High-throughput sequencing-based analysis of the composition and diversity of the endophyte community in roots of Stellera chamaejasme.

Stellera chamaejasme (S. chamaejasme) is an important medicinal plant with heat-clearing, detoxifying, swelling and anti-inflammatory effects. At the same time, it is also one of the iconic plants of natural grassland degradation in northwest China, playing a key role in the invasion process. Plant endophytes live in healthy plant tissues and can synthesize substances needed for plant growth, induce disease resistance in host plants, and enhance plant resistance to environmental stress. Therefore, studying the root endophytes of S. chamaejasme is of great significance for mining beneficial microbial resources and biological prevention and control of S. chamaejasme. This study used Illumina MiSeq high-throughput sequencing technology to analyze the composition and diversity of endophytes in the roots of S. chamaejasme in different alpine grasslands (BGC, NMC and XGYZ) in Tibet. Research results show that the main phylum of endophytic fungi in the roots of S. chamaejasme in different regions is Ascomycota, and the main phyla of endophytic bacteria are Actinobacteria, Proteobacteria and Firmicutes (Bacteroidota). Overall, the endophyte diversity of the NMC samples was significantly higher than that of the other two sample sites. Principal coordinate analysis (PCoA) and permutational multivariate analysis of variance (PERMANOVA) results showed significant differences in the composition of endophytic bacterial and fungal communities among BGC, NMC and XGYZ samples. Co-occurrence network analysis of endophytes showed that there were positive correlations between fungi and some negative correlations between bacteria, and the co-occurrence network of bacteria was more complex than that of fungi. In short, this study provides a vital reference for further exploring and utilizing the endophyte resources of S. chamaejasme and an in-depth understanding of the ecological functions of S. chamaejasme endophytes.

Association of survival with adjuvant chemotherapy in patients with stage IB gastric cancer: a multicentre, observational, cohort study.

Background: Recurrence following radical resection in patients with stage IB gastric cancer (GC) is not uncommon. However, whether postoperative adjuvant chemotherapy could reduce the risk of recurrence in stage IB GC remains contentious. Methods: We collected data on 2110 consecutive patients with pathologic stage IB (T1N1M0 or T2N0M0) GC who were admitted to 8 hospitals in China from 2009 to 2018. The survival of patients who received adjuvant chemotherapy was compared with that of postoperative observation patients using propensity score matching (PSM). Two survival prediction models were constructed to estimate the predicted net survival gain attributable to adjuvant chemotherapy. Findings: Of the 2110 patients, 1344 received adjuvant chemotherapy and 766 received postoperative observation. Following the 1-to-1 matching, PSM yielded 637 matched pairs. Among matched pairs, adjuvant chemotherapy was not associated with improved survival compared with postoperative observation (OS: hazard ratio [HR], 0.72; 95% CI, 0.52-1.00; DFS: HR, 0.91; 95% CI, 0.64-1.29). Interestingly, in the subgroup analysis, reduced mortality after adjuvant chemotherapy was observed in the subgroups with elevated serum CA19-9 (HR, 0.22; 95% CI, 0.08-0.57; P = 0.001 for multiplicative interaction), positive lymphovascular invasion (HR, 0.32; 95% CI, 0.17-0.62; P < 0.001 for multiplicative interaction), or positive lymph nodes (HR, 0.17; 95% CI, 0.07-0.38; P < 0.001 for multiplicative interaction). The survival prediction models mainly based on variables associated with chemotherapy benefits in the subgroup analysis demonstrated good calibration and discrimination, with relatively high C-indexes. The C-indexes for OS were 0.74 for patients treated with adjuvant chemotherapy and 0.70 for patients treated with postoperative observation. Two nomograms were built from the models that can calculate individualized estimates of expected net survival gain attributable to adjuvant chemotherapy. Interpretation: In this cohort study, pathologic stage IB alone was not associated with survival benefits from adjuvant chemotherapy compared with postoperative observation in patients with early-stage GC. High-risk clinicopathologic features should be considered simultaneously when evaluating patients with stage IB GC for adjuvant chemotherapy. Funding: National Natural Science Foundation of China; the National Key R&D Program of China.

A mannitol-modified emodin nano-drug restores the intestinal barrier function and alleviates inflammation in a mouse model of DSS-induced ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is an inflammatory disease of the colon that is characterized by mucosal ulcers. Given its increasing prevalence worldwide, it is imperative to develop safe and effective drugs for treating UC. Emodin, a natural anthraquinone derivative present in various medicinal herbs, has demonstrated therapeutic effects against UC. However, low bioavailability due to poor water solubility limits its clinical applications. METHODS: Emodin-borate nanoparticles (EmB) were synthesized to improve drug solubility, and they modified with oligomeric mannitol into microgels (EmB-MO) for targeted delivery to intestinal macrophages that express mannose receptors. UC was induced in a mouse model using dextran sulfate sodium (DSS), and different drug formulations were administered to the mice via drinking water. The levels of inflammation-related factors in the colon tissues and fecal matter were measured using enzyme-linked immunosorbent assay. Intestinal permeability was evaluated using fluorescein isothiocyanate dextran. HE staining, in vivo imaging, real-time PCR, and western blotting were performed to assess intestinal barrier dysfunction. RESULTS: Both EmB and EmB-MO markedly alleviated the symptoms of UC, including body weight loss, stool inconsistency, and bloody stools and restored the levels of pro- and anti-inflammatory cytokines. However, the therapeutic effects of EmB-MO on the macroscopic and immunological indices were stronger than those of EmB and similar to those of 5-aminosalicylic acid. Furthermore, EmB-MO selectively accumulated in the inflamed colon epithelium and restored the levels of the gut barrier proteins such as ZO-1 and Occludin. CONCLUSIONS: EmB-MO encapsulation significantly improved water solubility, which translated to greater therapeutic effects on the immune balance and gut barrier function in mice with DSS-induced UC. Our findings provide novel insights into developing emodin-derived drugs for the management of UC.

[Large- scale prospective clinical study on prophylactic intervention of COVID-19 in community population using Huoxiang Zhengqi Oral Liquid and Jinhao Jiere Granules].

To scientifically evaluate the intervention effect of Chinese medicine preventive administration(combined use of Huo-xiang Zhengqi Oral Liquid and Jinhao Jiere Granules) on community population in the case of coronavirus disease 2019(COVID-19), a large cohort, prospective, randomized, and parallel-controlled clinical study was conducted. Total 22 065 subjects were included and randomly divided into 2 groups. The non-intervention group was given health guidance only, while the traditional Chinese medicine(TCM) intervention group was given two coordinated TCM in addition to health guidance. The medical instructions were as follows. Huoxiang Zhengqi Oral Liquid: oral before meals, 10 mL/time, 2 times/day, a course of 5 days. Jinhao Jiere Granules: dissolve in boiling water and take after meals, 8 g/time, 2 times/day, a course of 5 days, followed up for 14 days, respectively. The study found that with the intake of medication, the incidence rate of TCM intervention group was basically maintained at a low and continuous stable level(0.01%-0.02%), while the non-intervention group showed an overall trend of continuous growth(0.02%-0.18%) from 3 to 14 days. No suspected or confirmed COVID-19 case occurred in either group. There were 2 cases of colds in the TCM intervention group and 26 cases in the non-intervention group. The incidence of colds in the TCM intervention group was significantly lower(P<0.05) than that in the non-intervention group. In the population of 16-60 years old, the incidence rate of non-intervention and intervention groups were 0.01% and 0.25%, respectively. The difference of colds incidence between the two groups was statistically significant(P<0.05). In the population older than 60 years old, they were 0.04% and 0.21%, respectively. The incidence of colds in the non-intervention group was higher than that in the intervention group, but not reaching statistical difference. The protection rate of TCM for the whole population was 91.8%, especially for the population of age 16-60(95.0%). It was suggested that TCM intervention(combined use of Huoxiang Zhengqi Oral Liquid and Jinhao Jiere Granules) could effectively protect community residents against respiratory diseases, such as colds, which was worthy of promotion in the community. In addition, in terms of safety, the incidence of adverse events and adverse reactions in the TCM intervention group was relatively low, which was basically consistent with the drug instructions.

Control of the polymyxin analog ratio by domain swapping in the nonribosomal peptide synthetase of Paenibacillus polymyxa.

Polymyxins are used as the last-line therapy against multidrug-resistant bacteria. However, their further clinical development needs to solve problems related to the presence of heterogeneous analogs, but there is still no platform or methods that can regulate the biosynthesis of polymyxin analogs. In this study, we present an approach to swap domains in the polymyxin gene cluster to regulate the production of different analogs. Following adenylation domain swapping, the proportion of polymyxin B1 increased from 41.36 to 52.90%, while that of B1-1 decreased from 18.25 to 3.09%. The ratio of polymyxin B1 and B3 following starter condensation domain swapping changed from 41.36 and 16.99 to 55.03 and 6.39%, respectively. The two domain-swapping strains produced 62.96% of polymyxin B1, 6.70% of B3 and 3.32% of B1-1. This study also revealed the presence of overflow fluxes between acetoin, 2,3-butanediol and polymyxin. To our best knowledge, this is the first report of engineering the polymyxin synthetase gene cluster in situ to regulate the relative proportions of polymyxin analogs. This research paves a way for regulating lipopeptide analogs and will facilitate the development of novel lipopeptide derivatives.

Quantitative analysis by reversed-phase high-performance liquid chromatography and retinal neuroprotection after topical administration of moxonidine.

AIM: To determine moxonidine in aqueous humor and iris-ciliary body by reversed-phase high performance liquid chromatography (RP-HPLC), and to evaluate the retinal neuroprotective effect after topical administration with moxonidine in a high intraocular pressure (IOP) model. METHODS: The eyes of albino rabbits were administered topically and ipsilaterally with 0.2% moxonidine. A RP-HPLC method was employed for the identification and quantification of moxonidine between 2 and 480min, which presented in the aqueous humor and iris-ciliary body. Flash electroretinography (F-ERG) amplitude and superoxide dismutase (SOD) level were measured between day 1 and day 15 after topical administration with moxonidine in a rabbit model of high IOP. Histological and ultrastructural observation underwent to analyze the changes of retinal morphology, the inner retinal layers (IRL) thickness, and retinal ganglion cell (RGC) counting. RESULTS: Moxonidine was detectable between 2 and 480min after administration, and the peak concentration developed both in the two tissues at 30min, 0.51 µg/mL in aqueous humor and 1.03 µg/g in iris-ciliary body. In comparison to control, F-ERG b-wave amplitude in moxonidine eyes were significantly differences between day 3 and day 15 (P<0.01) in the high IOP model; SOD levels were significantly higher at all time-points (P<0.01) with a maximum level of 20.29 U/mgprot at day 15; and RGCs were significantly higher (P<0.05). CONCLUSION: Moxonidine is a viable neuroprotective agent with application to high IOP model. All layers of retina, including RGC layer, retinal nerve fiber layer and INL, are more preserved after moxonidine administration. SOD plays a neuroprotective role in ocular hypertension-mediated RGC death.

Bio-removal of tetracycline antibiotics under the consortium with probiotics Bacillus clausii T and Bacillus amyloliquefaciens producing biosurfactants.

The contamination of the aquatic environments by tetracycline antibiotics (TCs) is an increasingly pressing issue. Here, we used the addition of exogenous surfactants and in situ biosynthesis of biosurfactants to remove tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), and their mixtures using the co-culture of probiotic Bacillus clausii T and Bacillus amyloliquefaciens HM618 producing surfactin. The addition of exogenous biosurfactants to remove TCs was superior to nonionic surfactants. The maximal bio-removal efficiencies for OTC and CTC among mixed antibiotics under the co-culture of B. clausii T and B. amyloliquefaciens HM618 were 76.6% and 88.9%, respectively, which were both better than the efficiency of the pure culture of B. clausii T. TCs were removed mainly through biotransformation rather than absorption and hydrolysis. The removal efficiency was in the order CTC > OTC > TC. The co-culture of B. clausii T and B. amyloliquefaciens HM618 alleviated the cytotoxicity of OTC and CTC. The toxicity of the biotransformation products was lower than that of the parent compounds. Demethylation, hydroxylation, and dehydration are likely the major mechanisms of TC biotransformation. These results illustrate the potential of using surfactants in the bioremediation of tetracycline antibiotics, and provide new avenues for further exploration of the bioremediation of antibiotics pollution.

Analysis of the fingerprint profile of bioactive constituents of traditional Chinese medicinal materials derived from animal bile using the HPLC-ELSD and chemometric methods: An application of a reference scaleplate.

Traditional Chinese medicinal materials derived from animal bile are widely applied in clinical therapy for thousands of years in several Southeast Asian countries. Although the constituents are similar, these crude drugs exhibit different pharmacological activities; bile acids are the main bioactive constituent. Depending on the source, the price of these crude drugs differs significantly. Therefore, a reliable fingerprint method is needed to analyze and distinguish these crude drugs with a similar composition. In this milieu, we aimed to establish a fingerprint chromatography method that can separate and detect several bile acids simultaneously. A high-performance liquid chromatography separation method was established with evaporative light scattering detection to detect the analytes. The main bioactive constituents of pig bile, cattle bile, sheep bile, bear bile, and three types of cow bezoar were analyzed using the proposed method. The fingerprint chromatography profile of 35 samples were obtained and analyzed using chemometric methods. Considering the differences among samples, a reference scaleplate method was used in the peak alignment procedure. Unsupervised methods (hierarchical cluster analysis and principle component analysis) and supervised methods (K-nearest neighbor, partial least squares discriminant analysis, support vector machine discriminant analysis, and soft independent modeling of class analogy) were used in the chemometric analysis. The results indicated that the fingerprint chromatograms of the seven crude drugs had fingerprint specificity and that they can be well distinguished. In addition, the reference scaleplate method using the chromatogram of a mixed standard solution is practically applicable for peak alignment in the analysis of samples with a large difference in chromatographic peaks. Overall, 17 bile acids can be separated and detected simultaneously using this method and some frequently used TCMMs derived from animal bile can be distinguished accurately.

[Expression of Forkhead boxO1 and Runt-related transcription factor 2 in periodontal tissue during orthodontic movement of teeth in rats].

PURPOSE: To investigate the expression of FoxO1 and Runx2 in periodontal tissue and their effect during orthodontic teeth movement(OTM) in rats. METHODS: Forty 8-week old male Sprague-Dawley (SD) rats were used to establish animal models of orthodontic teeth movement and divided into 5 groups randomly. The right side of jaws of each rat was set as experimental side, and the left side as control side. At 1, 3, 5, 7, 14 d after orthodontic treatment, the rats were sacrificed and the maxillary bone containing the first molar was dissected. H-E staining and immunohistochemical staining were used to detect the morphological changes, the expression of FoxO1 and Runx2 of the periodontal tissue at different points. Computer image analysis was used to evaluate the expression of FoxO1 and Runx2 in the periodontal tissues of the rats. The differences were analyzed by using SPSS19.0 software package. RESULTS: The expression of FoxO1 in periodontal ligament was mainly in osteoblasts and cementoblasts; and the expression of Runx2 was mainly in osteoblasts, fibroblasts and cementoblasts. In the experimental group,the expressions of FoxO1 and Runx2 in the periodontal tissues of rats increased significantly, reached the peak within 3-5 days, then decreased. There was no significant difference between the experimental group and the control group at 14th day (P>0.05), but significant difference was found between other group and control group (P<0.05). CONCLUSIONS: FoxO1 and Runx2 play a role in the reconstruction of periodontal tissue during orthodontic movement of teeth, and they are mainly involved in the process of osteoblast formation and bone formation.

SKOV-3 cell imaging by paramagnetic particles labeled with hairpin cell-penetrating peptides.

BACKGROUND: The hairpin cell-penetrating peptides (hCPPs) demonstrate an interesting characteristic of conditioned activation by molecules. We hypothesized that hCPPs have the potential to selectively deliver a paramagnetic gadolinium probe into the matrix metalloproteinase 2 (MMP-2) positive human ovary adenocarcinoma cell lines, SKOV-3. METHODS: hCPPs were synthesized and labeled with 1,4,7,10-tetraazacyclododecane-N,N',N'',N''' tetraacetic acid gadolinium (III) (Gd-DOTA) and fluorescein isothiocyanate (FITC) by f-moc strategy using a standard solid phase peptide synthesis protocol. MMP-2 expression and activity were demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) and zymography. Internalization and location of hCPPs in SKOV-3 cells were observed by fluorescein imaging and flow cytometery. Selective delivery of Gd-DOTA in SKOV-3 cells was observed by magnetic resonance imaging (MRI) and transmission electron microscopy (TEM). RESULTS: The uptake of hCPPs by SKOV-3 cells depended on the activity of MMP-2. T1WI signals of SKOV-3 cells treated with Gd-DOTA-hCPPs suggested the uptake of Gd-DOTA-hCPPs increased in a time- (r = 0.990, P < 0.01) and concentration-dependent manner (r = 0.964, P < 0.001), but was inhibited by a MMP-2 inhibitor. Electron-dense particles observed in the cytoplasm and nucleus by transmission electron microscopy proved the intracellular penetration of gadolinium. CONCLUSIONS: hCPPs can be used as an effective vector for an MRI molecular probe to assess the activity of MMP-2.

Applications of HPLC/MS in the analysis of traditional Chinese medicines.

In China, traditional Chinese medicines (TCMs) have been used in clinical applications for thousands of years. The successful hyphenation of high-Performance liquid chromatography (HPLC) and mass spectrometry (MS) has been applied widely in TCMs and biological samples analysis. Undoubtedly, HPLC/MS technique has facilitated the understanding of the treatment mechanism of TCMs. We reviewed more than 350 published papers within the last 5 years on HPLC/MS in the analysis of TCMs. The present review focused on the applications of HPLC/MS in the component analysis, metabolites analysis, and pharmacokinetics of TCMs etc. 50% of the literature is related to the component analysis of TCMs, which show that this field is the most populär type of research. In the metabolites analysis, HPLC coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry has been demonstrated to be the powerful tool for the characterization of structural features and fragmentation behavior patterns. This paper presented a brief overview of the applications of HPLC/MS in the analysis of TCMs. HPLC/MS in the fingerprint analysis is reviewed elsewhere.

[Study on estimation of radiation dose using FTIR in mice thymus].

Fourier transform infrared spectroscopy (FTIR) was applied to study the biochemical changes in the radiation damaged mouse thymus which increased with radiation dose and provided a new method for the estimation of the radiation dose of radiation damaged patients. The results demonstrated that with the dose increasing, the peak positions like 1 550, 1 400, 1 400 and 1 640 cm(-1) at the dose of 2, 3 and 5 Gy showed some difference, and there was obvious variance in the intensity: (1) The intensity ratio of 1 085 to 1 236 cm(-1) related to nucleic acid tended to decrease. (2) The intensity ratio of 1 640/1 550 decreased. (3) The intensity at 2 958, 2 925, 1 460 and 1 400 cm(-1) showed no significant difference. The results suggest that it may be possible for FTIR to become an effective method to estimate the radiation dose in clinic.

[A preliminary study on screening for Porphyromonas gingivalis outer membrane protein antigen with two-dimensional liquid phase fractionation and matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry].

OBJECTIVE: To screen a variety of Porphyromonas gingivalis (Pg) common outer membrane proteins with two-dimensional liquid phase fractionation (PF2D) and matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) and provide candidate target antigen for the design of vaccines with cross protection against a variety of Pg. METHODS: The outer membrane proteins of Pg301, PgATCC33277 and PgW83 were extracted through ultracentrifugation, and then they were separated by ProteomeLab PF2D protein fractionation system. After separation, the outer membrane proteins were obtained through comparison, and the primary structure of the proteins was identified by MALDI-TOF/TOF-MS and database. RESULTS: Ninety-nine protein samples out of 3 strains of Pg were obtained after the high performance chromato focusing (HPCF) separation process. B7 fractions of 3 strains of Pg were separated by the reversed-phase high performance liquid chromatography (RP-HPCL) separation process. After comparison of peak and retention time of chromatogram, the 8 common protein peaks of 3 strains of Pg were confirmed. The protein samples were identified by MALDI-TOF/TOF-MS, and one of them was known protein arg-gingipain A. CONCLUSIONS: PF2D protein fractionation system is of good reproducibility and high resolution. A combination of PF2D and MALDI-TOF/TOF-MS can be used to identify the common outer membrane proteins of Pg.

[Metabolic kinetic of puerarin in beagle liver microsomal by HPLC-ESI-MS].

OBJECTIVE: To develop a HPLC-ESI-MS method for the determination of puerarin and its metabolite and study the metabolic kinetics in beagle dog liver microsomes. METHOD: Beagle dog liver microsomes were prepared by using ultracentrifugation method. Chromatography was performed on a Shimadzu C18 column (2.0 mm x 150 mm, 5 microm). Amethanol-water gradient system was used. ESI interface was applied in the positive, and SIM m/z 417 was puerarin and m/z 531 was daidzein. RESULT: The puerarin was metabolized by NADPH regenerating system in beagle dog microsomes. The Michaelis-Menten parameters Km and Vmax in beagle dog microsomes were initially estimated by analyzing Lineweave-Brurk plot. The Vmax Km of puerarin were (0.047 +/- 0.006) mg x min(-1) x g(-1), (1.22 +/- 0.53) mg x L(-1). CONCLUSION: The puerarin and daidzein can be rapidly determined by HPLC-MS in beagle dog microsomes and the puerarin was metabolized to daidzein by CY P450. The study can give help for Baige capsule.

[Analysis of supercritical fluid extracts of Radix caulophylli with gas chromatography-mass spectrometry].

To analyze the constituents in supercritical fluid CO2 extraction (SFE-CO2) of Radix caulophylli, the Radix caulophylli was extracted with SFE-CO2, and analyzed by gas chromatography-mass spectrometry (GC-MS). The GC-MS analysis with a DB-5MS capillary column (30 mm x 0.32 mm ID, 0.25 microm film thickness) was used. The inlet temperature was maintained at 280 degrees C. The column oven was held at 80 degrees C for 2 min, then programmed from 80 to 280 degrees C at 5 degrees C x min(-1) and, finally, held for 4 min. Helium at a constant flow rate of 2.0 mL x min(-1) was used as the carrier gas. The mass spectrometry conditions were as follows: ionization energy, 70 eV; ion source temperature, 200 degrees C. The mass selective detector was operated in the TIC mode (m/z was from 40 - 500). For the first time 49 peaks were separated and identified, the compounds were quantitatively determined by normalization method, and the identified compounds represent 97.44% of total GC peak areas. Viz, n-hexadecanoic acid (31.4%), (E, E) -9, 12-octadecadienoic acid (26.54%), (Z)-7-tetradecenal (9.4%), hexadecenoic acid (3.23%), 10-undecenal (3.22%), octadecanoic acid (2.25%), and caulophylline (1.76%) etc. The results will provide important foundation for understanding the constituents and further exploitation of Radix caulophylli.

[Characteristics and in vitro imaging study of matrix metalloproteinase-2 targeting activable cell-penetrating peptide].

OBJECTIVE: To study invasively imaging MMP2-positive tumor cell by paramagnetic Gadolinium or fluorescein carried by a activable cell penetrating peptides. METHODS: To label Fluorescein-5-isothiocyanate (FITC) and gadopentetate with the activable cell-penetrating peptides by a solid-phase synthesis method. Identification by TOF mass spectrum (TOF-MS). Isoelectric point of the activable cell penetrating peptides is determined by disc electrophoresis. T1 relacion of gadopentetate labeled with the activable cell-penetrating peptides (B) in water were determined on 400 MHZ NMR. Human lung cancer cell lines: A549 were respectively stained by FITC labeled with ACPPs (A) or FITC alone. MMP2 expression and activity were determined by zymography. T1WI signal of A549 incubated with 120 nmol/ml B or Diethylenetriaminepentaacetic Acid-Gadolinium (Gd-DTPA) for different times were obtained by 1.5T MRI. The location of B in A549 was detected by Transmission Electron Microscopy. Visualization analysis and half-quantitative analysis were used to determine the signal characteristics. The ANOVA analysis and the paired samples t test were performed by SPSS 13.0. RESULTS: MALDI TOF-MS molecular weigh of A and B respectively is 3789.74 and 3911.93. Isoelectric point is 11.005T1 Relacion of 0.5 mmol/L Gd-DTPA and B at 17 degrees C respectively is (0.052 +/- 0.01) sec and (0.050 +/- 0.001) sec. Fluorescein uptake assays showed that A translocated into A549 but would be inhibited by MMP2 antibody. Zymography showed both active-MMP2 (67000) and pro-MMP2 (72000) expressed byA549. MR imaging showed A549 incubating with B had a high T(1) signal, and the signal of A549 incubating with Gd-DTPA is similar with that of the control group. Furthermore, ANOVA suggested that the T(1) signal intensity of A549 incubating with B was effected by incubating time (F = 267.569, P < 0.001) and increasing in a time-dependent fashion at the observed time. There is no difference between the T(1) signal intensity of A549 incubating with Gd-DTPA and the control group (P > 0.05). TEM showed A in cytoplasm and nucleus. CONCLUSION: The study in vitro suggests that the MMP-2 activable cell-penetrating peptides bearing contrast media can detect the MMP2-positive tumor cell.

Intercellular imaging by a polyarginine derived cell penetrating peptide labeled magnetic resonance contrast agent, diethylenetriamine pentaacetic acid gadolinium.

BACKGROUND: The cellular plasma membrane represents a natural barrier to many exogenous molecules including magnetic resonance (MR) contrast agent. Cell penetrating peptide (CPP) is used to internalize proteins, peptides, and radionuclide. This study was undertaken to assess the value of a new intracellular MR contrast medium, CPP labeled diethylenetriamine pentaacetic acid gadolinium (Gd-DTPA) in molecular imaging in vitro. METHODS: Fluorescein-5-isothiocyanate (FITC) and Gd-DTPA respectively labeled with CPP (FITC-CPP, Gd-DTPA-CPP) were synthesized by the solid-phase method. Human hepatic cancer cell line-HepG2 was respectively stained by FITC-CPP and FITC to observe the uptake and intracellular distribution. HepG2 was respectively incubated with 100 nmol/ml Gd-DTPA-CPP for 0, 10, 30, 60 minutes, and imaged by MR for studying the relationship between the incubation time and T(1)WI signal. The cytotoxicity to NIH3T3 fibroblasts cells was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide reduction assay (MTT). RESULTS: The molecular weights of CPP labeled imaging agents, which were determined by MALDI mass spectrometry (FITC-CPP MW = 2163.34, Gd-DTPA-CPP MW = 2285.99), were similar to the calculated molecular weights. Confocal microscopy suggested HepG2 translocated FITC-CPP in cytoplasm and nucleus independent with the incubation temperature. MR images showed HepG2 uptaken Gd-DTPA-CPP had a higher T(1) weighted imaging (T(1)WI) signal, and that the T(1)WI signal intensity was increasing in a time-dependent manner (r = 0.972, P = 0.001), while the signal intensity between the cells incubated by Gd-DTPA for 60 minutes and the controlled cells was not significantly different (P = 0.225). By MTT, all concentrations from 50 nmol/ml to 200 nmol/ml had no significant (F = 0.006, P = 1.000) effect on cell viability of mouse NIH3T3 fibroblasts, compared with the control group. CONCLUSIONS: The newly constructed CPP based on polyarginine can translocate cells by carrying FITC and MR contrast agent Gd-DTPA, and the intracellular concentrations are readily detectable by MR imaging, suggesting a new way for MR molecular imaging.

Application of cell penetrating peptide in magnetic resonance imaging of bone marrow mesenchymal stem cells.

Tracking the distribution and differentiation of stem cells by high-resolution imaging techniques would have significant clinical and research implications. In this study, a model cell-penetrating peptide was used to carry gadolinium particles for magnetic resonance imaging (MRI) of mesenchymal stem cells (MSCs). MSCs were isolated from rat bone marrow and identified by osteogenic differentiation in vitro. The cell-penetrating peptide labeled with fluorescein-5-isothiocyanate (FITC) and gadolinium was synthesized by a solid-phase peptide synthesis method. Fluorescein imaging analysis confirmed that this new peptide could internalize into the cytoplasm and nucleus at room temperature, 4 degrees and 37 degrees . Gadolinium were efficiently internalized into mesenchymal stem cells by the peptide in a time or concentration-dependent manner, resulting in intercellular shortening of longitudinal relaxation enhancements, which were obviously detected by 1.5 Tesla Magnetic Resonance Imaging. Cytotoxicity assay and flow cytometric analysis showed that the intercellular contrast medium incorporation did not affect cell viability at the tested concentrations. The in vitro experiment results suggested that the new constructed peptides could be a vector for tracking MSCs.

Salt loading on plasma asymmetrical dimethylarginine and the protective role of potassium supplement in normotensive salt-sensitive asians.

Asymmetrical dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase. Because endothelial NO pathway is compromised in patients with salt-sensitive hypertension, we investigated whether the plasma ADMA can be modulated by chronic salt loading in normotensive salt-sensitive persons and its relationship with NO, and we further determined whether or not dietary potassium supplementation can reverse them. Sixty normotensive subjects (aged 20 to 60 years) were selected from a rural community of Northern China. All of the people were sequentially maintained on a low-salt diet for 7 days (3 g/day, NaCl), then a high-salt diet for 7 days (18 g/day), and high-salt diet with potassium supplementation for another 7 days (4.5 g/day, KCl). After salt loading, the plasma ADMA concentrations increased significantly in salt-sensitive subjects (0.89+/-0.02 micromol/L versus 0.51+/-0.02 micromol/L; P<0.05), whereas the plasma NOx levels reduced considerably (41.8+/-2.1 micromol/L versus 63.5+/-2.1 micromol/L; P<0.01). All of the abnormalities normalized when dietary potassium were supplemented (0.52+/-0.03 micromol/L versus 0.89+/-0.02 micromol/L for ADMA and 58.1+/-0.9 micromol/L versus 41.8+/-2.1 micromol/L for NOx). Statistically significant correlations were found among plasma ADMA level, the mean blood pressure, and the level of NO after salt loading in normotensive salt sensitive individuals. Our study indicates that high dietary potassium intake reduces blood pressure and ADMA levels while increasing NO bioactivity in normotensive salt-sensitive but not salt-resistant Asian subjects after salt loading.

Paramagnetic particles carried by cell-penetrating peptide tracking of bone marrow mesenchymal stem cells, a research in vitro.

The ability to track the distribution and differentiation of stem cells by high-resolution imaging techniques would have significant clinical and research implications. In this study, a model cell-penetrating peptide was used to carry gadolinium particles for magnetic resonance imaging of the mesenchymal stem cells. The mesenchymal stem cells were isolated from rat bone marrow by Percoll and identified by osteogenic differentiation in vitro. The cell-penetrating peptides labeled with fluorescein-5-isothiocyanate and gadolinium were synthesized by a solid-phase peptide synthesis method and the relaxivity of cell-penetrating peptide-gadolinium paramagnetic conjugate on 400 MHz nuclear magnetic resonance was 5.7311 +/- 0.0122 m mol(-1) s(-1), higher than that of diethylenetriamine pentaacetic acid gadolinium (p < 0.05). Fluorescein imaging confirmed that this new peptide could internalize into the cytoplasm and nucleus. Gadolinium was efficiently internalized into mesenchymal stem cells by the peptide in a time- or concentration-dependent fashion, resulting in intercellular T1 relaxation enhancement, which was obviously detected by 1.5 T magnetic resonance imaging. Cytotoxicity assay and flow cytometric analysis showed the intercellular contrast medium incorporation did not affect cell viability and membrane potential gradient. The research in vitro suggests that the newly constructed peptides could be a vector for tracking mesenchymal stem cells.