Effects of insulin-like peptide 7 in Bemisia tabaci MED on tomato chlorosis virus transmission.

BACKGROUND: Tomato chlorosis virus (ToCV) is a semi-persistent plant virus that is primarily transmitted by the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae). It causes a serious disease that lowers tomato yield. Insulin-like peptide (ILP), an insulin homolog, regulates trehalose metabolism in a variety of insects. In a previous study, we discovered that trehalose metabolism is required for whiteflies to transmit ToCV effectively. Furthermore, transcriptome sequencing revealed that the BtILP7 gene was highly expressed in B. tabaci infected with ToCV. Therefore, the whitefly ILP7 gene may facilitate the transmission of ToCV and be an attractive target for the control of whiteflies and subsequently ToCV. RESULTS: The ToCV content in B. tabaci MED was found to be correlated with BtILP7 gene expression. Subsequent RNA interference (RNAi) of the BtILP7 gene had a significant impact on B. tabaci MED's trehalose metabolism and reproductive capacity, as well as ability to transmit ToCV. CONCLUSIONS: These results indicate that the BtILP7 gene was closely related to ToCV transmission by regulating trehalose metabolism and reproduction behavior, thus providing a secure and environmentally friendly management strategy for the control of whiteflies and ToCV-caused disease. © 2022 Society of Chemical Industry.

RNA-Seq Identification of Cd Responsive Transporters Provides Insights into the Association of Oxidation Resistance and Cd Accumulation in Cucumis sativus L.

Greenhouse vegetable production (GVP) has grown rapidly and has become a major force for cucumber production in China. In highly intensive GVP systems, excessive fertilization results in soil acidification, increasing Cd accumulation and oxidative stress damage in vegetables as well as increasing health risk of vegetable consumers. Therefore, enhancing antioxidant capacity and activating the expression level of Cd transporter genes seem to be feasible solutions to promote plant resistance to Cd stress and to reduce accumulated Cd concentration. Here, we used transcriptomics to identify five cucumber transporter genes (CsNRAMP1, CsNRAMP4, CsHMA1, CsZIP1, and CsZIP8) in response to cadmium stress, which were involved in Cd transport activity in yeast. Ionomics, gene expression, and REDOX reaction level association analyses have shown that the transcript of CsNRAMP4 was positively correlated with Cd accumulation and antioxidant capacity of cucumber roots. The expression level of CsHMA1 was negatively correlated with Cd-induced antioxidant capacity. The overexpression of CsHMA1 significantly relieved Cd stress-induced antioxidant activities. In addition, shoots with high CsHMA2 expression remarkably presented Cd bioaccumulation. Grafting experiments confirmed that CsHMA1 contributed to the high antioxidant capacity of cucumber, while CsHMA2 was responsible for the transport of Cd from the roots to the shoots. Our study elucidated a novel regulatory mechanism for Cd transport and oxidative damage removal in horticultural melons and provided a perspective to regulate Cd transport artificially by modulating Cd accumulation and resistance in plants.

Efficient markerless gene deletions in Pseudomonas protegens Pf-5 using a upp-based counterselective system.

OBJECTIVES: Developing a counterselective system for efficient markerless gene deletions in biocontrol strain P. protegens Pf-5. RESULTS: We successfully implemented a markerless deletion of upp in Pf-5 to obtain the 5-FU resistant strain Pf5139. With this strain, we performed markerless gene deletions for each component of Gac/Rsm system and a 17 kb DNA fragment with the deletion ratio of 20 to 50%, and efficiently constructed a strain with triple deletions based on the suicide plasmid pJQ200UPP. In addition, there is no obvious connection between the deleted fragment length and the deletion ratio. CONCLUSION: The upp-based counterselective system in this study is efficient and valuable for markerless gene deletions in Pf-5, indicating that it has great potential in the study of gene function and in the application of genome reduction for Pseudomonas strains.

Postnatal Expression Patterns of Estrogen Receptor Subtypes and Choline Acetyltransferase in Different Regions of the Papez Circuit.

The Papez circuit is crucial for several brain functions, including long-term memory and emotion. Estradiol modulates cognitive functions based on the expression pattern of its receptor subtypes including estrogen receptor (ER) α, ß, and G protein-coupled receptor 30 (GPR30). Similarly, the activity in the cholinergic system correlates with several brain functions, such as learning and memory. In this study, we used immunofluorescence to examine the expression patterns of ERß and Western blotting to analyze GPR30 and choline acetyltransferase (ChAT) expression, in different regions of the Papez circuit, including the prefrontal cortex, hippocampus, hypothalamus, anterior nucleus of the thalamus, and cingulum in female rats at postnatal days (PND) 1, 10, and 56. Our main finding was that the highest expression of ERß and GPR30 was noted in each brain area of the Papez circuit in the PND1 rats, whereas the expression of ChAT was the highest in PND10 rats. These results provide vital information on the postnatal expression patterns of ER subtypes and ChAT in different regions of the Papez circuit.

Genistein inhibits Aß25-35 -induced SH-SY5Y cell damage by modulating the expression of apoptosis-related proteins and Ca2+ influx through ionotropic glutamate receptors.

In this study, we investigated the protective effects of genistein against SH-SY5Y cell damage induced by ß-amyloid 25-35 peptide (Aß25-35 ) and the underlying mechanisms. Aß-induced neuronal death, apoptosis, glutamate receptor subunit expression, Ca2+ ion concentration, amino acid transmitter concentration, and apoptosis-related factor expression were evaluated to determine the effects of genistein on Aß-induced neuronal death and apoptosis. The results showed that genistein increased the survival of SH-SY5Y cells and decreased the level of apoptosis induced by Aß25-35 . In addition, genistein reversed the Aß25-35 -induced changes in amino acid transmitters, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, and N-methyl-d-aspartate (NMDA) receptor subunits in SH-SY5Y cells. Aß25-35 -induced changes in Ca2+ and B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X (Bax) protein and gene levels in cells were also reversed by genistein. Our data suggest that genistein protects against Aß25-35 -induced damage in SH-SY5Y cells, possibly by regulating the expression of apoptosis-related proteins and Ca2+ influx through ionotropic glutamate receptors.

Zn2+ reduction induces neuronal death with changes in voltage-gated potassium and sodium channel currents.

In the present study, cultured rat primary neurons were exposed to a medium containing N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a specific cell membrane-permeant Zn2+ chelator, to establish a model of free Zn2+ deficiency in neurons. The effects of TPEN-mediated free Zn2+ ion reduction on neuronal viability and on the performance of voltage-gated sodium channels (VGSCs) and potassium channels (Kvs) were assessed. Free Zn2+ deficiency 1) markedly reduced the neuronal survival rate, 2) reduced the peak amplitude of INa, 3) shifted the INa activation curve towards depolarization, 4) modulated the sensitivity of sodium channel voltage-dependent inactivation to a depolarization voltage, and 5) increased the time course of recovery from sodium channel inactivation. In addition, free Zn2+ deficiency by TPEN notably enhanced the peak amplitude of transient outward K+ currents (IA) and delayed rectifier K+ currents (IK), as well as caused hyperpolarization and depolarization directional shifts in their steady-state activation curves, respectively. Zn2+ supplementation reversed the effects induced by TPEN. Our results indicate that free Zn2+ deficiency causes neuronal damage and alters the dynamic characteristics of VGSC and Kv currents. Thus, neuronal injury caused by free Zn2+ deficiency may correlate with its modulation of the electrophysiological properties of VGSCs and Kvs.

An in situ crosslinked compression coat comprised of pectin and calcium chloride for colon-specific delivery of indomethacin.

The use of pectin for colon-specific drug delivery has been extensively investigated; however, when used alone, pectin is often compromised due to its high solubility. This study explored the feasibility of using an in situ compression-coated crosslinking system, composed of pectin and calcium chloride, for colon-specific drug delivery. A pectin/calcium chloride (P/Ca) coating was compressed onto a core tablet. The colon specificity of the compression-coated tablet was verified by dissolution, pharmacokinetics and scintigraphy with (99m)Tc labeling. The in situ pectin and calcium chloride gel slowed the release of indomethacin. The lag time varied between 3 h and 7 h depending on the amount of calcium chloride and the coating weight. Pectinase triggered the release of indomethacin from the compression-coated tablet, which was then accelerated by the calcium chloride in the coating layer. The compression-coated tablet had a prolonged tmax and apparent t1/2, as well as a decreased Cmax and AUC0-t, compared with the core tablet counterpart. Evaluation with γ-scintigraphy verified colon-specific delivery of the compression-coated tablet. In conclusion, the P/Ca in situ crosslinking system worked well for colon-specific drug delivery.

Single- and multiple-dose pharmacokinetics of a novel tetramethylpyrazine reservoir-type transdermal patch versus tetramethylpyrazine phosphate oral tablets in healthy normal volunteers, and in vitro/in vivo correlation.

A novel reservoir-type transdermal system of 2,3,5,6-tetramethylpyrazine (TMP) was developed containing eucalyptus oil as a penetration enhancer. The single and multiple-dose pharmacokinetic profiles of TMP administrated by TMP transdermal patch were characterized in healthy volunteers using an in vivo, randomized, open-label, two-way crossover design. 2,3,5,6-Tetramethylpyrazine phosphate (TMPP) oral tablets were chosen as reference. Following single/multiple oral administration of 200/100 mg TMPP tablets, a TMP C(max) of 1284/613.5 ng/mL was observed within 0.75 h. Single/multiple applications of the TMP patch yielded mean C(max) of 309/325 ng/mL at a median T(max) of 5/4 h, with steady state achieved at second application. The mean C(min) of the patch was 131±30.38 ng/mL, contrasting to nearly zero for the tablet. Multiple applications of patch produced an accumulative effect over single application. At steady state 250 mg/20 cm(2) TMP patch given daily provided comparable exposure to 100 mg TMPP tablets three times daily (3753.91 versus 3563.67 ng·h/mL). TMP tablets and patch yielded similar steady-state plasma concentrations: C(av) (148.48±51.27, 156.41±40.31 ng/mL). The results demonstrated that TMP patch can achieve a therapeutic effect that is comparable to oral administration, exhibited prolonged and sustained plasma levels, fewer drug fluctuations, lower adverse effects, more convenience, and improved patient compliance. In-vitro permeation through human skin demonstrated zero-order kinetics with the flux of 364 µg/cm(2)/h. The predicted C(av) (163.9 ng/mL) was in agreement with the observed C(av) (156.4 ng/mL).

Development of a reservoir-type transdermal delivery system containing eucalyptus oil for tetramethylpyrazine.

The purpose of this study was to formulate a reservoir-type transdermal delivery system (TDS) for 2,3,5,6-tetramethylpyrazine (TMP) to enable the delivery of a sufficient dose through human skin to achieve an effective therapeutic plasma concentration. To improve the penetration of TMP in the reservoir-type TDS, several chemical penetration enhancers were investigated using in vitro rat dorsal skin permeation studies. Eucalyptus oil was found to enhance the permeation of TMP to the greatest extent, with the optimal concentration being 5% and the flux being 542.6 ± 49.7 µg/cm(2)/h, which was 4.5-fold greater than control with no enhancer (p < 0.01). The flux of the optimized reservoir-type TDS permeated through the human epidermis was 346.0 ± 27.7 µg/cm(2)/h. Based on the in vitro human skin permeation flux and the pharmacokinetics parameters observed, the clinical surface area of the TDS patch was predicted to be 20 cm(2). The in vivo study conducted in rabbits showed that the TMP TDS patch containing 5% eucalyptus oil had a more favorable pharmacokinetic profile, with a lower C(max) and prolonged T(max) and mean residence time than that observed with the oral administration of TMP. The TMP reservoir-type TDS was shown to be a promising alternative route to oral administration or intravenous infusion of TMP.

Guar gum/ethylcellulose coated pellets for colon-specific drug delivery.

The aim of this work was to investigate guar gum/ethylcellulose mix coated pellets for potential colon-specific drug delivery. The coated pellets, containing 5-fluorouracil as a model drug, were prepared in a fluidized bed coater by spraying the aqueous/ethanol dispersion mixture of guar gum and ethylcellulose. The lag time of drug release and release rate were adjustable by changing the ratio of guar gum to ethylcellulose and coat weight gain. In order to find the optimal coating formulation that was able to achieve drug targeting to the colon, the effect of two independent variables (the ratio of guar gum to ethylcellulose and the coat weight gain) on drug release characteristics was studied using 3 x 4 factorial design and response surface methodology. Results indicated that drug release rate decreased as the proportion of ethylcellulose in the hybrid coat and the coat weight gain increased. When the ratio of guar gum to ethylcellulose was kept in the range of 0.2 to 0.7, and the coat weight gain in the range of 250% to 500%, the coated pellets can keep intact for about 5 h in upper gastrointestine and achieve colon-specific drug delivery. The pellets prepared under optimal conditions resulted in delayed-release sigmoidal patterns with T(5%) (time for 5% drug release) of 5.1 - 7.8 h and T(90%) (time for 90% drug release) of 9.8 - 16.3 h. Further more, drug release was accelerated and T(90%) of the optimum formulation pellets decreased to 9.0 - 14.5 h in pH 6.5 phosphate buffer with hydrolase. It is concluded that mixed coating of guar gum and ethylcellulose is able to provide protection of the drug load in the upper gastrointestinal tract, while allowing enzymatic breakdown of the hybrid coat to release the drug load in the colon.

In vitro evaluation and pharmacokinetics in dogs of guar gum and Eudragit FS30D-coated colon-targeted pellets of indomethacin.

A pH- and enzyme-dependent colon-targeted multi-unit delivery system of indomethacin was developed by coating guar gum and Eudragit FS30D sequentially onto drug-loaded pellets in a fluidized bed coater. In vitro studies showed that smaller coating weight gain of guar gum resulted in reduced release lag time t10 (10% release time), but favored degradation by enzymes (galactomannanase). A cumulative weight gain (CWG) of 44% provided sufficient enzymatic sensitivity and protection of the core. Under gradient pH conditions (pH = 1.2, 6.8, 7.4 and 6.5 for 2, 2, 1 and 15 h, respectively), indomethacin was released from Eudragit FS30D-coated pellets quickly after changing pH to 7.4. For guar gum/Eudragit FS30D double-coated pellets, only about 5% of the drug was released after another 1 h, showing retarding effect by guar gum coating. After changing pH to 6.5 and addition of galactomannanase, enzyme-dependent drug release was observed. Pharmacokinetic study in beagle dogs showed that fastest absorption with the smallest Tmax and Tlag was observed for uncoated pellets. The Tmax and Tlag of Eudragit FS30D-coated pellets were postponed to about 2.5 and 1 h, respectively. After a further guar gum coating, Tlag was further postponed to about 2.8 h, about 2 h of additional lag time on the basis of Eudragit FS30D coating. It is indicated that the guar gum/Eudragit FS30D-coated system has potential to be used to deliver drugs to the colon.

Electronic healthcare: issues and challenges.

The shift in the nation's attention, resources and interest toward internet-related healthcare activities, often called 'e-health', is dramatic and a most significant development in the healthcare environment. E-health, while exciting and promising, also presents new challenges, particularly in regard to acceptable standards, choice of technologies, overcoming traditional jurisdictional boundaries, up-front investment and privacy and confidentiality. This paper presents e-health architecture and various issues and challenges that need to be resolved before e-health becomes commonplace.

[Determination of ligustrazine in human plasma by high performance liquid chromatography with solid-phase extraction].

A high performance liquid chromatographic method was developed for the determination of ligustrazine in human plasma. The chromatographic separation was performed on a Luna C18 column (150 mm x 4.6 mm i.d., 5 microm) at column temperature of 40 degrees C. The mobile phase, a mixture of methanol-acetonitrile-acetate buffer of pH 5.0 (50:8:42, v/v), was delivered at a flow rate of 1.0 mL/min. The detection wavelength was 280 nm. Plasma samples were prepared with a C8 solid-phase extraction column. Linearity was confirmed in the mass concentration range of 25-5000 microg/L with the correlation coefficient of 0.9999. The extraction recovery of ligustrazine ranged from 96.72% to 100.90%. The relative standard deviations (RSDs) of intra- and inter-day assay at the mass concentrations of 50, 500 and 3000 microg/L were less than 8.64% and the accuracies were between 99.59%-103.26%. The limit of detection (LOD) was 10 microg/L. The results of this method validation satisfactorily meet the acceptance criteria of bioanalysis and the method is applicable to the pharmacokinetic studies of ligustrazine in human beings.

A pharmacokinetic study of intramuscular administration of bulleyaconitine A in healthy volunteers.

The pharmacokinetics of bulleyaconitine A (BLA) after a single dose of 0.2 mg intramuscular injection was evaluated in healthy volunteers. Physical exam, vital signs, clinical laboratory tests and electrocardiogram measurements were monitored to assess the safety and tolerance of the drug. The plasma levels of BLA in serial samples, collected over 15 h, were measured by a validated high-performance liquid chromatography (HPLC)-electrospray ionization tandem mass spectrometry (MS-MS) method. It was demonstrated that BLA was absorbed rapidly after intramuscular injection. The pharmacokinetic parameters were as follows: the t(max) value was 0.90+/-0.68 h, the C(max) value was 1.13+/-0.76 ng/ml, the AUC(0-t) was 5.16+/-2.05 ng.h/ml, and t(1/2) was found to be 4.88+/-0.97 h. No subject showed any drug-related clinically significant changes on physical examination, vital signs or laboratory tests. Eight of ten subjects reported a distinct feeling of pain at the site of injection starting approximately at the time of their peak plasma concentration and lasting for 2-6 h. The pain was tolerable, and no subject required additional treatment.

Determination of bulleyaconitine A in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry.

A sensitive and specific high-performance liquid chromatography (HPLC)-electrospray ionization tandem mass spectrometry (MS-MS) was developed for the determination of bulleyaconitine A (BLA) in human plasma. BLA and internal standard (I.S.) ketoconazole were extracted from the plasma by a liquid-liquid extraction. The supernatant was evaporated to complete dryness and reconstituted with acetonitrile containing 0.1% acetic acid before injecting into an ODS MS column. The gradient mobile phase was composed of a mixture of acetonitrile (containing 0.1% acetic acid, v/v) and 0.1% acetic acid aqueous solution eluted at 0.3 ml/min. BLA and I.S. were determined by multiple reaction monitoring using precursor-->product ion combinations at m/z 644.6-->584.3 and 531.2-->81.6, respectively. Linearity was established for the concentration range of 0.12-6 ng/ml. The recoveries of BLA ranged from 96.93 to 113.9% and the R.S.D. was within 20%. The method is rapid and applicable to the pharmacokinetic studies of BLA in human.

In vitro permeation of tetramethylpyrazine across porcine buccal mucosa.

AIM: To examine the in-vitro transport route of tetramethylpyrazine (TMP) across porcine buccal mucosa and to investigate the effects of drug concentration, pH in donor chamber, and 1-octanol/buffer partition coefficient on transbuccal permeation. METHODS: In-vitro permeation of TMP through porcine buccal mucosa was studied by using in-line flow through diffusion cells at 37 . The permeability of TMP was evaluated at different donor pH and drug concentration. Permeability of unionized (Pu) and ionized TMP (Pi) was calculated by using the Scientist software. RESULTS: The steady state flux of TMP increased linearly with the donor concentration (r2=0.96) at pH 7.4. The permeability and the partition coefficient increased with pH. Pu and Pi were 9.05 x 10(-6) cm.s(-1) and 2.99 x 10(-7) cm.s(-1), respectively. The total permeability coefficient increased with the fraction of unionized form. CONCLUSION: TMP permeated through buccal mucosa by a passive diffusion process. The partition coefficient and pH dependency of drug permeability indicated that the drug was transported mainly via the transcellular route by a partition mechanism.