Reusable thiol-modification Lactobacillus plantarum embedded in cellulose nanocrystals composite aerogel for efficient removal of Ochratoxin A in grape juice.

Ochratoxin A (OTA) contamination in grape juice has attracted widespread concern as OTA can lead to kidney disease and cause adverse neurological effects. An effective method to remove OTA is to make use of highly adsorbent materials that are able to remove the toxic contaminant. Recently, inactivated Lactobacillus plantarum-based biosorbents have shown to be an efficient, cost-effective and environmentally friendly bioremediation method in removing toxic pollutants such as OTA. We used five chemical thiol-modification methods to improve the adsorption efficiency of OTA in grape juice. The esterification of Lactobacillus plantarum (L-Es) significantly increased the sulfhydryl contents (-SH) by 251.33 µmol/g and >90% of OTA was removed. However, the inactivated microbial adsorbent was difficult to separate after adsorption and therefore, the prepared L-Es were embedded into the cellulose nanocrystals (L-Es@CNCs). Moreover, L-Es@CNCs significantly increased the adsorption rate of OTA in grape juice samples by 88.28% with negligible effects on juice quality due to the properties of easy re-use and excellent biodegradability. This showcases its potential application for OTA removal in the grape juice industry.

Sea Buckthorn Flavonoid Extracted by High Hydrostatic Pressure Inhibited IgE-Stimulated Mast Cell Activation through the Mitogen-Activated Protein Kinase Signaling Pathway.

Sea buckthorn (Hippophaë rhamnoides L.), as one of the Elaeagnaceae family, has the significant function of anti-tumor, anti-inflammation, anti-oxidation, and other physiological activities. High hydrostatic pressure (HHP) extraction has the advantages of being easy and efficient, while maintaining biological activity. In this study, sea buckthorn flavonoid (SBF) was extracted with HHP and purified sea buckthorn flavonoid (PSBF) was isolated by AB-8 macroporous resin column. HPLC analysis was used to quantified them. In addition, the effect of anti-allergy in RBL-2H3 cells by SBF, PSBF, and their flavonoid compounds was evaluated. The results demonstrate the conditions for obtaining the maximum flavonoid amount of SBF: 415 MPa for 10 min, 72% ethanol concentration, and a liquid to solid ratio of 40 mL/g, which increased the purity from 1.46% to 13.26%. Both SBF and PSBF included rutin, quercitrin, quercetin, isorhamnetin, and kaempferol. In addition, quercitrin, kaempferol, and SBF could regulate Th1/Th2 cytokine balance. Moreover, extracellular Ca2+ influx was reduced by quercitrin and PSBF. Furthermore, rutin, quercetin, iso-rhamnetin, and SBF could also inhibit P-p38 and P-JNK expression, thereby suppressing the phosphorylation of the MAPK signaling pathways. Overall, SBF is effective for relieving food allergy and might be a promising anti-allergic therapeutic agent.

Additive-optimized micro-structure in cellulose acetate butyrate-based reverse osmosis membrane for desalination.

Progress toward the high water flux of cellulose acetate butyrate (CAB)-based reverse osmosis (RO) membrane is a bottleneck for desalination and mitigation of fresh water shortage. Here, we develop an "optimization of formulation-induced structure" strategy using acetone (solvent), triethyl phosphate (pore-inducing agent), glycerin and n-propanol (boosters), which achieves a state-of-the-art salt rejection of 97.1% and permeate flux of 8.73 L m-2·h-1, ranking top among CAB-based RO membrane. Compared with reported literatures, it represents high separation performance for different concentrations (20-100 mg L-1) of Rhodamine B and Congo red, different ion types (NaCl and MgCl2), different time (600 min), and resistance to feed pressure changes. The key is the appropriate viscosity of the casting solution (995.52 mPa s), the synergy between the components and additives, contributing to the formation of "jellyfish"-like microscopic pore structure with the lowest surface roughness (Ra = 16.3) and good hydrophilicity. The proposed correlation mechanism between additive-optimized micro-structure and desalination provides a promising prospect for CAB-based RO membrane.

Purified Saponins in Momordica charantia Treated with High Hydrostatic Pressure and Ionic Liquid-Based Aqueous Biphasic Systems.

Momordica charantia L. (Cucurbitaceae) is rich in saponins, which have multiple biological effects. In this study, the total saponins of M. charantia were extracted by high hydrostatic pressure (HHP) technology. The optimal extraction process was determined (ethanol concentration 68%, pressure-holding time 8 min, ratio of material to solvent 1:35 and pressure 510 MPa), and the extraction amount of saponins reached 127.890 mg/g. On this basis, an ionic liquid-based aqueous biphasic system was constructed to purify the total saponins. Under the optimized conditions, the purity of M. charantia saponins was 76.06%. Liquid chromatography-mass spectrometry (LC/MS) was used to characterize the saponins in the purified extract of M. charantia. It was found that there were four kinds of saponins in the extract of M. charantia: kuguaglycoside A, momordicoside L, kuguacin B and kuguacin J, providing a basis for the study of the biological activity of saponins.

Optimized Extraction of cAMP From Jujube by Ultra-High Pressure Technology and the Anti-allergic Effect for Peanut Allergy Mouse.

Jujube contains abundant cyclic adenosine monophosphate (cAMP). In contrast, the extraction technology of cAMP from jujube is still to be explored. In this study, the ultra-high pressure extraction (UHPE) conditions for obtaining the maximum cAMP yield from jujube were optimized. Orthogonal array design (OAD) was applied to evaluate the effects of three variables (pressure, pressure-holding time, and liquid-to-solid ratio) by UHPE on cAMP yield. The results showed that the optimal cAMP yield (1223.2 µg/g) was derived at 300 MPa, 20 min duration, and a liquid-to-solid ratio of 2.5 ml/g. In addition, as an important functional ingredient in jujube, cAMP has potential anti-allergic effect. To develop the functional characteristics of jujube, the effect of cAMP was characterized in vivo with the Balb/c mouse model of peanut allergy, which was established by subcutaneous injection of crude peanut protein extract (PN). The results showed that treatment with cAMP in PN-sensitized mice suppressed the lesions in jejunal tissues and allergic symptoms and restored spleen index. Meanwhile, cAMP treatment reduced serum levels of specific immunoglobulin E (IgE), histamine, as well as interleukin-4 (IL-4) and stimulated the secretion of tumor necrosis factor-α (TNF-α), whereas the serum levels of interleukin-10 (IL-10) were not affected. Our results suggested that cAMP has an anti-allergic effect in PN-sensitized mice.

Long non-coding RNA NNT-AS1 positively regulates NPM1 expression to affect the proliferation of estrogen-mediated endometrial carcinoma by interacting.

Objective: This study aims to investigate the mechanism of long non-coding RNA NNT-AS1 in the proliferation of estrogen-mediated endometrial carcinoma (EC). Materials and methods: NNT-AS1, miR-30c, and Nucleophosmin 1 (NPM1) expressions were measured by quantitative real-time PCR and Western blotting. Cell Counting Kit-8 assay and 5-Ethynyl-2'-deoxyuridine (EdU) assay were used to detect the viability and proliferation of Ishikawa and HEC-1-A cells, respectively. RNA immunoprecipitation assay was used to confirm the interaction between NNT-AS1 and miR-30c. Luciferase reporter assay was performed to confirm the interaction between miR-30c and NPM1. Results: NNT-AS1 and NPM1 expressions in EC tissues and cell lines were higher than in benign endometrium and normal endometrial epithelial cells (EECs). miR-30c expression in EC tissues and cell lines was lower than in benign endometrium and normal EECs. NNT-AS1 interacted with miR-30c, and miR-30c negatively regulated NPM1 expression. Overexpression of NNT-AS1 increased NPM1 expression in EC cells, while overexpression of miR-30c reversed the effect. NNT-AS1 interference inhibited the mRNA level of NPM1, while the miR-30c inhibitor reversed the result. Estradiol (E2) promoted the proliferation of EC cells, small interfering RNA (siRNA) against NNT-AS1 inhibited EC cell proliferation, miR-30c inhibitor promoted cell proliferation, and NPM1 siRNA inhibited cell proliferation. E2 increased tumor volume, and NNT-AS1 interference reduced tumor volume in vivo. Conclusion: NNT-AS1 promoted the proliferation of estrogen-mediated EC by regulating miR-30c/NPM1.

N-methyladenosine reader YTHDF2-mediated long noncoding RNA FENDRR degradation promotes cell proliferation in endometrioid endometrial carcinoma.

Dysregulation of long noncoding RNA (LncRNA) FENDDR has been shown to be closely related to the progression of several cancers. However, its role and upstream regulatory mechanism in endometrioid endometrial carcinoma (EEC) remains unclear. This study was conducted using the cancerous tissues of EEC patients (n = 60), EEC cell lines, and a xenograft mouse model. The expression level of LncRNA FENDRR was decreased and the N-methyladenosine (m6A) methylation levels of LncRNA FENDRR was elevated in cancerous tissues of EEC patients. In vitro experiments demonstrated that YTH domain-containing 2 (YTHDF2), an m6A reader, recognized the abundance of m6A-modified LncRNA FENDRR in EEC cells and promoted its degradation. LncRNA FENDRR overexpression suppressed cell proliferation and facilitated cell apoptosis in the EEC cell line HEC-1B by reducing the protein level of SRY-related HMG box transcription factor 4 (SOX4). Interference of LncRNA FENDRR reversed the inhibitory effect of sh-YTHDF2 on cell proliferation and the promoting effect of sh-YTHDF2 on cell apoptosis in HEC-1B cells by silencing FENDRR. Finally, in vivo experiments confirmed that overexpression of LncRNA FENDRR retarded the growth of EEC cells. In conclusion, YTHDF2-mediated LncRNA FENDRR degradation promotes cell proliferation by elevating SOX4 expression in EEC.

Possible obliquity-forced warmth in southern Asia during the last glacial stage.

Orbital-scale global climatic changes during the late Quaternary are dominated by high-latitude influenced ~100,000-year global ice-age cycles and monsoon influenced ~23,000-year low-latitude hydroclimate variations. However, the shortage of highly-resolved land temperature records remains a limiting factor for achieving a comprehensive understanding of long-term low-latitude terrestrial climatic changes. Here, we report paired mean annual air temperature (MAAT) and monsoon intensity proxy records over the past 88,000 years from Lake Tengchongqinghai in southwestern China. While summer monsoon intensity follows the ~23,000-year precession beat found also in previous studies, we identify previously unrecognized warm periods at 88,000-71,000 and 45,000-22,000 years ago, with 2-3 °C amplitudes that are close to our recorded full glacial-interglacial range. Using advanced transient climate simulations and comparing with forcing factors, we find that these warm periods in our MAAT record probably depends on local annual mean insolation, which is controlled by Earth's ~41,000-year obliquity cycles and is anti-phased to annual mean insolation at high latitudes. The coincidence of our identified warm periods and intervals of high-frequent dated archaeological evidence highlights the importance of temperature on anatomically modern humans in Asia during the last glacial stage.

Changes in Physicochemical Properties and Volatiles of Kiwifruit Pulp Beverage Treated with High Hydrostatic Pressure.

Physicochemical properties and volatiles of kiwifruit pulp beverage treated with high hydrostatic pressure (HHP, 400-600 MPa/5-15 min) were investigated during 40-day refrigerated storage. Compared with heat treatment (HT), HHP ranged from 400-500 MPa was superior in retaining vitamin C, fresh-like color and volatiles, while soluble solids content and pH were not affected significantly. Furthermore, HHP improved brightness and inhibited browning of kiwifruit pulp beverage. Samples treated at 400 MPa for 15 min showed significantly higher vitamin C content and lower ∆E values over 40 days than heat-treated kiwifruit pulp beverage. The total content of alcohols, esters, acids, and ketones gradually increased, whereas the total aldehydes content decreased during storage. Interestingly, HHP treatment at 500 MPa for 15 min mostly retained important characteristic volatiles including hexanal and (E)-2-hexenal, indicating this treatment was more conducive to preserve the original fruity, fresh, grassy and green notes of kiwifruit pulp beverage than HT.

Comparison of High Hydrostatic Pressure, Ultrasound, and Heat Treatments on the Quality of Strawberry-Apple-Lemon Juice Blend.

Changes in the microbial, physicochemical, and sensory properties of blended strawberry-apple-lemon juice were investigated to comparatively assess the influence of three processing treatments, namely high hydrostatic pressure (HHP) (500 MPa/15 min/20 °C), ultrasound (US) (376 W/10 min/35 °C), and heat treatment (HT) (86 °C/1 min) over 12 days of storage at 4 °C. The results showed that the total aerobic bacteria (TAB) counts in the HHP-, US-, and HT-treated juice blends were less than 2 log10 CFU/mL, the yeast and mold (Y & M) counts were less than 1.3 log10 CFU/mL, and the coliforms most probable number (MPN/100 mL) was less than 3 after 10 days at 4 °C. Anthocyanins were maintained by HHP, but decreased by 16% and 12% after US and HT, respectively. Total phenols increased by 18% and 7% after HHP and US, respectively, while they were maintained by the HT. Furthermore, better maintenance of total phenols, total anthocyanins, ascorbic acid, antioxidant capacity, color, and sensory values were observed in the HHP-treated juice blend stored for 10 days at 4 °C, compared to both the US- and HT-treated samples. Therefore, HHP was proposed to be a better processing technology for juice blend.

Inactivation of Escherichia coli by Ultrasound Combined with Nisin.

The aim of this study was to investigate the inactivation of nonpathogenic Escherichia coli in nutrient broth and milk through the use of either ultrasound (US) alone or US combined with nisin (US + nisin) treatments. The E. coli cells were treated at 0 to 55°C, 242.04 to 968.16 W/cm2 for 0 to 15 min. The results showed that the inactivation of E. coli by US and US + nisin increased when the temperature, US power density, and treatment time were increased. The inactivation kinetics of E. coli in nutrient broth by US and US + nisin both conformed to linear models. The largest reductions of 2.89 and 2.93 log cycles by US and US + nisin, respectively, were achieved at 968.16 W/cm2 and at 25°C for 15 min. The suspension media of the E. coli cells influenced the inactivation effect of US, while the growth phases of E. coli cells did not affect their resistance to US. Under all experiment conditions of this study, the differences between US and US + nisin in their respective inactivation effects on E. coli were not obvious. The results suggested that nisin had either no effect at all or a weak synergistic effect with US and that the E. coli cells were inactivated mainly by US, thus indicating that the inactivation of E. coli by US is an "all or nothing" event.

Development and evaluation of a speech-generating AAC mobile app for minimally verbal children with autism spectrum disorder in Mainland China.

BACKGROUND: Mobile touchscreen devices are currently being used as speech-generating devices (SGDs) and have been shown to promote the communication skills, particularly the requesting skills of children with autism spectrum disorders (ASD) who have limited spoken language. However, no augmentative and alternative communication (AAC) mobile app has been developed and evaluated in the Chinese language in Mainland China. METHODS: We developed an AAC mobile app, which is the first in Mainland China, to our knowledge, named Yuudee (Chinese name (xiaoyudi)). Yuudee was developed using the Objective-C and Java programming languages. A five-phase training protocol for making requests using Yuudee was developed based on the Picture Exchange Communication System. We trained ten minimally verbal children with ASD to make requests using Yuudee and evaluated the effectiveness of the training. RESULTS: Yuudee has a built-in library of over 400 pictures with corresponding spoken phrases that are divided into 39 categories ranging from making simple requests to expressing emotions. An additional important feature of Yuudee is its customization functions that allow a parent or trainer to easily select pictures and phrases to display, create new pictures and phrases, and change the layouts and orders of the pictures to fit the personal needs of each child. Yuudee is freely available in an iOS version from the iTunes App Store (https://itunes.apple.com/cn/app/xiao-yu-di/id794832934?mt=8) and in an Android version from Google Play (https://play.google.com/store/apps/details?id=com.supersuperstar.yuudee.vue) and domestic Chinese Android App stores. Three consecutive unprompted successful responses, which were defined as an initial training success, were achieved in at least three of the five phases for all ten of the evaluated children. The accuracy rate of a given phase was calculated for each child who achieved three consecutive unprompted successful responses in the phase. Seven children achieved at least 50% accuracy in at least two of the five phases. The other three children achieved at least 50% accuracy in only one phase. Two children achieved at least 50% accuracy in all of the phases in which they were trained. CONCLUSIONS: Our data suggest that Yuudee is a useful tool for helping minimally verbal children with ASD make requests.

Human and Bovine Viruses and Bacteria at Three Great Lakes Beaches: Environmental Variable Associations and Health Risk.

Waterborne pathogens were measured at three beaches in Lake Michigan, environmental factors for predicting pathogen concentrations were identified, and the risk of swimmer infection and illness was estimated. Waterborne pathogens were detected in 96% of samples collected at three Lake Michigan beaches in summer, 2010. Samples were quantified for 22 pathogens in four microbial categories (human viruses, bovine viruses, protozoa, and pathogenic bacteria). All beaches had detections of human and bovine viruses and pathogenic bacteria indicating influence of multiple contamination sources at these beaches. Occurrence ranged from 40 to 87% for human viruses, 65-87% for pathogenic bacteria, and 13-35% for bovine viruses. Enterovirus, adenovirus A, Salmonella spp., Campylobacter jejuni, bovine polyomavirus, and bovine rotavirus A were present most frequently. Variables selected in multiple regression models used to explore environmental factors that influence pathogens included wave direction, cloud cover, currents, and water temperature. Quantitative Microbial Risk Assessment was done for C. jejuni, Salmonella spp., and enteroviruses to estimate risk of infection and illness. Median infection risks for one-time swimming events were approximately 2 × 10(-5), 8 × 10(-6), and 3 × 10(-7) [corrected] for C. jejuni, Salmonella spp., and enteroviruses, respectively. Results highlight the importance of investigating multiple pathogens within multiple categories to avoid underestimating the prevalence and risk of waterborne pathogens.

Long-term effect of agricultural reclamation on soil chemical properties of a coastal saline marsh in Bohai Rim, northern China.

Over the past six decades, coastal wetlands in China have experienced rapid and extensive agricultural reclamation. In the context of saline conditions, long-term effect of cultivation after reclamation on soil chemical properties has not been well understood. We studied this issue using a case of approximately 60-years cultivation of a coastal saline marsh in Bohai Rim, northern China. The results showed that long-term reclamation significantly decreased soil organic carbon (SOC) (-42.2%) and total nitrogen (TN) (-25.8%) at surface layer (0-30 cm) as well as their stratification ratios (SRs) (0-5 cm:50-70 cm and 5-10 cm:50-70 cm). However, there was no significant change in total phosphorus (TP) as well as its SRs under cultivation. Cultivation markedly reduced ratios of SOC to TN, SOC to TP and TN to TP at surface layer (0-30 cm) and their SRs (0-5 cm:50-70 cm). After cultivation, electrical conductivity and salinity significantly decreased by 60.1% and 55.3% at 0-100 cm layer, respectively, suggesting a great desalinization. In contrast, soil pH at 20-70 cm horizons notably increased as an effect of reclamation. Cultivation also changed compositions of cations at 0-10 cm layer and anions at 5-100 cm layer, mainly decreasing the proportion of Na+, Cl- and SO4(2-). Furthermore, cultivation significantly reduced the sodium adsorption ratio and exchangeable sodium percentage in plow-layer (0-20 cm) but not residual sodium carbonate, suggesting a reduction in sodium harm.

[Stabilities of two kinds of Plasmodium antigen substrate slides under different storage temperatures and time].

OBJECTIVE: To observe and compare the inspection effects of antigen substrate slides of Plasmodium cynomolgi (P. c) and Plasmodium falciparum (P. f) on the malaria antibody titer under different storage temperatures and time. METHODS: The densities of Plasmodium of P. c and P. f antigen slides were counted through a microscope, and the average numbers of Plasmodium in each field of vision were calculated. The pooled serum of patients with tertian malaria and falciparum malaria were treated as antibody serum, and the dilutions were from 1:5 to 1:1 280. The two kinds of antibody slides were placed under the temperature of 4-6, 25-27, 33-35 degrees C, and two slides of each kind were selected at Day 3, 5, 7, 10 post-storage to detect the end point antibody titer of malaria by the indirect fluorescent antibody test. Meanwhile, the P. c and P. f antigen slides storing under -20 degrees C for 1 year and 2 years were placed under the aforementioned 3 temperature conditions for 3 d, and the changes of the antibody titers were compared. RESULTS: The densities of Plasmodium in blood cells of P. c and P. f antigen slides were 2.00 x 10(5)/microl and 1.89x 10(5)/microl, respectively, and the average numbers of Plasmodium in each field of vision were 157 +/- 13 and 142 +/- 9, respectively. The end point titers of antibody of P. c and P. f antigen slides placed under the temperatures of 4-6, 25-27, 33-35 degrees C were all on a downward trend after storing for 5 d. The average antibody titers of the two kinds of slides under temperature of 4-6 degrees C and above were 1:440 and 1:80, respectively, and there was a significantly statistic difference between them(t = 1.940, P < 0.05). When P. c and P. f antigen slides storing under -20 degrees C for 1 year were placed under 4-6 degrees C for 3 d, the end point antibody titers were both 1:640, while for those storing under -20 degrees C for 2 years, the end point antibody titers were 1:320 and 1:160, respectively, both the differences were statistically significant (t(P. c) =11.362, P(P. c) < 0.01; t(P. f) = 38.845, P(P. f) < 0.001). CONCLUSION: The end point antibody titers for malaria detection of P. c and P. f antigen slides decrease gradually with the raise of temperature and the prolonging of time for storage.

[Correlation of IL-4 and IL-13 gene polymorphisms with asthma and total serum IgE levels].

OBJECTIVE: To investigate the correlation between the single nucleotide polymorphisms (SNP) of +33C/T in the promoter region of IL-4 gene and +1923C/T in intron-3 region of IL-13 gene and the susceptibility of asthma, and to study the impact of these polymorphisms upon total serum IgE levels. METHODS: The 2 polymorphisms were determined by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP), in 150 asthmatic subjects (asthma group) and 160 healthy controls (healthy control group) of the Han nationality in Shandong province enrolled from December 2003 to June 2007. The total serum IgE levels were determined by ELISA. RESULTS: The genotype frequencies of CC, CT and TT in +33C/T sites of IL-4 gene were 43% (68/160), 35% (56/160), 22% (36/160) respectively in the controls, and 18% (27/150), 36% (54/150), 46% (69/150) respectively in the asthmatic subjects. The genotype frequencies of CC, CT and TT in +1923C/T sites of IL-13 gene were 41% (66/160), 43% (68/160), 16% (26/160) respectively in the controls, and 21% (31/150), 38% (57/150), 41% (61/150) respectively in the asthmatic subjects. The distribution of genotype in each sites between the 2 groups was significantly different (chi(2) = 27.821, 26.544 respectively, all P < 0.01). The CT and TT genotypes carried higher risks for asthma than CC genotypes (chi(2) = 21.870, 14.206 respectively, all P < 0.01). The total serum IgE levels of CC, CT and TT in +33C/T sites of IL-4 gene were (92 +/- 37), (122 +/- 45), (146 +/- 44) KU/L respectively in the controls, and (179 +/- 40), (294 +/- 51), (341 +/- 80) KU/L respectively in the asthmatic subjects. The total serum IgE levels of CC, CT and TT in +1923C/T sites of IL-13 gene were (85 +/- 31), (102 +/- 38), (144 +/- 49) KU/L respectively in the controls, and (186 +/- 65), (297 +/- 87), (363 +/- 140) KU/L respectively in the asthmatic subjects. In these 2 sites, the total serum IgE level of asthmatics was higher than that of the controls with the same genotype between the 2 groups (t = 4.653, 6.547, 7.754; and 4.673, 6.784, 8.157 respectively, all P < 0.01). The total serum IgE levels of CT, TT genotypes were higher than CC genotypes in the same group (t = 5.748, 6.253 respectively, all P < 0.01). CONCLUSIONS: There was a significant correlation between the polymorphisms of +33C/T sites of IL-4 and +1923C/T sites of IL-13 gene and susceptibility to asthma and increase of the total serum IgE. The IL-4 gene and IL-13 gene may be important candidate genes for asthma.

In situ reaction on Cu(OH)2 nanoribbons for controlling growth of nanorods arrays of copper oxalate.

Cu(OH)2 nanoribbons template provides a simple way for producing arrays of copper oxalate nanorods and assemblies with controllable size and shape of the arrays of copper oxalate in situ reaction on Cu(OH)2 nanoribbons. The observations of scanning electron microscopy and characterization of X-ray powder diffraction and Fourier transmission infrared spectrometry show formation of the arrays of cubic column nanorods, nanotubes, and corn-like nanorods of CuC2O4 by controlling the concentration of oxalate solution and reaction time. We believe this approach can also be applied for synthesis of inorganic and inorganic/organic material architectures.

[Establishment of in vitro microtest for determining sensitivity of Plasmodium falciparum to pyronaridine].

OBJECTIVE: To establish an in vitro microtest for determining the sensitivity of Plasmodium falciparum to pyronaridine. METHODS: Pyronaridine-coated plate and culture medium which is easy to use in the field were prepared. P. falciparum parasites from in vitro continuous passage culture (FCC1/HN) were used for experimental tests in the laboratory. When they were proved stable and reliable through repeated determinations, field trials were made in Hainan and Yunnan Provinces during the malaria transmission season with blood samples from clinical falciparum malaria cases. A 4-week in vivo test was carried out as a control. RESULTS: The pyronaridine-coated plate and culture medium were proved to be stable. The effective period of pyronaridine-coated plate, the ampule sealed liquid culture medium and the bottled lyophilized culture medium, all stored at 4 degrees C was 6 months, 2 months and 2 years respectively. Through several years field determinations, the baseline data of pyronaridine-sensitivity of P. falciparum in the country were collected and the sensitivity of P. falciparum to pyronaridine was also revealed to have decreased gradually. The mean drug concentration for in vitro complete inhibition of schizont formation raised by 2-4 times although the clinical therapeutic efficacy of pyronaridine was still satisfactory at the present time. CONCLUSION: The developed in vitro microtest can be used for determination of the sensitivity of P. falciparum to pyronaridine, and it is more convenient and sensitive than the 4-week in vivo method.

[The point mutations in Pfcrt and Pfmdr1 genes in Plasmodium falciparum isolated from Hainan Province].

OBJECTIVE: To evaluate the point mutations in Pfcrt and Pfmdr1 genes in Plasmodium falciparum isolated from Hainan Province. METHODS: Nested polymerase chain reaction and restriction fragment length polymorphism were used to detect the point mutations at codon 76 of Pfcrt and at codon 86, 1246 of Pfmdr1 in P. falciparum isolates. Chloroquine resistance was measured by the in vitro microtest recommended by WHO. RESULTS: In 36 samples tested, 28 were successfully amplified for Pfcrt, 64.3% of them carried mutant allele at codon 76, 21.4% with wild allele K76 and 14.3% with mixed allele mutation. While for Pfmdr1, 3.4% isolates displayed the 86Y mutation, 89.7% with wild allele N86 and 6.9% with the mixed alleles in 29 isolates which were amplified successfully for N86Y. No point mutation in Pfmdr1 at codon 1246 was found in 13 isolates from the total 36 samples. By the in vitro test, 72.2% (26/36) showed resistance to chloroquine. The 76T and 86Y mutant alleles were present in both in vitro susceptible and resistant isolates. There was a significant difference between susceptible and resistant isolates carrying 76T mutant codon (P < 0.05), but no significant difference was found in Pfmdr1 (P < 0.05). CONCLUSION: There is a significant difference of the 76T prevalence in Pfcrt gene between the susceptible isolate and resistant one of P. falciparum to chloroquine in vitro. The Pfcrt 76T may be used as a predictive marker for chloroquine resistance surveillance.