Endogenous circadian reporters reveal functional differences of PERIOD paralogs and the significance of PERIOD:CK1 stable interaction.

Adverse consequences from having a faulty circadian clock include compromised sleep quality and poor performance in the short-term, and metabolic diseases and cancer in the long-term. However, our understanding of circadian disorders is limited by the incompleteness of our molecular models and our dearth of defined mutant models. Because it would be prohibitively expensive to develop live animal models to study the full range of complicated clock mechanisms, we developed PER1-luc and PER2-luc endogenous circadian reporters in a validated clock cell model, U-2 OS, where the genome can be easily manipulated, and functional consequences of mutations can be accurately studied. When major clock genes were knocked out in these cells, circadian rhythms were modulated similarly compared with corresponding mutant mice, validating the platform for genetics studies. Using these reporter cells, we uncovered critical differences between two paralogs of PER. Although PER1 and PER2 are considered redundant and either one can serve as a pacemaker alone, they were dramatically different in biochemical parameters such as stability and phosphorylation kinetics. Consistently, circadian phase was dramatically different between PER1 and PER2 knockout reporter cells. We further showed that the stable binding of casein kinase1δ/ε to PER is not required for PER phosphorylation itself, but is critical for delayed timing of phosphorylation. Our system can be used as an efficient platform to study circadian disorders associated with pathogenic mutations and their underlying molecular mechanisms.

Development of a film-based immunochromatographic microfluidic device for malaria diagnosis.

In this study, a novel film-based immunochromatographic microfluidic device (IMD) has been developed for malaria diagnosis. A microfluidic channel was patterned on a polyethylene terephthalate (PET) double-sided adhesive film using a plotting cutter and was assembled with a polycarbonate (PC) film. The PC film used for the probe immobilization layer was activated using oxygen plasma treatment to modify the film surface with avidin-biotin linker to immobilize a capture antibody. A fluorescent labeled Pan type mAb conjugate was prepared for signal indicator after undergoing a sandwich enzyme-linked immunosorbent assay (ELISA). Target antigens include Plasmodium falciparum (P. falciparum) lactate dehydrogenase (LDH) and Plasmodium vivax (P. vivax) LDH which were injected into the sample inlet. Target antigens combined with the conjugate and then flowed to the detection chamber where two test dots and a control dot (Ctrl) exist. In the presence of P. falciparum LDH, three detection dots including test dot 1 (T1), test dot 2 (T2) and Ctrl revealed fluorescence signals where P. falciparum mAb, Pan type pLDH mAb and goat anti-mouse IgG were immobilized, respectively. When P. vivax LDH was present, T2 and Ctrl dots showed fluorescence signals while no signal was detected with the negative control. P. falciparum LDH and P. vivax LDH were successfully detected on the IMD with a detection limit of 50 ng/mL and 100 ng/mL, respectively. The IMD provides a point-of-care diagnosis platform which is able to analyze pathogenic bacteria and viruses that can be applied in the field of clinical diagnosis and food safety testing.

Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9.

CRISPR-Cas9 is a powerful gene editing technique that can induce mutations in a target gene of interest in almost any mammalian cell line. However, its practicality can be limited if target cell lines are difficult to transfect and do not proliferate. In the current study, we have developed a streamlined approach for CRISPR-based gene knockouts with three key advantages, which allows phenotypic assay of gene knockouts without clonal selection and expansion. First, it integrates into a single, all-in-one vector transgenes for Cas9, sgRNA, and a fluorescence marker. Second, we used the Gateway system to rapidly clone specific sgRNAs into the all-in-one vector through PCR and in vitro recombination, without conventional enzyme digestion and ligation. Third, it uses adenovirus for the capacity to package the all-in-one vector, and for its high efficiency of transduction. We tested the all-in-one adenoviral CRISPR-Cas9 in a circadian clock model cell line U2OS, and demonstrated that essential clock genes such as Bmal1 and Per1 were knocked out so efficiently that functional assays could be performed from the heterogenic population without any clonal selection and expansion. This streamlined approach may prove invaluable for rapid functional assays of candidate genes in diverse biological pathways, including the circadian clock.

Direct infusion MS-based lipid profiling reveals the pharmacological effects of compound K-reinforced ginsenosides in high-fat diet induced obese mice.

The serum lipid metabolites of lean and obese mice fed normal or high-fat diets were analyzed via direct infusion nanoelectrospray-ion trap mass spectrometry followed by multivariate analysis. In addition, lipidomic biomarkers responsible for the pharmacological effects of compound K-reinforced ginsenosides (CK), thus the CK fraction, were evaluated in mice fed high-fat diets. The obese and lean groups were clearly discriminated upon principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) score plot, and the major metabolites contributing to such discrimination were triglycerides (TGs), cholesteryl esters (CEs), phosphatidylcholines (PCs), and lysophosphatidylcholines (LPCs). TGs with high total carbon number (>50) and low total carbon number (<50) were negatively and positively associated with high-fat diet induced obesity in mice, respectively. When the CK fraction was fed to obese mice that consumed a high-fat diet, the levels of certain lipids including LPCs and CEs became similar to those of mice fed a normal diet. Such metabolic markers can be used to better understand obesity and related diseases induced by a hyperlipidic diet. Furthermore, changes in the levels of such metabolites can be employed to assess the risk of obesity and the therapeutic effects of obesity management.

Ginsenoside Rb1 is transformed into Rd and Rh2 by Microbacterium trichothecenolyticum.

Ginsenosides are the most important ingredient of ginseng and are known to possess many pharmacological and biological effects. Rb1, a major protopanaxadiol ginsenoside, is the most abundant ginsenoside in Panax ginseng C.A Meyer and can be hydrolyzed into more pharmaceutically potent minor ginsenosides. To identify a microorganism that is capable of converting Rb1 into other ginsenosides, we screened 12 Microbacterium spp., and M. trichothecenolyticum was identified as a likely candidate. M. trichothecenolyticum converted Rb1 into Rd and then into Rh2 based on TLC and HPLC analyses of reaction products. This biotransformation method can be easily applied for mass production of Rd and Rh2 by using Rb1.

Red ginseng saponin extract attenuates murine collagen-induced arthritis by reducing pro-inflammatory responses and matrix metalloproteinase-3 expression.

Ginseng, the root of Panax ginseng C. A. MEYER, has been used as a food product and medicinal ingredient. In this study, we assessed the anti-arthritic effects of red ginseng saponin extract (RGSE), including ginsenosides Rg3, Rk1 and Rg5 as major components, on a murine type II collagen (CII)-induced arthritis (CIA), which is a valid animal model of human arthritis. Oral administration of RGSE at 10 mg/kg reduced the clinical arthritis score and paw swelling in the CIA mice, and inhibited joint space narrowing and histological arthritis, illustrating the severity of synovial hyperplasia, inflammatory cell infiltration, pannus formation, and erosion of cartilage. RGSE inhibited the expression of matrix metalloproteinase-3 and nitrotyrosine formation, and recovered the expression of superoxide dismutase in the joints of the CIA mice. Orally administered RGSE also reduced the levels of serum tumor necrosis factor-alpha and interleukin-1beta in the CIA mice. CII- or lipopolysaccharide-stimulated cytokine production, in addition to CII-specific proliferation, was reduced in the spleen cells of the RGSE-treated CIA mice, as compared with those from vehicle-treated CIA mice. Furthermore, RGSE administration protected against CIA-induced oxidative tissue damage by restoring the increased malondialdehyde levels and the decreased glutathione levels and catalase activities almost to control levels. Therefore, RGSE may be a beneficial supplement which can improve human arthritis.

Generation and characterization of monoclonal antibody to ginsenoside rg3.

While studying the mechanism of ginsenoside Rg3 (G-Rg3) on tumor inhibition, we produced monoclonal antibody to G-Rg3 for more specific investigation. We immunized Balb/c mice to G-Rg3 conjugated bovine serum albumin (BSA) by intraperitoneal injection and hybridized splenocytes from those immunized mice and myeloma cells. From those fusion cell lines, we selected productive monoclonal clones and obtained culture media containing monoclonal antibody to G-Rg3. After purification, we performed enzyme-linked immunosorbent assay (ELISA) to verify the sensitivity and specificity of the antibody. When compared with G-Rh2 having a very similar structure as a metabolite of G-Rg3, the antibody worked only with G-Rg3 in a concentration-dependent manner. We confirmed that the monoclonal antibody to G-Rg3 can be applied to immunocytochemistry for detection of the treated G-Rg3 inside A549 human lung adenocarcinomas. Thus, the monoclonal antibody to G-Rg3 would be a useful tool for measuring the bioactivity of G-Rg3 in various fields.

Effects of in vitro-digested ginsenosides on lipid accumulation in 3T3-L1 adipocytes.

Ginseng, the root of Panax ginseng C. A. Meyer, is frequently used in traditional oriental medicines. The major active components of ginseng are the saponins, which are also called ginsenosides and are known for their pharmacological and biological activities. In this study, the effects of ginsenosides on lipid accumulation in 3T3-L1 adipocytes were investigated after the ginsenosides were in vitro-digested with artificial gastric and intestinal fluids. Ginseng extract was incubated with an artificial digestive fluid, and the changes were analyzed by HPLC, after which the effects of the digest on 3T3-L1 adipocytes were observed. Polar ginsenosides were transformed into less-polar ginsenosides at the low pH of the gastric acid, without any influence from the digestive enzymes. Additionally, the artificially digested ginsenosides showed inhibitory effects on lipid accumulation in 3T3-L1 adipocytes. When the 3T3-L1 adipocytes were treated with various ginseng samples that possessed different polarities, the less polar ginsenosides were more effective in reducing lipid accumulation. Furthermore, when the Rg3, Rk1, and Rg5 ginsenosides were used to treat the cells individually, Rg3 ginsenoside was the most effective at inhibiting lipid accumulation. These results suggest that the less polar ginsenosides, particularly ginsenoside Rg3, effectively reduce lipid accumulation in adipocytes. Accordingly, our results suggest that ginsenoside Rg3 should be developed as an antiobesity treatment.

Simultaneous quantification of 14 ginsenosides in Panax ginseng C.A. Meyer (Korean red ginseng) by HPLC-ELSD and its application to quality control.

A new method of high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD) was developed for the simultaneous quantification of 14 major ginsenosides, which are the marker compounds of Panax ginseng C.A. Meyer (Korean red ginseng). Various types of ginseng samples were extracted, and the amounts of the 14 ginsenosides (Rg1, Re, Rf, Rh1, Rg2, Rb1, Rc, Rb2, Rb3, Rd, Rg3, Rk1, Rg5, and Rh2) were determined by reverse-phase HPLC-ELSD using digoxin as an internal standard. The mobile phase consisted of a programmed gradient of aqueous acetonitrile. Calibration curves for each ginsenoside were determined for the quantification. The method was validated for linearity, precision, accuracy, limit of detection, and limit of quantification. This quantification method was applied to several finished ginseng products including white ginseng, red ginseng powder, and red ginseng concentrate. The amounts of the 14 ginsenosides in the various ginseng samples could be analyzed simultaneously. This validated HPLC method is expected to provide a new basis for the quality assessment of ginseng products.

Preparative isolation of four ginsenosides from Korean red ginseng (steam-treated Panax ginseng C. A. Meyer), by high-speed counter-current chromatography coupled with evaporative light scattering detection.

Ginseng (Panax ginseng C. A. Meyer) has been well known to have a variety of ginsenosides that show diverse biological activities. Especially, the components of ginsenosides are quite different depending on the processing method. Recently, there have been several reports showing that less polar ginsenosides from Korean red ginseng (steam-treated Panax ginseng) have potent biological activities such as radical scavenging, vasodilating and anti-tumor activities. In this study, we have isolated four known ginsenosides Rg3, Rk1, Rg5 and F4 from Korean red ginseng by high-speed counter-current chromatography (HSCCC) coupled with evaporative light scattering detection (ELSD). The enriched saponin fraction (350 mg) was separated by using methylene chloride-methanol-water-isopropanol (6:6:4:1, v/v) as the two-phase solvent system and yielded 28.6 mg of Rg5, 26.6 mg of Rk1, 32.2 mg of Rg3 and 8.1 mg of F4. The purity of these ginsenosides was assessed by HPLC-ELSD to be over 95%, and their structures were characterized by electrospray ionization mass spectrometry (ESI-MS), (1)H NMR and (13)C NMR.

Loss of At4 function impacts phosphate distribution between the roots and the shoots during phosphate starvation.

Plants display a range of adaptive responses to phosphate (Pi) starvation including an increase in the proportion of Pi allocated to the roots, which enhances lateral root development and consequently Pi acquisition. The mechanisms by which plants sense Pi and signal Pi reallocation are largely unknown. Previously, we cloned At4, a gene predicted to contain multiple short open-reading frames (ORFs), whose expression is strongly induced by Pi starvation. At4 is a member of a small gene family whose members, AtIPS1 and two additional genes reported here, At4.1 and At4.2, share little conservation among the predicted ORFs but high conservation of a 22-nt sequence located in the 3' half of the transcript. Here, we show that under Pi-starvation conditions, At4 is expressed in the vascular tissue and transcript levels are regulated by both cytokinin and ABA. at4, an At4 loss-of-function mutant fails to redistribute Pi to the roots correctly in response to Pi deprivation and At4 shoots continue to accumulate a greater proportion of Pi relative to wild type. Consistent with this, the primary root growth rate in at4 is faster than wild type in low-Pi conditions. The conserved sequence found in all members of the At4 gene family hybridizes to a small RNA present in Pi-starved roots. These data support a role for At4 in the internal allocation of Pi and suggest that the At4 gene is not only subject to Pi-starvation-inducible expression, but that transcript levels may be adjusted at a post-transcriptional level by the activity of an miRNA.

Complex regulation of Arabidopsis AGR1/PIN2-mediated root gravitropic response and basipetal auxin transport by cantharidin-sensitive protein phosphatases.

Polar auxin transport, mediated by two distinct plasma membrane-localized auxin influx and efflux carrier proteins/complexes, plays an important role in many plant growth and developmental processes including tropic responses to gravity and light, development of lateral roots and patterning in embryogenesis. We have previously shown that the Arabidopsis AGRAVITROPIC 1/PIN2 gene encodes an auxin efflux component regulating root gravitropism and basipetal auxin transport. However, the regulatory mechanism underlying the function of AGR1/PIN2 is largely unknown. Recently, protein phosphorylation and dephosphorylation mediated by protein kinases and phosphatases, respectively, have been implicated in regulating polar auxin transport and root gravitropism. Here, we examined the effects of chemical inhibitors of protein phosphatases on root gravitropism and basipetal auxin transport, as well as the expression pattern of AGR1/PIN2 gene and the localization of AGR1/PIN2 protein. We also examined the effects of inhibitors of vesicle trafficking and protein kinases. Our data suggest that protein phosphatases, sensitive to cantharidin and okadaic acid, are likely involved in regulating AGR1/PIN2-mediated root basipetal auxin transport and gravitropism, as well as auxin response in the root central elongation zone (CEZ). BFA-sensitive vesicle trafficking may be required for the cycling of AGR1/PIN2 between plasma membrane and the BFA compartment, but not for the AGR1/PIN2-mediated root basipetal auxin transport and auxin response in CEZ cells.

Phosphate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from both low- and high-phosphate environments.

Of the mineral nutrients essential for plant growth, phosphorus plays the widest diversity of roles and a lack of phosphorus has profound effects on cellular metabolism. At least eight members of the Arabidopsis Pht1 phosphate (Pi) transporter family are expressed in roots and Pht1;1 and Pht1;4 show the highest transcript levels. The spatial and temporal expression patterns of these two genes show extensive overlap. To elucidate the in planta roles of Pht1;1 and Pht1;4, we identified loss-of-function mutants and also created a double mutant, lacking both Pht1;1 and Pht1;4. Consistent with their spatial expression patterns, membrane location and designation as high-affinity Pi transporters, Pht1;1 and Pht1;4 contribute to Pi transport in roots during growth under low-Pi conditions. In addition, during growth under high-Pi conditions, the double mutant shows a 75% reduction in Pi uptake capacity relative to wildtype. Thus, Pht1;1 and Pht1;4 play significant roles in Pi acquisition from both low- and high-Pi environments.