Large-scale separation of alkaloids from Corydalis decumbens by pH-zone-refining centrifugal partition chromatography and their anticomplement activity.

Centrifugal partition chromatography in the pH-zone-refining mode was successfully applied to the separation of alkaloids from the crude extract of Corydalis decumbens. The experiment was performed with a two-phase solvent system composed of petroleum ether-ethyl acetate-ethanol-water (5:5:3:7, v/v/v/v) where triethylamine (10 mM) was added to the stationary phase and hydrochloric acid (10 mM) to the mobile phase. From 1.6 g of the crude extract, 43 mg protopine, 189 mg (+)-egenine, and 158 mg tetrahydropalmatine were obtained with a purity of 98.2%, 94.6%, and 96.7%, respectively. Tetrahydropalmatine showed an interesting anticomplement effect with CH50 0.11 and AP50 0.25 mg/mL, respectively. In a mechanistic study, tetrahydropalmatine interacted with C1, C3, C4, and C5 components in the complement activation cascade.

Research Progresses and Applications of Fluorescent Protein Antibodies: A Review Focusing on Nanobodies.

Since the discovery of fluorescent proteins (FPs), their rich fluorescence spectra and photochemical properties have promoted widespread biological research applications. FPs can be classified into green fluorescent protein (GFP) and its derivates, red fluorescent protein (RFP) and its derivates, and near-infrared FPs. With the continuous development of FPs, antibodies targeting FPs have emerged. The antibody, a class of immunoglobulin, is the main component of humoral immunity that explicitly recognizes and binds antigens. Monoclonal antibody, originating from a single B cell, has been widely applied in immunoassay, in vitro diagnostics, and drug development. The nanobody is a new type of antibody entirely composed of the variable domain of a heavy-chain antibody. Compared with conventional antibodies, these small and stable nanobodies can be expressed and functional in living cells. In addition, they can easily access grooves, seams, or hidden antigenic epitopes on the surface of the target. This review provides an overview of various FPs, the research progress of their antibodies, particularly nanobodies, and advanced applications of nanobodies targeting FPs. This review will be helpful for further research on nanobodies targeting FPs, making FPs more valuable in biological research.

Screening and Characterization of Shark-Derived VNARs against SARS-CoV-2 Spike RBD Protein.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is the major target for antibody therapeutics. Shark-derived variable domains of new antigen receptors (VNARs) are the smallest antibody fragments with flexible paratopes that can recognize protein motifs inaccessible to classical antibodies. This study reported four VNARs binders (JM-2, JM-5, JM-17, and JM-18) isolated from Chiloscyllium plagiosum immunized with SARS-CoV-2 RBD. Biolayer interferometry showed that the VNARs bound to the RBD with an affinity KD ranging from 38.5 to 2720 nM, and their Fc fusions had over ten times improved affinity. Gel filtration chromatography revealed that JM-2-Fc, JM-5-Fc, and JM-18-Fc could form stable complexes with RBD in solution. In addition, five bi-paratopic VNARs, named JM-2-5, JM-2-17, JM-2-18, JM-5-18, and JM-17-18, were constructed by fusing two VNARs targeting distinct RBD epitopes based on epitope grouping results. All these bi-paratopic VNARs except for JM-5-18 showed higher RBD binding affinities than its component VNARs, and their Fc fusions exhibited further enhanced binding affinities, with JM-2-5-Fc, JM-2-17-Fc, JM-2-18-Fc, and JM-5-18-Fc having KD values lower than 1 pM. Among these Fc fusions of bi-paratopic VNARs, JM-2-5-Fc, JM-2-17-Fc, and JM-2-18-Fc could block the angiotensin-converting enzyme 2 (ACE2) binding to the RBD of SARS-CoV-2 wildtype, Delta, Omicron, and SARS-CoV, with inhibition rates of 48.9~84.3%. Therefore, these high-affinity VNAR binders showed promise as detectors and therapeutics of COVID-19.

Bioavailability of oral methylnaltrexone increases with a phosphatidylcholine-based formulation.

OBJECTIVE: Methylnaltrexone (MNTX), a peripherally restricted opioid antagonist with mu-opioid receptor selectivity, can reduce opioid activity in the gastrointestinal tract while sparing the pain relief afforded by opioids. Since the bioavailability of oral MNTX is low, it is necessary to explore the oral formulations of MNTX that increase its bioavailability. MATERIALS AND METHODS: An MNTX-phosphatidylcholine complex (MNTX-PC) formulation was prepared. The physicochemical properties of MNTX-PC were analyzed, and its bioavailability was evaluated in rats. After 250 mg/kg of oral MNTX-PC, plasma samples were collected up to 9 h. The concentrations of the compound in rat plasma were quantified using LC/MS/MS. RESULTS: Two MNTX plasma concentration peaks were observed at 120 and 180 min for the MNTX-PC group and control (MNTX in a water solution). Tmax was 180 min, C(max) was 1083.7 ± 293.9 ng/mL, and T(½) was 496 min for the MNTX-PC group. For control, T(max) was 180 min, C(max) was 448.4 ± 126.0 ng/mL, and T(½) was 259 min. The AUC0₋540 min for the MNTX-PC group was 5758.2 ± 1474.2 ngh/mL; for control, 1405.9 ± 447.8 ngh/mL. Thus, the relative bioavailability after the oral administration of MNTX-PC was 410% compared to that of control. CONCLUSION: MNTX-PC formulation significantly enhanced the oral bioavailability of MNTX.

Red nucleus mGluR1 and mGluR5 facilitate the development of neuropathic pain through stimulating the expressions of TNF-α and IL-1ß.

Our previous study has identified that glutamate in the red nucleus (RN) facilitates the development of neuropathic pain through metabotropic glutamate receptors (mGluR). Here, we further explored the actions and possible molecular mechanisms of red nucleus mGluR Ⅰ (mGluR1 and mGluR5) in the development of neuropathic pain induced by spared nerve injury (SNI). Our data indicated that both mGluR1 and mGluR5 were constitutively expressed in the RN of normal rats. Two weeks after SNI, the expressions of mGluR1 and mGluR5 were significantly boosted in the RN contralateral to the nerve injury. Administration of mGluR1 antagonist LY367385 or mGluR5 antagonist MTEP to the RN contralateral to the nerve injury at 2 weeks post-SNI significantly ameliorated SNI-induced neuropathic pain. However, unilateral administration of mGluRⅠ agonist DHPG to the RN of normal rats provoked a significant mechanical allodynia, this effect could be blocked by LY367385 or MTEP. Further studies indicated that the expressions of TNF-α and IL-1ß in the RN were also elevated at 2 weeks post-SNI. Administration of mGluR1 antagonist LY367385 or mGluR5 antagonist MTEP to the RN at 2 weeks post-SNI significantly inhibited the elevations of TNF-α and IL-1ß. However, administration of mGluR Ⅰ agonist DHPG to the RN of normal rats significantly enhanced the expressions of TNF-α and IL-1ß, these effects were blocked by LY367385 or MTEP. These results suggest that activation of red nucleus mGluR1 and mGluR5 facilitate the development of neuropathic pain by stimulating the expressions of TNF-α and IL-1ß. mGluR Ⅰ maybe potential targets for drug development and clinical treatment of neuropathic pain.

Selection, identification and crystal structure of shark-derived single-domain antibodies against a green fluorescent protein.

Shark variable domain of new antigen receptors (VNARs) are the smallest naturally occurring binding domains with properties of low complexity, small size, cytoplasmic expression, and ease of engineering. Green fluorescent protein (GFP) molecules have been analyzed in conventional microscopy, but their spectral characteristics preclude their use in techniques offering substantially higher resolution. Besides, the GFP molecules can be quenched in acidic environment, which makes it necessary to develop anti-GFP antibody to solve these problems. In view of the diverse applications of GFP and unique physicochemical features of VNAR, the present study aims to generate VNARs against GFP. Here, we identified 36 VNARs targeting eCGP123, an extremely stable GFP, by phage display from three immunized sharks. These VNARs bound to eCGP123 with affinity constant KD values ranging from 6.76 to 605 nM. Among them, two lead VNARs named aGFP-14 and aGFP-15 with nanomolar eCGP123-binding affinity were selected for in-depth characterization. aGFP-14 and aGFP-15 recognized similar epitopes on eCGP123. X-ray crystallography studies clarified the mechanism by which aGFP14 interacts with eCGP123. aGFP-14 also showed cross-reaction with EGFP, with KD values of 47.2 nM. Finally, immunostaining analyses demonstrated that aGFP-14 was able to bind effectively to the EGFP expressed in both cultured cells and mouse brain tissues, and can be used as a fluorescence amplifier for EGFP. Our research demonstrates a feasible idea for the screening and production of shark-derived VNARs. The two high-affinity VNARs developed in the study contribute to the diversity of GFP sdAbs and may enhance the applications of GFP.

Exosome-Like Vesicles as New Mediators and Therapeutic Targets for Treating Insulin Resistance and ß-Cell Mass Failure in Type 2 Diabetes Mellitus.

Exosome-like vesicles (ELVs), the smallest class of extracellular vesicles released from cells, function in cellular crosstalk and therefore profoundly affect physiologic responses and pathologic progression. A growing body of evidence supports a novel role for ELVs as important mediators and therapeutic targets due to their effects on regulation of both insulin signaling and ß-cell mass. Pathologic conditions associated with type 2 diabetes (such as high blood glucose, inflammation, hypoxia, and fatty acids) can alter the quantity and components of ELVs secreted from the pancreas or peripheral insulin-targeting tissues. These released ELVs can either enter the blood circulation or be taken up by neighboring cells or macrophages, which can lead to insulin resistance or ß-cell apoptosis. This review focuses on the roles of ELVs in insulin resistance and ß-cell failure and also highlights the potential use of ELVs and exosome-based delivery systems in therapeutic interventions aimed at treating type 2 diabetes mellitus as well as the challenges associated with exosome-targeting therapeutics.

Good hemostatic effect of platelets stored at 4°C in an in vitro model of massive blood loss and thrombocytopenia.

This study compared the corrective effects of storage of platelets at 4°C and at 22°C in an in vitro model of massive blood loss and thrombocytopenia to provide an experimental basis for the storage of platelets for clinical applications.In vitro model of massive blood loss and thrombocytopenia were constructed by the in vitro hemodilution method and cell washing method. Using storage of platelets at 4°C (1, 3, 5, 7, 10, 14 days) and at 22°C (1, 3, 5 days) to correct the coagulation condition of the different models, by thromboelastography and by routine blood indices.①Platelets stored at 4°C (1, 3, 5,7, 10, 14 days) and at 22°C (1, 3, 5 days) to correct the in vitro model of massive blood loss. Platelet count results improved from 17 to 27 × 10/L to greater than 120 × 10/L for 4°C storage, and 20 to 27 × 10/L to greater than 120 × 10/L for 22°C storage. Thromboelastography maximum amplitude (TEG-MA) results improved from 8.8 to 15.4 mm to greater than 43 mm for 4°C storage, and 12.2 to 14.4 mm to greater than 44.8 mm for 22°C storage. Thromboelastography reaction time values decreased from 9.9-24.9 minutes to 3.8-5.5 minutes for 4°C storage, and 9.9-22.7 minutes to 4.3-4.5 minutes for 22°C storage. ②Platelets stored at 4°C (1, 3, 5,7, 10, 14 days) and at 22°C (1, 3, 5 days) to correct the in vitro model of thrombocytopenia. Platelet count results improved from 12 to 34 × 10/L to greater than 99 × 10/L for 4°C storage, and 12 to 34 × 10/L to greater than 120 × 10/L for 22°C storage. TEG-MA results improved from 21.4 to 32.1 mm to greater than 49.1 mm for 4°C storage, and 21.4 to 31.6 mm to greater than 50.5 mm for 22°C storage.Platelets stored at 4°C and 22°C have the same correcting effect for 1, 3, and 5 days. Platelets stored at 4°C for 7 to 14 days have similarly hemostatic effect on the in vitro model of massive blood loss and thrombocytopenia.

Degradation of phenolic compounds with simultaneous bioelectricity generation in microbial fuel cells: Influence of the dynamic shift in anode microbial community.

This study evaluated the feasibility of microbial fuel cells (MFCs) for simultaneous electricity generation and degradation of phenolic compounds. The voltage generation was inhibited by 36.18-63.90%, but the degradation rate increased by 146.15-392.31% when the initial concentration of syringic acid (SA), vanillic acid (VA), and 4-hydroxybenzoic acid (HBA) increased from 0.3 to 3.0 g/L. The collaboration among the functional microbes significantly enhanced the degradation rate of parent compounds and their intermediates in MFCs systems, while the accumulated intermediates severely inhibited their complete mineralization in fermentative systems. High-throughput sequencing showed that the growth of fermentative bacteria prevailed, but electrogenic bacteria were inhibited in the anode microbial community (AMC) under high concentrations of phenolic compounds (3.0 g/L). These findings provide a better understanding of the dynamic shift and synergy effects of the AMC to evaluate its potential for the treatment of phenolic-containing wastewater.

Downregulation of ß1,4-galactosyltransferase 5 improves insulin resistance by promoting adipocyte commitment and reducing inflammation.

Protein glycosylation is an important post-translational modification. Aberrant glycosylation has been implicated in many diseases because of associated changes in protein distribution and biological function. We showed that the expression of ß1, 4-galactosyltransferase 5 (B4GalT5) was positively correlated with diabetes and obesity. In vivo, B4GalT5 knockdown in subcutaneous adipose tissue alleviated insulin resistance and adipose tissue inflammation, and increased adipogenesis in high-fat diet (HFD)-fed mice and ob/ob mice. Downregulation of B4GalT5 in preadipocyte cells induced commitment to the adipocyte lineage in the absence of bone morphogenetic protein (BMP) 2/4 treatment, which is typically essential for adipogenic commitment. RNAi silencing experiments showed B4GalT5 knockdown activated Smad and p38 MPAK signaling pathways through both type 1A and 2 BMP receptors. Remarkably, B4GalT5 knockdown decreased BMPRIA glycosylation but increased BMPRIA stability and cellular location, thus leading to redistribution of BMPRIA and activation of the BMP signaling pathway. Meanwhile, downregulation of B4GalT5 decreased the infiltration of macrophages and the markers of M1 macrophages in subcutaneous adipose tissue of HFD mice and ob/ob mice. In bone marrow-derived macrophages (BMDMs) and RAW264.7cells, B4GalT5 knockdown also repressed the markers of M1 by reducing NFκB and JNK signaling. These results demonstrated B4GalT5 downregulation improved insulin resistance by promoting adipogenic commitment and decreasing M1 macrophage infiltration.

Preparative separation and quantitative determination of two kaurenoic acid isomers in root barks of Acanthopanax gracilistylus.

The kaurenoic acid-type diterpenoids in Acanthopanacis Cortex have been reported to be the major active components. However, the diterpenoids are present as position isomers that exacerbate the challenges in obtaining standards compounds. Little work has been done on the quantitative analysis of the diterpenoids in the herb. In the present study, two diterpenoid isomers ent-16ßH,17-isovalerate-kauran-19-oic acid (1) and ent-16ßH,17-methyl butanoate-kauran-19-oic acid (2) with high purity were separated by analytical HPLC, followed by recrystallization in acetone. Furthermore, an HPLC-ELSD method was developed and validated for simultaneous determination of 1 and 2 in 9 batches of Acanthopanacis Cortex samples. The HPLC separation and quantification was achieved in 40 min using an Agela Promosil C18 column eluted with a gradient of water and acetonitrile. The calibration curves showed good linearity (r2 ≥ 0.999 9) within the test ranges. The LOD ranged from 0.407 2 to 0.518 0 µg and LOQ ranged from 1.018 0 to 1.295 0 µg. The precisions (%RSD) were within 1.47% for the two isomers. The recovery of the assay was in the range of 98.78%-99.11% with RSD values less than 2.76%. It is the first time to establish a quantitative HPLC method for the analysis of the bioactive kaurenoic acid isomers in the herb.