Recent Advances and Multiple Strategies of Monoterpenoid Overproduction in Saccharomyces cerevisiae and Yarrowia lipolytica.

Monoterpenoids are an important subclass of terpenoids that play important roles in the energy, cosmetics, pharmaceuticals, and fragrances fields. With the development of biotechnology, microbial synthesis of monoterpenoids has received great attention. Yeasts such Saccharomyces cerevisiae and Yarrowia lipolytica are emerging as potential hosts for monoterpenoids production because of unique advantages including rapid growth cycles, mature gene editing tools, and clear genetic background. Recently, advancements in metabolic engineering and fermentation engineering have significantly enhanced the accumulation of monoterpenoids in cell factories. First, this review introduces the biosynthetic pathway of monoterpenoids and comprehensively summarizes the latest production strategies, which encompass enhancing precursor flux, modulating the expression of rate-limited enzymes, suppressing competitive pathway flux, mitigating cytotoxicity, optimizing substrate utilization, and refining the fermentation process. Subsequently, this review introduces four representative monoterpenoids. Finally, we outline the future prospects for efficient construction cell factories tailored for the production of monoterpenoids and other terpenoids.

Establishment of a selectable marker recycling system for iterative gene editing in Fusarium fujikuroi.

Gibberellic acid (GA3) is a vital plant growth hormone widely used in agriculture. Currently, GA3 production relies on liquid fermentation by the filamentous fungus Fusarium fujikuroi. However, the lack of an effective selection marker recycling system hampers the application of metabolic engineering technology in F. fujikuroi, as multiple-gene editing and positive-strain screening still rely on a limited number of antibiotics. In this study, we developed a strategy using pyr4-blaster and CRISPR/Cas9 tools for recycling orotidine-5'-phosphate decarboxylase (Pyr4) selection markers. We demonstrated the effectiveness of this method for iterative gene integration and large gene-cluster deletion. We also successfully improved GA3 titers by overexpressing geranylgeranyl pyrophosphate synthase and truncated 3-hydroxy-3-methyl glutaryl coenzyme A reductase, which rewired the GA3 biosynthesis pathway. These results highlight the efficiency of our established system in recycling selection markers during iterative gene editing events. Moreover, the selection marker recycling system lays the foundation for further research on metabolic engineering for GA3 industrial production.

Preparation and In Vitro and In Vivo Evaluation of Rectal In Situ Gel of Meloxicam Hydroxypropyl-ß-cyclodextrin Inclusion Complex.

Meloxicam (MLX) is one of the most effective NSAIDs, but its poor water solubility and low bioavailability limit its clinical application. In this study, we designed a thermosensitive in situ gel of the hydroxypropyl-ß-cyclodextrin inclusion complex (MLX/HP-ß-CD-ISG) for rectal delivery to improve bioavailability. The best method for preparing MLX/HP-ß-CD was the saturated aqueous solution method. The optimal inclusion prescription was optimized using an orthogonal test, and the inclusion complex was evaluated via PXRD, SEM, FTIR and DSC. Then, MLX/HP-ß-CD-ISG was characterized regarding the gel properties, release in vitro, and pharmacokinetics in vivo. The inclusion rate of the inclusion complex obtained via the optimal preparation process was 90.32 ± 3.81%. The above four detection methods show that MLX is completely embedded in the HP-ß-CD cavity. The developed MLX/HP-ß-CD-ISG formulation has a suitable gelation temperature of 33.40 ± 0.17 °C, a gelation time of 57.33 ± 5.13 s, pH of 7.12 ± 0.05, good gelling ability and meets the requirements of rectal preparations. More importantly, MLX/HP-ß-CD-ISG significantly improved the absorption and bioavailability of MLX in rats, prolonging the rectal residence time without causing rectal irritation. This study suggests that the MLX/HP-ß-CD-ISG can have a wide application prospect with superior therapeutic benefits.

Preparation, In Vitro and In Vivo Evaluation of Nanoemulsion In Situ Gel for Transnasal Delivery of Traditional Chinese Medicine Volatile Oil from Ligusticum sinense Oliv.cv. Chaxiong.

Ischemic stroke is a difficult-to-treat brain disease that may be attributed to a limited therapeutic time window and lack of effective clinical drugs. Nasal-brain administration is characterized by low systemic toxicity and is a direct and non-invasive brain targeting route. Preliminary studies have shown that the volatile oil of Chaxiong (VOC) has an obvious anti-ischemic stroke effect. In this work, we designed a nanoemulsion thermosensitive in situ gel (VOC-NE-ISG) loaded with volatile oil of Chaxiong for ischemia via intranasal delivery to rat brain treatment of cerebral ischemic stroke. The developed VOC-NE-ISG formulation has a suitable particle size of 21.02 ± 0.25 nm and a zeta potential of -20.4 ± 1.47 mV, with good gelling ability and prolonged release of the five components of VOC. The results of in vivo pharmacokinetic studies and brain targeting studies showed that intranasal administration of VOC-NE-ISG could significantly improve the bioavailability and had excellent brain-targeting efficacy of nasal-to-brain delivery. In addition, the results of pharmacodynamics experiments showed that both VOC-NE and VOC-NE-ISG could reduce the neurological deficit score of model rats, reducing the size of cerebral infarction, with a significant effect on improving ischemic stroke. Overall, VOC-NE-ISG may be a promising intranasal nanomedicine for the effective treatment of ischemic stroke.

Preparation, in vitro and in vivo Evaluation of Thermosensitive in situ Gel Loaded with Ibuprofen-Solid Lipid Nanoparticles for Rectal Delivery.

Background: Ibuprofen (IBU), a nonsteroidal anti-inflammatory drug, shows poor gastrointestinal absorption due to its low solubility, which limits its clinical application. Objective: In the present study, we aimed to develop thermosensitive gel-mediated ibuprofen-solid lipid nanoparticles (IBU-SLN-ISG) to improve the dissolution and bioavailability of IBU after rectal delivery. Methods: IBU-loaded SLNs (IBU-SLNs) were developed and optimized applying Box-Behnken design. The optimized IBU-SLNs were characterized by physicochemical parameters and morphology. Then, the optimized IBU-SLNs was incorporated into the gel and characterized for gel properties and rheology and investigated its release in vitro, pharmacokinetics in vivo, rectal irritation and rectal retention time. Results: The optimized SLNs had an EE of 90.74 ± 1.40%, DL of 11.36 ± 1.20%, MPS of 166.77 ± 2.26 nm, PDI of 0.27 ± 0.08, and ZP of -21.00 ± 0.59 mV. The FTIR spectra confirmed successful encapsulation of the drug inside the nanoparticle as only peaks responsible for the lipid could be identified. This corroborated well with XRD spectra, which showed a completely amorphous state of the IBU-SLNs as compared to the crystalline nature of the pure drug. The gelation temperature of the prepared IBU-SLN-ISG was 33.30 ± 0.78°C, the gelation time was 14.67 ± 2.52 s, the gel strength was 54.00 ± 1.41 s, and the mucoadhesion was (11.54±0.37) × 102dyne/cm2. The in vitro results of IBU-SLNs and IBU-SLN-ISG showed a biphasic release pattern with initial burst release followed by sustained release. More importantly, IBU-SLN-ISG produced much better absorption of IBU and improved bioavailability in rats. In addition, IBU-SLN-ISG caused no irritation or damage to rectal tissues, and could be retained in the rectum for a long time. Conclusion: Thermosensitive in situ gel loaded with IBU-solid lipid nanoparticles might be further developed as a more convenient and effective rectal dosage form.

Pharmacokinetics of five phthalides in volatile oil of Ligusticum sinense Oliv.cv. Chaxiong, and comparison study on physicochemistry and pharmacokinetics after being formulated into solid dispersion and inclusion compound.

BACKGROUNDS: The dried rhizome of Ligusticum sinense Oliv.cv. Chaxiong has been used to treat cardiovascular and cerebrovascular diseases, atherosclerosis, anemia and stroke. A high purity extract from chaxiong (VOC, brownish yellow oil) was extracted and separated. Its main components were senkyunolide A (SA, 33.81%), N-butylphthalide (NBP, 1.38%), Neocnidilide (NOL, 16.53%), Z-ligustilide (ZL, 38.36%), and butenyl phthalide (BP, 2.48%), respectively. Little is known about the pharmacokinetics of these phthalides in Chaxiong, and different preparations to improve the physicochemistry and pharmacokinetics of VOC have not been investigated. METHODS: At different predetermined time points after oral administration or intravenous administration, the concentrations of SA, NBP, NOL, ZL and BP in the rat plasma were determined using LC-MS/MS, and the main PK parameters were investigated. VOC-P188 solid dispersion and VOC-ß-CD inclusion compound were prepared by melting solvent method and grinding method, respectively. Moreover, the physicochemical properties, dissolution and pharmacokinetics of VOC-P188 solid dispersion and VOC-ß-CD inclusion compound in rats were assessed in comparison to VOC. RESULTS: The absorptions of SA, NBP, NOL, ZL and BP in VOC were rapid after oral administration, and the absolute bioavailability was less than 25%. After the two preparations were prepared, dissolution rate was improved at pH 5.8 phosphate buffer solution. Comparing VOC and physical mixture with the solid dispersion and inclusion compound, it was observed differences occurred in the chemical composition, thermal stability, and morphology. Both VOC-P188 solid dispersion and VOC-ß-CD inclusion compound had a significantly higher AUC and longer MRT in comparison with VOC. CONCLUSION: SA, NBP, NOL, ZL and BP in VOC from chaxiong possessed poor absolute oral bioavailability. Both VOC-P188 solid dispersion and VOC-ß-CD inclusion compound could be prospective means for improving oral bioavailability of SA, NBP, NOL, ZL and BP in VOC.

[Pharmacokinetic study on five components in phthalide target areas of Chaxiong and its ß-CD inclusion compounds based on UPLC-MS/MS].

This study aims to establish a method for the determination of the concentration of five main components of phthalide target areas of Chaxiong(CPTA) and its inclusion of ß-CD in the plasma of rats, and determine the pharmacokinetic parameters, absolute bioavailability and relative bioavailability of CPTA/ß-CD inclusion compound in vivo. The plasma concentrations of senkyunolide A, N-butylphthalide, new osthol lactone, Z-ligustilide and butenyl phthalide were determined with UPLC-MS/MS. The content determination was conducted at the chromatographic conditions as follows: Shim-pack GIST C_(18)-AQ HP column(2.1 mm×100 mm, 3 µm), mobile phase of 0.1% formic acid solution(A)-acetonitrile(B), gradient elution, flow rate of 0.3 mL·min~(-1), column temperature of 35 ℃ and injection volume of 2 µL. The mass spectra were obtained with electrospray ion source(ESI), positive ion mode and multi reaction monitoring. CPTA/ß-CD inclusion compound was prepared by grinding method, DAS 2.0 software was used to model the data, and the absolute bioavailability of CPTA and relative bioavailability of inclusion compound were calculated. Finally, the methods for the determination of five components of senkyunolide A, N-butylphthalide, new osthol lactone, Z-ligustilide and butenyl phthalide in CPTA, were successfully established. The linear relationship among the five components was good within their respective ranges, r>0.99. The absolute bioavailability of the five components in rats was 22.30%, 16.32%, 21.90%, 10.16% and 12.43%, respectively. After CPTA/ß-CD inclusion was prepared, the relative bioavailability of the five components was 138.69%, 198.39%, 218.01%, 224.54% and 363.55%, respectively, significantly improved. This method is rapid, accurate and sensitive, so it is suitable for the pharmacokinetic study of extracts in traditional Chinese medicine and their preparations.

Effects of 5 MeV Proton Irradiation on Nitrided SiO2/4H-SiC MOS Capacitors and the Related Mechanisms.

In this paper the effects of 5 MeV proton irradiation on nitrided SiO2/4H-SiC metal-oxide-semiconductor (MOS) capacitors are studied in detail and the related mechanisms are revealed. The density of interface states (Dit) is increased with the irradiation doses, and the annealing response suggests that the worse of Dit is mainly caused by displacement effect of proton irradiation. However, the X-rays photoelectron spectroscopy (XPS) measurement shows that the quantity proportion of breaking of Si≡N induced by displacement is only 8%, which means that the numbers of near interface electron traps (NIETs) and near interface hole traps (NIHTs) are not significantly changed by the displacement effect. The measurements of bidirectional high frequency (HF) C-V characteristics and positive bias stress stability show that the number of un-trapped NIETs and oxide electron traps decreased with increasing irradiation doses because they are filled by electrons resulted from the ionization effect of proton irradiation, benefiting to the field effective mobility (µFE) and threshold voltage stability of metal-oxide-semiconductor field-effect transistors (MOSFETs). The obviously negative shift of flat-band voltage (VFB) resulted from the dominant NIHTs induced by nitrogen passivation capture more holes produced by ionization effect, which has been revealed by the experimental samples with different nitrogen content under same irradiation dose.

Simple, Rapid and Sensitive Detection of Pseudomonas aeruginesa by Colorimetric Multiple Cross Displacement Amplification.

Pseudomonas aeruginosa (P. aeruginosa) is a major opportunistic pathogen in hospital-acquired infections. Thus, early diagnosis is the best strategy for fighting against these infections. In this report, we incorporated multiple cross displacement amplification (MCDA) combined with the malachite green (MG) for rapid, sensitive, specific and visual detection of P. aeruginosa by targeting the oprl gene. The MCDA-MG assay was conducted at 67°C for only 40 min during the amplification stage, and then products were directly detected by using colorimetric indicators (MG), eliminating the use of an electrophoresis instrument or amplicon analysis equipment. The entire process, including specimen processing (35 min), amplification (40 min) and detection (5 min), can be finished within 80 min. All 30 non-P. aeruginosa strains tested negative, indicating the high specificity of the MCDA primers. The analytical sensitivity of the MCDA-MG assay was 100 fg of genomic templates per reaction in pure culture, which was in complete accordance with MCDA by gel electrophoresis and real-time turbidity. The assay was also successfully applied to detecting P. aeruginosa in stool samples. Therefore, the rapidity, simplicity, and nearly equipment-free platform of the MCDA-MG technique make it possible for clinical diagnosis, and more.

Design, synthesis, biological evaluation, homology modeling and docking studies of (E)-3-(benzo[d][1,3]dioxol-5-ylmethylene) pyrrolidin-2-one derivatives as potent anticonvulsant agents.

A series of (E)-3-(benzo[d][1,3]dioxol-5-ylmethylene)pyrrolidin-2-one derivatives were designed, synthesized, and evaluated for their anticonvulsant activities. In the preliminary screening, compounds 5, 6a-6f and 6h-6i showed promising anticonvulsant activities in MES model, while 6f and 6g represented protection against seizures at doses of 100 mg/kg and 0.5 h in scPTZ model. The most active compound 6d had a high-degree protection against the MES-induced seizures with ED50 value of 4.3 mg/kg and TD50 value of 160.9 mg/kg after intraperitoneal (i.p.) injection in mice, which provided 6d in a high protective index (TD50/ED50) of 37.4 comparable to the reference drugs. Beyond that, 6d has been selected and evaluated in vitro experiment to estimate the activation impact. Apparently, 6d clearly inhibits the Nav1.1 channel. Our preliminary results provide new insights for the development of small-molecule activators targeting specifically Nav1.1 channels to design potential drugs for treating epilepsy. The computational parameters, such as homology modeling, docking study, and ADME prediction, were made to exploit the results.

Detection of nucleic acids and elimination of carryover contamination by using loop-mediated isothermal amplification and antarctic thermal sensitive uracil-DNA-glycosylase in a lateral flow biosensor: application to the detection of Streptococcus pneumoniae.

The authors report on a loop-mediated isothermal amplification (LAMP) scheme that uses antarctic thermally sensitive uracil-DNA-glycosylase (AUDG) for simultaneous detection of nucleic acids and elimination of carryover contamination. It was applied in a lateral flow assay (LFA) format. The assay has attractive features in that it does not require the use of labeled primers or probes, and can eliminate false-positive results generated by unwanted hybridization between two labeled primers or between a labeled primer and probe. LAMP amplification and AUDG digestion are conducted in a single pot, and the application of a closed-tube reaction prevents false-positives due to carryover contamination. The method was applied to the detection of the human pathogen Streptococcus pneumoniaein in pure cultures and spiked blood samples. This LFA can detect S. pneumoniae in pure cultures with a 25 fg.µL-1 detection limit and in spiked blood samples with a 470 cfu.mL-1 detection limit. Conceivably, this assay can be applied to the detection of various other targets if the specific LAMP primers are available. Graphical abstract ᅟ.

Rapid and Sensitive Detection of Vibrio parahaemolyticus and Vibrio vulnificus by Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification Technique.

Vibrio parahaemolyticus and Vibrio vulnificus are two marine seafood-borne pathogens causing severe illnesses in humans and aquatic animals. In this study, a recently developed novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully developed and evaluated for simultaneous detection of V. parahaemolyticus and V. vulnificus strains in only a single reaction. Two MERT-LAMP primer sets were designed to specifically target toxR gene of V. parahaemolyticus and rpoS gene of V. vulnificus. The MERT-LAMP reactions were conducted at 62 °C, and the positive results were produced in as short as 19 min with the genomic DNA templates extracted from the V. parahaemolyticus and V. vulnificus strains. The two target pathogens present in the same sample could be simultaneously detected and correctly differentiated based on distinct fluorescence curves in a real-time format. The sensitivity of MERT-LAMP assay was 250 fg and 125 fg DNA per reaction with genomic templates of V. parahaemolyticus and V. vulnificus strains, which was in conformity with conventional LAMP detection. Compared with PCR-based techniques, the MERT-LAMP technology was 100- and 10-fold more sensitive than that of PCR and qPCR methods. Moreover, the limit of detection of MERT-LAMP approach for V. parahaemolyticus isolates and V. vulnificus isolates detection in artificially-contaminated oyster samples was 92 CFU and 83 CFU per reaction. In conclusion, the MERT-LAMP assay presented here was a rapid, specific, and sensitive tool for the detection of V. parahaemolyticus and V. vulnificus, and could be adopted for simultaneous screening of V. parahaemolyticus and V. vulnificus in a wide variety of samples.

Multiple Cross Displacement Amplification Combined with Gold Nanoparticle-Based Lateral Flow Biosensor for Detection of Vibrio parahaemolyticus.

Vibrio parahaemolyticus (V. parahaemolyticus) is a marine seafood-borne pathogen causing severe illnesses in humans and aquatic animals. In the present study, multiple cross displacement amplification was combined with a lateral flow biosensor (MCDA-LFB) to detect the toxR gene of V. parahaemolyticus in DNA extracts from pure cultures and spiked oyster homogenates. Amplification was carried out at a constant temperature (62°C) for only 30 min, and amplification products were directly applied to the biosensor. The entire process, including oyster homogenate processing (30 min), isothermal amplification (30 min) and results indicating (∼2 min), could be completed within 65 min. Amplification product was detectable from as little as 10 fg of pure V. parahaemolyticus DNA and from approximately 4.2 × 102 CFU in 1 mL of oyster homogenate. No cross-reaction with other Vibrio species and with non-Vibrio species was observed. Therefore, the MCDA-LFB method established in the current report is suitable for the rapid screening of V. parahaemolyticus in clinical, food, and environmental samples.

The Novel Multiple Inner Primers-Loop-Mediated Isothermal Amplification (MIP-LAMP) for Rapid Detection and Differentiation of Listeria monocytogenes.

Here, a novel model of loop-mediated isothermal amplification (LAMP), termed multiple inner primers-LAMP (MIP-LAMP), was devised and successfully applied to detect Listeria monocytogenes. A set of 10 specific MIP-LAMP primers, which recognized 14 different regions of target gene, was designed to target a sequence in the hlyA gene. The MIP-LAMP assay efficiently amplified the target element within 35 min at 63 °C and was evaluated for sensitivity and specificity. The templates were specially amplified in the presence of the genomic DNA from L. monocytogenes. The limit of detection (LoD) of MIP-LAMP assay was 62.5 fg/reaction using purified L. monocytogenes DNA. The LoD for DNA isolated from serial dilutions of L. monocytogenes cells in buffer and in milk corresponded to 2.4 CFU and 24 CFU, respectively. The amplified products were analyzed by real-time monitoring of changes in turbidity, and visualized by adding Loop Fluorescent Detection Reagent (FD), or as a ladder-like banding pattern on gel electrophoresis. A total of 48 pork samples were investigated for L. monocytogenes by the novel MIP-LAMP method, and the diagnostic accuracy was shown to be 100% when compared to the culture-biotechnical method. In conclusion, the MIP-LAMP methodology was demonstrated to be a reliable, sensitive and specific tool for rapid detection of L. monocytogenes strains.

Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification.

We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61-65 °C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primers annealed to the template strands without a denaturing step to initiate the synthesis. For the subsequent isothermal amplification step, a series of primer binding and extension events yielded several single-stranded DNAs and single-stranded single stem-loop DNA structures. Then, these DNA products enabled the strand-displacement reaction to enter into the exponential amplification. Three mainstream methods, including colorimetric indicators, agarose gel electrophoresis and real-time turbidity, were selected for monitoring the MCDA reaction. Moreover, the practical application of the MCDA assay was successfully evaluated by detecting the target pathogen nucleic acid in pork samples, which offered advantages on quick results, modest equipment requirements, easiness in operation, and high specificity and sensitivity. Here we expounded the basic MCDA mechanism and also provided details on an alternative (Single-MCDA assay, S-MCDA) to MCDA technique.

Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification: A Novel Analytically Rapid, Sensitive, Multiplex Loop-Mediated Isothermal Amplification Detection Technique.

Loop-mediated isothermal amplification (LAMP) is restricted to detecting a single target, limiting the usefulness of this method. To achieve multiplex LAMP-based detection, we developed a novel approach we called the multiple endonuclease restriction real-time-LAMP assay. In this system, the LAMP forward or backward inner primers contain 5' end short sequences that are recognized by the restriction endonuclease Nb.BsrDI, and the new forward or backward inner primers were modified at the 5' end with a fluorophore and in the middle with a dark quencher. Nb.BsrDI digests the newly synthesized double-stranded terminal sequences (5' end short sequences and their complementary sequences), which releases the quenching, resulting in a gain of signal. The assay permitted real-time detection of single or multiple target sequences in a single tube, and the positive results can be obtained in as short as 12 minutes. The novel methodology is highly efficient and specific, detecting down to 250 fg of DNA per reaction of Listeria DNA tested, and was successful in evaluating raw meat samples. The multiple endonuclease restriction real-time-LAMP technology, which is an extension of LAMP to accommodate robust, target-specific, and multiplex detection, provides a molecular diagnostic tool with less detection time and high sensitivity and specificity compared with those of LAMP and quantitative real-time PCR.

Formulations, hemolytic and pharmacokinetic studies on saikosaponin a and saikosaponin d compound liposomes.

The aim of this study was to develop and optimise a saikosaponin a and saikosaponin d compound liposome (SSa-SSd-Lip) formulation with reduced hemolysis and enhanced bioavailability. A screening experiment was done with Plackett-Burman design, and response surface methodology of five factors (EPC/SSa-SSd ratio, EPC/Chol ratio, water temperature, pH of PBS, and ultrasound time) was employed to optimise the mean diameter, entrapment efficiency of SSa and SSd, and the reduction of hemolysis for SSa-SSd-Lip. Under the optimal process conditions (EPC/SSa-SSd ratio, EPC/Chol ratio, water temperature and pH of PBS were 26.71, 4, 50 °C and 7.4, respectively), the mean diameter, the entrapment efficiency of SSa, the entrapment efficiency of SSd and the hemolysis were 203 nm, 79.87%, 86.19%, 25.16% (SSa/SSd 12.5 mg/mL), respectively. The pharmacokinetic studies showed that the SSa-SSd-Lip had increased circulation time, decreased Cl, and increased AUC, MRT and T1/2ß (p < 0.05) for both SSa and SSd after intravenous administration in comparison with solution.

Pharmacokinetics, tissue distribution and excretion of peimisine in rats assessed by liquid chromatography-tandem mass spectrometry.

Peimisine, the common ingredient of "zhebeimu" groups and "chuanbeimu" groups, is responsible for the expectorant and cough relieving effects. The aim of this study was to investigate the pharmacokinetics, tissue distribution and excretion of peimisine in male and female SD (Sprague-Dawley) rats by a rapid and sensitive LC-MS/MS (liquid chromatography-tandem mass spectrometry) method used carbamazepine as the internal standard after oral administration, carbamazepine was stated as an IS. The results showed that peimisine was slowly distributed, and eliminated from rat plasma and manifested linear dynamics in a dose range of 0.26-6.5 mg/kg. Tested by ANOVA, there were gender differences in the pharmacokinetic parameters of AUC(0-t), AUC(0-∞) among a single dose of 0.26, 1.3, 6.5 mg/kg (P < 0.05). Drug blood and tissue levels in male rats were significantly higher than the female counterparts after oral administration, while both the males and the females showed high drug levels in spleen, kidney, lung, liver and heart. On the other hand, the peimisine levels that can be reached in uterus, ovary, testis and brain is low. The excretion study showed that little administered peimisine (<0.7%) was recovered in the male and female bile. Approximately 13.46 and 15.05% were recovered in female urine and feces, while 43.07 and 7.49% were recovered in male urine and feces, respectively, which indicated that the major elimination route of male rats was urine excretion. In addition, there was significant differences in total cumulative excretive ratio of peimisine in feces (P < 0.05) and no significant differences in the urine (P > 0.05) at a dose of 1.3 mg/kg.

Rapid and sensitive detection of Listeria monocytogenes by cross-priming amplification of lmo0733 gene.

Listeria monocytogenes is a food-borne pathogen that causes severe opportunistic infection in humans and animals. This study reports the development of single cross-priming amplification (S-CPA) and double CPA (D-CPA) assays targeting species-specific gene lmo0733 for identifying L. monocytogenes strains. The CPA assays were performed at a constant temperature 64 °C using seven specific primers and evaluated for specificity and sensitivity. The color change of positive amplification was directly observed by Loopamp® Fluorescent Detection Reagent (FD), and the DNA products were visualized as a ladder-like banding pattern on 2.5% gel electrophoresis. Moreover, the positive reactions were also detected by real-time measurement of turbidity. 50 L. monocytogenes and 46 non-L. monocytogenes strains were used for the method verification, and the specificity was 100%. The limit of detection (LoD) of the S-CPA and D-CPA assays was 2.5 pg DNA per reaction and 10-fold more sensitive than PCR. A total of 60 pork samples were tested for L. monocytogenes using the S-CPA assay developed in the study, and the accuracy of the S-CPA and the culture-biotechnical method was 100% identical. The results suggested that the S-CPA assay was a rapid, sensitive, and valuable tool for detection of L. monocytogenes in food products.

Retarded dissolution of ibuprofen in gelatin microcapsule by cross-linking with glutaradehyde.

Ibuprofen-loaded gelatin microcapsule, a solid form of microcapsules simultaneously containing ethanol and ibuprofen in water-soluble gelatin shell was previously reported to improve the dissolution of drug. In this study, to retard the initial high dissolution of ibuprofen from gelatin microcapsule, the ibuprofen-loaded cross-linked gelatin microcapsule was prepared by treating an ibuprofen-loaded gelatin microcapsule with glutaraldehyde and its dissolution was evaluated compared to ibuprofen powder and gelatin microcapsule. The ibuprofen-loaded cross-linked microcapsule treated with glutaraldehyde for 10 and 60 sec gave significantly higher dissolution rates than did ibuprofen powder. Furthermore, the dissolution rate of ibuprofen from the cross-linked microcapsule treated for 10 sec was similar to that from gelatin microcapsule. However, the dissolution rate of ibuprofen from the cross-linked microcapsule treated for 60 sec decreased significantly compared to gelatin microcapsule, suggesting that the treatment of gelatin microcapsule with glutaraldehyde for 60 sec could cross-link the gelatin microcapsule. Furthermore, the cross-linking of gelatin microcapsule markedly retarded the release rate of ibuprofen in pH 1.2 simulated gastric fluid compared to gelatin microcapsule. However, the cross-linking of gelatin microcapsule with glutaraldehyde hardly changed the size of gelatin microcapsules, ethanol and ibuprofen contents encapsulated in gelatin microcapsule. Thus, the ibuprofen-loaded cross-linked gelatin microcapsule could retard the initial high dissolution of poorly water-soluble ibuprofen.