Several Affinity Tags Commonly Used in Chromatographic Purification

Affinity tags have become powerful tools from basic biological research to structural and functional proteomics. They were widely used to facilitate the purification and detection of proteins of interest, as well as the separation of protein complexes. Here, we mainly discuss the benefits and drawbacks of several affinity or epitope tags frequently used, including hexahistidine tag, FLAG tag, Strep II tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, and c-Myc tag. In some cases, a large-size affinity tag, such as GST or MBP, can significantly impact on the structure and biological activity of the fusion partner protein. So it is usually necessary to excise the tag by protease. The most commonly used endopeptidases are enterokinase, factor Xa, thrombin, tobacco etch virus, and human rhinovirus 3C protease. The proteolysis features of these proteases are described in order to provide a general guidance on the proteolytic removal of the affinity tags

Neural Dysfunction Underlying Working Memory Processing at Different Stages of the Illness Course in Schizophrenia:A Comparative Meta-analysis

Schizophrenia (SCZ), as a chronic and persistent disorder, exhibits working memory deficits across various stages of the disorder, yet the neural mechanisms underlying these deficits remain elusive with inconsistent neuroimaging findings. We aimed to compare the brain functional changes of working memory in patients at different stages: clinical high risk (CHR), first-episode psychosis (FEP), and long-term SCZ, using meta-analyses of functional magnetic resonance imaging (fMRI) studies. Following a systematic literature search, fifty-six whole-brain task-based fMRI studies (15 for CHR, 16 for FEP, 25 for long-term SCZ) were included. The separate and pooled neurofunctional mechanisms among CHR, FEP and long-term SCZ were generated by Seed-based d Mapping toolbox. The CHR and FEP groups exhibited overlapping hypoactivation in the right inferior parietal lobule, right middle frontal gyrus, and left superior parietal lobule, indicating key lesion sites in the early phase of SCZ. Individuals with FEP showed lower activation in left inferior parietal lobule than those with long-term SCZ, reflecting a possible recovery process or more neural inefficiency. We concluded that SCZ represent as a continuum in the early stage of illness progression, while the neural bases are inversely changed with the development of illness course to long-term course

Effect of Eucommia ulmoides extract on osteoblast proliferation

Purpose: To evaluate the effect of Eucommia ulmoides extract (EUE) on osteoblast proliferation as well as investigate its probable mechanisms of action.Methods: EUE was pharmacologically evaluated at three doses. Osteoblast cells were divided as follows: Group I: negative control; groups II–IV: received EUE (180, 360 and 540 μg/ml, respectively). We performed 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay to determine osteoblast viability following treatment. Alkaline phosphatase (ALP), osteocalcin, and collagen I levels in osteoblasts were quantified using commercially available kits. Thereafter, mRNA and protein expression of ALP, collagen I, osteocalcin, transforming growth factor-β1 (TGF-β1) were measured using real-time quantitative PCR (qPCR) and western blot, respectively.Results: EUE significantly (p < 0.01) promoted osteoblast proliferation at three treatment doses (180, 360, and 540 μg/mL). Furthermore, ALP, osteocalcin, collagen I and TGF-β1 expression at both mRNA and protein levels increased significantly (all p < 0.05) following EUE treatment.Conclusion: The results suggest that EUE may promote osteoblast cell proliferation and that ALP, osteocalcin, collagen I and TGF-β1 gene expression may be involved in the mechanism of action.Keywords: qPCR, collagen I, Bone, Liver, Eucommia ulmoides extrac

Roles of microRNA on cancer cell metabolism

Advanced studies of microRNAs (miRNAs) have revealed their manifold biological functions, including control of cell proliferation, cell cycle and cell death. However, it seems that their roles as key regulators of metabolism have drawn more and more attention in the recent years. Cancer cells display increased metabolic autonomy in comparison to non-transformed cells, taking up nutrients and metabolizing them in pathways that support growth and proliferation. MiRNAs regulate cell metabolic processes through complicated mechanisms, including directly targeting key enzymes or transporters of metabolic processes and regulating transcription factors, oncogenes / tumor suppressors as well as multiple oncogenic signaling pathways. MiRNAs like miR-375, miR-143, miR-14 and miR-29b participate in controlling cancer cell metabolism by regulating the expression of genes whose protein products either directly regulate metabolic machinery or indirectly modulate the expression of metabolic enzymes, serving as master regulators, which will hopefully lead to a new therapeutic strategy for malignant cancer. This review focuses on miRNA regulations of cancer cell metabolism,including glucose uptake, glycolysis, tricarboxylic acid cycle and insulin production, lipid metabolism and amino acid biogenesis, as well as several oncogenic signaling pathways. Furthermore, the challenges of miRNA-based strategies for cancer diagnosis, prognosis and therapeutics have been discussed

Maternal control of axial–paraxial mesoderm patterning via direct transcriptional repression in zebrafish

AbstractAxial–paraxial mesoderm patterning is a special dorsal–ventral patterning event of establishing the vertebrate body plan. Though dorsal–ventral patterning has been extensively studied, the initiation of axial–paraxial mesoderm pattering remains largely unrevealed. In zebrafish, spt cell-autonomously regulates paraxial mesoderm specification and flh represses spt expression to promote axial mesoderm fate, but the expression domains of spt and flh initially overlap in the entire marginal zone of the embryo. Defining spt and flh territories is therefore a premise of axial–paraxial mesoderm patterning. In this study, we investigated why and how the initial expression of flh becomes repressed in the ventrolateral marginal cells during blastula stage. Loss- and gain-of-function experiments showed that a maternal transcription factor Vsx1 is essential for restricting flh expression within the dorsal margin and preserving spt expression and paraxial mesoderm specification in the ventrolateral margin of embryo. Chromatin immunoprecipitation and electrophoretic mobility shift assays in combination with core consensus sequence mutation analysis further revealed that Vsx1 can directly repress flh by binding to the proximal promoter at a specific site. Inhibiting maternal vsx1 translation resulted in confusion of axial and paraxial mesoderm markers expression and axial–paraxial mesoderm patterning. These results demonstrated that direct transcriptional repression of the decisive axial mesoderm gene by maternal ventralizing factor is a crucial regulatory mechanism of initiating axial–paraxial mesoderm patterning in vertebrates

Effect of dietary soy isoflavones on bone loss in ovariectomized rats

Purpose: To determine the effect of dietary soy isoflavone supplementation on bone loss in ovariectomized (OVX) rats.Methods: Forty-eight rats were assigned randomly to groups of OVX rats receiving soy isoflavones (20, 30, or 40 mg/kg of body weight daily), untreated OVX rats, or untreated intact rats. After 8 weeks, bone mineral density (BMD), mineral (Ca, P, Mn, Mg, and Zn) concentrations, and the expression of osteoblast-related genes were measured in femur tissue samples.Results: Eight weeks after OVX, there was a significant decrease in body weight, serum levels of osteocalcin, alkaline phosphatase, and oestradiol, BMD and mineral elements, as well as the expressions of Ctnnb1, Runx2, and Sp7 (all p < 0.05). These decreases were accompanied by reduced maximum load capacity of lumbar vertebrae. Daily supplementation with soy isoflavones dosedependently ameliorated these effects (all p < 0.05). Western blotting revealed that these effects were likely due to the reversal of the OVX-induced decrease in Notch1 proteins in bone and muscle.Conclusion: Soy isoflavone treatment represents a potential therapy for preventing postmenopausal bone loss by stimulating the Notch signalling.Keywords: Mineral elements, Alkaline phosphatase, Isoflavones, Bone loss, Notch pathwa

Comparisons of three polyethyleneimine-derived nanoparticles as a gene therapy delivery system for renal cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Polyethyleneimine (PEI), which can interact with negatively charged DNA through electrostatic interaction to form nanocomplexes, has been widely attempted to use as a gene delivery system. However, PEI has some defects that are not fit for keeping on gene expression. Therefore, some modifications against PEI properties have been done to improve their application value in gene delivery. In this study, three modified PEI derivatives, including poly(ε-caprolactone)-pluronic-poly(ε-caprolactone) grafted PEI (PCFC-g-PEI), folic acid-PCFC-isophorone diidocyanate-PEI (FA-PEAs) and heparin-PEI (HPEI), were evaluated in terms of their cytotoxicity and transfection efficiency <it>in vitro </it>and <it>in vivo </it>in order to ascertain their potential application in gene therapy.</p> <p>Methods</p> <p>MTT assay and a marker GFP gene, encoding green fluorescent protein, were used to evaluate cell toxicity and transfection activity of the three modified PEI <it>in vitro</it>. Renal cell carcinoma (RCC) models were established in BALB/c nude mice inoculated with OS-RC-2 cells to detect the gene therapy effects using the three PEI-derived nanoparticles as gene delivery vehicles. The expression status of a target gene Von Hippel-Lindau (VHL) in treated tumor tissues was analyzed by semiquantitative RT-PCR and immunohistochemistry.</p> <p>Results</p> <p>Each of three modified PEI-derived biomaterials had an increased transfection efficiency and a lower cytotoxicity compared with its precursor PEI with 25-kD or 2-kD molecule weight <it>in vitro</it>. And the mean tumor volume was obviously decreased 30% by using FA-PEAs to transfer VHL plasmids to treat mice RCC models. The VHL gene expression was greatly improved in the VHL-treated group. While there was no obvious tumor inhibition treated by PCFC-g-PEI:VHL and HPEI:VHL complexes.</p> <p>Conclusions</p> <p>The three modified PEI-derived biomaterials, including PCFC-g-PEI, FA-PEAs and HPEI, had an increased transfection efficiency <it>in vitro </it>and obviously lower toxicities compared with their precursor PEI molecules. The FA-PEAs probably provide a potential gene delivery system to treat RCC even other cancers in future.</p

Effect of Zishen Jiangtang Pill, a Chinese Herbal Product, on Rats with Diabetic Osteoporosis

Diabetic osteoporosis (DO) is a complication of diabetes. Zishen Jiangtang Pill (ZJP) is a Chinese herbal product which has been used in clinic to maintain blood glucose level and bone density for decades. However, the evidence about its mechanism on diabetes and osteoporosis is still unknown. The aim of this study is to investigate therapeutic effect of ZJP on DO in streptozotocin- (STZ-) induced rats. Rats were randomly assigned to 4 groups: one control group (CON), one model group (MOD), and two ZJP treatment groups (1.5 and 3.0 g/kg/d). All rats were treated for 8 weeks. Results showed that ZJP decreased the blood glucose level during OGTT and prevented the changes of FBG and Fins. Similarly, ZJP inhibited the changes of BCa, P, TRACP-5b, CTX-1, BALP, and BGP and the reduction of BMD. In parallel, 1H-NMR metabolomic studies showed that ZJP significantly altered the metabolic fingerprints of blood and urine level. These findings suggest that ZJP can effectively improve glucose metabolism, abnormal bone metabolism, and metabolic disorders in DO rats, which may be a useful alternative medicine for DO therapy

An n-of-1 Trial Service in Clinical Practice: Testing the Effectiveness of Liuwei Dihuang Decoction for Kidney-Yin Deficiency Syndrome

Objective. To describe the clinical use of n-of-1 RCTs for kidney-Yin deficiency syndrome that is a traditional Chinese medicine syndrome in publicly clinical practice in China. Methods. Our study included patients with kidney-Yin deficiency syndrome, using a within-patient, randomized, double-blind, crossover comparison of Liuwei Dihuang decoction versus placebo. Outcome Measures. Primary outcome measures included number of individual completion rates, response rate, and post-n-of-1 RCTs decisions. Secondary measures were the whole group score of individual Likert scale, SF-36 questionnaire. Results. Fifty patients were recruited and 3 were not completed. Forty-seven patients completed 3 pairs of periods, 3 (6.38%) were responders, 28 (59.57%) were nonresponders, and 16 (34.05%) were possible responders. Doctors and patients used the trial results to making decision. Three responders stayed on the medication management, 28 nonresponders ceased the LDD, 7 patients of the 16 possible responders could not give clear decision, and the others kept the same medication station. Among the whole group, neither the individual Likert score nor the SF-36 showed any statistical differences between LDD and placebo. Discussion. More attention should be paid to choose experienced TCM doctor as investigator and keep the simulant same with test medication in n-of-1 RCTs of TCM and sufficiently biological half-life period of Chinese medicine compound

Shielding Protection by Mesoporous Catalysts for Improving Plasma-Catalytic Ambient Ammonia Synthesis

[Image: see text] Plasma catalysis is a promising technology for decentralized small-scale ammonia (NH(3)) synthesis under mild conditions using renewable energy, and it shows great potential as an alternative to the conventional Haber–Bosch process. To date, this emerging process still suffers from a low NH(3) yield due to a lack of knowledge in the design of highly efficient catalysts and the in situ plasma-induced reverse reaction (i.e., NH(3) decomposition). Here, we demonstrate that a bespoke design of supported Ni catalysts using mesoporous MCM-41 could enable efficient plasma-catalytic NH(3) production at 35 °C and 1 bar with >5% NH(3) yield at 60 kJ/L. Specifically, the Ni active sites were deliberately deposited on the external surface of MCM-41 to enhance plasma–catalyst interactions and thus NH(3) production. The desorbed NH(3) could then diffuse into the ordered mesopores of MCM-41 to be shielded from decomposition due to the absence of plasma discharge in the mesopores of MCM-41, that is, “shielding protection”, thus driving the reaction forward effectively. This promising strategy sheds light on the importance of a rational design of catalysts specifically for improving plasma-catalytic processes