A novel fully human anti-NT-ANGPTL3 antibody from phage display library exhibits potent ApoB, TG, and LDL-C lowering activities in hyperlipidemia mice.

Dyslipidemia is characterized by elevated plasma levels of low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), and TG-rich lipoprotein (TGRLs) in circulation, and is closely associated with the incidence and development of cardiovascular disease. Angiopoietin-like protein 3 (ANGPTL3) deficiency has been identified as a cause of familial combined hypolipidemia in humans, which allows it to be an important therapeutic target for reducing plasma lipids. Here, we report the discovery and characterization of a novel fully human antibody F1519-D95aA against N-terminal ANGPTL3 (NT-ANGPTL3), which potently inhibits NT-ANGPTL3 with a KD as low as 9.21 nM. In hyperlipidemic mice, F1519-D95aA shows higher apolipoprotein B (ApoB) and TG-lowering, and similar LDL-C reducing activity as compared to positive control Evinacumab (56.50% vs 26.01% decrease in serum ApoB levels, 30.84% vs 25.28% decrease in serum TG levels, 23.32% vs 22.52% decrease in serum LDLC levels, relative to vehicle group). Molecular docking and binding energy calculations reveal that the F1519-D95aA-ANGPTL3 complex (10 hydrogen bonds, -65.51 kcal/mol) is more stable than the Evinacumab-ANGPTL3 complex (4 hydrogen bonds, -63.76 kcal/mol). Importantly, F1519-D95aA binds to ANGPTL3 with different residues in ANGPTL3 from Evinacumab, suggesting that F1519-D95aA may be useful for the treatment of patients resistant to Evinacumab. In conclusion, F1519-D95aA is a novel fully human anti-NT-ANGPTL3 antibody with potent plasma ApoB, TG, and LDL-C lowering activities, which can potentially serve as a therapeutic agent for hyperlipidemia and relevant cardiovascular diseases.

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Traditional antibody drug conjugates (ADC) combine monoclonal antibodies with cytotoxic drugs to accurately strike cancer cells, but there are still many shortcomings in stability, targeting, efficacy, and safety. Novel ADC, such as bi-specific, site-specific, dual-payload, and pro-drug type ADC, can be optimized by simultaneously binding 2 different antigens or epitopes, selecting more stable linkers, coupling with specific amino acid sites of antibodies, carrying different drug payloads, and adopting prodrug strategies, while retaining the characteristics of traditional ADC. Significantly improving the stability, targeting, efficacy and safety of drugs can better meet the needs of clinical treatment. Novel ADC will play a more important role in cancer treatment in the future. Discussing the progress of novel ADC in cancer treatment and analyzing their advantages and challenges can provide theoretical support for the development of anti-cancer strategies and provide directions for drug research and development.

Lunasin attenuates oxidant-induced endothelial injury and inhibits atherosclerotic plaque progression in ApoE-/- mice by up-regulating heme oxygenase-1 via PI3K/Akt/Nrf2/ARE pathway.

Oxidative stress-induced vascular endothelial cell (VEC) injury is a major mechanism in the initiation and development of atherosclerosis. Lunasin, a soybean-derived 43-aa peptide, has been previously shown to possess potent antioxidant and anti-inflammatory activities other than its established anticancer activities. This study investigated the effects of lunasin on protecting VECs from oxidative damage and inhibiting atherosclerotic plaque progression in apolipoprotein E-deficient (ApoE-/-) mice and explored its underlying mechanism. Biochemical and histologic analyses were performed by using EA.hy926 human VECs and a high-fat diet (HFD) ApoE-/- mouse atherosclerosis model. Our data indicated that lunasin attenuated H2O2-induced, mitochondria-dependent endothelial apoptosis via down-regulating Bax and up-regulating Bcl-2, inhibiting the mitochondrial depolarization, and reducing the release of cytochrome c, as well as decreasing the activation of caspase-9 and caspase-3 in vitro and in vivo. Mechanic studies showed that lunasin significantly up-regulated heme oxygenase-1 via the PI3K/Akt/nuclear factor erythroid 2-related factor 2/antioxidant response element pathway, and reduced H2O2-induced ROS production in VECs, thereby attenuating oxidant-induced endothelial injury and inhibiting atherosclerotic plaque progression in ApoE-/- mice. In conclusion, our in vitro and in vivo data suggest that lunasin protects VECs from oxidative damage by enhancing heme oxygenase-1 expression via activation of the PI3K/Akt/nuclear factor erythroid 2-related factor 2/antioxidant response element pathway and inhibiting mitochondria-dependent apoptosis, thereby effectively attenuating atherosclerosis in HFD-fed ApoE-/- mice. Lunasin may act as a potential therapeutic agent for the prevention and treatment of atherosclerosis.-Gu, L., Ye, P., Li, H., Wang, Y., Xu, Y., Tian, Q., Lei, G., Zhao, C., Gao, Z., Zhao, W., Tan, S. Lunasin attenuates oxidant-induced endothelial injury and inhibits atherosclerotic plaque progression in ApoE-/- mice by up-regulating heme oxygenase-1 via PI3K/Akt/Nrf2/ARE pathway.

Lead compounds and key residues of ribosomal protein S1 in drug-resistant Mycobacterium tuberculosis.

Ribosomal protein S1 (RpsA) has been identified as a novel target of pyrazinoic acid (POA), which is the active form of pyrazinamide (PZA), in vivo. RpsA plays a crucial role in trans-translation, which is widespread in microbes. In our investigation, we first described the discovery of promising RpsA antagonists for drug-resistant mycobacterium (MtRpsAd438A) and M. smegmatis, as well as wild-type M. tuberculosis. These antagonists were discovered via structure/ligand-based virtual screening approaches. A total of 21 targeted compounds were selected by virtual screening, combined scores, affinity, similarities and rules for potential as drugs. Next, the affinities of these compounds for three targeted proteins were tested in vitro by applying various technologies, including fluorescence quenching titration (FQT), saturation transfer difference (STD), and chemical shift perturbation (CSP) assays. The results showed that seven compounds had a high affinity for the targeted proteins. Our discovery set the stage for discovering new chemical entities (NCEs) for PZA-resistant tuberculosis and providing key residues for rational drug design to target RpsA.

Lunasin Improves the LDL-C Lowering Efficacy of Simvastatin via Inhibiting PCSK9 Expression in Hepatocytes and ApoE-/- Mice.

Statins are the most popular therapeutic drugs to lower plasma low density lipoprotein cholesterol (LDL-C) synthesis by competitively inhibiting hydroxyl-3-methyl-glutaryl-CoA (HMG-CoA) reductase and up-regulating the hepatic low density lipoprotein receptor (LDLR). However, the concomitant up-regulation of proprotein convertase subtilisin/kexin type 9 (PCSK9) by statin attenuates its cholesterol lowering efficacy. Lunasin, a soybean derived 43-amino acid polypeptide, has been previously shown to functionally enhance LDL uptake via down-regulating PCSK9 and up-regulating LDLR in hepatocytes and mice. Herein, we investigated the LDL-C lowering efficacy of simvastatin combined with lunasin. In HepG2 cells, after co-treatment with 1 µM simvastatin and 5 µM lunasin for 24 h, the up-regulation of PCSK9 by simvastatin was effectively counteracted by lunasin via down-regulating hepatocyte nuclear factor 1α (HNF-1α), and the functional LDL uptake was additively enhanced. Additionally, after combined therapy with simvastatin and lunasin for four weeks, ApoE-/- mice had significantly lower PCSK9 and higher LDLR levels in hepatic tissues and remarkably reduced plasma concentrations of total cholesterol (TC) and LDL-C, as compared to each monotherapy. Conclusively, lunasin significantly improved the LDL-C lowering efficacy of simvastatin by counteracting simvastatin induced elevation of PCSK9 in hepatocytes and ApoE-/- mice. Simvastatin combined with lunasin could be a novel regimen for hypercholesterolemia treatment.

A Novel Fully Human Agonistic Single Chain Fragment Variable Antibody Targeting Death Receptor 5 with Potent Antitumor Activity In Vitro and In Vivo.

Agonistic antibodies, which bind specifically to death receptor 5 (DR5), can trigger apoptosis in tumor cells through the extrinsic pathway. In this present study, we describe the use of a phage display to isolate a novel fully human agonistic single chain fragment variable (scFv) antibody, which targets DR5. After five rounds of panning a large (1.2 × 108 clones) phage display library on DR5, a total of over 4000 scFv clones were screened by the phage ELISA. After screening for agonism in a cell-viability assay in vitro, a novel DR5-specific scFv antibody TR2-3 was isolated, which inhibited COLO205 and MDA-MB-231 tumor cell growth without any cross-linking agents. The activity of TR2-3 in inducing apoptosis in cancer cells was evaluated by using an Annexin V-PE apoptosis detection kit in combination with flow cytometry and the Hoechst 33342 and propidium iodide double staining analysis. In addition, the activation of caspase-dependent apoptosis was evaluated by Western blot assays. The results indicated that TR2-3 induced robust apoptosis of the COLO205 and MDA-MB-231 cells in a dose-dependent and time-dependent manner, while it remarkably upregulated the cleavage of caspase-3 and caspase-8. Furthermore, TR2-3 suppressed the tumor growth significantly in the xenograft model. Taken together, these data suggest that TR2-3 exhibited potent antitumor activity both in vitro and in vivo. This work provides a novel human antibody, which might be a promising candidate for cancer therapy by targeting DR5.

Hirudin as a novel fusion tag for efficient production of lunasin in Escherichia coli.

Fusion expression provides an effective means for the biosynthesis of longer peptides in Escherichia coli. However, the commonly used fusion tags are primarily suitable for laboratory scale applications due to the high cost of commercial affinity resins. Herein, a novel approach exploiting hirudin as a multipurpose fusion tag in combination with tobacco etch virus (TEV) protease cleavage has been developed for the efficient and cost-effective production of a 43-amino acid model peptide lunasin in E. coli at preparative scale. A fusion gene which allows for lunasin to be N-terminally fused to the C-terminus of hirudin through a flexible linker comprising a TEV protease cleavage site was designed and cloned in a secretion vector pTASH. By cultivation in a 7-L bioreactor, the fusion protein was excreted into the culture medium at a high yield of ~380 mg/L, which was conveniently recovered and purified by inexpensive HP20 hydrophobic chromatography at a recovery rate of ~80%. After polishing and cleavage with TEV protease, the finally purified lunasin was obtained with ≥95% purity and yield of ~86 mg/L culture medium. Conclusively, this hirudin tagging strategy is powerful in the production of lunasin and could be applicable for the production of other peptides at preparative scale.

Engineering lentivirus envelope VSV-G for liver targeted delivery of IDOL-shRNA to ameliorate hypercholesterolemia and atherosclerosis.

Lentiviral vectors (LVs) have been widely used as a tool for gene therapies. However, tissue-selective transduction after systemic delivery remains a challenge. Inducible degrader of low-density lipoprotein receptor is an attractive target for treating hypercholesterolemia. Here, a liver-targeted LV, CS8-LV-shIDOL, is developed by incorporating a hepatocyte-targeted peptide derived from circumsporozoite protein (CSP) into the lentivirus envelope for liver-targeted delivery of IDOL-shRNA (short hairpin RNA) to alleviate hypercholesterolemia. Tail-vein injection of CS8-LV-shIDOL results in extremely high accumulation in liver and nearly undetectable levels in other organs in mice. In addition, it shows superior therapeutic efficacy in lowering serum low-density lipoprotein cholesterol (LDL-C) and reducing atherosclerotic lesions over unmodified LV-shIDOL in hyperlipidemic mice. Mechanically, the envelope-engineered CS8-LV-shIDOL can enter liver cells via low-density lipoprotein receptor-related protein (LRP). Thus, this study provides a novel approach for liver-targeted delivery of IDOL-shRNA to treat hypercholesterolemia by using an envelope-engineered LV, and this delivery system has great potential for liver-targeted transgene therapy.

Biological Effects and Biomedical Applications of Areca Nut and Its Extract.

The dried, mature fruit of the palm tree species Areca catechu L. is known as the areca nut (AN) or betel nut. It is widely cultivated in the tropical regions. In many nations, AN is utilized for traditional herbal treatments or social activities. AN has historically been used to address various health issues, such as diarrhea, arthritis, dyspepsia, malaria, and so on. In this review, we have conducted a comprehensive summary of the biological effects and biomedical applications of AN and its extracts. Initially, we provided an overview of the constituents in AN extract. Subsequently, we summarized the biological effects of AN and its extracts on the digestive system, nervous system, and circulatory system. And we elucidated the contributions of AN and its extracts in antidepressant, anti-inflammatory, antioxidant, and antibacterial applications. Finally, we have discussed the challenges and future perspectives regarding the utilization of AN and its extracts as emerging pharmaceuticals or valuable adjuncts within the pharmaceutical field.

Clinical significance and biological function of interferon regulatory factor 1 in non-small cell lung cancer.

The clinical application and biological function of interferon regulatory factor 1 (IRF1) in non-small cell lung cancer (NSCLC) patients undergoing chemoimmunotherapy remain elusive. The aim of this study was to investigate the predictive and prognostic significance of IRF1 in NSCLC patients. We employed the cBioPortal database to predict frequency changes in IRF1 and explore its target genes. Bioinformatic methods were utilized to analyze the relationship between IRF1 and immune regulatory factors. Retrospective analysis of clinical samples was conducted to assess the predictive and prognostic value of IRF1 in chemoimmunotherapy. Additionally, A549 cells with varying IRF1 expression levels were constructed to investigate its effects on NSCLC cells, while animal experiments were performed to study the role of IRF1 in vivo. Our findings revealed that the primary mutation of IRF1 is deep deletion and it exhibits a close association with immune regulatory factors. KRAS and TP53 are among the target genes of IRF1, with interferon and IL-2 being the predominantly affected pathways. Clinically, IRF1 levels significantly correlate with the efficacy of chemoimmunotherapy. Patients with high IRF1 levels exhibited a median progression-free survival (mPFS) of 9.5 months, whereas those with low IRF1 levels had a shorter mPFS of 5.8 months. IRF1 levels positively correlate with PD-L1 distribution and circulating IL-2 levels. IL-2 enhances the biological function of IRF1 and recapitulates its role in vivo in the knockdown group. Therefore, IRF1 may possess predictive and prognostic value for chemoimmunotherapy in NSCLC patients through the regulation of the IL-2 inflammatory pathway.

Restoration of miR-299-3p promotes macrophage phagocytosis and suppresses malignant phenotypes in breast cancer carcinogenesis via dual-targeting CD47 and ABCE1.

Immunoevasion has been a severe obstacle for the clinical treatment of breast cancer (BC). CD47, known as an anti-phagocytic molecule, plays a key role in governing the evasion of tumor cells from immune surveillance by interacting with signal-regulated protein α (SIRPα) on macrophages. Here, we report for the first time that miR-299-3p is a direct regulator of CD47 with tumor suppressive effects both in vitro and in vivo. miRNA expression profiles and overall survival of BC cohorts from the Cancer Genome Atlas, METABRIC, or GSE19783 datasets showed that miR-299-3p is downregulated in BC tissues and that BC patients with low levels of miR-299-3p have poorer prognoses. Using dual-luciferase reporter, qRT-PCR, Western blot, and phagocytosis assays, we proved that restoration of miR-299-3p can suppress CD47 expression by directly targeting the predicted seed sequence "CCCACAU" in its 3'-UTR, leading to phagocytosis of BC cells by macrophages, whereas miR-299-3p inhibition or deletion reversed this effect. Additionally, Gene Ontology (GO) analysis and a variety of confirmatory experiments revealed that miR-299-3p was inversely correlated with cell proliferation, migration, and the cell cycle process. Mechanistically, miR-299-3p can also directly target ABCE1, an essential ribosome recycling factor, alleviating these malignant phenotypes of BC cells. In vivo BC xenografts based on nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice further proved that restoration of miR-299-3p resulted in a significant suppression of tumorigenesis and a promotion of macrophage activation and infiltration. Overall, our study suggested that miR-299-3p is a potent inhibitor of CD47 and ABCE1 to exhibit bifunctional BC-suppressing effects through immune activation conjugated with malignant behavior inhibition in breast carcinogenesis and thus can potentially serve as a novel therapeutic target for BC.

Deep Eutectic Solvents for Efficient and Selective Extraction of α-Glucosidase Inhibitors from Waste Seeds of Refined Betel Nuts.

During the production process of refined betel nuts in China, a large amount of processing by-product, betel nut waste seeds, is generated. Betel nut waste seeds are rich in bioactive elements, but they have not been effectively utilized yet. In this study, an ultrasonic-assisted deep eutectic solvent method (DES) was used to selectively extract α-glucosidase inhibitors from waste seeds. Compared with traditional extraction solvents such as water and ethanol, the extraction efficiency of specific DESs is higher, and the content of alkaloids in the extracts is lower. However, it should be noted that some pure DESs exhibit inhibitory activity towards α-glucosidase. DESs, based on choline chloride/urea, were selected due to the high extraction efficiency of α-glucosidase inhibitors and their low alkaloid content as well as low inhibitory activity. The optimal extraction conditions were determined using single-factor experiments as follows: 30% (v/v) water content, a choline chloride/urea ratio of 5:3, a solid-liquid ratio of 1:10, extraction temperature of 40 °C, and a duration of 30 min. Through recovery experiments, it was found that the DES can be reused four times under these conditions, maintaining an inhibition rate comparable to alcohol extraction methods. The IC50 value of the extract was measured at 0.0066 mg/mL, superior to acarbose. In summary, this research has successfully developed an efficient and selective method for extracting α-glucosidase inhibitors from betel nut waste seeds, thereby presenting a promising avenue for future applications.

Inhibitory effect of r-hirudin variant III on streptozotocin-induced diabetic cataracts in rats.

The in vivo inhibitory effect of r-hirudin variant III (rHV3) on streptozotocin (STZ)-induced diabetic cataracts in rats was investigated. SD-rats were firstly made diabetic by a single intraperitoneal injection of 2% (W/V) STZ (65 mg/kg). Two weeks later, cataract formation was examined by slit lamp microscope, and the cataracted animals were randomly grouped. The animals in the treated groups received rHV3 drops administration to the eyes with various doses. After 4 weeks treatment, the animals were sacrificed to evaluate the biochemical changes of aldose reductase (AR), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels in the eye lens. Meanwhile, the cataract progression was monitored by slit lamp microscope. As a result, rHV3 drops treatment significantly increased the activities of SOD and GSH-Px in the lens in a dose-dependent manner, whereas AR activity and MDA level in the lens were dramatically decreased. Also, the morphological observation further confirmed the inhibition of the development of STZ-induced diabetic cataracts by the rHV3 drops treatment. Thus, our data suggest that rHV3 drops are pharmacologically effective for the protection against STZ-induced diabetic cataracts in rats.

Identification of novel microRNAs in primates by using the synteny information and small RNA deep sequencing data.

Current technologies that are used for genome-wide microRNA (miRNA) prediction are mainly based on BLAST tool. They often produce a large number of false positives. Here, we describe an effective approach for identifying orthologous pre-miRNAs in several primates based on syntenic information. Some of them have been validated by small RNA high throughput sequencing data. This approach uses the synteny information and experimentally validated miRNAs of human, and incorporates currently available algorithms and tools to identify the pre-miRNAs in five other primates. First, we identified 929 potential pre-miRNAs in the marmoset in which miRNAs have not yet been reported. Then, we predicted the miRNAs in other primates, and we successfully re-identified most of the published miRNAs and found 721, 979, 650 and 639 new potential pre-miRNAs in chimpanzee, gorilla, orangutan and rhesus macaque, respectively. Furthermore, the miRNA transcriptome in the four primates have been re-analyzed and some novel predicted miRNAs have been supported by the small RNA sequencing data. Finally, we analyzed the potential functions of those validated miRNAs and explored the regulatory elements and transcription factors of some validated miRNA genes of interest. The results show that our approach can effectively identify novel miRNAs and some miRNAs that supported by small RNA sequencing data maybe play roles in the nervous system.

The Evaluation of Prognostic Value and Immune Characteristics of Ferroptosis-Related Genes in Lung Squamous Cell Carcinoma.

Background The purpose of our study was to construct a prognostic model based on ferroptosis-related gene signature to improve the prognosis prediction of lung squamous carcinoma (LUSC). Methods The mRNA expression profiles and clinical data of LUSC patients were downloaded. LUSC-related essential differentially expressed genes were integrated for further analysis. Prognostic gene signatures were identified through random forest regression and univariate Cox regression analyses for constructing a prognostic model. Finally, in a preliminary experiment, we used the reverse transcription-quantitative polymerase chain reaction assay to verify the relationship between the expression of three prognostic gene features and ferroptosis. Results Fifty-six ferroptosis-related essential genes were identified by using integrated analysis. Among these, three prognostic gene signatures (HELLS, POLR2H, and POLE2) were identified, which were positively affected by LUSC prognosis but negatively affected by immune cell infiltration. Significant overexpression of immune checkpoint genes occurred in the high-risk group. In preliminary experiments, we confirmed that the occurrence of ferroptosis can reduce three prognostic gene signature expression. Conclusions The three ferroptosis-related genes could predict the LUSC prognostic risk of antitumor immunity.

A bifunctional anti-PCSK9 scFv/Exendin-4 fusion protein exhibits enhanced lipid-lowering effects via targeting multiple signaling pathways in HFD-fed mice.

Fusion protein which encompasses more than one functional component, has become one of the most important representatives of macromolecular drugs for disease treatment since that monotherapy itself might not be effective enough to eradicate the disease. In this study, we sought to construct a bifunctional antibody fusion protein by fusing anti-PCSK9 scFv with Exendin-4 for simultaneously lowering both LDL-C and TG. Firstly, three Ex4-anti-PCSK9 scFv fusion proteins were constructed by genetically connecting the C-terminal of Exendin-4 to the N-terminal of anti-PCSK9 scFv through various flexible linker peptides (G4S)n (n = 2, 3, 4). After soluble expression in E. coli, the most potent Ex4-(G4S)4-anti-PCSK9 scFv fusion protein was selected based on in vitro activity assays. Then, we investigated the in vivo therapeutic effects of Ex4-(G4S)4-anti-PCSK9 scFv on the serum lipid profile and bodyweight changes as well as underlying molecular mechanism in HFD-fed C57BL/6 mice. The data showed that Ex4-(G4S)4-anti-PCSK9 scFv exhibits enhanced effects of lowering both LDL-C and TG in serum, reducing food intake and body weight via blocking PCSK9/LDLR, activating AMPK/SREBP-1 pathways, and up-regulating sirt6. Conclusively, Ex4-(G4S)4-anti-PCSK9 has the potential to serve as a promising therapeutic agent for effectively treating dyslipidemia with high levels of both LDL-C and TG.

Extracellular secretion of anticoagulant peptide hirudin in Lactococcus lactis using SP310mut2 signal peptide.

Hirudin can be used as an oral anticoagulant and antithrombotic agent. The hirudin variant III gene, derived from the medicinal leech, Hirudo medicinalis, was fused to SP310mut2 signal sequence and expressed by a nisin-controlled gene expression system in Lactococcus lactis which was then grown in a 7 l fermenter. After induction with 8 ng nisin ml(-1), the product was secreted into the culture medium and accumulated up to ~2.7 mg l(-1). MALDI-TOF/MS and anticoagulant activity analyses on the purified product confirmed its authenticity. This is the first demonstration that hirudin can be extracellularly secreted and correctly processed in L. lactis.

Robust preparative-scale extracellular production of hirudin in Escherichia coli and its purification and characterization.

Hirudin variant III (HV3) is potentially useful in the prevention and treatment of cataracts. To prepare sufficient amounts of rHV3 for further preclinical studies, we developed an effective process for robust preparative-scale extracellular production of rHV3 in Escherichia coli. In a 7-l bioreactor, under the optimal fed-batch fermentation conditions, rHV3 was excreted into the culture supernatant and yielded up to 915 mg l(-1). Then, a four-step purification procedure was applied to the product, which included ultrafiltration, hydrophobic chromatography, anion-exchange chromatography, and preparative reversed-phase fast protein liquid chromatography (FPLC). The overall maximum recovery attained was 56 %, the purity reached at least 99 % as evaluated by HPLC analysis, the molecular weight was determined to be 7,011.10 Da by matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF/MS) analysis, and the pI was 4.46 as analyzed by isoelectric focusing. The N- and C-terminal sequence analysis confirmed the product homogeneity. The final product contained at most 10 pg of residual DNA per dose (0.2 mg) of rHV3 by high-sensitivity hybridization assay and at most 3 EU endotoxin protein/mg by limulus amebocyte lysate assay. Taken together, the rHV3 produced in multigram quantities in E. coli by this bioprocess meets the regulatory criteria for biopharmaceuticals and can be used as a drug candidate for preclinical studies.

Generation of a Novel High-Affinity Antibody Binding to PCSK9 Catalytic Domain with Slow Dissociation Rate by CDR-Grafting, Alanine Scanning and Saturated Site-Directed Mutagenesis for Favorably Treating Hypercholesterolemia.

Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has become an attractive therapeutic strategy for lowering low-density lipoprotein cholesterol (LDL-C). In this study, a novel high affinity humanized IgG1 mAb (named h5E12-L230G) targeting the catalytic domain of human PCSK9 (hPCSK9) was generated by using CDR-grafting, alanine-scanning mutagenesis, and saturated site-directed mutagenesis. The heavy-chain constant region of h5E12-L230G was modified to eliminate the cytotoxic effector functions and mitigate the heterogeneity. The biolayer interferometry (BLI) binding assay and molecular docking study revealed that h5E12-L230G binds to the catalytic domain of hPCSK9 with nanomolar affinity (KD = 1.72 nM) and an extremely slow dissociation rate (koff, 4.84 × 10-5 s-1), which interprets its quite low binding energy (-54.97 kcal/mol) with hPCSK9. Additionally, h5E12-L230G elevated the levels of LDLR and enhanced the LDL-C uptake in HepG2 cells, as well as reducing the serum LDL-C and total cholesterol (TC) levels in hyperlipidemic mouse model with high potency comparable to the positive control alirocumab. Our data indicate that h5E12-L230G is a high-affinity anti-PCSK9 antibody candidate with an extremely slow dissociation rate for favorably treating hypercholesterolemia and relevant cardiovascular diseases.

Perfluorodecanoic acid induces meiotic defects and deterioration of mice oocytes in vitro.

Perfluorodecanoic acid (PFDA) is a member of the perfluoroalkyl substances, which are toxic to organic functions. Recently, it has been found in follicular fluid, seriously interfering with reproduction. Follicular fluid provides the oocyte with necessary resources during the process of oocytes maturation. However, the effects of PFDA on the oocyte need investigation. Our study evaluated the impacts of PFDA on the meiosis and development potential of mouse oocytes by exposing oocytes to PFDA in vitro at 350, 400, and 450 µM concentrations. The results showed that exposure to PFDA resulted in the first meiotic prophase arrest by obstructing the function of the maturation-promoting factor. It also induced the dysfunction of the spindle assembly checkpoint, expedited the progression of the first meiotic process, and increased the risk of aneuploidy. The oocytes treated with PFDA had a broken cytoskeleton which also contributed to meiotic maturation failure. Besides, PFDA exposure caused mitochondria defections, increased the reactive oxygen species level in oocytes, and consequently induced oocyte apoptosis. Moreover, PFDA produced epigenetic modifications in oocytes and increased the frequency of mature oocytes with declined development potential. In summary, our data indicated that PFDA disturbs the meiotic process and induces oocyte quality deterioration.