Differences between immunodeficient mice generated by classical gene targeting and CRISPR/Cas9-mediated gene knockout.

Immunodeficient mice are widely used for pre-clinical studies to understand various human diseases. Here, we report the generation of four immunodeficient mouse models using CRISPR/Cas9 system without inserting any foreign gene sequences such as NeoR cassettes and their characterization. By eliminating any possible effects of adding a NeoR cassette, our mouse models may allow us to better elucidate the in vivo functions of each gene. Our FVB-Rag2-/-, B6-Rag2-/-, and BALB/c-Prkdc-/- mice showed phenotypes similar to those of the earlier immunodeficient mouse models, including a lack of mature B cells and T cells and an increase in the number of CD45+DX-5+ natural killer cells. However, B6-Il2rg-/- mice had a unique phenotype, with a lack of mature B cells, increased number of T cells, and decreased number of natural killer cells. Additionally, serum immunoglobulin levels in all four immunodeficient mouse models were significantly reduced when compared to those in wild-type mice with the exception of IgM in B6-Il2rg-/- mice. These results indicate that our immunodeficient mouse models are a robust tool for in vivo studies of the immune system and will provide new insights into the variation in phenotypic outcomes resulting from different gene-targeting methodologies.

Polygoni Rhizoma inhibits inflammatory response through inactivation of nuclear factor-kappaB and mitogen activated protein kinase signaling pathways in RAW264.7 mouse macrophage cells.

The objective of this study was to determine the antiinflammatory effects of Polygoni Rhizoma (PR), an Oriental medicinal herb, in interleukin 1 beta (IL-1ß) and lipopolysaccharide (LPS)-stimulated RAW264.7 mouse macrophage cells. PR significantly reduced the production of pro-inflammatory cytokines such as IL-6, tumor necrosis factor alpha (TNF-α) and pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase 2 (COX-2) and prostaglandin E2 (PGE2) even at a concentration of 1 µg/mL in the cells. In addition, PR inhibited the transcriptional activity of NF-κB as well as the degradation and phosphorylation of inhibitory kappa B alpha (IκBα). Furthermore, PR suppressed the phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2), p38 and c-Jun N-terminal kinase 1/2 (JNK1/2) in IL-1ß and LPS-treated RAW264.7. The results suggest that PR exerts an antiinflammatory property by inhibiting iNOS, COX-2, TNF-α and IL-6 production in association with inactivation of the NF-κB and MAPK signaling pathways in RAW 264.7 cells.

Analgesic and anti-inflammatory effects of ethyl acetate fraction of Polygonum cuspidatum in experimental animals.

Polygonum cuspidatum (PC) has been used for the treatment of arthritis and urinary diseases in traditional medicine. Despite recent evidence that PC has anti-oxidant, anti-tumoral, and anti-inflammatory effects, analgesic and anti-inflammatory effects of PC have not been elucidated yet in vivo. Thus, in the present study, analgesic and anti-inflammatory effects of ethyl acetate extract of PC (EAPC) were investigated in vivo for the first time. Hot plate test and tail-flick test revealed that EAPC at 200 mg/kg exerts analgesic effect (p < 0.05). In contrast, EAPC did not show significant analgesic effect in acetic acid-induced writhing test. Serotonin-induced paw edema model and Freund's complete adjuvant (FCA)-induced adjuvant arthritis model were used to examine anti-inflammatory effect of EAPC in vivo. In serotonin-induced paw edema model, EAPC suppressed swelling inflammatory response within 12 min after serotonin injection, at both 100- and 200-mg/kg dose (p < 0.05). Consistently, in FCA-induced adjuvant arthritis model, FCA at 200 mg/kg significantly suppressed FCA-induced joint swelling within 3 days (p < 0.05), whereas FCA at 100 mg/kg showed the similar result within 5 days (p < 0.05). Furthermore, EAPC effectively inhibited positive responses of c-reactive protein and rheumatoid factor compared to untreated control. Taken together, our findings suggest that EAPC can be a potent candidate for rheumatoid arthritis treatment.

Tebuconazole Fungicide Induces Lipid Accumulation and Oxidative Stress in HepG2 Cells.

Tebuconazole (TEB), a triazole fungicide, is frequently applied to agriculture for the increase of food production. Although TEB causes liver toxicity, its effects on cellular lipid accumulation are rarely investigated. Therefore, this study aimed to study the effects of TEB on lipid metabolism and accumulation in HepG2 cells. HepG2 cells were exposed to 0-320 µM TEB for 1-24 h. TEB (20-80 µM, 24 h)-treated cells showed lipid accumulation. Further, TEB (20-80 µM, 1-12 h) increased the nuclear translocation of peroxisome proliferator-activated receptors and the expression of lipid uptake and oxidation-related markers such as cluster of differentiation 36, fatty acid transport protein (FATP) 2, FATP5, and carnitine palmitoyltransferase 1. Oxidative stress levels in TEB-treated cells (20-80 µM, 24 h) were higher, compared to those in the control. TEB (20-80 µM, 24 h) also induced the loss of mitochondrial membrane potential and lower levels of microsomal triglyceride transfer protein in the cells. Thus, TEB can induce lipid accumulation by altering the expression of lipid-metabolizing molecules and can therefore impair lipid metabolism. Our data suggest that human exposure to TEB may be a risk factor for non-alcoholic fatty liver disease.

Emerging Paradigm of Crosstalk between Autophagy and the Ubiquitin-Proteasome System.

Cellular protein homeostasis is maintained by two major degradation pathways, namely the ubiquitin-proteasome system (UPS) and autophagy. Until recently, the UPS and autophagy were considered to be largely independent systems targeting proteins for degradation in the proteasome and lysosome, respectively. However, the identification of crucial roles of molecular players such as ubiquitin and p62 in both of these pathways as well as the observation that blocking the UPS affects autophagy flux and vice versa has generated interest in studying crosstalk between these pathways. Here, we critically review the current understanding of how the UPS and autophagy execute coordinated protein degradation at the molecular level, and shed light on our recent findings indicating an important role of an autophagy-associated transmembrane protein EI24 as a bridging molecule between the UPS and autophagy that functions by regulating the degradation of several E3 ligases with Really Interesting New Gene (RING)-domains.

(-)-Epigallocatechin-3-O-gallate (EGCG) attenuates the hemodynamics stimulated by caffeine through decrease of catecholamines release.

A human study of the effects on hemodynamics of caffeine and epigallocatechin-3-O-gallate (EGCG) was performed. Caffeine tablets (200 mg) were orally administered to healthy males aged between 25 and 35 years 30 min after oral administration of EGCG tablets (100 and 200 mg). The increase in BP induced by caffeine was inhibited when co-administrated with EGCG. We found that caffeine slightly decreased heart rate (HR) in the volunteers. Although EGCG enhanced HR reduction, the effect was not significant. In addition, caffeine increased blood catecholamine levels, but EGCG inhibited the increase in noradrenaline, adrenaline and dopamine levels induced by caffeine. Whether EGCG decreases the elevated HR and systolic perfusion pressure, and ventricular contractility induced by adrenergic agonists in the isolated rat heart was investigated. The modified Krebs-Henseleit solution was perfused through a Langendorff apparatus to the isolated hearts of rats. HR, systolic perfusion pressure, and developed maximal rates of contraction (+dP/dtmax) and relaxation (-dP/dtmax) were increased by epinephrine (EP) and isoproterenol (IP). In contrast, EGCG decreased the elevated HR, systolic perfusion pressure, and left ventricular ±dp/dtmax induced by EP and/or IP. In conclusion, EGCG could attenuate the hemodynamics stimulated by caffeine through decreasing catecholamine release.

Force spectroscopy of multivalent binding of riboflavin-conjugated dendrimers to riboflavin binding protein.

Putative riboflavin receptors are considered as biomarkers due to their overexpression in breast and prostate cancers. Hence, these receptors can be potentially exploited for use in targeted drug delivery systems where dendrimer nanoparticles with multivalent ligand attachments can lead to greater specificity in cellular interactions. In this study, the single molecule force spectroscopy technique was used to assess the physical strength of multivalent interactions by employing a riboflavin (RF)-conjugated generation 5 PAMAM dendrimer G5(RF)n nanoparticle. By varying the average RF ligand valency (n = 0, 3, 5), the rupture force was measured between G5(RF)n and the riboflavin binding protein (RFBP). The rupture force increased when the valency of RF increased. We observed at the higher valency (n = 5) three binding events that increased in rupture force with increasing loading rate. Assuming a single energy barrier, the Bell-Evans model was used to determine the kinetic off-rate and barrier width for all binding interactions. The analysis of our results appears to indicate that multivalent interactions are resulting in changes to rupture force and kinetic off-rates.

Effects of berberine on angiotensin-converting enzyme and NO/cGMP system in vessels.

The present study was designed to examine the relaxant and anticonstrictive effects of berberine in the isolated thoracic aorta in rats. Intravenous injection of berberine lowered the mean arterial pressure (MAP) of anesthesized rats in a dose-dependent manner. The angiotensin-converting enzyme (ACE) activities were inhibited significantly by the addition of berberine in a dose-dependent manner of which the IC50 value of berberine for ACE was 42 micrograms/ml (125 microM). In the endothelium-intact rings, angiotensin I-induced contraction was markedly attenuated by prior exposure of aortic rings to berberine. Treatment of the intact aortic rings with berberine (10 micrograms/ml) increased the NOx and cGMP productions relative to the vehicle-treated group. Berberine induced a dose-dependent relaxation in phenylephrine-precontracted aortic rings, but NG-nitro-L-arginine methyl ester (L-NAME)-pretreated intact aortic rings or functional removal of the endothelium attenuated the berberine-induced relaxation without an effect on maximum response. These results suggest that berberine has a hypotensive effect, at least in part, via the inhibition of ACE and direct release of NO/cGMP in the vascular tissues.

Effects of bulbus Fritillaria water extract on blood pressure and renal functions in the L-NAME-induced hypertensive rats.

A pharmacological inhibition of nitric oxide synthase (NOS) in rats produces renal vasoconstriction, renal dysfunction, and hypertension. The present study was aimed at investigating whether Bulbus Fritillaria water extract (BFWE) ameliorates NG-nitro-L-arginine methylester (L-NAME)-induced hypertension. Treatment of rats with L-NAME (60 mg/l drinking water, 4 weeks) caused a sustained increase in systolic blood pressure (SBP). The NO concentration in plasma and NO productions in the vascular tissues of the L-NAME-treated group were significantly reduced as compared with those in the control, whereas the expressions of NOS proteins were not altered. BFWE restored SBP to normal level in the L-NAME-treated hypertensive rats. Moreover, BFWE was able to preserve the vascular NO production and plasma NO metabolites concentration without changes of the expression NOS proteins. The renal functional parameters including urinary volume, sodium excretion, and creatinine clearance (Ccr) were significantly restored in rats co-treated with BFWE and L-NAME compared to the L-NAME-treated group. Taken together, these results suggest that BFWE prevents the increase of SBP in the L-NAME-induced hypertension that may have been caused by enhanced generation of vascular NO and amelioration of renal functions.

Effects of bulb of Fritillaria ussuriensis maxim. on angiotensin converting enzyme and vascular release of NO/cGMP in rats.

The present study was designed to investigate whether the Bulbus Fritillaria shows a hypotensive effect via the angiotensin converting enzyme (ACE) inhibition and elicit NO/cGMP release in rat aortic rings. Intravenous injection of Bulbus Fritillaria water extract lowered the mean arterial pressure of anesthetized rats in a dose-dependent manner. The ACE activities were inhibited significantly by the additions of ethylacetate and butanol extracts from Bulbus Fritillaria, of which IC(50) values were 292 and 320 microg/ml, respectively. Moreover, angiotensin I-induced vasoconstriction was also strongly inhibited by the additions of ethylacetate and butanol extracts from Bulbus Fritillaria. In order to assess whether NO production was induced by Bulbus Fritillaria extracts, we directly measured NO and cGMP production levels from the aortic ring elicited by extracts of Bulbus Fritillaria. Our results showed that the hexane, butanol, and water extracts of Bulbus Fritillaria increased NO and cGMP productions in intact vascular tissue. These findings suggest that Bulbus Fritillaria extracts have a hypotensive effect in rats via the inhibition of ACE activity and direct release of NO/cGMP in the vascular tissue.

Effects of Wonli Acupuncture Procedure in Patients with LSS: A Clinical, Retrospective Study.

Background. Lumbar spinal stenosis (LSS) is a disease with increasing prevalence due to prolongation of average life span. Despite various treatment methods, many limitations remain unsolved. Objective. We are reporting cases of patients who have been treated with Wonli Acupuncture, a method of treating LSS by directly approaching the intervertebral foramen and interlaminar space with acupuncture needles different from those used in original acupuncture. Methods. A total of 82 patients with LSS were treated with Wonli Acupuncture, and out of those, 47 patients without exclusion criteria were selected for the following research. We compared the pretreatment VAS and ODI scores based on 1-year follow-up measurements. Results. The ODI value dropped by 15.3 ± 24.8 on average (from 35.2 ± 19.9 at the baseline to 19.8 ± 20.6 at the reading) (P < 0.01) and the average VAS also dropped by 19.2 ± 37.2 (from 60.7 ± 23.1 at baseline to 41.5 ± 31.9 at the reading) (P < 0.01). Conclusions. Wonli Acupuncture was found to have clinical efficacy for lumbar spinal stenosis.

Forged seal detection based on the seal overlay metric.

This paper describes a method for verifying the authenticity of a seal impression imprinted on a document based on the seal overlay metric, which refers to the ratio of an effective seal impression pattern and the noise in the neighborhood of the reference impression region. A reference seal pattern is obtained by taking the average of a number of high-quality impressions of a genuine seal. A target seal impression to be examined, often on paper with some background texts and lines, is segmented out from the background by an adaptive threshold applied to the histogram of color components. The segmented target seal impression is then spatially aligned with the reference by maximizing the count of matching pixels. Then the seal overlay metric is computed for the reference and the target. If the overlay metric of a target seal is below a predetermined limit for the similarity to the genuine, then the target is classified as a forged seal. To further reduce the misclassification rate, the seal overlay metric is adjusted by the filling rate, which reflects the quality of inked pattern of the target seal. Experiment results demonstrate that the proposed method can detect elaborate seal impressions created by advanced forgery techniques such as lithography and computer-aided manufacturing.

Alcohol-fermented soybean increases the expression of receptor-interacting protein 2 and IκB kinase ß in mouse peritoneal macrophages.

Soybean is a useful component of traditional Korean medicine with well-documented health-promoting effects. We investigated the effects of alcohol-fermented soybean (AFS) on immune function. When AFS treatment was used in combination with recombinant interferon-γ (rIFN-γ), there was a marked cooperative induction of nitric oxide (NO) and tumor necrosis factor (TNF)-α production in mouse peritoneal macrophages. AFS increased the expression of inducible NO synthase mRNA and protein in rIFN-γ-primed macrophages. Treating macrophages with pyrrolidine dithiocarbamate, an inhibitor of nuclear factor-κB (NF-κB), decreased the synergistic effects of AFS. In addition, AFS in combination with rIFN-γ increased the phosphorylation of p38 and c-Jun N-terminal kinase (JNK) but not extracellular signal-regulated kinase. However, AFS had no effect on phosphorylation of mitogen-activated protein kinases by itself. The p38 inhibitor SB203580 or the JNK inhibitor SP600125 inhibited the AFS-induced NO and TNF-α production. When AFS was used in combination with rIFN-γ, there was a co-operative activation of NF-κB and receptor-interacting protein 2 (Rip2)/IκB kinase (IKK)-ß. Our results indicate that AFS increases the production of NO and TNF-α through the activation of Rip2/IKK-ß in rIFN-γ-primed macrophages.