synchronization of oocyte maturation on IVF rates. Mice were superovulated and orally treated with 7.5 mg cilostazol (CLZ), a phosphodiesterase 3A (PDE3A) inhibitor, to induce ovulation of immature oocytes at different stages depending on frequency and time of administration of CLZ. Mice treated with CLZ ovulated germinal vesicle (GV) or metaphase I (MI) oocytes that underwent maturation in vitro or in vivo (i.e. in the oviduct) followed by IVF. Superovulated control mice ovulated mature oocytes that underwent IVF directly upon collection. Ovulated MI oocytes matured in vitro or in vivo had similar maturation rates but significantly higher IVF rates, 2–4 cell embryos, than those observed in control oocytes. Ovulated GV oocytes matured in vitro showed similar maturation rates but significantly higher IVF rates than those observed in control oocytes. However, ovulated GV oocytes matured in vivo had significantly lower IVF rates than those noted in control oocytes. It is concluded that CLZ is able to synchronize oocyte maturation and improve IVF rates in superovulated mice. CLZ may be capable of showing similar effects in humans, especially since temporal arrest of human oocyte maturation with other PDE3A inhibitors in vitro was found to improve oocyte competence level. The capability of a clinically approved PDE3A inhibitor to improve oocyte fertilization rates in mice at doses extrapolated from human therapeutic doses suggests the potential scenario of the inclusion of CLZ in superovulation programs. This may improve IVF outcomes in infertile patients.     

Metformin inhibits MAPK signaling and rescues pancreatic aquaporin 7 expression to induce insulin secretion in type 2 diabetes mellitus

Metformin is the first-line antidiabetic agent for type 2 diabetes mellitus (T2DM) treatment. Although accumulated evidence has shed light on the consequences of metformin action, the precise mechanisms of its action, especially in the pancreas, are not fully understood. Aquaporin 7 (AQP7) acts as a critical regulator of intraislet glycerol content, which is necessary for insulin production and secretion. The aim of this study was to investigate the effects of different doses of metformin on AQP7 expression and explore the possible mechanism of its protective effects in the pancreatic islets. We used an in vivo model of high-fat diet in streptozocin-induced diabetic rats and an in vitro model of rat pancreatic β-cells (INS-1 cells) damaged by hyperglycemia and hyperlipidemia. Our data showed that AQP7 expression levels were decreased, whereas p38 and JNK mitogen-activated protein kinases (MAPKs) were activated in vivo and in vitro in response to hyperglycemia and hyperlipidemia. T2DM rats treated with metformin demonstrated a reduction in blood glucose levels and increased regeneration of pancreatic β-cells. In addition, metformin upregulated AQP7 expression as well as inhibited activation of p38 and JNK MAPKs both in vivo and in vitro. Overexpression of AQP7 increased glycerol influx into INS-1 cells, whereas inhibition of AQP7 reduced glycerol influx, thereby decreasing subsequent insulin secretion. Our findings demonstrate a new mechanism by which metformin suppresses the p38 and JNK pathways, thereby upregulating pancreatic AQP7 expression and promoting glycerol influx into pancreatic β-cells and subsequent insulin secretion in T2DM.     

A ratiometric dual luciferase reporter for quantitative monitoring of pre-mRNA splicing efficiency in vivo

Precursor messenger RNA (pre-mRNA) splicing is critical for cell growth and development, and errors in RNA splicing frequently cause cellular dysfunction, abnormal gene expression, and a variety of human diseases. However, there is currently a lack of reliable systems to noninvasively monitor the mRNA splicing efficiency in cells and animals. Here, we described the design of a genetically engineered ratiometric dual luciferase reporter to continuously quantify the changes in mRNA splice variants in vivo. This reporter system is encoded within a single polypeptide but on separate exons, thus generating two distinct luciferase signals derived from spliced and unspliced mRNAs. With this reporter, the two kinds of luciferase in the same individual can minimize the influence of indirect factors on splicing, and the ratio of these two luciferase intensities represents the dynamic splicing efficiency of pre-mRNA. Our study offers a convenient and robust tool for the screening and identification of small molecules or trans-acting factors that affect the efficiency of specific splicing reactions.     

Anti-breast cancer action of carbonic anhydrase IX inhibitor 4-[4-(4-Benzo[1,3]dioxol-5-ylmethyl-piperazin-1-yl)-benzylidene-hydrazinocarbonyl]-benzenesulfonamide (BSM-0004): in vitro and in vivo studies

Anti-breast cancer action of novel human carbonic anhydrase IX (hCA IX) inhibitor BSM-0004 has been investigated using in vitro and in vivo models of breast cancer. BSM-0004 was found to be a potent and selective hCA IX inhibitor with a Ki value of 96 nM. In vitro anticancer effect of BSM-0004 was analysed against MCF 7 and MDA-MA-231 cells, BSM-0004 exerted an effective cytotoxic effect under normoxic and hypoxic conditions, inducing apoptosis in MCF 7 cells. Additionally, this compound significantly regulates the expression of crucial biomarkers associated with apoptosis. The investigation was extended to confirm the efficacy of this hCA IX inhibitor against in vivo model of breast cancer. The results specified that the treatment of BSM-0004 displayed an effective in vivo anticancer effect, reducing tumour growth in a xenograft cancer model. Hence, our investigation delivers an effective anti-breast cancer agent that engenders the anticancer effect by inhibiting hCA IX.     

Single-Cell Quantitative Proteomic Analysis of Human Oocyte Maturation Revealed High Heterogeneity in In Vitro–Matured Oocytes

Oocyte maturation is pertinent to the success of in vitro maturation (IVM), which is used to overcome female infertility, and produced over 5000 live births worldwide. However, the quality of human IVM oocytes has not been investigated at single-cell proteome level. Here, we quantified 2094 proteins in human oocytes during in vitro and in vivo maturation (IVO) by single-cell proteomic analysis and identified 176 differential proteins between IVO and germinal vesicle oocytes and 45 between IVM and IVO oocytes including maternal effect proteins, with potential contribution to the clinically observed decreased fertilization, implantation, and birth rates using human IVM oocytes. IVM and IVO oocytes showed separate clusters in principal component analysis, with higher inter-cell variability among IVM oocytes, and have little correlation between mRNA and protein changes during maturation. The patients with the most aberrantly expressed proteins in IVM oocytes had the lowest level of estradiol per mature follicle on trigger day. Our data provide a rich resource to evaluate effect of IVM on oocyte quality and study mechanism of oocyte maturation.     

Engineering a genome‐reduced bacterium to eliminate Staphylococcus aureus biofilms in vivo

Bacteria present a promising delivery system for treating human diseases. Here, we engineered the genome‐reduced human lung pathogen Mycoplasma pneumoniae as a live biotherapeutic to treat biofilm‐associated bacterial infections. This strain has a unique genetic code, which hinders gene transfer to most other bacterial genera, and it lacks a cell wall, which allows it to express proteins that target peptidoglycans of pathogenic bacteria. We first determined that removal of the pathogenic factors fully attenuated the chassis strain in vivo. We then designed synthetic promoters and identified an endogenous peptide signal sequence that, when fused to heterologous proteins, promotes efficient secretion. Based on this, we equipped the chassis strain with a genetic platform designed to secrete antibiofilm and bactericidal enzymes, resulting in a strain capable of dissolving Staphylococcus aureus biofilms preformed on catheters in vitro, ex vivo, and in vivo. To our knowledge, this is the first engineered genome‐reduced bacterium that can fight against clinically relevant biofilm‐associated bacterial infections.     

FSH mediates estradiol synthesis in hypoxic granulosa cells by activating glycolytic metabolism through the HIF-1α–AMPK–GLUT1 signaling pathway

Owing to the avascular environment within ovarian follicles, granulosa cells (GCs) are believed to live in a hypoxic niche. Follicle-stimulating hormone (FSH)-mediated steroidogenesis is crucial for normal growth and maturation of ovarian follicles, but it remains unclear how FSH stimulates estradiol (E2) synthesis under hypoxic conditions. Here, we aimed to explore whether FSH affects the ATP production required for estrogen synthesis from the perspective of glucose metabolism. It was observed that the levels of both E2 and HIF-1α were markedly increased in a dose-dependent manner in mouse ovarian GCs after the injection of FSH in vivo, indicating that hypoxia/HIF-1α may be relevant to FSH-induced E2 synthesis. By treating hypoxic GCs with FSH in vitro, we further revealed that the activation of the AMP-activated protein kinase (AMPK)–GLUT1 pathway, which in turn stimulates ATP generation, may be essential for FSH-mediated E2 production during hypoxia. In contrast, inhibition of AMPK or GLUT1 with siRNAs/antagonist both repressed glycolysis, ATP production, and E2 synthesis despite FSH treatment. Moreover, blocking HIF-1α activity using siRNAs/PX-478 suppressed AMPK activation, GLUT1 expression, and E2 levels in FSH-treated GCs. Finally, the in vitro findings were verified in vivo, which showed markedly increased AMPK activity, GLUT1 expression, glycolytic flux, ATP levels, and E2 concentrations in ovarian GCs following FSH injection. Taken together, these findings uncovered a novel mechanism for FSH-regulating E2 synthesis in hypoxic GCs by activating glycolytic metabolism through the HIF-1α–AMPK–GLUT1 pathway.     

Lipid A-based affinity biosensor for screening anti-sepsis components from herbs

LPS (lipopolysaccharide), an outer membrane component of Gram-negative bacteria, plays an important role in the pathogenesis of sepsis and lipid A is known to be essential for its toxicity. Therefore it could be an effective measure to prevent sepsis by neutralizing or destroying LPS. Numerous studies have indicated that many traditional Chinese medicines are natural antagonists of LPS in vitro and in vivo. The goal of this study is to develop a rapid method to screen anti-sepsis components from Chinese herbs by use of a direct lipid A-based affinity biosensor technology based on a resonant mirror. The detergent OG (n-octyl β-D-glucopyranoside) was immobilized on a planar non-derivatized cuvette which provided an alternative surface to bind the terminal hydrophilic group of lipid A. A total of 78 herbs were screened based on the affinity biosensor with a target of lipid A. The aqueous extract of PSA (Paeonia suffruticosa Andr) was found to possess the highest capability of binding lipid A. Therefore an aqueous extraction from this plant was investigated further by our affinity biosensor, polyamide chromatography and IEC–HPLC. Finally, we obtained a component (PSA-I-3) from Paeonia suffruticosa Andr that was evaluated with the affinity biosensor. We also studied the biological activities of PSA-I-3 against sepsis in vitro and in vivo to further confirm the component we screened with the biosensor. In vitro, we found that PSA-I-3 could decrease TNFα (tumour necrosis factor α) release from RAW264.7 cells induced by LPS in a dose-dependent manner. In vivo, it increased remarkably the survival of KM (KunMing) mice by challenging both lethal-dose LPS and heat-killed Escherichia coli compared with control groups. Our results suggest that the constructed affinity biosensor can successfully screen the anti-sepsis component from Chinese herbs.     

Membrane-dependent relief of translation elongation arrest on pseudouridine- and N1-methyl-pseudouridine-modified mRNAs

Expression of therapeutically important proteins has benefited dramatically from the advent of chemically modified mRNAs that feature decreased lability and immunogenicity. This had a momentous effect on the rapid development of COVID-19 mRNA vaccines. Incorporation of the naturally occurring pseudouridine (Ψ) or N¹-methyl-pseudouridine (N1mΨ) into in vitro transcribed mRNAs prevents the activation of unwanted immune responses by blocking eIF2α phosphorylation, which inhibits translation. Here, we report that Ψs in luciferase (Luc) mRNA exacerbate translation pausing in nuclease-untreated rabbit reticulocyte lysate (uRRL) and promote the formation of high-order-ribosome structures. The major deceleration of elongation occurs at the Ψ-rich nucleotides 1294–1326 of Ψ-Luc mRNA and results in premature termination of translation. The impairment of translation is mainly due to the shortage of membranous components. Supplementing uRRL with canine microsomal membranes (CMMs) relaxes the impediments to ribosome movement, resolves collided ribosomes, and greatly enhances full-size luciferase production. CMMs also strongly stimulated an extremely inefficient translation of N1mΨ-Luc mRNA in uRRL. Evidence is presented that translational pausing can promote membrane recruitment of polysomes with nascent polypeptides that lack a signal sequence. Our results highlight an underappreciated role of membrane binding to polysomes in the prevention of ribosome collision and premature release of nascent polypeptides.     

miR-134-5p inhibits osteoclastogenesis through a novel miR-134-5p/Itgb1/MAPK pathway

Osteoporosis affects approximately 200 million people and severely affects quality of life, but the exact pathological mechanisms behind this disease remain unclear. Various miRNAs have been shown to play a predominant role in the regulation of osteoclast formation. In this study, we explored the role of miR-134-5p in osteoclastogenesis both in vivo and in vitro. We constructed an ovariectomized (OVX) mouse model and performed microarray analysis using bone tissue from OVX mice and their control counterparts. Quantitative RT-PCR data from bone tissue and bone marrow macrophages (BMMs) confirmed the decreased expression of miR-134-5p in OVX mice observed in microarray analysis. In addition, a decrease in miR-134-5p was also observed during induced osteoclastogenesis of BMMs collected from C57BL/6N mice. Through transfection with miR-134-5p agomirs and antagomirs, we found that miR-134-5p knockdown significantly accelerated osteoclast formation and cell proliferation and inhibited apoptosis. Furthermore, a luciferase reporter assay showed that miR-134-5p directly targets the integrin surface receptor gene Itgb1. Cotransfection with Itgb1 siRNA reversed the effect of the miR-134-5p antagomir in promoting osteoclastogenesis. Moreover, the abundance levels of MAPK pathway proteins phosphorylated-p38 (p-p38) and phosphorylated-ERK (p-ERK) were significantly increased after transfection with the miR-134-5p antagomir but decreased after transfection with the miR-134-5p agomir or Itgb1 siRNA, which indicated a potential relationship between the miR-134-5p/Itgb1 axis and the MAPK pathway. Collectively, these results revealed that miR-134-5p inhibits osteoclast differentiation of BMMs both in vivo and in vitro and that the miR-134-5p/Itgb1/MAPK pathway might be a potential target for osteoporosis therapy.     

Enzyme activity effects of N-terminal His-tag attached to catalytic sub-unit of phosphoinositide-3-kinase

NTT (N-terminal tags) on the catalytic (p110) sub-unit of PI 3-K (phosphoinositol 3-kinase) have previously been shown to increase cell signalling and oncogenic transformation. Here we test the impact of an NT (N-terminal) His-tag on in vitro lipid and protein kinase activity of all class-1 PI 3-K isoforms and two representative oncogenic mutant forms (E545K and H1047R), in order to elucidate the mechanisms behind this elevated signalling and transformation observed in vivo. Our results show that an NT His-tag has no impact on lipid kinase activity as measured by enzyme titration, kinetics and inhibitor susceptibility. Conversely, the NT His-tag did result in a differential effect on protein kinase activity, further potentiating the elevated protein kinase activity of both the helical domain and catalytic domain oncogenic mutants with relation to p110 phosphorylation. All other isoforms also showed elevated p110 phosphorylation (although not statistically significant). We conclude that the previously reported increase in cell signalling and oncogenic-like transformation in response to p110 NTT is not mediated via an increase in the lipid kinase activity of PI 3-K, but may be mediated by increased p110 autophosphorylation and/or other, as yet unidentified, intracellular protein/protein interactions. We further observe that tagged recombinant protein is suitable for use in in vitro lipid kinase screens to identify PI 3-K inhibitors; however, we recommend that in vivo (including intracellular) experiments and investigations into the protein kinase activity of PI 3-K should be conducted with untagged constructs.     

The conserved C2 phospholipid‐binding domain in Delta contributes to robust Notch signalling

Accurate Notch signalling is critical for development and homeostasis. Fine‐tuning of Notch–ligand interactions has substantial impact on signalling outputs. Recent structural studies have identified a conserved N‐terminal C2 domain in human Notch ligands which confers phospholipid binding in vitro. Here, we show that Drosophila ligands Delta and Serrate adopt the same C2 domain structure with analogous variations in the loop regions, including the so‐called β1‐2 loop that is involved in phospholipid binding. Mutations in the β1‐2 loop of the Delta C2 domain retain Notch binding but have impaired ability to interact with phospholipids in vitro. To investigate its role in vivo, we deleted five residues within the β1‐2 loop of endogenous Delta. Strikingly, this change compromises ligand function. The modified Delta enhances phenotypes produced by Delta loss‐of‐function alleles and suppresses that of Notch alleles. As the modified protein is present on the cell surface in normal amounts, these results argue that C2 domain phospholipid binding is necessary for robust signalling in vivo fine‐tuning the balance of trans and cis ligand–receptor interactions.     

Bone morphogenetic protein 6–mediated crosstalk between endothelial cells and hepatocytes recapitulates the iron-sensing pathway in vitro

Liver sinusoidal endothelial cell–derived bone morphogenetic protein 6 (BMP6) and the BMP6–small mothers against decapentaplegic homolog (SMAD) signaling pathway are essential for the expression of hepcidin, the secretion of which is considered the systemic master switch of iron homeostasis. However, there are continued controversies related to the strong and direct suppressive effect of iron on hepatocellular hepcidin in vitro in contrast to in vivo conditions. Here, we directly studied the crosstalk between endothelial cells (ECs) and hepatocytes using in vitro coculture models that mimic hepcidin signaling in vivo. Huh7 cells were directly cocultured with ECs, and EC conditioned media (CM) were also used to culture Huh7 cells and primary mouse hepatocytes. To explore the reactions of ECs to surrounding iron, they were grown in the presence of ferric ammonium citrate and heme, two iron-containing molecules. We found that both direct coculture with ECs and EC-CM significantly increased hepcidin expression in Huh7 cells. The upstream SMAD pathway, including phosphorylated SMAD1/5/8, SMAD1, and inhibitor of DNA binding 1, was induced by EC-CM, promoting hepcidin expression. Efficient blockage of this EC-mediated hepcidin upregulation by an inhibitor of the BMP6 receptor ALK receptor tyrosine kinase 2/3 or BMP6 siRNA identified BMP6 as a major hepcidin regulator in this coculture system, which highly fits the model of hepcidin regulation by iron in vivo. In addition, EC-derived BMP6 and hepcidin were highly sensitive to levels of not only ferric iron but also heme as low as 500 nM. We here establish a hepatocyte–endothelial coculture system to fully recapitulate iron regulation by hepcidin using EC-derived BMP6.     

In?vitro and in?vivo study on the effect of antifungal agents on hematopoietic cells in mice

Liposomal amphotericin B, voriconazole, and caspofungin are currently used for systemic and severe fungal infections. Patients with malignant diseases are treated with granulocyte-colony stimulating factor (G-CSF) for the recovery of granulocytes after chemotherapy or hematopoietic cell (HC) transplantation. Since they have a high incidence of fungal infections, they inevitably receive antifungal drugs for treatment and prophylaxis. Despite their proven less toxicity for various cell types comparatively with amphotericin B and the decrease in the number of leukocytes that has been reported as a possible complication in clinical studies, the effect of liposomal amphotericin B, voriconazole, and caspofungin on HCs has not been clarified. The present study aimed to examine the in vitro and in vivo effect of these three modern antifungals on HCs. Colony-forming unit (CFU) assays of murine bone marrow cells were performed in methylcellulose medium with or without cytokines and in the presence or absence of various concentrations of liposomal amphotericin B, voriconazole, and caspofungin. In the in vivo experiments, the absolute number of granulocytes was determined during leukocyte recovery in sublethally irradiated mice receiving each antifungal agent separately, with or without G-CSF. In vitro, all three antifungal drugs were nontoxic and, interestingly, they significantly increased the number of CFU-granulocyte-macrophage colonies in the presence of cytokines, at all concentrations tested. This was contrary to the concentration-dependent toxicity and the significant decrease caused by conventional amphotericin B. In vivo, the number of granulocytes was significantly higher with caspofungin plus G-CSF treatment, higher and to a lesser extent higher, but not statistically significantly, with voriconazole plus G-CSF and liposomal amphotericin B plus G-CSF treatments, respectively, as compared with G-CSF alone. These data indicate a potential synergistic effect of these antifungals with the cytokines, in vitro and in vivo, with subsequent positive effect on hematopoiesis.     

Ceramide levels in blood plasma correlate with major depressive disorder severity and its neutralization abrogates depressive behavior in mice

Major depressive disorder (MDD) is a severe disease of unknown pathogenesis that will affect ∼10% of people during their lifetime. Therapy for MDD requires prolonged treatment and often fails, predicating a need for novel treatment strategies. Here, we report increased ceramide levels in the blood plasma of MDD patients and in murine stress-induced models of MDD. These blood plasma ceramide levels correlated with the severity of MDD in human patients and were independent of age, sex, or body mass index. In addition, intravenous injection of anti-ceramide antibodies or neutral ceramidase rapidly abrogated stress-induced MDD, and intravenous injection of blood plasma from mice with MDD induced depression-like behavior in untreated mice, which was abrogated by ex vivo preincubation of the plasma with anti-ceramide antibodies or ceramidase. Mechanistically, we demonstrate that ceramide accumulated in endothelial cells of the hippocampus of stressed mice, evidenced by the quantitative measurement of ceramide in purified hippocampus endothelial cells. We found ceramide inhibited the activity of phospholipase D (PLD) in endothelial cells in vitro and in the hippocampus in vivo and thereby decreased phosphatidic acid in the hippocampus. Finally, we show intravenous injection of PLD or phosphatidic acid abrogated MDD, indicating the significance of this pathway in MDD pathogenesis. Our data indicate that ceramide controls PLD activity and phosphatidic acid formation in hippocampal endothelial cells and thereby mediates MDD. We propose that neutralization of plasma ceramide could represent a rapid-acting targeted treatment for MDD.