Virtual Screening of Potential RoxS Inhibitors and Evaluation of Their Antimicrobial Activity in Combination with Antibiotics against Clinically Resistant Bacteria.

Pseudomonas aeruginosa with difficult-to-treat resistance has been designated as an urgent or serious threat by the CDC in the United States; therefore, novel antibacterial drugs and combination strategies are urgently needed. The sensor kinase RoxS is necessary for the aerobic growth of Pseudomonas aeruginosa. This study aimed to screen candidate RoxS inhibitors and evaluate their efficacy in treating multi-drug-resistant and extensively drug-resistant Pseudomonas aeruginosa in combination with meropenem and amikacin to identify promising combination strategies. RoxS protein structures were constructed using homology modeling and potential RoxS inhibitors, including Ezetimibe, Deferasirox, and Posaconazole, were screened from the FDA-approved ZINC drug database using molecular docking and molecular dynamics simulations. MIC and checkerboard assays were used to determine the in vitro antimicrobial efficacy of the three drugs in combination with antibiotics. The results of in vitro experiments showed an additive effect of 100 µg/mL Deferasirox or 16 µg/mL Posaconazole in combination with meropenem and a synergistic effect of 1.5 µg/mL Deferasirox and amikacin. In summary, these three drugs are potential inhibitors of RoxS, and their combination with meropenem or amikacin is expected to reverse the resistance of P. aeruginosa, providing new combination strategies for the treatment of clinically difficult-to-treat Pseudomonas aeruginosa.

Meloxicam inhibits STING phosphorylation and alleviates intracellular DNA-mediated autoimmune responses.

BACKGROUND: Cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is critical for cytosolic DNA-sensing and the subsequent immune responses. The inappropriate activation of this pathway leads to DNA-induced autoimmune response. Understanding the precise regulation of cGAS-STING pathway is important for developing therapeutics to treat several autoimmune diseases caused by self-DNA. RESULTS: We report that Meloxicam (MXC) inhibits intracellular DNA-, but not RNA-induced immune responses. We find that MXC inhibits the phosphorylation of STING by examining in different cells with various DNA stimulations. We further find that MXC significantly dampens the expression levels of interferon-stimulated genes (ISGs) by using DNA 3' repair exonuclease 1 (TREX1)-deficient cell, an experimental model for self-DNA-induced autoimmune disease. Importantly, we demonstrate that MXC could promote the survival in Trex1-/- mouse model for Aicardi-Goutières syndrome (AGS). CONCLUSIONS: Our study identified a non-steroidal anti-inflammatory drug, MXC, that exhibits potential effect in treating the autoimmunity caused by self-DNA.

Leptin activates the JAK/STAT pathway to promote angiogenesis in RF/6A cells in vitro.

AIM: To investigate the effect of leptin on the angiogenesis of RF/6A cells (monkey retinal choroidal endothelial cells) in vitro and test the cellular signaling in the mechanism. METHODS: RF/6A cells were cultured in vitro and randomly divided into four groups: normal control, with leptin at 50, 100, 200 ng/mL for cell counting kit-8 (CCK8). RF/6A cell proliferation and migration were examined by Transwell assays, while RF/6A cell tube formation by Matrigel assay. JAK2, p-JAK2, STAT3, and p-STAT3 protein expression was measured by Western blotting. Cells were then divided into the following treatment groups: control, 100 ng/mL leptin and AG-490 (100 ng/mL leptin+10 µmol/L AG-490) for examinations of RF/6A cellular behaviour again. Analysis of differences was carried out using one-way ANOVA and least significant difference (LSD). RESULTS: RF/6A cell proliferation, migration and cell tube formation were promoted significantly by leptin in a dose-dependent manner (P<0.05). Western blotting showed that leptin up-regulated p-JAK2 and p-STAT3 expression levels. Treatment with the JAK/STAT pathway inhibitor, AG-490, decreased leptin-induced p-JAK2 and p-STAT3 expression, and inhibited cell proliferation, migration and cell tube formation induced by leptin (P<0.05). CONCLUSION: Leptin can promote RF/6A cell angiogenesis in vitro via activation of the JAK2/STAT3 signaling pathway.

Synchronous concomitant pancreatic acinar cell carcin and gastric adenocarcinoma: A case report and review of literature.

BACKGROUND: Multiple primary malignant tumors are two or more malignancies in an individual without any relationship between the neoplasms. In recent years, an increasing number of cases have been reported. However, concomitant primary gastric and pancreatic cancer reported a relatively small incidence, involving no pancreatic acinar cell carcinoma reports. Here, we present the first case of concomitant pancreatic acinar cell carcinoma and gastric adenocarcinoma. CASE SUMMARY: A 69-year-old male presented to our department with a history of vomiting, epigastric pain, and weight loss. Imaging revealed space-occupying lesions in the stomach and the tail of the pancreas, respectively. The patient underwent laparoscopic radical gastrectomy and pancreatectomy simultaneously. The pathologies of surgical specimens were completely different: The resected gastric specimen was moderate to poorly differentiated adenocarcinoma, whereas the pancreatic tumor was consistent with acinar cell carcinoma. The patient was treated with six cycles of oxaliplatin and S-1 chemotherapy. As of March 2021, the patient was healthy without any recurrence or metastasis. After thoroughly reviewing the literature on simultaneous pancreatic and gastric cancers at home and abroad, we discussed the clinical characteristics of these rare synchronous double cancers. Most of the cases had undergone surgery and adjuvant chemotherapy, and all of the cases were pathologically confirmed by the postoperative specimen. CONCLUSION: Synchronous pancreatic acinar cells and gastric adenocarcinoma can occur and should be considered when tumors are found in these organs.

Chemical and Biological Study of Novel Aplysiatoxin Derivatives from the Marine Cyanobacterium Lyngbya sp.

Since 1970s, aplysiatoxins (ATXs), a class of biologically active dermatoxins, were identified from the marine mollusk Stylocheilus longicauda, whilst further research indicated that ATXs were originally metabolized by cyanobacteria. So far, there have been 45 aplysiatoxin derivatives discovered from marine cyanobacteria with various geographies. Recently, we isolated two neo-debromoaplysiatoxins, neo-debromoaplysiatoxin G (1) and neo-debromoaplysiatoxin H (2) from the cyanobacterium Lyngbya sp. collected from the South China Sea. The freeze-dried cyanobacterium was extracted with liquid-liquid extraction of organic solvents, and then was subjected to multiple chromatographies to yield neo-debromoaplysiatoxin G (1) (3.6 mg) and neo-debromoaplysiatoxin H (2) (4.3 mg). They were elucidated with spectroscopic methods. Moreover, the brine shrimp toxicity of the aplysiatoxin derivatives representing differential structural classifications indicated that the debromoaplysiatoxin was the most toxic compound (half inhibitory concentration (IC50) value = 0.34 ± 0.036 µM). While neo-aplysiatoxins (neo-ATXs) did not exhibit apparent brine shrimp toxicity, but showed potent blocking action against potassium channel Kv1.5, likewise, compounds 1 and 2 with IC50 values of 1.79 ± 0.22 µM and 1.46 ± 0.14 µM, respectively. Therefore, much of the current knowledge suggests the ATXs with different structure modifications may modulate multiple cellular signaling processes in animal systems leading to the harmful effects on public health.

CD59 receptor targeted delivery of miRNA-1284 and cisplatin-loaded liposomes for effective therapeutic efficacy against cervical cancer cells.

Co-delivery of two different therapeutics (miRNA-1284 and cisplatin (CDDP)) into the cancer cells in a single nanocarrier provides new dimension to the cancer treatment. In this study, we have designed the CD59sp-conjugated miRNA-1284/cisplatin(CDDP)-loaded liposomes for the enhanced therapeutic effect against cervical cancers. Compared with miRNA-1284/CDDP-loaded liposomes (LP-miCDDP), CD59 antibody-conjugated LP-miCDDP (CD/LP-miCDDP) showed a significantly higher cytotoxicity in HeLa cells. Notably, MiR-1284 showed a typical concentration-dependent cell killing effect in the cervical cancer cells owing to the downregulation of HMGB1. Flow cytometer analysis showed that CD/LP-miCDDP resulted in maximum apoptosis effect (~ 60%) compared to CDDP (~ 20%) or miR-1284 (~ 12%) treated cells indicating the superior anticancer effect in the cancer cells. Importantly, CD/LP-miCDDP significantly prolonged the blood circulation of encapsulated drug in rats with AUC(o-t) of CD/LP-miCDDP showed a 6.9 fold higher value than that of free CDDP. Similarly, CD/LP-miCDDP showed an eightfold decrease in the clearance (CL) and 3.6-fold higher t1/2 compared to that of free CDDP. Overall, results demonstrated that targeted and synergistic co-delivery of therapeutic components could be promising in cervical cancer therapy.

Neo-debromoaplysiatoxin C, with new structural rearrangement, derived from debromoaplysiatoxin.

Neo-debromoaplysiatoxin C (1), a new member of the aplysiatoxin family, was isolated from the marine cyanobacterium Lyngbya sp. The structure of 1 was elucidated based on spectroscopic data, and its stereochemistry was determined from NOESY spectrum and biosynthetic considerations. This new compound presents an intriguing 10-membered lactone ring skeleton derived from debromoaplysiatoxin by structural rearrangement, which is the first example in the aplysiatoxin family. Its biological properties were evaluated for cytotoxicity, PKCδ activation and inhibitory effects on potassium channel.

Wheat Gluten Regulates Cholesterol Metabolism by Modulating Gut Microbiota in Hamsters with Hyperlipidemia.

The objective of this research was to evaluate the effect of wheat gluten on gut microbiota from hamsters and also analyse whether alterations in microbiota could result in wheat gluten's lipid-lowering properties. Four weeks male hamsters were divided into 3 groups (n=10). Two hypercholesterolemic groups were fed for 35 days with hypercholesterolemic diet, containing 20% (w/w) wheat gluten or casein. Wheat gluten significantly reduced serum total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) concentrations, and also decreased the liver total cholesterol (TC), free cholesterol (FC), cholesterol ester (CE), triglycerides (TG) concentrations. Wheat gluten group had a higher fecal lipids, total cholesterol (TC) and bile acids (BA) than that of casein group (p < 0.05). Moreover, wheat gluten significantly increased total short-chain fatty acids (SCFA) concentrations in feces. Sequencing of 16S rRNA gene revealed that intake of wheat gluten decreased the relative abundances of Firmicutes and Erysipelotrichaceae, but to increased the relative abundances of Bateroidetes, Bacteroidales_S24-7_group and Ruminococcaceae. The lipid lowering properties of wheat gluten was associated with the lower ratio of Firmicutes/Bateroidetes, the lower of the bacterial taxa Erysipelotrichaceae and the higher of the bacterial taxa Bacteroidales_S24-7_group and Ruminococcaceae. These results suggest that wheat gluten modulate cholesterol metabolism by altering intestinal microflora.

Effects of tail docking and/or teeth clipping on behavior, lesions, and physiological indicators of sows and their piglets.

To evaluate effects of tail docking and/or teeth clipping on sows and their piglets, a total of 24 sows and their 302 piglets at 3 days of age were randomly allocated to one of four treatments: teeth clipping and tail docking (TCTD), teeth clipping (TC), tail docking (TD), or intact teeth and tail (Intact). Behavior of piglets and sows, lesions on the body and tail of piglets and sows' teats were inspected. Heart rates of processed piglets were increased (p < .01) during the procedures. Teeth clipping decreased body surface temperature (p < .01) of piglets during and after the procedures but tail docking did not (p > .01). Processed piglets spent more (p < .05) time lying alone and playing/fighting than sham-processed piglets. Tail docked piglets spent less (p < .01) time standing than tail sham-docked piglets. Intact teeth increased (p < .05) the avoidance behaviors of sows. Teeth clipping decreased (p < .05) the lesion scores on the anterior, middle, and posterior teats. Taken together, piglet teeth clipping had more impact on sows and their piglets than tail docking did in the lactation period based on our findings.

Effect of Oat and Tartary Buckwheat - Based Food on Cholesterol - Lowering and Gut Microbiota in Hypercholesterolemic Hamsters.

The nutritional components in oat and tartary buckwheat had been assessed to have cholesterollowering effects. However, The effect of oat and tartary buckwheat based-food (OF) on cholesterol-lowering and gut microbiota in hypercholesterole hamsters was still limited studied because they are usually consumed in whole gran as well as after being processed. In this study, normal diets, high fat diet (HFD) with/without OF were fed to hamsters for 30 days respectively and growth parameters, metabolic parameters, and gut microbiota were investigated, respectively. It was found that OF significantly decreased plasma total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-cholesterol), lowered liver TC, cholesterol ester (CE), and triglycerides (TG) concentrations, and increased fecal weight and bile acids (BA) concentrations, compared with HFD (p < 0.05). Moreover, the concentrations of acetate, propionate, butyrate and total short-chain fatty acids (SCFAs) were significantly increased in hamsters fed with OF, compared with HFD (p < 0.05). OF changed the overall structure of gut microbiota. The relative abundances of Erysipelotrichaceae, Ruminococcaceae, and Lachnospiraceae were decreased and the relative abundance of Eubacteriaceae was increased, compared with HFD. These results suggested that OF could reduce the concentrations of plasma lipid by inhibiting cholesterol absorption in liver and promoting excretions of fecal lipid and BA. And it also increased SCFAs and modulated the gut microbiota effectively to exert the hypocholesterolemic effects.

Effects of Simulated Heat Wave and Ozone on High Fat Diet ApoE Deficient Mice.

OBJECTIVE: To discuss the cardiac toxicities of a heat waves and ozone exposure on cardiovascular diseases (CVDs) and explore a possible mechanism. METHODS: The incidence of ozone exposure combined with heat wave was simulated in the Shanghai Meteorological and Environmental Animal Exposure System (Shanghai-METAS). A total of 64 ApoE-/- mice, matched by weight, were randomly divided into 8 groups and exposed to heat wave conditions or ozone. The levels of creatine kinase (CK), D-lactate dehydrogenase (D-LDH), intercellular adhesion molecule 1 (sICAM-1), tumor necrosis factor alpha (TNF-α), nitric oxide (NO), endothelin-1 (ET-1), D-dimer (D2D), plasminogen activator inhibitor-1 (PAI-1) and blood lipid in plasma and heat shock protein-60 (HSP60), hypoxia inducible factor 1 alpha (HIF-1α), interleukin-6 (IL-6), C-reactive protein (CRP), superoxide dismutase (SOD), and malondialdehyde (MDA) in hearts were measured after exposure. RESULTS: The levels of all indicators, except for SOD, increased with the ozone-only exposure. However, cardiac damage was most significant when the heat wave conditions were combined with severe ozone exposure. Moreover, the levels of CK, D-LDH, NO, PAI-1, sICAM-1, and TNF-α in plasma increased significantly (P < 0.05), and the contents of HSP60, HIF-1α, CRP, and MDA in hearts increased considerably (P < 0.05), but the activity of SOD decreased significantly. In addition, the levels of four blood lipid items remarkably increased (except the level of HDL-C which decreased significantly) with ozone exposure. CONCLUSION: A short-term exposure to a heat wave and ozone causes severe toxic effects on the heart. Cardiac damage was most significant under combined heat wave and severe ozone exposure simulations.

Modeling Analysis of Potential Target of Dolastatin 16 by Computational Virtual Screening.

Dolastatin 16 is a cyclic depsipeptide isolated from the marine invertebrates and cyanobacterium Lyngbya majuscula, however, its bioactivity has been a historical question. In this study, peptidyl-prolyl cis-trans isomerase FKBP1A (FKBP12) was predicted as a potential target of dolastatin 16 via PharmMapper as well as verified using chemical-protein interactome (CPI) and molecular docking. FKBP1A has been previously identified as a target for the natural polyketide FK506 (tacrolimus), an immune suppressor inhibiting the rejection of organ transplantation in clinical use. The comparison study via the reverse pharmacophore screening and molecular docking of dolastatin 16 and FK506 indicated the good consistency of analysis with the computational approach. As the results, the lowest binding energy of dolastatin 16-FKBP1A complex was -7.4 kcal/mol and FK506-FKBP1A complex was -8.7 kcal/mol. The ligand dolastatin 16 formed three hydrogen bonds vs. four of FK506, as well as seven hydrophobic interactions vs. six of FK506 within the active site residues. These functional residues are highly repetitive and consistent with previously reported active site of model of FK506-FKBP1A complex, and the pharmacophore model was shown feasibly matching with the molecular feature of dolastatin 16.

Two Marine Cyanobacterial Aplysiatoxin Polyketides, Neo-debromoaplysiatoxin A and B, with K+ Channel Inhibition Activity.

The isolation and structure elucidation of two cyanobacterial debromoaplysiatoxin (DAT) analogues, neo-debromoaplysiatoxin A (1) and neo-debromoaplysiatoxin B (2), were reported and found to possess 6/10/6 and 6/6/6 fused-ring systems, respectively, which are rarely seen among aplysiatoxins. Both compounds exhibited potent blocking activity against Kv1.5 with IC50 values of 6.94 ± 0.26 and 0.30 ± 0.05 µM, respectively. These findings suggest the potential of aplysiatoxin analogues in modulating ionic channels and also provide links between the DAT target, protein kinase C, and cell regulation.

Anterior mediastinum invasion by multiple myeloma: A case report.

Multiple myeloma (MM) is a clonal proliferation of malignant plasma cells in the bone marrow (BM) that secretes monoclonal paraproteins in the blood serum and urine. Bone marrow MM cells can invade and damage the functions of other tissues and organs, such as the lungs, spleen, liver, pancreas, kidneys and lymph nodes. However, the invasion of MM cells primarily located in the BM to the anterior mediastinum at the site of the thymus is an extremely rare event. The current study reports the case of a 53-year-old female who presented with MM with involvement of the anterior mediastinum. The diagnosis was based on clinical imaging analyses and the results from BM and laboratory examinations, local biopsy pathology and immunohistochemistry. The patient was administered two courses of chemotherapy (epirubicin, dexamethasone and thalidomide). As a result, the tumor reduced in size, but the laboratory examination indicated no significant change. Next, the patient was switched to one course of PAD chemotherapy (bortezomib, epirubicin and dexamethasone). The original tumor was significantly reduced in size following this chemotherapy, and all the indicators improved. The present study suggests that invasion of the thymus by MM may lead to immune disturbance arising from the abnormal thymus gland. In the clinic, extramedullary plasmacytoma in the thymus should be carefully distinguished from thymoma.

Effect of oxidative stress on heme oxygenase-1 expression in patients with gestational diabetes mellitus.

The anti-oxidative stress effect of heme oxygenase-1 (HO-1) expression is being increasingly studied. However, few studies regarding HO-1 have been conducted in patients with gestational diabetes mellitus (GDM). In the present study, HO-1 expression was compared in peripheral blood mononuclear cells at 24-28 weeks of pregnancy in patients with GDM and healthy females, to investigate the correlation between HO-1 and oxidative stress by calculation of MDA content in the peripheral blood serum (thiobarbituric acid method), tested ROS (flow cytometry method), HO-1mRNA (RT-PCR method), and HO-1 protein (western blotting method) of Mononuclear cells. The results show that the levels of serum malonaldehyde (MDA), reactive oxygen species (ROS), HO-1 mRNA and HO-1 protein expression in peripheral blood mononuclear cells were higher in the GDM group than in the control group. Correlation analysis showed that the expression levels of HO-1 protein were positively correlated with the HO-1 mRNA expression levels (r=0.680; P=0.000), and the levels of ROS (r=0.572; P=0.000) and MDA (r=0.614; P=0.000). HO-1 mRNA expression levels were found to positively correlate with the levels of MDA (r=0.451; P=0.010) and fasting plasma glucose (FPG; r=0.337; P=0.039). Partial correlation analysis demonstrated that, after removing the effects of body mass index, FPG and 2-h plasma glucose, HO-1 protein expression levels were positively correlated with the levels of HO-1 mRNA expression (r=0.611; P=0.005), ROS (r=0.526; P=0.021) and MDA (r=0.519; P=0.015). These findings indicate that pregnant females with GDM may be protected against oxidative injury due to the induction of adaptive and compensatory expression of HO-1 to guard against oxidative stress induced by high glucose levels.

Primary extramedullary plasmacytoma of the penis: a case report.

Extramedullary plasmacytoma involving the penis is extremely rare. Here, we describe a case of primary extramedullary plasmacytoma of the penis in a 64-year-old man who presented with a palpable penile mass. Nuclear magnetic resonance imaging revealed the presence of a large, round non-encapsulated mass in the perineum. A contrast-enhanced computed tomography scan of the pelvis showed that the mass was located in the tunica albuginea and corpora cavernosa at the base of the penis. The mass encased the urethra and demonstrated no marked enhancement during the arterial phase. The patient underwent successful surgical resection of the tumor. Histologically, the tumor was composed primarily of neoplastic plasma cells that were positive for CD38, vimentin and Ki 67. Postoperatively, the patient recovered well and exhibited no evidence of development of multiple myeloma, local recurrence or distant metastasis at 2 months post-surgery. To the best of our knowledge, our case represents the first documented case of human primary extramedullary plasmacytoma of the penis.

[Identification of serum biomarkers for assessing minimal residual disease in acute leukemia by serum peptide pattern].

OBJECTIVE: To screen serum biomarkers for minimal residual disease (MRD) monitoring according to differential peptidomics profile in the serum from the patients with acute leukemia (AL) and healthy controls. METHODS: Serum polypeptides from 90 AL patients, 60 healthy controls and 20 patients with benign hematological disorders were enriched by copper chelate magnetic beads, and the peptidomics profile was obtained by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis. And the intensities of differential peptides were calculated to assess MRD level. RESULTS: The diagnostic models by using support vector machine (SVM) algorithm according to differential peptides between AL patients and healthy controls with P<0.01 by t-test were established. The sensitivity and specificity of distinguishing AL patients from healthy controls were 98% and 99%, respectively. The model obtained a sensitivity of 98% and a specificity of 96% for distinguishing newly-diagnosed AL patients from AL patients with hematological complete remission (AL-HCR). Then a sensitivity of 92% and a specificity of 93% were obtained for distinguishing patients with AL-CR from AL patients with molecular complete remission (AL- MR). The intensity of peptide with m/z (mass-to-charge ratio) 4468 was significantly higher in newly- diagnosed AL patients compared to healthy controls, and gradually decreased with the increase of remission degree, and it was not found increase in patients with benign hematological disorders. CONCLUSION: The SVM diagnostic model established by differential serum peptide profile could be used to discriminate AL patients with different stages of remission and to evaluate the treatment efficacy. The peptide of m/z 4468 could be used for MRD assessment, and continuous monitoring of its expression level will play an important role in the individual treatment and recurrence prediction.

Immobilized capillary tyrosinase microreactor for inhibitor screening in natural extracts by capillary electrophoresis.

A method for creating an immobilized capillary tyrosinase (TRS) reactor based on a layer-by-layer (LBL) assembly for inhibitor screening is described. Tyrosinase was immobilized on the surface of fused-silica capillary via ionic binding technique with cationic polyelectrolyte hexadimethrine bromide (HDB). Then, HDB solution with the same plug length as the TRS was injected again into the capillary to cover the immobilized enzyme by forming HDB-TRS-HDB sandwich-like structure. Then, the substrate of l-tyrosine was introduced into the capillary and on-line enzyme inhibition study was performed by capillary electrophoresis (CE). The enzyme activity was determined by the quantification of peak area of the product of l-DOPA. Enzyme inhibition can be read out directly from the reduced peak area of the product in comparison with a reference electropherogram obtained in the absence of any inhibitor. The immobilized enzyme could withstand 25 consecutive assays by only losing 12% activity. A known TRS inhibitor, kojic acid was employed as a model compound for the validation of the inhibitor screening method. Finally, screening 19 natural extracts of traditional Chinese drugs was demonstrated. The results indicated that inhibition activity could be straightforwardly identified with the system.

High-density extraction solvent-based solvent de-emulsification dispersive liquid-liquid microextraction combined with MEKC for detection of chlorophenols in water samples.

For the first time, the high-density solvent-based solvent de-emulsification dispersive liquid-liquid microextraction (HSD-DLLME) was developed for the fast, simple, and efficient determination of chlorophenols in water samples followed by field-enhanced sample injection with reverse migrating micelles in CE. The extraction of chlorophenols in the aqueous sample solution was performed in the presence of extraction solvent (chloroform) and dispersive solvent (acetone). A de-emulsification solvent (ACN) was then injected into the aqueous solution to break up the emulsion, the obtained emulsion cleared into two phases quickly. The lower layer (chloroform) was collected and analyzed by field-enhanced sample injection with reverse migrating micelles in CE. Several important parameters influencing the extraction efficiency of HSD-DLLME such as the type and volume of extraction solvent, disperser solvent and de-emulsification solvent, sample pH, extraction time as well as salting-out effects were optimized. Under the optimized conditions, the proposed method provided a good linearity in the range of 0.02-4 µg/mL, low LODs (4 ng/mL), and good repeatability of the extractions (RSDs below 9.3%, n = 5). And enrichment factors for three phenols were 684, 797, and 233, respectively. This method was then utilized to analyze two real environmental samples from wastewater and tap water and obtained satisfactory results. The obtained results indicated that the developed method is an excellent alternative for the routine analysis in the environmental field.

Effect of the Miaoyao Fanggan sachet-derived isorhamnetin on TLR2/4 and NKp46 expression in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Miaoyao Fanggan Sachets (MFS) has long been used as a folk medicine for the prevention of influenza in Southeast of Guizhou Province, China. However, the precise immunological mechanisms by which MFS confers protection have not been defined. STUDY AIM: To explore the effects of MFS on innate immune system responses using a cold stress-induced immune impairment model as a means of examining MFS-mediated influenza prevention. We investigated the effects of MFS on toll-like receptor 2 and 4 (TLR2/4) gene and protein expression levels and on the percentage of NKp46(+) cells present in serum. No overt toxicity was observed following continuous administration of MFS at high doses. METHODS: Kunming male mice (n=40) were randomly divided into 4 groups consisting of the continuous inhalation Sachet group, Yu-Ping-Feng powder (YPF-P) gavage positive control group, discontinuous inhalation MFS group and untreated controls. After 4 weeks, mice were sacrificed and lungs harvested. The expression of toll-like receptors 2 and 4 (TLR2/4) gene and protein levels was assessed using real-time polymerase chain reaction (RT-PCR) and Western blot analyses, respectively. An additional 60 Kunming mice were randomly divided into 6 groups comprised of a blank control group, continuous MFS inhalation group, an immune-compromised continuous MFS inhalation group, an immuno-compromised group, an immune-compromised MFS discontinuous inhalation group and an immune-compromised positive control group. Immune suppression was induced by cold stress (4 °C/4 h daily for 3 days) and mice were treated with MFS or YPF-P before cold stress treatments. Immuno-compromised mice were treated with MFS continuously or intermittently, or treated with YPF-P. Blood samples were collected and examined for natural killer cells based on positive NKp46 staining. The isorhamnetin associated with MFS-induced immune modulation was obtained from 'wo ga le' which is considered to be a major component of MFS, and analyzed by HPLC. RESULTS: Mice continuously inhaling MFS showed a moderate increase in TLR2/4 mRNA and protein levels compared to mice in the control and discontinuous inhalation groups. MFS significantly increased the TLR2/4 expression in a dose-dependent manner. Furthermore, there was also a slightly significant increase in the number of NKp46(+) cells in the continuous inhalation group compared to controls and discontinuous inhalation group. Pretreatment with MFS partially prevented cold stress-induced inhibition of NKp46(+) cells. HPLC analysis of the 'wo ga le' associated with immune function identified the major component to be isorhamnetin. CONCLUSIONS: Taken together, these data suggested that MFS significantly enhanced TLR2/4 expression levels and the number of NKp46(+) cells in mice and moderately affected innate immune responses associated with protection against influenza, suggesting that isorhamnetin in the MFS enhanced innate immune potency. The use of MFS for the prevention of various respiratory tract infections can be attributed to its antimicrobial properties. In a pilot study, a large quantity (40 g) was administered over a prolonged period did not produce apparent toxicity.