Search

The emergence of new therapeutic modalities requires complementary tools for their efficient syntheses. Availability of methodologies for site-selective modification of biomolecules remains a long-standing challenge, given the inherent complexity and the presence of repeating residues that bear functional groups with similar reactivity profiles. We describe a bioconjugation strategy for modification of native peptides relying on high site selectivity conveyed by enzymes. We engineered penicillin G acylases to distinguish among free amino moieties of insulin (two at amino termini and an internal lysine) and manipulate cleavable phenylacetamide groups in a programmable manner to form protected insulin derivatives. This enables selective and specific chemical ligation to synthesize homogeneous bioconjugates, improving yield and purity compared to the existing methods, and generally opens avenues in the functionalization of native proteins to access biological probes or drugs.

An efficient method of recovering and recycling solvent for counter-current chromatography was established by which zeaxanthin was separated from Lycium barbarum L. fruits. A column with activated carbon combined with high performance counter-current chromatography formed the recovering and recycling solvent system. Using the solvent system of n-hexane-ethyl acetate-ethanol-water (8:2:7:3, v/v) from the references, five injections were performed with an almost unchanged purity of zeaxanthin (80.9, 81.2, 81.5, 81.3, and 80.2% respectively) in counter-current chromatography separation. Meanwhile, the mobile phase reduced by half than conventional counter-current chromatography. By this present method, an effective improvement of counter-current chromatography solvent utilization was achieved.

The chiral separation by counter-current chromatography has made great progress in the past three decades. It has become increasingly popular in the field of chiral separation, and many applications have been introduced during the last years. This review mainly focuses on the current topics, applications, and trends in chiral separation by counter-current chromatography. It contains the development of modern counter-current chromatography apparatus, theory of counter-current chromatography, overview of applications of chiral counter-current chromatography enantioseparation, its current situation, and challenges. At last, some conclusions and perspectives also have been discussed in this review.

Mesenchymal Stem Cells (MSCs) are distributed in many parts of the human body, including the bone marrow, placenta, umbilical cord, fat, and nasal mucosa. One of the unique features of MSCs is their multidirectional differentiation potential, including the ability to undergo osteogenesis, adipogenesis, and chondrogenesis, and to produce neurons, endothelial cells, Schwann cells, medullary nucleus cells, cardiomyocytes, and alveolar epithelial cells. MSCs have thus become a hot research topic in recent years. Numerous studies have investigated the differentiation of MSCs into various types of cells in vitro and their application to numerous fields. However, most studies have cultured MSCs under atmospheric oxygen tension with an oxygen concentration of 21%, which does not reflect a normal physiological state, given that the oxygen concentration generally used in vitro is four to ten times that to which MSCs would be exposed in the body. We therefore review the growing number of studies exploring the effect of hypoxic preconditioning on the differentiation of MSCs.

Microglial cells are the first line immune cells that initiate inflammatory responses following cerebral ischemia/reperfusion(I/R) injury. Microglial cells are also associated with a novel subtype of pro-inflammatory programmed cell death known as pyroptosis. Research has been directed at developing treatments that modulate inflammatory responses and protect against cell death caused by cerebral I/R. Key among such treatments include mesenchymal stem cell (MSC) therapy. A unique type of MSC termed olfactory mucosa mesenchymal stem cell (OM-MSC) confers neuroprotection by promoting the secretion of paracrine factors, and neuroprotection. This study investigated whether hypoxic OM-MSCs could inhibit microglial cell death upon I/R insult in vitro. A traditional oxygen-glucose deprivation/reperfusion (OGD/R) model, analogous to I/R, was established. Results showed that OGD/R induced apoptosis and pyroptosis in microglial cells while hypoxia in OM-MSCs significantly attenuated these effects. Moreover, the effects of OM-MSCs were mediated by Hypoxia-inducible factor 1-alpha (HIF-1α). Taken together, these findings reveal that hypoxia-preconditioned OM-MSC inhibits pyroptotic and apoptotic death of microglial cell in response to cerebral ischemia/reperfusion insult by activating HIF-1α in vitro.

Maximal efficiency from enzyme cascades Enzymes are highly selective catalysts that can be useful for specific transformations in organic synthesis. Huffman et al. combined designer enzymes in a multistep cascade reaction (see the Perspective by O'Reilly and Ryan). The approach eliminates purification steps, recycles expensive cofactors, and couples favorable and unfavorable reactions. With the target molecule islatravir, an experimental HIV drug, they optimized five enzymes by directed evolution to be compatible with unnatural substrates and stable in the reaction conditions. Stereochemical purity was amplified at every enzymatic step, and the final synthesis was both atom economical and efficient. Science , this issue p. 1255 ; see also p. 1199

Mesenchymal stem cells (MSCs) play important roles in tissue repair and regeneration, such as the induction of angiogenesis, particularly under hypoxic conditions. However, the molecular mechanisms underlying hypoxic MSC activation remain largely unknown. MSC-derived extracellular vesicles (EVs) are vital mediators of cell-to-cell communication and can be directly utilized as therapeutic agents for tissue repair and regeneration. Here, we explored the effects of EVs from human hypoxic olfactory mucosa MSCs (OM-MSCs) on angiogenesis and its underlying mechanism. EVs were isolated from normoxic (N) OM-MSCs (N-EVs) and hypoxic (H) OM-MSCs (H-EVs) using differential centrifugation and identified by transmission electron microscopy and flow cytometry. In vitro and in vivo, both types of OM-MSC-EVs promoted the proliferation, migration, and angiogenic activities of human brain microvascular endothelial cells (HBMECs). In addition, angiogenesis-stimulatory activity in the H-EV group was significantly enhanced compared to the N-EV group. MicroRNA profiling revealed a higher abundance of miR-612 in H-EVs than in N-EVs, while miR-612 inactivation abolished the N-EV treatment benefit. To explore the roles of miR-612, overexpression and knock-down experiments were performed using a mimic and inhibitor or agomir and antagomir of miR-612. The miR-612 target genes were confirmed using the luciferase reporter assay. Gain- and loss-of-function studies allowed the validation of miR-612 (enriched in hypoxic OM-MSC-EVs) as a functional messenger that stimulates angiogenesis and represses the expression of TP53 by targeting its 3′-untranslated region. Further functional assays showed that hypoxic OM-MSC-EVs promote paracrine Hypoxia-inducible factor 1-alpha (HIF-1α)-Vascular endothelial growth factor (VEGF) signaling in HBMECs via the exosomal miR-612-TP53-HIF-1α-VEGF axis. These findings suggest that hypoxic OM-MSC-EVs may represent a promising strategy for ischemic disease by promoting angiogenesis via miR-612 transfer. Graphical Abstract

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence

A target-guidance separation strategy composed of activity screening process with high-performance liquid chromatography and separating process with high-speed countercurrent chromatography (HSCCC)...

In this work, to further verify and develop the novel counter-current chromatography modified column separate mode, a melamine modified counter-current chromatography column was prepared. Meanwhile, the modified counter-current chromatography column was used to separate stevioside and rebaudioside A with the same partition coefficient in chosen solvent system to evaluate its separation efficiency. The results show that because of the presence of intermolecular forces between melamine and model compounds, better separation could be achieved on the modified column while it's almost impossible to be separated on the unmodified column. So the results of this research further show that column modified method is a possible approach to further increase the separation ability of counter-current chromatography. Take advantage of large sample handing capacity of counter-current chromatography, the mothed may have great potential to be an efficient method of separation and preparation enantiomer compounds.

Cerebral ischemia/reperfusion injury causes a series of intricate cascade reactions in brain tissue causing apoptosis and proinflammatory programmed cell death known as pyroptosis of nerve cells. The dysfunction of target organelle mitochondria plays a key role in the process of neuronal apoptosis and pyroptosis. Mesenchymal stem cells (MSCs) have been widely used in the experimental or clinical treatment of various ischemic diseases, but the therapeutic efficacy of MSCs on cerebral ischemia-reperfusion injury need to be improved. We successfully cultured olfactory mucosa MSCs (OM-MSCs) to obtain a better source of seed cells. In this way, the therapeutic potential of OM-MSCs transplantation has been evaluated for ischemic stroke using an optimized culture scheme in vitro. Ischemic-hypoxic preconditioned OM-MSCs (IhOM-MSCs) were used to treat a neuron model of oxygen-glucose deprivation/reperfusion and the middle cerebral artery occlusion in rats. These results demonstrated that IhOM-MSCs mediated the upregulation of the downstream target genes GRP78 and Bcl-2 by miR-181a to protect mitochondrial function and inhibit apoptosis and pyroptosis of neurons in the ischemia/reperfusion injury model. Thus, IhOM-MSCs transplantation may be an effective therapy of ischemic stroke in the future.

Mesenchymal stromal cells (MSCs) have been used for the treatment of neuronal injury and neurodegenerative diseases. Their underlying mechanism may involve increased secretion of paracrine factors, which promotes tissue repair. Presently, exosomes have been regarded as important components of paracrine secretion and paracrine factors. MSC exosomes represent a promising opportunity to develop novel cell-free therapy approaches. In this study, exosomes from nasal olfactory mucosa MSCs (OM-MSCs) were extracted and purified using ultracentrifugation, resulting in exosome diameters of 40–130 nm. Similar to other exosomes, OM-MSC exosomes were CD63- and CD81-positive and calnexin-negative. Functionally, OM-MSC exosomes promoted human brain microvascular endothelial cell (HBMEC) proliferation and migration. The present study analyzed the OM-MSC exosome paracrine proteome. A total of 304 exosome-associated proteins were identified by LC–MS/MS, including plasminogen activator inhibitor 1 (SERPINE 1), insulin-like growth factor binding protein family members (IGFBP 4 and 5), epidermal growth factor receptor (EGFR), neurogenic locus notch homolog protein 2 (NOTCH 2), apolipoprotein E (APOE), and heat shock protein HSP90-beta (HSP90AB1). These molecules are known to be important in neurotrophic, angiogenesis, cell growth, differentiation, apoptosis, and inflammation and are highly correlated with the mechanism of tissue repair and neural restoration. These observations may provide a basis for further evaluation of OM-MSC exosome potential as a novel therapeutic modality.

Elution-extrusion counter current chromatography extrudes the most solute retained in the column with the highest possible peak resolution. It can greatly improve the hydrophobic window. In recent years, elution-extrusion counter current chromatography has received extensive attention in the separation of complex samples. This article first reviews the development and application of elution-extrusion counter current chromatography, including its origin, mechanism, advantages and disadvantages, and some representative applications. At the same time, this review also shared our visions and ideas on how to improve the elution-extrusion mode. This article aims to provide certain reference for the research of this technology.

An efficient combination strategy based on high-speed shear dispersing emulsifier technique and high-performance countercurrent chromatography was developed for on-line extraction and isolation of carotenoids from the fruits of Lycium barbarum. In this work, the high-speed shear dispersing emulsifier technique has been employed to extract crude extracts using the upper phase of high-performance countercurrent chromatography solvent system composed of n-hexane-dichloromethane-acetonitrile (10:4:6.5, v/v) as the extraction solvent. At the separation stage, the high-performance counter-current chromatography process adopts elution-extrusion mode and the upper phase of the solvent system as stationary phase (reverse-phase mode). As a result, three compounds including zeaxanthin, zeaxanthin monopalmitate, and zeaxanthin dipalmitate with purities of 89, 90, and 93% were successfully obtained in one extraction-separation operation within 120 min. The targeted compounds were analyzed and identified by high-performance liquid chromatography, mass spectrometry, and NMR spectroscopy. The results indicated that the present on-line combination method could serve as a simple, rapid, and effective way to achieve weak polar and unstable compounds from natural products.

The stationary phase retention is one of the most important parameters in countercurrent chromatography. In this work, a simple gradient equilibrium method was developed to further improve the stationary phase retention based on the optimized condition in the traditional equilibrium model. Meanwhile, this novel gradient equilibrium method was used to separate three flavone model compounds and compared with the conventional isocratic equilibrium method to evaluate the separation efficiency. The results show that better resolution or shorter separation time could be achieved with gradient equilibrium compared to isocratic equilibrium. So this novel equilibrium method has enormous potential for obtaining a better separation or saving the separating time in the preparative separation of target compounds.

Background : Pre-hospital emergency airway management plays an important role in pre-hospital care. Laryngeal masks are increasingly employed for the airway management of pre-hospital critical patients and have achieved promising results. Although several randomized controlled trials have reported benefits, the efficacy of laryngeal masks in pre-hospital emergency airway management compared to endotracheal intubation have not been systematically reviewed. Methods: Electronic databases (PubMed, Cochrane Library, Embase, Scopus and CNKI) were searched up to April 2019 for related randomized studies. Outcome indicators included overall intubation success rates, the success rates of the first intubation, insertion time, resuscitation efficiency rates, SpO2 rise time, the blood gas index and adverse events. Two investigators selected the trials, extracted the data according to inclusion and exclusion criteria, and assessed the quality of the literature according to the Jada score. The meta-analysis was performed using stata14.0 software. Results: We included 31 human studies. Compared to endotracheal intubation, the application of laryngeal mask for pre-hospital emergencies enhanced the resuscitation efficiency rates [RR=1.20, 95% CI (1.06, 1.35), P

Objective To investigate the efficacy of poly (lactic acid co castor oil) microspheres containing ropivacaine for sciatic nerve block of mice. Methods A total of 150 Kunming male mice were randomly assigned into 3 groups, namely placebo microspheres (lactic acid co castor oil) group (group A, n=50), ropivacaine injection group (group B, n=50) and ropivacaine microspheres group (group C, n=50). After sevoflurane anesthesia, the mouse was fixed on the operating table and the bilateral sciatic nerve was exposed. The corresponding preparations were implanted or injected near the sciatic nerve. Five mice were randomly selected from each group for the next experiments. Paw withdrawal thermal latency, the ability to splay and flex of the hind paw and plasma ropivacaine concentration were measured 10min, 30min, 1h, 3h, 5h, 7h, 10h, 15h, 30h and 48h after drug administration. Results The anesthetic effect of group C began to work at 3h. Compared with group B, the duration of sciatic nerve sensory block of group C was significantly longer and the effect of motor block was weaker. No anesthetic effect was observed in group A. The sensory and motor block of group B reached the peak at 1h, and the pharmacodynamics subsided at 7h. Compared with group B, the concentration of ropivacaine in group C increased slowly, and the peak value at 10h after administration was gradually decreased. Conclusions Ropivacaine loading poly (lactic acid co castor oil) microspheres can significantly extend the effect of ropivacaine on sciatic nerve sensory block. Compared with ropivacaine injection, motor block effect of ropivacaine loading poly (lactic acid co castor oil) microspheres is reduced and its plasma ropivacaine concentration fluctuation range is small. DOI: 10.11855/j.issn.0577-7402.2018.04.06

While the formation of colloidal aggregates leads to artifacts in early drug discovery, their composition makes them attractive as nanoparticle formulations for targeted drug delivery as the entire nanoparticle is composed of drug. The typical transient stability of colloidal aggregates has inhibited exploiting this property. To overcome this limitation, we investigated a series of proteins to stabilize colloidal aggregates of the chemotherapeutic, fulvestrant, including the following: bovine serum albumin, a generic human immunoglobulin G, and trastuzumab, a therapeutic human epidermal growth factor receptor 2 antibody. Protein coronas reduced colloid size to

Colloidal aggregates of small molecules are the most common artifact in early drug discovery, sequestering and inhibiting target proteins without specificity. Understanding their structure and mechanism has been crucial to developing tools to control for, and occasionally even exploit, these particles. Unfortunately, their polydispersity and transient stability have prevented exploration of certain elementary properties, such as how they pack. Dye-stabilized colloidal aggregates exhibit enhanced homogeneity and stability when compared to conventional colloidal aggregates, enabling investigation of some of these properties. By small-angle X-ray scattering and multiangle light scattering, pair distance distribution functions suggest that the dye-stabilized colloids are filled, not hollow, spheres. Stability of the coformulated colloids enabled investigation of their preference for binding DNA, peptides, or folded proteins, and their ability to purify one from the other. The coformulated colloids showed little ability to bind DNA. Correspondingly, the colloids preferentially sequestered protein from even a 1600-fold excess of peptides that are themselves the result of a digest of the same protein. This may reflect the avidity advantage that a protein has in a surface-to-surface interaction with the colloids. For the first time, colloids could be shown to have preferences of up to 90-fold for particular proteins over others. Loaded onto the colloids, bound enzyme could be spun down, resuspended, and released back into buffer, regaining most of its activity. Implications of these observations for colloid mechanisms and utility will be considered.

Ting Yuan,1* Yi Zhuo,1* Chaying Su,2 Xuan Li,1 Da Duan,1 Lite Ge,1 Pei Wu,1 Hao Wang,1 Yujia Deng,1 Ming Lu1 1Department of Neurosurgery, Second Affiliated Hospital of Hunan Normal University (163 Hospital of the People’s Liberation Army), Changsha, 2Department of Pediatrics, 169 Hospital of the People’s Liberation Army, Hengyang, Peoples’ Republic ofChina * These authors contributed equally to this work Abstract: Human olfactory mucosa mesenchymal-like stem cells (hOM-MSCs) secrete paracrine factors that may exert a protective effect on the cerebral ischemia. This study was done to determine the hypoxic and ischemic effects on the mRNA and protein expression level of paracrine factors by hOM-MSCs. The hOM-MSCs were cultured with 5% or 20% serum and under either normoxic (21% O2) or hypoxic (3% O2) conditions. Expression levels of mRNA and protein for vascular endothelial growth factor (VEGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and matrix metalloproteinase-2 (MMP-2) were determined by quantitative reverse transcription polymerase chain reaction and Western blot, respectively. Hypoxia reduced gene expression for VEGF (5% serum), GDNF, BDNF (5% serum) and NGF (5% serum) and increased it for BDNF (20% serum), MMP-2 (5% serum) and NGF (20% serum). Ischemia lowered gene expression for VEGF (hypoxia), GDNF, BDNF (hypoxia), MMP-2 (normoxia) and NGF (hypoxia) and increased it for VEGF (normoxia), BDNF (normoxia), MMP-2 (hypoxia) and NGF (normoxia).The protein level of these factors was almost in line with the gene level. These data demonstrate that serum and oxygen levels have a significant effect on the gene and protein expression levels of paracrine factors by hOM-MSCs, which will affect how hOM-MSCs interact in vivo during cerebral ischemia. Keywords: human olfactory mucosa mesenchymal-like stem cells, paracrine factors, hypoxia, ischemia, cerebral ischemia

Countercurrent chromatography is a kind of continuous and effective liquid-liquid partition chromatography with many unique characteristics such as large load capacity, no irreversible adsorption, high recovery rate, low risk of sample denaturation and so on, which has irreplaceable advantages in the separation of proteins and peptides. This review presents the advances of several kinds of new technology of countercurrent chromatography in the separation of proteins and peptides. The development prospect of this field is also discussed.

Transplantation of olfactory ensheathing cells (OECs) has been shown to enhance synapse formation. However, the mechanisms underlying this effect are not completely understood. We performed profiling of the OEC and astrocyte secretomes via a proteomics approach, in case hevin secreted by astrocytes might be involved in the formation of synapses. Semi-quantitative proteomic analysis revealed that 25 proteins were highly expressed, and 22 were weakly expressed in OEC conditioned medium compared with astrocyte conditioned medium. These molecules are highly associated with neural differentiation and regeneration, enzyme regulatory activity, and growth factor binding. The quantification data of clusterin, fibronectin, hevin, insulin-like growth factor binding protein 2 and secreted protein acidic and rich in cysteine were further confirmed by western blotting. Moreover, the addition of hevin in the culture medium improved neurite sprouting and outgrowth of differentiated neural stem cells. The greater expression of hevin in OEC conditioned medium than in astrocyte conditioned medium was associated with a greater capacity of synaptic formation. Thus, our results indicate that soluble factors secreted by OECs provide a permissive environment for nerve repair, and hevin is one of the key molecules facilitating neurite sprouting and outgrowth.

Small molecule aggregates are considered nuisance compounds in drug discovery, but their unusual properties as colloids could be exploited to form stable vehicles to preserve protein activity. We investigated the co-aggregation of seven molecules chosen because they had been previously intensely studied as colloidal aggregators, co-formulating them with bis-azo dyes. The co-formulation reduced colloid sizes to

A combinative method using the 2,2-diphenyl-1-picrylhydrazyl (DPPH)-high performance liquid chromatography (HPLC)-diode array detector (DAD) and high-speed counter-current chromatography (HSCCC) has been developed to screen and separate radical scavengers from the water extract of Cynomorium songaricum Rupr. Under the target guidance of the DPPH-HPLC-DAD experiment, 3 compounds were found to possess potential antioxidant activities. In order to identify the chemical structures of those compounds, the HSCCC method with a two-phase solvent system composed of ethyl acetate–water (1 : 1; v/v) was developed to isolate and purify the active compounds. Three compounds, 14.3 ± 0.7 mg of (+)-catechin (1), 15.2 ± 0.6 mg of (−)-epicatechin (2) and 11.3 ± 0.4 mg oleuropein (3) were obtained from 600 mg of crude sample with purities of 97.8%, 98.3% and 95.6%, respectively, as determined by HPLC. Their structures were elucidated by electrospray ionization-mass spectrometry (ESI-MS), 1H nuclear magnetic resonance (NMR) and 13C NMR. Among them, oleuropein was obtained from C. songaricum for the first time. The peak order of the three compounds in the HSCCC chromatogram was completely different from that in the HPLC chromatogram. Even both HSCCC and HPLC methods that are employed in this paper were in reversed phase mode. The elution order of HSCCC was determined by the hydrophobicity of the compounds, while the elution order of HPLC may be affected by some other interactions between the stationary phase and the compounds such as hydrogen bonding and steric effects. In addition, antioxidant activities of the three compounds were evaluated by the methods of DPPH radical scavenging assay. All of them showed high radical scavenging activities with the EC50 values being 16.97 ± 0.02, 11.15 ± 0.09 and 41.32 ± 0.20 μg mL−1 for (+)-catechin, (−)-epicatechin and oleuropein, respectively.

Nasal olfactory mucosa mesenchymal stem cells (OM-MSCs) have the ability to promote regeneration in the nervous systemin vivo. Moreover, with view to the potential for clinical application, OM-MSCs have the advantage of being easily accessible from patients and transplantable in an autologous manner, thus eliminating immune rejection and contentious ethical issues. So far, most studies have been focused on the role of OM-MSCs in central nervous system replacement. However, the secreted proteomics of OM-MSCs have not been reported yet. Here, proteins secreted by OM-MSCs cultured in serum-free conditions were separated on SDS-PAGE and identified by LC-MS/MS. As a result, a total of 274 secreted proteins were identified. These molecules are known to be important in neurotrophy, angiogenesis, cell growth, differentiation, and apoptosis, and inflammation which were highly correlated with the repair of central nervous system. The proteomic profiling of the OM-MSCs secretome might provide new insights into their nature in the neural recovery. However, proteomic analysis for clinical biomarkers of OM-MSCs needs to be further studied.

Antioxidants play an essential role in human health, as they have been found to be capable of lowering the incidence of many diseases, such as cancer and angiocardiopathy. Currently, more attention is paid to natural antioxidants because of the possible insecurity of synthetic antioxidants. Thus, the development of efficient techniques or methods to separate antioxidants from natural sources is requested urgently. High-speed counter current chromatography (HSCCC) is a unique support-free liquid-liquid chromatographic technique and has been widely applied in the field of separation of natural products. In this review, we summarize and analyze the related researches on the application of HSCCC in the separation of various natural antioxidants so far. The purpose of the article is to provide a certain theoretical support for the separation of natural antioxidants by HSCCC, and to make full use of advantages of HSCCC in the separation of bioactive components. In particular, some key problems associated with the separation strategies, the structural categories of natural antioxidants, solvent system choices, and the application of different elution modes in HSCCC separation, are summarized and commented. We expect that the content reviewed can offer more evidence for the development of the field of natural antioxidants separation, so as to achieve large-scale preparation of natural antioxidants.