Pharmacokinetics and Safety of Estradiol Valerate Tablet and Its Generic: A Phase 1 Bioequivalence Study in Healthy Chinese Postmenopausal Female Subjects.

Purpose: Estradiol valerate (Progynova®) is used as hormone therapy to supplement estrogen deficiency. This study aimed to assess the bioequivalence of an estradiol valerate tablet and its generic form, under fasting and fed conditions. Methods: A randomized, open-label, single-dose, 2-period crossover study was conducted on healthy postmenopausal Chinese female volunteers under fasting and fed conditions. For each period, the subjects received either a 1 mg tablet of estradiol valerate or its generic. Blood samples were collected before dosing and up to 72 hours after administration. Plasma levels of total estrone, estradiol, and unconjugated estrone were quantified using a validated liquid chromatography-tandem mass spectrometry method. Results: A total of 54 volunteers were enrolled in this study. The primary pharmacokinetic parameters, including Cmax, AUC0-t, and AUC0-∞, were similar for the two drugs under both fasting and fed conditions, with 90% confidence intervals for the geometric mean ratios of these parameters, all meeting the bioequivalence criterion of 80-125%. A total of 48 adverse events (AEs) were reported in the fed study compared with 24 AEs in the fasting study. Conclusion: Estradiol valerate and its generic form were bioequivalent and well tolerated under both fasting and fed conditions.

Screening and construction of nanobodies against human CD93 using phage libraries and study of their antiangiogenic effects.

Background: Cluster of Differentiation 93 (CD93) plays an important role in angiogenesis and is considered an important target for inhibiting tumor angiogenesis, but there are currently no therapeutic antibodies against CD93 in the clinic. Thus, we describe the screening of novel nanobodies (Nbs) targeting human CD93 from a phage library of shark-derived Nbs. Methods: Screening and enrichment of phage libraries by enzyme-linked immunosorbent assay (ELISA). Anti-CD93 Nbs were purified by expression in E. coli. The binding affinity of anti-CD93 Nbs NC81/NC89 for CD93 was examined by flow cytometry (FC) and ELISA. The thermal stability of NC81/NC89 was examined by ELISA and CD spectroscopy. Afterward, the anti-angiogenic ability of NC81/NC89 was examined by MTT, wound healing assay, and tube formation assay. The expression level of VE-cadherin (VE-Ca) and CD93 was detected by Western Blot (WB). The binding sites and binding forms of NC81/NC89 to CD93 were analyzed by molecular docking. Results: The anti-CD93 Nbs were screened in a phage library, expressed in E. coli, and purified to >95% purity. The results of FC and ELISA showed that NC81/NC89 have binding ability to human umbilical vein endothelial cells (HUVECs). The results of ELISA and CD spectroscopy showed that NC81/NC89 retained the ability to bind CD93 at 80°C and that the secondary structure remained stable. In vitro, the results showed that NC81 and NC89 significantly inhibited the proliferation and migration of human umbilical vein endothelial cells (HUVECs) as well as tube formation on Matrigel. Western Blot showed that NC81 and NC89 also inhibited the expression of VE-Ca thereby increasing vascular permeability. It was found during molecular docking that the CDR regions of NC81 and NC89 could be attached to CD93 by strong hydrogen bonds and salt bridges, and the binding sites were different. Conclusion: We have successfully isolated NC81 and NC89, which bind CD93, and both Nbs significantly inhibit angiogenesis and increase vascular permeability. These results suggest that NC81 and NC89 have potential clinical applications in angiogenesis-related therapies.

Gadd45g insufficiency drives the pathogenesis of myeloproliferative neoplasms.

Despite the identification of driver mutations leading to the initiation of myeloproliferative neoplasms (MPNs), the molecular pathogenesis of MPNs remains incompletely understood. Here, we demonstrate that growth arrest and DNA damage inducible gamma (GADD45g) is expressed at significantly lower levels in patients with MPNs, and JAK2V617F mutation and histone deacetylation contribute to its reduced expression. Downregulation of GADD45g plays a tumor-promoting role in human MPN cells. Gadd45g insufficiency in the murine hematopoietic system alone leads to significantly enhanced growth and self-renewal capacity of myeloid-biased hematopoietic stem cells, and the development of phenotypes resembling MPNs. Mechanistically, the pathogenic role of GADD45g insufficiency is mediated through a cascade of activations of RAC2, PAK1 and PI3K-AKT signaling pathways. These data characterize GADD45g deficiency as a novel pathogenic factor in MPNs.

Effect of Brucea javanica Oil on the Toxicity of ß-Cypermethrin Emulsifiable Concentrate Formulation.

Enhancing the performance of traditional pesticide formulations by improving their leaf surface wetting capabilities is a crucial approach for maximizing the pesticide efficiency. This study develops an emulsifiable concentrate (EC) of 4.5% ß-cypermethrin containing Brucea javanica oil (BJO). The incorporation of BJO aims to improve the leaf-wetting properties of the EC formulation and enhance its insecticidal effectiveness. The droplet size and emulsion characteristics of ß-CYP EC emulsion with varying concentrations of the emulsifier were evaluated, and changes after incorporating BJO were assessed to develop the optimal formulation. A comprehensive comparison was conducted among commercial 4.5% ß-cypermethrin EC (ß-CYP EC-1), 4.5% ß-cypermethrin EC with BJO (ß-CYP EC-2), and 4.5% ß-cypermethrin EC without BJO (ß-CYP EC-3). This comparison encompassed various factors including storage stability, insecticidal activity, cytotoxicity, and wetting performance on cabbage leaves. The results indicated that the ideal emulsifier concentration was 15% emulsifier 0201B. ß-CYP EC-2 demonstrated superior wetting properties on cabbage leaves (the wetting performance of ß-CYP EC-2 emulsion on cabbage leaves is 2.60 times that of the ß-CYP EC-1 emulsion), heightened insecticidal activity against the third larvae of Plutella xylostella [diamondback moth (DBM)] [the insecticidal activity of the ß-CYP EC-2 emulsion against the third larvae of DBM is 1.93 times that of the ß-CYP EC-1 emulsion (12 h)], and more obvious inhibitory effects on the proliferation of DBM embryo cells than the other tested formulations. These findings have significant implications for advancing pest control strategies and promoting sustainable and effective agricultural practices.

Twist family BHLH transcription factor 1 is required for the maintenance of leukemia stem cell in MLL-AF9+ acute myeloid leukemia.

Leukemia stem cells (LSC) are a rare population capable of limitless self-renewal and are responsible for the initiation, maintenance, and relapse of leukemia. Elucidation of the mechanisms underlying the regulation of LSC function could provide novel treatment strategies. Here, we show that TWIST1 is extremely highly expressed in the LSC of MLL-AF9+ acute myeloid leukemia (AML), and its upregulation is positively regulated by KDM4C in a H3K9me3 demethylation-dependent manner. We further demonstrate that TWIST1 is essential for the viability, dormancy, and self-renewal capacities of LSC, and that it promotes the initiation and maintenance of MLL-AF9-mediated AML. In addition, TWIST1 directly interacts and collaborates with HOXA9 in inducing AML in mice. Mechanistically, TWIST1 exerts its oncogenic function by activating the WNT5a/RAC1 axis. Collectively, our study uncovers a critical role of TWIST1 in LSC function and provides new mechanistic insights into the pathogenesis of MLL-AF9+ AML.

n-3 PUFA Promotes Ferroptosis in PCOS GCs by Inhibiting YAP1 through Activation of the Hippo Pathway.

Polycystic ovary syndrome (PCOS) is an endocrine disorder characterized by hyperandrogenemia with multiple suspended sinus follicles, thickened cortical tissue, and excessive proliferation of ovarian granulosa cells that severely affects the fertility and quality of life of women. The addition of n-3 PUFA to the diet may slightly reduce body weight and greatly alleviate disturbed blood hormone levels in PCOS mice. We treated KGN as a cell model for n-3 PUFA addition and showed that n-3 PUFA inhibited the proliferation of GCs and promoted ferroptosis in ovarian granulosa cells. We used CCK-8, fluorescence quantitative transmission electron microscopy experiments and ferroptosis marker gene detection and other methods. Furthermore, n-3 PUFA was found to promote YAP1 exocytosis by activating Hippo and weakening the cross-talk between YAP1 and Nrf2 by activating the Hippo signaling pathway. In this study, we found that n-3 PUFA inhibited the over proliferation of granulosa cells in ovarian follicles by activating Hippo, promoting YAP1 exocytosis, weakening the cross-talk between YAP1 and Nrf2, and ultimately activating the ferroptosis sensitivity of ovarian granulosa cells. We demonstrate that n-3 PUFA can alleviate the hormonal and estrous cycle disorder with PCOS by inhibiting the YAP1-Nrf2 crosstalk that suppresses over proliferating ovarian granulosa cells and promotes iron death in GCs. These findings reveal the molecular mechanisms by which n-3 PUFA attenuates PCOS and identify YAP1-Nrf2 as a potential therapeutic target for regulation granulosa cells in PCOS.

Protein-Targeted Glycan Editing on Living Cells Disrupts KRAS Signaling.

The frequent mutation of KRAS oncogene in some of the most lethal human cancers has spurred incredible efforts to develop KRAS inhibitors, yet only one covalent inhibitor for the KRASG12C mutant has been approved to date. New venues to interfere with KRAS signaling are desperately needed. Here, we report a "localized oxidation-coupling" strategy to achieve protein-specific glycan editing on living cells for disrupting KRAS signaling. This glycan remodeling method exhibits excellent protein and sugar specificity and is applicable to different donor sugars and cell types. Attachment of mannotriose to the terminal galactose/N-acetyl-D-galactosamine epitopes of integrin αv ß3 , a membrane receptor upstream of KRAS, blocks its binding to galectin-3, suppresses the activation of KRAS and downstream effectors, and mitigates KRAS-driven malignant phenotypes. Our work represents the first successful attempt to interfere with KRAS activity by manipulating membrane receptor glycosylation.

Regulation of SIRT1 in Ovarian Function: PCOS Treatment.

The sirtuin family, a group of NAD+-dependent class 3 histone deacetylases (HDACs), was extensively studied initially as a group of longevity genes that are activated in caloric restriction and act in concert with nicotinamide adenine dinucleotides to extend the lifespan. Subsequent studies have found that sirtuins are involved in various physiological processes, including cell proliferation, apoptosis, cell cycle progression, and insulin signaling, and they have been extensively studied as cancer genes. In recent years, it has been found that caloric restriction increases ovarian reserves, suggesting that sirtuins may play a regulatory role in reproductive capacity, and interest in the sirtuin family has continued to increase. The purpose of this paper is to summarize the existing studies and analyze the role and mechanism of SIRT1, a member of the sirtuin family, in regulating ovarian function. Research and review on the positive regulation of SIRT1 in ovarian function and its therapeutic effect on PCOS syndrome.

Multistage targeting and dual inhibiting strategies based on bioengineered tumor matrix microenvironment-mediated protein nanocages for enhancing cancer biotherapy.

Regulation of the apoptotic pathway plays a critical role in inducing tumor cell death and circumventing drug resistance. Survivin protein is the strongest inhibitor of apoptosis found so far. It is highly expressed in several cancers and is a promising target for cancer therapy. However, clinical applications are limited by incomplete inhibition of survivin expression. Here, we present a novel strategy that extended the release of YM155 (an effective survivin inhibitor that works by inhibiting the activity of survivin promoter) and TATm-survivin (T34A) (TmSm) protein (survivin protein mutant with penetrating peptide, a potential anticancer protein therapeutic) via tumor matrix microenvironment-mediated ferritin heavy chain nanocages (FTH1 NCs), enabling significant inhibition of survivin activity at both transcript and protein levels. FTS (FTH1-matrix metalloproteinase-2-TmSm)/YM155 NC synthesis was easily scaled up, and these NCs could sequentially release TmSm protein through matrix metalloproteinase-2 and promote YM155 to enter the nucleus via transferrin receptor 1 (TfR1) binding, which increased the cytotoxicity and apoptosis of Capan-2 and A549 cells compared to that with individual drugs. Moreover, FTS/YM155 NCs enhanced drug accumulation at tumor sites and had a higher tumor inhibition rate (88.86%) than the compounds alone in A549 tumor-bearing mice. In addition, FTS/YM155 NCs exerted significant survivin downregulation (4.43-fold) and caspase-3 upregulation (4.31-fold) and showed better therapeutic outcomes without inducing organ injury, which highlights their promising future clinical application in precision therapy. This tumor microenvironment-responsive platform could be harnessed to develop an effective therapy via multilevel inhibition of cancer targets.

Upregulated NLRP3 inflammasome activation is attenuated by anthocyanins in patients with nonalcoholic fatty liver disease: A case-control and an intervention study.

OBJECTIVES: Despite the recent attention focused on the roles of the NLRP3 inflammasome in the pathogenesis of metabolic and inflammatory diseases, little is known about the activation status of NLRP3 inflammasome in patients with nonalcoholic fatty liver disease (NAFLD). The present study aimed to investigate whether inflammasomes activation is upregulated in patients with NAFLD and the upregulation can be attenuated by anthocyanins, which are polyphenols with known anti-inflammatory activities. METHODS: This study included a case-control study and a randomized controlled intervention trial. In the first part, NAFLD patients and healthy controls were recruited from a cohort of railroad workers. In the second part, NAFLD patients were randomly assigned to receive either capsules of anthocyanins (320 mg daily) or placebo for 12 weeks. A series of genes and factors associated with activation of NLRP3 inflammasome in subjects' plasma and peripheral blood mononuclear cells (PBMCs) were analyzed. RESULTS: Compared with healthy controls, the mRNA levels of NLRP3 inflammasome components (NLRP3, caspase-1, interleukin (IL)-1ß, and IL-18) were significantly upregulated in the PBMCs of NAFLD patients. Consistently, plasma levels of mature IL-1ß and IL-18 in NAFLD patients were significantly higher than in controls. After anthocyanin administration, both mRNA expression of NLRP3 inflammasome components (caspase-1, IL-1ß, and IL-18) in PBMCs and plasma levels of IL-1ß and IL-18 decreased dramatically in NAFLD patients compared with controls. CONCLUSIONS: This study has demonstrated that the activation of NLRP3 inflammasome is highly increased in NAFLD patients, but it can be markedly suppressed by anthocyanins, which provides a rationale for the development of anti-inflammatory therapies in NAFLD.

A nanobody-based molecular toolkit for ubiquitin-proteasome system explores the main role of survivin subcellular localization.

Targeted protein degradation is a powerful tool for determining the function of specific proteins nowadays. Survivin is the smallest member of the inhibitor of the apoptosis protein (IAP) family. It exists in the cytoplasm and nucleus of cells, but the exact function of survivin in different subcellular locations retained unclear updates due to the lack of effective and simple technical means. In this study, we created a novel nanoantibody-based molecular toolkit, namely, the ubiquitin-proteasome system (Nb4A-Fc-T2A-TRIM21), that can target to degrade survivin localized in cytoplasmic and cell nuclear by ubiquitinating, and by which to verify the potential roles of survivin subcellular localization. Also, the results showed that the cytoplasmic survivin mainly plays an anti-apoptotic function by directly or indirectly inhibiting the caspase pathway, and the nuclear survivin mainly promotes cell proliferation and participates in the regulation of the cell cycle. In addition, the Nb4A-Fc-T2A-TRIM21 system can degrade the endogenous survivin protein in a large amount by the ubiquitin-proteasome pathway, and the system can provide theoretical support for ubiquitination degradation targeting other endogenous proteins.

Mitochondrial Iron Metabolism: The Crucial Actors in Diseases.

Iron is a trace element necessary for cell growth, development, and cellular homeostasis, but insufficient or excessive level of iron is toxic. Intracellularly, sufficient amounts of iron are required for mitochondria (the center of iron utilization) to maintain their normal physiologic function. Iron deficiency impairs mitochondrial metabolism and respiratory activity, while mitochondrial iron overload promotes ROS production during mitochondrial electron transport, thus promoting potential disease development. This review provides an overview of iron homeostasis, mitochondrial iron metabolism, and how mitochondrial iron imbalances-induced mitochondrial dysfunction contribute to diseases.

Factors associated with red blood cell transfusion among hospitalized patients: a cross-sectional study.

Red blood cell (RBC) transfusion is a clinically effective therapy in anemia, for example in patients with malignancies (Shander et al., 2020), bleeding (Odutayo et al., 2017), and preoperative anemia (Padmanabhan et al., 2019). The past few decades have witnessed a shortage of blood for transfusion due to limited health insurance coverage for blood use and the rapid expansion of hospitals (Chen et al., 2011; Shi et al., 2014). Blood donation levels may easily be affected by general changes in the environment, policy, major events such as disasters, and public sentiment (Hu et al., 2019). Meanwhile, the transfusion of allogeneic RBC is a double-edged sword, increasing the possibility of infectious and immunological complications, and also leading to higher morbidity and mortality after transfusion (Frank et al., 2012). Considering that the continual shortfall has been increasingly prominent, identifying the factors associated with RBC transfusion could help blood transfusion departments to improve their supply of blood products as well as their inventory management (O'Donnell et al., 2018).

Reprogramming of human peripheral blood mononuclear cells from a patient suffering from recurrent hydatidiform mole to an iPSC line FAHZUi001-A carrying a homozygous p.Gln421Ter mutation in NLRP7 gene.

Recurrent hydatidiform mole (RHM) is characterized by the occurrence of at least twice hydatidiform mole. Unlike sporadic complete hydatidiform moles (CHMs), which are androgenetic with 2 paternal chromosomes, CHMs associated with familial recurrence are genetically biparental with a maternal and a paternal chromosome. NLRP7 mutations have been reported in 55% of RHM cases. Here, we generated induced pluripotent stem cells (iPSCs) from a patent with NLRP7 gene mutation c.1261C > T by reprogramming peripheral blood mononuclear cells by non-integrated method. The resulting iPSCs carrying NLRP7 mutation, had normal karyotype, expressed pluripotency markers, and could differentiate into three germ layersin vivo.

GADD45g acts as a novel tumor suppressor, and its activation suggests new combination regimens for the treatment of AML.

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy for which there is an unmet need for novel treatment strategies. Here, we characterize the growth arrest and DNA damage-inducible gene gamma (GADD45g) as a novel tumor suppressor in AML. We show that GADD45g is preferentially silenced in AML, especially in AML with FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations and mixed-lineage leukemia (MLL)-rearrangements, and reduced expression of GADD45g is correlated with poor prognosis in patients with AML. Upregulation of GADD45g impairs homologous recombination DNA repair, leading to DNA damage accumulation, and dramatically induces apoptosis, differentiation, and growth arrest and increases sensitivity of AML cells to chemotherapeutic drugs, without affecting normal cells. In addition, GADD45g is epigenetically silenced by histone deacetylation in AML, and its expression is further downregulated by oncogenes FLT3-ITD and MLL-AF9 in patients carrying these genetic abnormalities. Combination of the histone deacetylase 1/2 inhibitor romidepsin with the FLT3 tyrosine kinase inhibitor AC220 or the bromodomain inhibitor JQ1 exerts synergistic antileukemic effects on FLT3-ITD+ and MLL-AF9+ AML, respectively, by dually activating GADD45g. These findings uncover hitherto unreported evidence for the selective antileukemic role of GADD45g and provide novel strategies for the treatment of FLT3-ITD+ and MLL-AF9+ AML.

Designing and constructing a phage display synthesized single domain antibodies library based on camel VHHs frame for screening and identifying humanized TNF-α-specific nanobody.

Tumor necrosis factor (TNF-α) is an important clinically tested cytokine that could induce autoimmune diseases and inflammation. Therefore, the anti-TNF-α therapy strategy was developed and used therapeutically in various diseases, especially in the cytokine storm associated chimeric antigen receptor (CAR) T-cell therapy and antiviral therapy. Compare with other anti-TNF-α inhibitors, anti-TNF-α Nb (nanobody) has many unique advantages. Herein, we reported a novel humanized scaffold for library construction, which could be soluble and expressed in Escherichia coli (E.coli), and the efficiency capacity could reach as high as 2.01 × 109. Meanwhile, an anti-TNF-α Nb was selected for further study after 4 rounds of screening, NT-3, as the optimal Nb could effectively inhibit TNF-mediated cytotoxicity. The IC50 of NT-3 was determined as 0.804 µM, and its apoptosis inhibition rate was 62.47 % in L929 cells. Furthermore, the molecular docking results showed that complementarity-determining regions (CDRs) of NT-3 could connect to TNF for blocking function through strong hydrogen bonds and salt bridges. In general, our study not only provided a good Nb screening platform in vitro without animal immunization, but also generated a series of novel humanized anti-TNF-α Nb candidates with potential applications.

Dietary betaine prevents obesity through gut microbiota-drived microRNA-378a family.

Betaine is a natural compound present in commonly consumed foods and may have a potential role in the regulation of glucose and lipids metabolism. However, the underlying molecular mechanism of its action remains largely unknown. Here, we show that supplementation with betaine contributes to improved high-fat diet (HFD)-induced gut microbiota dysbiosis and increases anti-obesity strains such as Akkermansia muciniphila, Lactobacillus, and Bifidobacterium. In mice lacking gut microbiota, the functional role of betaine in preventing HFD-induced obesity, metabolic syndrome, and inactivation of brown adipose tissues are significantly reduced. Akkermansia muciniphila is an important regulator of betaine in improving microbiome ecology and increasing strains that produce short-chain fatty acids (SCFAs). Increasing two main members of SCFAs including acetate and butyrate can significantly regulate the levels of DNA methylation at host miR-378a promoter, thus preventing the development of obesity and glucose intolerance. However, these beneficial effects are partially abolished by Yin yang (YY1), a common target gene of the miR-378a family. Taken together, our findings demonstrate that betaine can improve obesity and associated MS via the gut microbiota-derived miR-378a/YY1 regulatory axis, and reveal a novel mechanism by which gut microbiota improve host health.

Thermally Triggered, Cell-Specific Enzymatic Glyco-Editing: In Situ Regulation of Lectin Recognition and Immune Response on Target Cells.

In situ glyco-editing on the cell surface can endow cellular glycoforms with new structures and properties; however, the lack of cell specificity and dependence on cells' endogenous functions plague the revelation of cellular glycan recognition properties and hamper the application of glyco-editing in complicated authentic biosystems. Herein, we develop a thermally triggered, cell-specific glyco-editing method for regulation of lectin recognition on target live cells in both single- and cocultured settings. The method relies on the aptamer-mediated anchoring of microgel-encapsulated neuraminidase on target cells and subsequent thermally triggered enzyme release for localized sialic acid (Sia) trimming. This temperature-based enzyme accessibility modulation strategy exempts genetic or metabolic engineering operations and, thus for the first time, enables tumor-specific desialylation on complicated tissue slices. The proposed method also provides an unprecedented opportunity to potentiate the innate immune response of natural killer cells toward target tumor cells through thermally triggered cell-specific desialylation, which paves the way for in vivo glycoimmune-checkpoint-targeted cancer therapeutic intervention.

Adropin regulates hepatic glucose production via PP2A/AMPK pathway in insulin-resistant hepatocytes.

Adropin as a secretory peptide has shown a protective role on the disorders of glucose and lipid metabolism. However, the role and mechanism of this peptide on the hepatic glucose production has remained unclear. Adropin knockout (KO) mice were generated to explore its effects on the enhanced hepatic glucose production in obesity. We found that compared to wild-type (WT) mice, adropin-KO mice developed the unbalanced enhanced hepatic glucose production in advance of the whole-body insulin resistance (IR) by high-fat diet (HFD). Mechanistically, adropin dissociated CREB-CRTC2 and FoxO1-PGC1α complex and reduced their binding to the promoters of G6Pase and PEPCK to decrease glucose production in IR. However, these effects were not observed in insulin-sensitive hepatocytes. Furthermore, in IR hepatocytes, dampened AMPK signaling was re-activated by adropin treatment via inhibition of PP2A. To further authenticate AMPK role in vivo, we administrated HFD-fed mice with AAV8-CA AMPKα and found that AMPK activation functionally restored the aberrant glucose production and IR induced by adropin-deficiency. This study provides evidence that adropin activates the AMPK pathway via inhibition of PP2A and decreases the liver glucose production in IR context. Therefore, adropin may represent a novel target for the prevention and treatment of diabetes.