Prognostic significance of prognostic nutritional index and systemic immune-inflammation index in patients after curative breast cancer resection: a retrospective cohort study.

BACKGROUND: Nutritional status and inflammation are closely associated with poor outcome in malignant tumors. However, the prognostic impact of postoperative in these variables on breast cancer (BC) remains inconclusive. We aimed to determine whether prognostic nutritional index (PNI), systemic immune-inflammation index (SII), neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) affect two long-term outcomes among patients after curative resection of BC. METHODS: We retrospectively reviewed 508 patients with BC treated with curative surgery between February 5, 2013 and May 26, 2020. All patients were divided into 3 groups based on tertiles (T1-T3) of PNI, SII, NLR, and PLR. The effects of four indexes on disease-free survival (DFS) and overall survival (OS) have been evaluated using Cox proportional hazards models and Kaplan-Meier method. RESULTS: Compared with PNI-lowest cases, patients with highest PNI showed significantly longer DFS (multivariate adjusted hazard ratio [HR] = 0.37, 95% confident interval [CI] 0.19-0.70, P for trend = 0.002), whereas higher PLR seemed to be marginally associated with poorer DFS (P for trend = 0.086 and 0.074, respectively). Subgroup analyses indicate the potential modification effects of family history of BC and radiotherapy on the prognosis value of PNI to DFS in BC patients (P for interaction = 0.004 and 0.025, respectively). In addition, the levels of three inflammatory indices, namely SII, NLR, and PLR might be positively related with increased age at diagnosis (all P for trend < 0.001). CONCLUSIONS: A high PNI was associated with better DFS, supporting its roles as prognostic parameters for patients with BC. The nutritional status and systemic immune may exert great effects on patient prognosis. Further studies are warrant to explore the prognosis value of PLR.

Up-regulation expression and prognostic significance of Syntaxin4 in kidney renal clear cell carcinoma.

BACKGROUND: Syntaxin4 (STX4) gene encodes the protein STX4, a member of soluble N-ethylmaleimide-sensitive factor attachment protein receptors protein, playing a vital role in cell invadopodium formation and invasion, which is associated with the malignant progression of various human cancers. However, the expression and prognostic significance of STX4 in kidney renal clear cell carcinoma (KIRC) remain to be investigated. METHODS: In this study, we collected the mRNA expression of STX4 in 535 KIRC patients from The Cancer Genome Atlasthrough the University of California Santa Cruz Xena database platform. Then we explored the expression of STX4 in KIRC, and the relationship with clinicopathological characteristics and prognostic value. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes function enrichment analyses were used to explore the potential mechanism of STX4 in KIRC. qRT-PCR analysis was performed toverify the above results with real world tissue specimens. RESULTS: The results indicated that STX4 was up-expressed in KIRC, and were associated with higher histological grade, advanced stage, and poorer prognosis. Moreover, elevated STX4 expression is an independent risk factor for KIRC. qRT-PCR analysis showed that STX4 was significantly elevated in 10 paired of KIRC samples compared to normal samples. Functional enrichment analysis indicated that endo/exocytosis, autophagy, mTOR signaling pathway, and NOD-like receptor signaling pathway were enriched. CONCLUSIONS: In summary, STX4 is constantly up-expressed in KIRC tissues, associated with a poor prognosis. We suggest that it can be an effective biomarker for the prognosis of KIRC and may be a novel therapeutic target in KIRC.

Cost-effectiveness analyses of sacubitril-valsartan for heart failure.

The objective of this study was to evaluate the pharmacoeconomic value of sacubitril-valsartan for the treatment of heart failure (HF). PubMed, Embase, Cochrane Library, ScienceDirect, Scopus, CNKI, Wanfang, and VIP databases were searched systematically and the retrieval time ended in August 2019. According to the criteria of inclusion and exclusion, the quality of studies included was evaluated as per the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) scale, and the results were extracted and analyzed systematically. The total of 11 cost-effectiveness studies was identified, 10 were performed in the developed countries and 1 in Thailand. All the patients in the studies had chronic heart failure with reduced ejection fraction (HFrEF). Totally, the quality of all the 11 studies was reported to be of an average score of 20.5. Study perspective and time horizons were described in the 11 studies. All included studies discounted the cost or effectiveness. Only 1 study estimated direct and indirect costs; 10 studies evaluated direct cost. The incremental cost-effectiveness ratio (ICER) of sacubitril-valsartan treating HFrEF was $13,150 per quality-adjusted life-years (QALY) in Thailand and $86,735 in Singapore. In European countries, the ICER was from $21,786 to $34,576 per QALY and mean value was $25,410.6 per QALY. In the USA, ICER values ranged from $47,099 to $143,891 per QALY, and mean value was $73,383.5 per QALY; ICER was $30,090 per QALY in Colombia. With the exception of Thailand and Singapore, the ICER of other countries in the included literature was below the implemented country-specific thresholds. Based on existing literatures, with the exception of Thailand and Singapore, sacubitril-valsartan for the treatment of HFrEF is a better cost-effective therapy with ICER basically below the implemented country-specific thresholds. Sacubitril-valsartan was not considered a cost-effective treatment for heart failure with reduced ejection fraction in Thailand and Singapore with the current economic evaluation evidences, but with the willingness-to-pay (WTP) of other counties, sacubitril-valsartan was found to be a cost-effective treatment compared with comparator. Drug cost, time horizon, and hospitalization were the most influential variables across studies. Four studies indicated that with the longer time horizon, the lower ICER value would gain. Further studies are warranted to better evaluate comprehensive utility value of sacubitril-valsartan on heart failure.

Application of Immunoassays for Rapid Monitor of Carbofuran Residue in Vegetables.

Carbofuran residue in vegetables is a concern to human health. Direct competitive enzyme-linked immunosorbent assay (dcELISA) and dipstick immunoassay were developed in the present study. The dcELISA showed a 50% inhibition concentration (IC50 ) and working range of 1.3 and 0.2 to 7.5 ng/mL, respectively, while the cutoff value of dipstick immunoassay was 20 ng/mL. Applying the two immunoassays, we achieved the goal of rapid screening of carbofuran residue in commercial vegetables with a simple sample processing method. Among 46 leek, 39 potato, and 39 sweet potato samples, carbofuran residue was detected in 22% of the leek samples, and two samples exceeded the maximum residue limit of China (0.02 mg/kg). In addition, carbofuran residue was found at less than 2.5 ng/g in one potato and one sweet potato samples. The residual level of carbofuran measured by immunoassays agreed well with those determined by ultra-performance liquid chromatography tandem mass spectrometry. To ensure food safety and human health, it is greatly necessary and meaningful to monitor carbofuran residue in commercial vegetables. PRACTICAL APPLICATION: Rapid monitoring of carbofuran residue in vegetables is very necessary and important for consumers, regulatory agencies, and food industry.

[Pharmacokinetics of eight constituents in rat plasma after oral administration of Modified Xiaochaihu Granules for gastric ulcer based on UPLC-MS/MS].

An ultra-performance liquid chromatography-tandem mass spectrometric method (UPLC-MS/MS) was developed in this study to simultaneously determine the contents of eight effective constituents in rat plasma, including baicalin, wogonoside, baicalein, liquiritin, glycyrrhizic acid, berberine hydrochloride, saikosaponin a and saikosaponin d in plasma of gastric ulcer rats, and investigate the pharmacokinetics of Modified Xiaochaihu Granules. Chromatographic separation was conducted on Zorbax SB-C18 column (2.1 mm×100 mm, 1.8 µm) with acetonitrile -0.1% formic acid aqueous solution as the mobile phase for gradient elution, at a flow rate of 0.4 mL·min⁻¹ and column temperature of 40 °C. Detection was performed in the multiple reaction monitoring (MRM) mode with ESI ion source. All calibration curves showed good linearity (r>0.996) over a wide concentration range for all constituents. RSDs of intra-day and inter-day precision were all within 15% and the extraction recoveries of all the constituents were in the range of 81.92% to 104.8%. The time to peak (tmax) of these eight constituents was (2.69±2.02), (5.17±2.04), (0.25±0), (0.83±0.26), (0.92±0.20), (0.92±0.20), (0.58±0.20), and (0.083±0) h, respectively; the half-life (t1/2) of them was (7.85±0.34), (10.16±2.21), (6.79±0.21), (8.32±0.48), (11.05±1.78), (11.56±3.46), (15.30±1.84), and (5.54±1.91) h, respectively; the peak concentration (Cmax) of them was (55.02±1.67), (213.66±4.62), (62.61±0.69), (68.43±1.42), (62.22±0.39), (30.17±1.89), (61.79±4.81), and (38.02±1.75) µg·L⁻¹, respectively. This established method is simple and accurate with good repeatability and strong specificity, which could provide modern experimental basis for modified Xiaochaihu granules in clinical treatment of gastric ulcer.

Quality Testing of Artemisinin-Based Antimalarial Drugs in Myanmar.

Artemisinin-based combination therapies are the frontline treatment of Plasmodium falciparum malaria. The circulation of falsified and substandard artemisinin-based antimalarials in Southeast Asia has been a major predicament for the malaria elimination campaign. To provide an update of this situation, we purchased 153 artemisinin-containing antimalarials, as convenience samples, in private drug stores from different regions of Myanmar. The quality of these drugs in terms of their artemisinin derivative content was tested using specific dipsticks for these artemisinin derivatives, as point-of-care devices. A subset of these samples was further tested by high-performance liquid chromatography (HPLC). This survey identified that > 35% of the collected drugs were oral artesunate and artemether monotherapies. When tested with the dipsticks, all but one sample passed the assays, indicating that the detected artemisinin derivative content corresponded approximately to the labeled contents. However, one artesunate injection sample was found to contain no active ingredient at all by the dipstick assay and subsequent HPLC analysis. The continued circulation of oral monotherapies and the description, for the first time, of falsified parenteral artesunate provides a worrisome picture of the antimalarial drug quality in Myanmar during the malaria elimination phase, a situation that deserves more oversight from regulatory authorities.

Pilot testing of dipsticks as point-of-care assays for rapid diagnosis of poor-quality artemisinin drugs in endemic settings.

BACKGROUND: Good-quality artemisinin drugs are essential for malaria treatment, but increasing prevalence of poor-quality artemisinin drugs in many endemic countries hinders effective management of malaria cases. METHODS: To develop a point-of-care assay for rapid identification of counterfeit and substandard artemisinin drugs for resource-limited areas, we used specific monoclonal antibodies against artesunate and artemether, and developed prototypes of lateral flow dipstick assays. In this pilot test, we evaluated the feasibility of these dipsticks under different endemic settings and their performance in the hands of untrained personnel. RESULTS: The results showed that the dipstick tests can be successfully performed by different investigators with the included instruction sheet. None of the artemether and artesunate drugs collected from public pharmacies in different endemic countries failed the test. CONCLUSION: It is possible that the simple dipstick assays, with future optimization of test conditions and sensitivity, can be used as a qualitative and semi-quantitative assay for rapid screening of counterfeit artemisinin drugs in endemic settings.

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the analysis of 6-benzylaminopurine and its ribose adduct in bean sprouts.

6-Benzylaminopurine (6-BA), a cytokinin plant growth regulator, has been banned for use in bean sprout production in China. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed with a specific monoclonal antibody (mAb 3E5). The assay showed a half-maximum inhibition concentration (IC50) and detection range of 18.9 ng/mL and 3.6-106 ng/mL, respectively. Recoveries of 6-BA spiked in home cultured bean sprout samples averaged from 75% to 89% with a correlation coefficient (R(2)) of 0.998 between the results determined by icELISA and those by liquid chromatography-electrospray ionization quadrupole Orbitrap mass spectrometry (LC-ESI-MS). LC-ESI-MS showed that 6-BA had been partially metabolized to 6-benzylaminopurine riboside (6-BAR) in the positive samples. The content of 6-BA determined by icELISA was about 5-70 times higher than that of LC-ESI-MS because mAb 3E5 had 315% cross-reactivity with 6-BAR. Such icELISA being ultra-sensitive to 6-BAR would allow quick monitoring of 6-BA by detecting 6-BAR as a potential biomarker.

Rapid evaluation of artesunate quality with a specific monoclonal antibody-based lateral flow dipstick.

Artesunate is a frontline antimalarial drug for treating Plasmodium falciparum malaria. To produce specific antibodies to artesunate, the carboxyl group of artesunate was directly conjugated to carrier protein as the immunogen. A specific monoclonal antibody (mAb) 3D82G6 against artesunate was obtained by high-throughput screening of positive hybridoma clones. This monoclonal antibody had 4.0, 0.5, and 0.9 % cross reactivities with artemisinin, dihydroartemisinin, and artemether, respectively. A dipstick immunoassay was developed, and the indicator range for artesunate was 1000-2000 ng mL(-1). No interference was observed with artemisinin, dihydroartemisinin, artemether, and other commonly used antimalarial drugs for up to 20,000 ng mL(-1). The dipsticks were used for determination of artesunate contents in commercial drugs, and the results were agreeable with those determined by high-performance liquid chromatography. This dipstick, with its specificity and sensitivity for artesunate and simplicity to use, makes it a potential point-of-care device for rapid quality evaluation of artesunate-containing antimalarial drugs. Graphical Abstract Specific monoclonal antibody-based lateral flow dipstick for artesunate.

Sonic hedgehog improves ischemia-induced neovascularization by enhancing endothelial progenitor cell function in type 1 diabetes.

The Sonic hedgehog (Shh) pathway is downregulated in type 1 diabetes, and it has been reported that augmentation of this pathway may alleviate diabetic complications. However, the cellular mechanisms underlying these protective effects are poorly understood. Recent studies indicate that impaired function of endothelial progenitor cells (EPCs) may contribute to cardiovascular problems in diabetes. We hypothesized that impaired Shh signaling contribute to endothelial progenitor cell dysfunction and that activating the Shh signaling pathway may rescue EPC function and promote diabetic neovascularization. Adult male C57/B6 mice and streptozotocin (STZ)-induced type 1 diabetic mice were used. Gli1 and Ptc1 protein levels were reduced in EPCs from diabetic mice, indicating inhibition of the Shh signaling pathway. EPC migration, tube formation ability, and mobilization were impaired in diabetic mice compared with non-diabetic controls (p < 0.05 vs control), and all were improved by in vivo administration of the Shh pathway receptor agonist SAG (p < 0.05 vs diabetes). SAG significantly increased capillary density and blood perfusion in the ischemic hindlimbs of diabetic mice (p < 0.05 vs diabetes). The AKT activity was lower in EPCs from diabetic mice than those from non-diabetic controls (p < 0.05 vs control). This decreased AKT activity led to an increased GSK-3ß activity and degradation of the Shh pathway transcription factor Gli1/Gli2. SAG significantly increased the activity of AKT in EPCs. Our data clearly demonstrate that an impaired Shh pathway mediated by the AKT/GSK-3ß pathway can contribute to EPC dysfunction in diabetes and thus activating the Shh signaling pathway can restore both the number and function of EPCs and increase neovascularization in type 1 diabetic mice.

TGF-ß Regulates Survivin to Affect Cell Cycle and the Expression of EGFR and MMP9 in Glioblastoma.

Transforming growth factor beta (TGF-ß) is suggestive of a molecular target for cancer therapy due to its involvement in cell cycle, differentiation, and morphogenesis. Meanwhile, survivin is identified as an apoptosis inhibitor and involved in tumorgenesis. Here, we aimed to investigate the potential associations between TGF-ß and survivin in glioblastoma U87 cell line. Survivin small interfering RNA (siRNA), Western blotting, and cell cycle analysis were introduced to detect relevant proteins in TGF-ß pathways. In this study, we observed a concentration- and time-dependent increase of survivin expression after treatment with TGF-ß1. However, the kinase inhibitors U0126 and LY294002 inhibited the upregulation of survivin in comparison with DMSO. In addition, survivin siRNA effectively abrogated survivin expression in U87 cells, therefore affected cells' entry into the S phase of cell cycle, and then repressed the expression of epidermal growth factor receptor (EGFR) and matrix metalloproteinase 9 (MMP9) in comparison with non-transfection. In conclusion, the present study shows that TGF-ß upregulates survivin expression via ERK and PI3K/AKT pathway, leading to glioblastoma cell cycle progression. Thus, the blockade of survivin will allow for the treatment of glioblastoma, partially attributing to the inhibition of EGFR and MMP9 expression.

Oxidative stress contributes to the impaired sonic hedgehog pathway in type 1 diabetic mice with myocardial infarction.

Our previous study demonstrated that an impaired sonic hedgehog (Shh) pathway contributed to cardiac dysfunction in type 1 diabetic mice with myocardial infarction (MI). The present study aimed to test the hypothesis that oxidative stress may contribute to the impaired Shh pathway and cardiac dysfunction in type 1 diabetic mice with MI. Streptozotocin-induced type 1 diabetic mice (C57/Bl6, male) and rat neonatal cardiomyocytes were used in the present study. Mice were randomly assigned to undergo ligation of the coronary artery or pseudosurgery. A potent antioxidant Tempol was administered in vivo and in vitro. Cardiac function was assessed by echocardiography, capillary density by immunohistochemisty, percentage of myocardial infarct using Massons trichrome staining, reactive oxygen species detection using dihydroethidium dye or 2,7-dichlorofluorescein diacetate probe and protein expression levels of the Shh pathway by western blot analysis. The antioxidant Tempol was shown to significantly increase myocardial protein expression levels of Shh and patched-1 (Ptc1) at 7-18 weeks and improved cardiac function at 18 weeks in type 1 diabetic mice, as compared with mice receiving no drug treatment. Furthermore, myocardial protein expression levels of Shh and Ptc1 were significantly upregulated on day 7 after MI, and capillary density was enhanced. In addition, the percentage area of myocardial infarct was reduced, and the cardiac dysfunction and survival rate were improved on day 21 in diabetic mice treated with Tempol. In vitro, treatment of rat neonatal cardiomyocytes with a mixture of xanthine oxidase and xanthine decreased protein expression levels of Shh and Ptc1 in a concentration-dependent manner, and Tempol attenuated this effect. These results indicate that oxidative stress may contribute to an impaired Shh pathway in type 1 diabetic mice, leading to diminished myocardial healing and cardiac dysfunction. Antioxidative strategies aimed at restoring the endogenous Shh pathway may offer a useful means for improving diabetic cardiac function.

Development of a Monoclonal Antibody-Based icELISA for the Detection of Ustiloxin B in Rice False Smut Balls and Rice Grains.

Rice false smut is an emerging and economically-important rice disease caused by infection by the fungal pathogen Villosiclava virens. Ustiloxin B is an antimitotic cyclopeptide mycotoxin isolated from the rice false smut balls that formed in the pathogen-infected rice spikelets. A monoclonal antibody (mAb) designated as mAb 1B5A10 was generated with ustiloxin B-ovalbumin conjugate. A highly-sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed. The median inhibitory concentration (IC50) of the icELISA was 18.0 ng/mL for the detection of ustiloxin B; the limit of detection was 0.6 ng/mL, and the calibration range was from 2.5 to 107.4 ng/mL. The LOD/LOQ values of the developed ELISA used for the determination of ustiloxin B in rice false smut balls and rice grains were 12/50 µg/g and 30/125 ng/g, respectively. The mAb 1B5A10 cross-reacted with ustiloxin A at 13.9% relative to ustiloxin B. Average recoveries of ustiloxin B ranged from 91.3% to 105.1% for rice false smut balls at spiking levels of 0.2 to 3.2 mg/g and from 92.6% to 103.5% for rice grains at spiking levels of 100 to 5000 ng/g. Comparison of ustiloxin B content in rice false smut balls and rice grains detected by both icELISA and high performance liquid chromatography (HPLC) demonstrated that the developed icELISA can be employed as an effective and accurate method for the detection of ustiloxin B in rice false smut balls, as well as rice food and feed samples.

Impaired autophagy contributes to adverse cardiac remodeling in acute myocardial infarction.

OBJECTIVE: Autophagy is activated in ischemic heart diseases, but its dynamics and functional roles remain unclear and controversial. In this study, we investigated the dynamics and role of autophagy and the mechanism(s), if any, during postinfarction cardiac remodeling. METHODS AND RESULTS: Acute myocardial infarction (AMI) was induced by ligating left anterior descending (LAD) coronary artery. Autophagy was found to be induced sharply 12-24 hours after surgery by testing LC3 modification and Electron microscopy. P62 degradation in the infarct border zone was increased from day 0.5 to day 3, and however, decreased from day 5 until day 21 after LAD ligation. These results indicated that autophagy was induced in the acute phase of AMI, and however, impaired in the latter phase of AMI. To investigate the significance of the impaired autophagy in the latter phase of AMI, we treated the mice with Rapamycin (an autophagy enhancer, 2.0 mg/kg/day) or 3-methyladenine (3MA, an autophagy inhibitor, 15 mg/kg/day) one day after LAD ligation until the end of experiment. The results showed that Rapamycin attenuated, while 3MA exacerbated, postinfarction cardiac remodeling and dysfunction respectively. In addition, Rapamycin protected the H9C2 cells against oxygen glucose deprivation in vitro. Specifically, we found that Rapamycin attenuated NFκB activation after LAD ligation. And the inflammatory response in the acute stage of AMI was significantly restrained with Rapamycin treatment. In vitro, inhibition of NFκB restored autophagy in a negative reflex. CONCLUSION: Sustained myocardial ischemia impairs cardiomyocyte autophagy, which is an essential mechanism that protects against adverse cardiac remodeling. Augmenting autophagy could be a therapeutic strategy for acute myocardial infarction.

Development of a colloidal gold-based lateral flow dipstick immunoassay for rapid qualitative and semi-quantitative analysis of artesunate and dihydroartemisinin.

BACKGROUND: Artemisinin-based combination therapy (ACT) plays an indispensable role in malaria control and elimination. However, the circulation of counterfeit, substandard drugs has greatly threatened malaria elimination campaigns. Most methods for the analysis of artemisinin and its derivatives require expensive equipment and sophisticated instrumentation. A convenient, easy-to-use diagnostic device for rapid evaluation of the quality of artemisinin drugs at the point-of-care is still lacking. In this study a lateral flow dipstick immunoassay was developed for qualitative and semi-quantitative analysis of artesunate (ATS) and dihydroartemisinin (DHA) in anti-malarial drugs. METHODS: This assay was based on a monoclonal antibody (mAb) raised against ATS. ATS-bovine serum albumin and goat anti-mouse IgG, used as the test capture reagent and the control capture reagent, were coated on the nitrocellulose membrane to form the test line and control line, respectively. The conjugate pad was saturated with the gold-labelled anti-ATS mAb. RESULTS: The indicator range of the dipsticks, defined as lowest concentration of the target analytes between which the test line was not visible, were 100-200 and 200-500 ng mL(-1) for ATS and DHA, respectively. No competitive inhibition was observed up to 5,000 ng mL(-1) of quinine, chloroquine diphosphate salt, primaquine phosphate, pyrimethamine, lumefantrine, amodiaquine, piperaquine tetraphosphate tetrahydrate or pyronaridine tetraphosphate. Semi-quantitative analysis of ATS and DHA in commercial drugs and raw drug materials with the dipsticks produced result agreeable with those determined by high performance liquid chromatography (HPLC). Storage test showed that the indicator range for artemisinins remained unchanged after a week at 37 °C and increased four-folds after six months of storage at 4 °C or ambient temperature. CONCLUSIONS: The new selected mAb 3D82G7 with high avidity and broad cross reactivity for artemisinins was used to develop and optimize a dipstick immunoassay for qualitative and semi-quantitative analysis of ATS and DHA in anti-malarial drugs. The semi-quantitative analysis of ATS and DHA in commercial drugs and raw drug materials, and the specificity test of the artemisinin-related drugs both proved the accurate performance of the developed dipsticks for semi-quantitation of ACT samples. The dipstick may be used as a point-of-care device for identifying substandard and counterfeit ATS- and DHA-containing anti-malarial drugs.

Development of a specific monoclonal antibody-based ELISA to measure the artemether content of antimalarial drugs.

Artemether is one of the artemisinin derivatives that are active ingredients in antimalarial drugs. Counterfeit and substandard antimalarial drugs have become a serious problem, which demands reliable analytical tools and implementation of strict regulation of drug quality. Structural similarity among artemisinin analogs is a challenge to develop immunoassays that are specific to artemisinin derivatives. To produce specific antibodies to artemether, we used microbial fermentation of artemether to obtain 9-hydroxyartemether, which was subsequently used to prepare a 9-O-succinylartemether hapten for conjugation with ovalbumin as the immunogen. A monoclonal antibody (mAb), designated as 2G12E1, was produced with high specificity to artemether. 2G12E1 showed low cross reactivities to dihydroartemisinin, artemisinin, artesunate and other major antimalarial drugs. An indirect competitive enzyme linked immunosorbent assay (icELISA) developed showed a concentration causing 50% of inhibition for artemether as 3.7 ng mL⁻¹ and a working range of 0.7-19 ng mL⁻¹. The icELISA was applied for determination of artemether content in different commercial drugs and the results were comparable to those determined by high-performance liquid chromatography analysis. In comparison with reported broad cross activity of anti-artemisinin mAbs, the most notable advantage of the 2G12E1-based ELISA is its high specificity to artemether only.

[Development of a lateral flow dipstick immunoassay for rapid detection of ginsenoside Re].

A sensitive antibody-based lateral flow dipstick was developed for ginsenoside Re (GRe) detection. The stick consisted of a sample pad, a conjugate pad, membrane and an absorbent pad. The membrane was coated with two capture reagents, GRe-BSA conjugate and goat anti-mouse antibodies, forming a test line and a control line, respectively. The conjugate pad was saturated with colloidal gold particles coated with affinity purified monoclonal anti-GRe antibody. The visual detection limit was 200 microg x L(-1) of GRe and the reaction time was 10 min. The Panax ginseng roots were identified after these samples (10 mg) were extracted with 5 mL tap water for 30 min at room temperature, and the extracts were tested by the dipsticks and ELISA kit. The true and false P. ginseng could be distinguished with dipsticks. The dipstick could be used to detect the quality of the P. ginseng samples when the extract was diluted 100-folds. The results were compared with those obtained using an indirect competitive enzyme-linked immunosorbent assay (icELISA). The dipstick assay proved to be a sensitive and rapid tool for quality control of P. ginseng.

Induction of autophagy contributes to the myocardial protection of valsartan against ischemia­reperfusion injury.

The mechanisms underlying the myocardial protection of valsartan against ischemia/reperfusion (I/R) injury are complicated and remain unclear. The aim of this study was to investigate whether autophagy machinery was involved in the protection against I/R injury that is induced by valsartan. In vivo rat hearts were subjected to ischemia by 30 min ligation of the left anterior descending coronary artery, followed by a 120 min reperfusion. 3­methyladenine (3­MA), a specific inhibitor on autophagic sequestration, was used to inhibit autophagy. The hemodynamics, infarct size of the ventricle and LC3B protein were measured. Western blot analysis was performed to investigate the mechanism by which autophagy was induced by valsartan. Valsartan preconditioning resulted in a significant decrease in infarct size and induced autophagy in the rat heart subjected to I/R injury. The hemodynamics assay showed that the valsartan­induced cardiac functional recovery was attenuated by 3­MA. By contrast, 3­MA decreased the improvement induced by valsartan on the histology and infarction of the rat heart subjected to I/R injury. Valsartan preconditioning induced autophagy via the AKT/mTOR/S6K pathway, independent of Beclin1. In conclusion, valsartan preconditioning induced autophagy via the AKT/mTOR/S6K pathway, which contributed to the myocardial protection against I/R injury.

Impaired sonic hedgehog pathway contributes to cardiac dysfunction in type 1 diabetic mice with myocardial infarction.

AIMS: The incidence and mortality of myocardial infarction (MI) in diabetic patients are higher than in non-diabetic patients; however, the mechanisms by which diabetes results in cardiac dysfunction are poorly understood. The present study tested the hypothesis that an impaired sonic hedgehog (Shh) pathway contributes to cardiac dysfunction in type 1 diabetic mice with MI. METHODS AND RESULTS: Adult male C57/B6 mice and streptozotocin-induced type 1 diabetic mice were used. Myocardial proteins of Shh, Patched-1 (Ptc1), and glioma-associated oncogene-1 (Gli1) were significantly decreased in type 1 diabetic mice at 10 weeks, and this was accompanied by cardiac dysfunction. Although myocardial proteins of Shh, Ptc1, and Gli1 were significantly increased 7 days after MI compared with the sham group in control mice, these proteins were markedly decreased in streptozotocin-induced diabetic mice. Treatment with Shh pathway agonist for 21 days significantly increased Ptc1 and Gli1 proteins, enhanced capillary density, reduced the percentage myocardial infarct, and then improved cardiac function in diabetic mice with MI compared with those with no drug treatment. This treatment had no effects in control mice with MI. Conversely, treatment with Shh pathway antagonist for 21 days significantly decreased Ptc1 and Gli1 proteins, reduced capillary density, enlarged the percentage myocardial infarct, and then exacerbated cardiac dysfunction in control mice with MI compared with those with no drug treatment. CONCLUSIONS: These findings indicate that in type 1 diabetic mice the myocardial Shh pathway is impaired and that the impaired Shh pathway contributes to cardiac dysfunction. Strategies that are aimed at augmenting the Shh pathway may offer useful means for improving diabetic cardiac dysfunction.