A platform for qualitative and quantitative characterization of α-Gal and NeuGc at the oligosaccharide level.

Glycosylation modification serves as a pivotal quality attribute in glycoprotein-based therapeutics, emphasizing its role in drug safety and efficacy. Prior studies have underscored the potential immunogenic risks posed by the presence of galactose-α-1,3-galactose (α-Gal) and N-glycolylneuraminic acid (NeuGc) in glycoprotein formulations. This accentuates the imperative for developing robust qualitative and quantitative analytical methods to monitor these immunogenic epitopes, thereby ensuring drug safety. In the present investigation, α-Gal and NeuGc were accurately quantified using UPLC-FLR-MS/MS at the oligosaccharide level. Remarkably, α-Gal could be identified when the ion intensity ratio or the mass-to-charge ratio (m/z) of 528.19 to 366.14 exceeded 1. Concurrently, NeuGc and N-acetylneuraminic acid (NeuAc) could be unambiguously identified through their respective fragment ions at m/z 673.23 and m/z 657.23. Furthermore, relative quantification of α-Gal and NeuGc was achieved using fluorescence signals. This study introduces a sensitive and reliable analytical approach for the qualitative and quantitative assessment of α-Gal and NeuGc in glycoprotein pharmaceuticals. The methodology offers significant potential for application in process control and optimization of glycoprotein production, aimed at minimizing immunogenicity and enhancing product quality.

Dual trigger for final oocyte maturation in expected normal responders with a high immature oocyte rate: a randomized controlled trial.

Objective: To evaluate whether dual trigger could improve reproductive outcomes in women with low oocyte maturation rates compare to human chorionic gonadotropin (hCG) trigger. Methods: This study included expected normal ovarian responders younger than 40 years old whose immature oocyte rate in the previous cycle was more than 50% at the reproductive center from July 2021 to November 2022. A total of 73 patients were enrolled at trigger, including 34 in the hCG trigger group and 39 in the dual trigger group (co-administration of gonadotrophin releasing hormone (GnRH) agonist and hCG, 40 and 34 h prior to oocyte retrieval, respectively). The primary outcome was oocyte maturation rate. Results: There was no significant difference in the number of oocytes retrieved between the two study groups, but the oocyte maturation rate was higher in dual trigger group (84.0% [14.0%] vs. 55.5% [19.8%], p < 0.001). Moreover, there were also higher cumulative pregnancy rate (69.4% vs. 40.0%, p = 0.035) and cumulative live birth rate (66.7% vs. 36.0%, p = 0.022) in dual trigger group. Conclusion: For normal responders with low oocyte maturation rates, the dual trigger may be more effective than the conventional hCG trigger. Clinical trial registration: ClinicalTrials.gov, identifier ChiCTR2100049292.

Universal protocol and standard-spiking strategy for profiling of host cell proteins in therapeutic growth hormone.

Liquid chromatography coupled to mass spectrometry (LC-MS) is widely used for host cell proteins (HCP) identification in antibody drug development because of its sensitivity, selectivity, and adaptability. However, LC-MS based identification of HCP in biotherapeutics produced from the prokaryotic Escherichia coli-derived growth hormone (GH) has rarely been reported. Herein, we developed a universal and powerful workflow by combining optimized sample preparation with one-dimension ultra-high performance LC-MS based shotgun proteomics to support HCP profiling in GH samples from downstream pools and the final product, which would be beneficial to direct the purification process development and compare the difference of impurity of different products for guiding the development of the biosimilar. A standard-spiking strategy was also developed to increase the depth of HCP identification. Spiking with standards enables additional identification of HCP species, which is promising for trace-level HCP analysis. Our universal and standard-spiking protocols would open an avenue for profiling HCP in biotherapeutics derived from prokaryotic host cells.

Comparison of pregnancy outcomes of letrozole-induced frozen-thawed embryo transfer cycles in PCOS women with two different abnormal ovulation patterns: A retrospective cohort study.

No studies have been conducted on the impact of different types of ovulatory dysfunction on the outcomes of frozen-thawed embryo transfers (FETs) in a letrozole-stimulated cycle in women with polycystic ovarian syndrome (PCOS). This study aimed to compare whether pregnancy outcomes of the letrozole-induced protocol in FET cycles differed between oligo-ovulatory and anovulatory women with PCOS. In a retrospective cohort study, women with PCOS who had undergone letrozole-induced FET at a university-affiliated fertility clinic from February 2014 to October 2020 were identified. The primary end point was live birth rate (LBR) per embryo transfer. Propensity score matching and multivariate logistic regression analyses were performed to control for the relevant confounders. A total of 652 women with PCOS undergoing letrozole-induced FET were included in the final analysis. Three hundred sixty-three of these women had oligo-ovulatory periods, while 289 had anovulatory periods. Propensity score matching analysis showed that LBR did not differ between groups (36.8% in oligo-ovulatory group vs 32.8% in anovulatory group, P = .431). Nevertheless, after controlling for potential confounding factors, LBR was significantly lower in anovulatory than oligo-ovulatory women (adjusted odds ratio 1.57, 95% confidence interval 1.08-2.29, P = .018). Furthermore, the pregnancy loss rate among the oligo-ovulatory group remained lower than those among the anovulatory group (adjusted odds ratio 0.23, 95% confidence interval 0.12-0.44, P < .001). Despite adjustment for confounding factors, those with oligo-ovulatory PCOS had a higher LBR and lower pregnancy loss rate compared with those with anovulatory PCOS. This may indicate that when oligo-ovulation is detected, PCOS patients should be intervened in time to conceive as soon as possible. Prospective studies must be conducted in the future to verify our findings.

GnRH Agonist and hCG (Dual Trigger) Versus hCG Trigger for Final Oocyte Maturation in Expected Normal Responders With a High Immature Oocyte Rate: Study Protocol for a Randomized, Superiority, Parallel Group, Controlled Trial.

Introduction: The choice of trigger drug for the controlled ovarian hyperstimulation (COH) protocol correlates with the outcome of in vitro fertilization/intracytoplasmic sperm injection embryo transfer (IVF/ICSI-ET). The co-administration of gonadotropin releasing hormone agonist (GnRH-a) and human chorionic gonadotropin (hCG), i.e., dual trigger, for final oocyte maturation, has received much attention in recent years. This trial was designed to determine whether a dual trigger approach by lengthening the time between trigger and ovum pick-up (OPU) improves the quantity and quality of mature oocytes/top-quality embryos and pregnancy outcomes in expected normal responders with a high immature oocyte rate. Methods and Analysis: We propose a study at the Affiliated Hospital of Shandong University of Chinese Medicine. A total of 90 individuals undergoing COH use a fixed GnRH antagonist protocol. They will be assigned randomly into two groups according to the trigger method and timing: recombinant hCG (6500 IU) will be injected only 36 hours before OPU for final oocyte maturation (hCG-only trigger); co-administration of GnRH-a and hCG for final oocyte maturation, 40 and 34 hours prior to OPU, respectively (Dual trigger). The primary outcome is metaphase-II (MII) oocytes rate. Secondary outcomes are number of oocytes retrieved, fertilization rate, top-quality embryos rate, blastula formation rate, embryo implantation rate, clinical pregnancy rate, miscarriage rate, live birth rate, cumulative pregnancy/live birth rates, and ovarian hyperstimulation syndrome (OHSS) rate. Ethics and Dissemination: The reproductive ethics committee of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine certified this study (Identifier: SDUTCM/2021.7.26) as ethical. All individuals will sign written informed consent. All data and biological samples will be protected according to law. The results of this study will be disseminated in a peer-reviewed scientific journal. Clinical Trial Registration: [chictr.gov.cn], identifier [ChiCTR2100049292].

Importin alpha 1 is required for the nucleus entry of Fowl Adenovirus serotype 4 Fiber-1 protein.

Fiber-1 protein (F1) is the structural protein of Fowl Adenovirus serotype 4 (FAdV-4), which could recondite the receptors of host cytomembrane. In this study, we firstly determined that F1 protein located in nucleus of LMH cells after infection with FAdV-4. We additionally revealed that F1 protein had a classic NLS, and the NLS was required for F1 nucleus entry, which was intently associated to the 26th Pro in NLS. The time rule result indicated that some F1 proteins firstly positioned in the nucleus 6 h posttranfection, and it entirely located in the nucleus 12 h posttranfection, then it ordinarily placed in cytoplasm 18 h posttranfection by means of microscopic fluorescence observation and Western Blotting. Then after transfection with pCI-neo-flag-F1 or infection with FAdV-4, the importin alpha 1 was once investigated whether or not it was required for F1 protein nucleus entry through immunofluorescence and/or Co-IP, results demonstrated that the F1 protein and importin alpha 1 co-located in the nucleus 6 h and 12 h posttranfection. The tiers of F1 protein vicinity in nucleus have been additionally investigated after knockdown expression or overexpression of importin alpha 1, and the results further revealed that importin alpha 1 used to be required for F1 protein nucleus entry. Finally, the function of F1 protein nucleus entry was investigated by qPCR, RT-PCR and Western Blotting, and the results revealed that F1 protein nucleus location was conducive to the proliferation of FAdV-4. In summary, we firstly reveal that the F1 protein of FAdV-4 locates in nucleus infected with FAdV-4, and confirm that importin alpha 1 binds to the NLS of F1 protein to nucleus localization, which promotes the proliferation of FAdV-4.

Anxiety and depression among pregnant women undergoing IVF-ET and WeChat group peer support during the COVID-19 pandemic: Study protocol for a randomized controlled trial.

INTRODUCTION: The psychological well-being of pregnant women following assisted reproductive has increasingly gained attention in recent years. Anxiety and depression may be associated to pregnancy outcomes. This study aims to determine whether peer support and the WeChat group platform will reduce anxiety and depression among in vitro fertilization and embryo transfer (IVF-ET) women. METHODS AND ANALYSIS: In the present randomized controlled study, 296 patients with confirmed clinical pregnancy following IVF-ET will be randomly assigned to receive standard intervention support or WeChat peer support on a 1:1 basis. The levels of anxiety and depression are the primary endpoints. Assessments will be performed at baseline measurements, first trimester, second trimester, and third trimester, and data will be collected. ETHICS AND DISSEMINATION: This study has been approved as ethical by the affiliated hospital of Shandong University of Traditional Chinese Medicine's Reproductive Ethics Committee. Each patient will sign a written statement of informed permission. All information and biological samples will be legally protected. A peer-reviewed academic journal will publish the findings of this investigation. DISCUSSION: Given the inconvenience of visits due to the current pandemic of COVID-19, this study addresses the patient's visit needs by combining WeChat, the most widely used social software in China, with peer support, while helping improve maternal anxiety, depression, and pregnancy outcomes following IVF-ET.

Caspase-Mediated Anti-Apoptotic Effect of Ginsenoside Rg5, a Main Rare Ginsenoside, on Acetaminophen-Induced Hepatotoxicity in Mice.

Frequent overdose of acetaminophen (APAP) is one of the most common and important incentives of acute hepatotoxicity. Prior to this work, our research group confirmed that black ginseng (Panax ginseng, BG) showed powerful protective effects on APAP-induced ALI. However, it is not clear which kind of individual ginsenoside from BG plays such a liver protection effect. The objective of the current investigation was to evaluate whether ginsenoside Rg5 (G-Rg5) protected against APAP-induced hepatotoxicity and the involved action mechanisms. Mice were administrated with G-Rg5 at two dosages of 10 or 20 mg/kg for 7 consecutive days. After the last treatment, all of the animals that received a single intraperitoneal injection of APAP (250 mg/kg) showed severe liver toxicity after 24 h, and the liver protection effects of G-Rg5 were examined. The results clearly indicated that pretreatment with G-Rg5 remarkably inhibited the production of serum tumor necrosis factor (TNF-α) and interleukin-1ß (IL-1ß) compared with the APAP group. Meanwhile, G-Rg5 decreased the hepatic malondialdehyde (MDA) content, the protein expression levels of 4-hydroxynonenal (4-HNE) and cytochrome P450 2E1 (CYP2E1) in the liver tissues. G-Rg5 decreased APAP caused the hepatic overexpression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Furthermore, analysis of immunohistochemistry and Western blotting also indicated that G-Rg5 pretreatment inhibited activation of apoptotic pathways mainly via increasing the expression of Bcl-2 protein, decreasing the expression of Bax protein, proliferating cell nuclear antigen (PCNA), cytochrome c, caspase-3, caspase-8, and caspase-9. Liver histopathological observation provided further evidence that pretreatment with G-Rg5 could significantly inhibit hepatocyte necrosis, inflammatory cell infiltration, and apoptosis caused by APAP. In conclusion, the present study clearly demonstrates that G-Rg5 exerts a liver protection effect against APAP-induced acute hepatotoxicity mainly via a caspase-mediated anti-apoptotic effect.

Ginsenoside Rg5 Ameliorates Cisplatin-Induced Nephrotoxicity in Mice through Inhibition of Inflammation, Oxidative Stress, and Apoptosis.

Although cisplatin is an effective anti-cancer agent that is widely used for treating various types of malignant solid tumors, the nephrotoxicity induced by cisplatin severely limits its clinical application. The present study was designed to explore the potential protective effect of ginsenoside Rg5, a rare ginsenoside generated during steaming ginseng, on cisplatin-induced nephrotoxicity in a mouse experimental model. The possible mechanisms underlying this nephroprotective effect were also investigated for the first time. Rg5 was given at doses of 10 and 20 mg/kg for 10 consecutive days. On Day 7, a single nephrotoxic dose of cisplatin (25 mg/kg) was injected to mice. Cisplatin administration resulted in renal dysfunction as evidenced by increase in serum creatinine (CRE) and blood urea nitrogen (BUN) levels. In addition, cisplatin increased the level of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), the makers of lipid peroxidation, and depleted glutathione (GSH) content and superoxide dismutase (SOD) activity in renal tissues. These effects were associated with the significantly increased levels of cytochrome P450 E1 (CYP2E1), 4-hydroxynonenal (4-HNE), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, nuclear factor-kappa B (NF-κB) p65, and cyclooxygenase-2 (COX-2) in renal tissues. However, pretreatment with ginsenoside Rg5 significantly attenuated the renal dysfunction, oxidative stress and inflammation response induced by cisplatin. Furthermore, ginsenoside Rg5 supplementation inhibited activation of apoptotic pathways through increasing Bcl-2 and decreasing Bax expression levels. Histopathological examination further confirmed the nephroprotective effect of Rg5. Collectively, these results clearly suggest that Rg5-mediated alleviation of cisplatin-induced nephrotoxicity may be related to its anti-oxidant, anti-apoptotic and anti-inflammatory effects.