Purification and characterization of cell-envelopeproteinase from Lactobacillus casei DI-1

Using a Ca2+-free method, the cell-envelope proteinase (CEP) of Lactobacillus casei DI-1 isolated from duck small intestine was released from cells and purified by ammonium sulfate precipitation, and by diethylaminoethyl (DEAE)-Sephadex A-25 and Sephadex G-100 gel chromatography. The purified CEP had a monomer structure with a molecular mass of about 35 kDa. Optimal activity occurred at pH 7.0 and 37°C. The purified CEP was a metallopeptidase, which was activated by Co2+, Ba2+, Mg2+ and Fe3+, and inhibited by Ca2+, Zn2+, K+, Ni2+, Mn2+, and ethylenediaminetetraacetic acid (EDTA). It was a serine proteinase which was inhibited by phenylmethylsulfonyl fluoride (PMSF). Its kinetic constant (Km) is 0.29 mM and the first 10 amino acids of the CEP’s N-terminal sequences were Asp-Asn-Asp-Phe-Glu-Ile-Phe-Glu-Ser-Ser. The hydrolysates of α-, β- and κ-casein produced by CEP showed different angiotensin-I-converting enzyme (ACE) inhibitory activity; the hydrolysates of β-casein displayed the greatest ACE inhibitory activity.Key words: Cell-envelope proteinase, purification, characterization

The Protective Effect of Magnesium Lithospermate B on Hepatic Ischemia/Reperfusion via Inhibiting the Jak2/Stat3 Signaling Pathway

Acute inflammation is an important component of the pathogenesis of hepatic ischemia/reperfusion injury (HIRI). Magnesium lithospermate B (MLB) has strong neuroprotective and cardioprotective effects. The purpose of this study was to determine whether MLB had underlying protective effects against hepatic I/R injury and to reveal the potential mechanisms related to the hepatoprotective effects. In this study, we first examined the protective effect of MLB on HIRI in mice that underwent 1 h ischemia followed by 6 h reperfusion. MLB pretreatment alleviated the abnormal liver function and hepatocyte damage induced by I/R injury. We found that serum inflammatory cytokines, including IL-6, IL-1β, and TNF-α, were significantly decreased by MLB during hepatic ischemia/reperfusion (I/R) injury, suggesting that MLB may alleviate hepatic I/R injury via inhibiting inflammatory signaling pathways. Second, we investigated the protein level of p-Jak2/Jak2 and p-Stat3/Stat3 using Western blotting and found that MLB could significantly inhibit the activation of the Jak2/Stat3 signaling pathway, which was further verified by AG490 in a mouse model. Finally, the effect of MLB on the Jak2/Stat3 pathway was further assessed in an in vitro model of RAW 264.7 cells; 1 µg/ml LPS induced the secretion of inflammatory mediators, including IL-6, TNF-α, and activation of the Jak2/Stat3 signaling pathway. MLB significantly inhibited the abnormal secretion of inflammatory factors and the activation of the Jak2/Stat3 signaling pathway in RAW264.7 cells. In conclusion, MLB was found for the first time to reduce inflammation induced by hepatic I/R via suppressing the Jak2/Stat3 pathway

Interleukin-35 Expression in Non-Small Cell Lung Cancer is Associated with Tumor Progression

Background/Aims: Lung cancer continues to be the leading cause of cancer related deaths worldwide due to its high incidence, malignant behavior and lack of major advancements in treatment strategy. The occurrence and development of lung cancer is closely related to inflammation. Thus, we conducted the present study to investigate the effects of IL-35 (Interleukin 35), a newly identified anti-inflammatory factor, on non-small cell lung cancer (NSCLC), which accounts for about 85% of all lung cancers. Methods: We first evaluated the IL-35 expression in 384 pairs of NSCLC samples and their adjacent normal mucosa by realtime PCR, ELISA (Enzyme-linked immunoassay) and tissue microarrays. Then the role of IL-35 on patient survival rates, cancer progression and their sensitivity to chemotherapy drugs were assessed. Results: IL-35 was barely expressed in the NSCLC tissues but highly expressed in the adjacent normal tissues. The down-regulation of IL-35 was significantly correlated with the results of American Joint Committee on Cancer stage, differentiation and it was also shown to be an independent prognostic indicator of disease-free survival and overall survival for patients with NSCLC. Overexpression of IL-35 in NSCLC cells suppressed cell migration, invasion, proliferation, colony formation through suppressing β-catenin. IL-35 inhibited NSCLC formation in the mice model and sensitize the cancer cells to chemotherapy drugs. Conclusion: Our results showed that IL-35 plays an inhibitory role in NSCLC development and function as a novel prognostic indicator and a potential therapeutic target

Efficacy and Safety of Glp-1 Analog Ecnoglutide in Adults With Type 2 Diabetes: A Randomized, Double-Blind, Placebo-Controlled Phase 2 Trial

Glucagon-like peptide-1 (GLP-1) analogs are important therapeutics for type 2 diabetes and obesity. Ecnoglutide (XW003) is a novel, long-acting GLP-1 analog. We conducted a Phase 2, randomized, double-blind, placebo-controlled study enrolling 145 adults with T2DM. Participants were randomized to 0.4, 0.8, or 1.2 mg ecnoglutide or placebo as once-weekly injections for 20 weeks. The primary objective was to evaluate the efficacy of ecnoglutide, as measured by HbA1c change from baseline at Week 20. Secondary endpoints included body weight, glucose and lipid parameters, as well as safety. We show that, at end of treatment, the 0.4, 0.8, and 1.2 mg groups had statistically significant HbA1c reductions from baseline of -1.81%, -1.90%, and -2.39%, respectively, compared to -0.55% for placebo (P \u3c 0.0001). At end of treatment, 71.9% of the 1.2 mg group had HbA1c ≤ 6.5% versus 9.1% on placebo, and 33.3% had body weight reductions ≥5% versus 3.0% for placebo. Ecnoglutide was generally safe and well tolerated. China Drug Trials Registry CTR20211014

Enhanced Anti-diabetic Effect of Berberine Combined With Timosaponin B2 in Goto-Kakizaki Rats, Associated With Increased Variety and Exposure of Effective Substances Through Intestinal Absorption

Objective: Inspired by the traditionally clinical application of herb pair Zhimu-Huangbo to treat diabetes, a combination of plant ingredients, timosaponin B2 (TB-2) and berberine (BBR), was evaluated for their anti-diabetic efficacy and cooperative mechanisms.Methods: The efficacy and pharmacokinetics of orally administered TB-2 (33.3 mg/kg/day), BBR (66.7 mg/kg/day), and TB-2+BBR (100 mg/kg/day) were evaluated in spontaneously non-obese diabetic Goto-Kakizaki (GK) rats, and metformin (200 mg/kg/day) was used as a positive control. The comparative exposure of the parent drugs, timosaponin A3 (TB-2 metabolite), and M1–M5 (BBR metabolites) was quantified in the portal vein plasma (before hepatic disposition), liver, and systemic plasma (after hepatic disposition) of normal rats on single and combination treatments. Cooperative mechanism of TB-2 and BBR on intestinal absorption and hepatic metabolism was investigated in Caco-2 cells and primary hepatocytes, respectively.Results: After a 6-week experiment, non-fasting and fasting blood glucose levels and oral glucose tolerance test results showed that TB-2+BBR treatments (100 mg/kg/day) displayed significantly anti-diabetic efficacy in GK rats, comparable to that on metformin treatments. However, no significant improvement was observed on TB-2 or BBR treatments alone. Compared to single treatments, combination treatments led to the increased circulating levels of BBR by 107% in GK rats. In normal rats, the hepatic exposure of BBR, timosaponin A3, and M1–M5 was several hundred folds higher than their circulating levels. Co-administration also improved the levels in the plasma and liver by 41–114% for BBR, 141–230% for TB-2, and 12–282% for M1–M5. In vitro, the interaction between TB-2 and BBR was mediated by intestinal absorption, rather than hepatic metabolism.Conclusion: Combining TB-2 and BBR enhanced the anti-diabetic efficacy by increasing the in vivo variety of effective substances, including the parent compounds and active metabolites, and improving the levels of those substances through intestinal absorption. This study is a new attempt to assess the effects of combined plant ingredients on diabetes by scientifically utilizing clinical experience of an herb pair

Amyloid and SCD jointly predict cognitive decline across Chinese and German cohorts.

INTRODUCTION Subjective cognitive decline (SCD) in amyloid-positive (Aβ+) individuals was proposed as a clinical indicator of Stage 2 in the Alzheimer's disease (AD) continuum, but this requires further validation across cultures, measures, and recruitment strategies. METHODS Eight hundred twenty-one participants from SILCODE and DELCODE cohorts, including normal controls (NC) and individuals with SCD recruited from the community or from memory clinics, underwent neuropsychological assessments over up to 6 years. Amyloid positivity was derived from positron emission tomography or plasma biomarkers. Global cognitive change was analyzed using linear mixed-effects models. RESULTS In the combined and stratified cohorts, Aβ+ participants with SCD showed steeper cognitive decline or diminished practice effects compared with NC or Aβ- participants with SCD. These findings were confirmed using different operationalizations of SCD and amyloid positivity, and across different SCD recruitment settings. DISCUSSION Aβ+ individuals with SCD in German and Chinese populations showed greater global cognitive decline and could be targeted for interventional trials. HIGHLIGHTS SCD in amyloid-positive (Aβ+) participants predicts a steeper cognitive decline. This finding does not rely on specific SCD or amyloid operationalization. This finding is not specific to SCD patients recruited from memory clinics. This finding is valid in both German and Chinese populations. Aβ+ older adults with SCD could be a target population for interventional trials

High-Resolution ISAR Imaging with Wideband V-FM Waveforms

V-FM waveforms, composed of two chirp signals with the opposite slopes, can also achieve high range resolution with wide bandwidth via intrapulse frequency modulation. In this paper, a framework for inverse synthetic aperture radar (ISAR) imaging of moving targets with V-FM waveforms is investigated, where the range compression of the received signals is achieved by the dual-channel dechirping and the azimuth compression is done via the traditional Fourier transform (FT). The two corresponding reconstructed temporary high-resolution range profiles (HRRPs) from the double channels are synthesized for the HRRPs of the target, in which one is flipped from left to right and added to the other. Then the final HRRPs are arranged into a two-dimensional (2D) array and the azimuth compression is done via FT to achieve the ISAR imaging after the motion compensation. Simulated trials, adopting the scattering center modeling of the Yak-42 plane, are used to validate the correctness of the analyses and the finally well-focused images greatly support the effectiveness of V-FM waveforms in ISAR imaging

Assessment of biliary clearance in early drug discovery using sandwich-cultured hepatocytes model (SCH)

It is challenging to predict biliary clearance for new chemical entities (NCEs) in the early stages of drug discovery. Sandwich-cultured hepatocytes (SCH) have been reported to be a valuable tool for characterizing the hepatobiliary disposition and drug-drug interaction (DDI) potential of drug candidates; however, there is no comprehensive report projecting in vivo biliary clearance potential using in vitro SCH model during the drug discovery stage. The objective of this study was to determine if SCH can be used as an in vitro flagging tool and to identify potential connections between various in vitro ADME profiling assays. In this study, the biliary clearance of 110 discovery compounds was evaluated using the rat SCH model. PAMPA, Caco-2, and rat liver microsomes were employed in parallel to explore the relationships among the in vitro biliary excretion, cellular permeability, and liver metabolism of test compounds. Selected compounds were further tested in bile duct cannulated rats for IVIVC purpose. It was found that the rat SCH model proved to be a valuable tool for ranking and predicting in vivo biliary clearance during drug discovery. Passive permeability and metabolism may influence test compound biliary excretion. For compounds with extremely low passive permeability and metabolism, rat SCH may underestimate in vivo biliary clearance. The combination of passive permeability, metabolic intrinsic clearance, and the SCH model could serve as an initial screening platform for bile excretion potential as well as a means for improving compound liabilities and properties. A preliminary evaluation strategy was proposed to highlight biliary excretion risk evaluation during the drug discovery process