ITRAQ-based quantitative proteomics reveals the proteome profiles of MDBK cells infected with bovine viral diarrhea virus.

BACKGROUND: Bovine viral diarrhea (BVD) which is caused by Bovine viral diarrhea virus (BVDV), is an acute, contagious disease. In spite of the use of vaccines and elimination projects, BVDV still causes severe economic losses to the cattle industry for the past few years. The current study presents a preliminary analysis of the pathogenic mechanisms from the perspective of protein expression levels in infected host cells at different points in time to elucidate the infection process associated with BVDV. METHODS: We used the isobaric tags for relative and absolute quantitation (iTRAQ) technology coupled with liquid chromatography-tandem mass spectrometric (LC-MS/MS) approach for a quantitative proteomics comparison of BVDV NADL-infected MDBK cells and non-infected cells. The functions of the proteins were deduced by functional annotation and their involvement in metabolic processes explored by KEGG pathway analysis to identify their interactions. RESULTS: There were 357 (47.6% downregulated, 52.4% upregulated infected vs. control), 101 (52.5% downregulated, 47.5% upregulated infected vs. control), and 66 (21.2% downregulated, 78.8% upregulated infected vs. control) proteins were differentially expressed (fold change > 1.5 or < 0.67) in the BVDV NADL-infected MDBK cells at 12, 24, and 48 h after infection. GO analysis showed that the differentially expressed proteins (DEPs) are mainly involved in metabolic processes, biological regulation and localization. KEGG enrichment analysis showed that some signaling pathways that involved in the regulation of BVDV NADL-infection and host resistance are significantly (P < 0.05) enriched at different stages of the BVDV NADL-infection, such as Endocytosis signaling pathway, FoxO signaling pathway, Homologous recombination signaling pathway and Lysosome pathway. CONCLUSIONS: These results revealed that the DEPs in BVDV NADL-infected MDBK cells have a wide range of regulatory effects; in addition, they provide a lot of resources for the study of host cell proteomics after BVDV infection.

Analysis of pituitary transcriptomics indicates that lncRNAs are involved in the regulation of sheep estrus.

Seasonal estrus is a key factor limiting animal fertility, and understanding the molecular mechanisms that regulate animal estrus is important for improving animal fertility. The pituitary gland, which is the most important endocrine gland in mammals, plays an important role in regulating the physiological processes such as growth, development, and reproduction of animals. Here, we used RNA-seq technology to study the expression profile of lncRNAs in the anterior pituitary of sheep during estrus and anestrus. In this study, we identified a total of 995 lncRNAs, of which 335 lncRNAs were differentially expressed in two states (including 38 up-regulated and 297 down-regulated lncRNAs). RT-qPCR verified the expression levels of several lncRNAs. Target predictive analysis revealed that these lncRNAs can act in cis or trans and regulate the expression of genes involved in the regulation of sheep estrus. Target gene enrichment analysis of differentially expressed lncRNAs indicates that these lncRNAs can regulate sheep estrus by regulating hormone metabolism and energy metabolism. Through our research, we provide the expression profile of lncRNAs in the pituitary of sheep, which provides a valuable resource for further understanding of the genetic regulation of seasonal estrus in sheep from the perspective of lncRNAs.

Design, synthesis and biological evaluation of novel triaryldimethylaminobutan-2-ol derivatives against Mycobacterium tuberculosis.

Bedaquiline (TMC207), a typical diarylquinoline anti-tuberculosis drug, has been approved by FDA to specifically treat MDR-TB. Herein we describe design, synthesis, and in vitro biological evaluation against Mycobacterium tuberculosis of a series of triaryldimethylaminobutan-2-ol derivatives obtaining from the structural modification of TMC207. Compounds 23, 25, 28, 32, 39 and 43 provided superior anti-mycobacterial activity than positive control PC01 which shows the same configuration and contains TMC207. Compounds 16, 20, 29, 34, 37, 45 and 47 exhibited the similar activity to positive control PC01. Most importantly, the series of compounds showed excellent activity against XDR-Mtb. The result of acute toxicity suggested that this class of triaryldimethylaminobutan-2-ol derivatives should be graded as low. Further SAR analysis indicates that a large steric bulk of triaryl and 7-Br, 3-OCH3 on 1-naphthyl are critical.

Asymmetric Synthesis and Absolute Configuration Assignment of a New Type of Bedaquiline Analogue.

Bedaquiline is the first FDA-approved new chemical entity to fight multidrug-resistant tuberculosis in the last forty years. Our group replaced the quinoline ring with a naphthalene ring, leading to a new type of triarylbutanol skeleton. An asymmetric synthetic route was established for our bedaquiline analogues, and the goal of assigning their absolute configurations was achieved by comparison of experimental and calculated electronic circular dichroism spectra, and was confirmed by the combined use of circular dichroism and NMR spectroscopy.

Synthesis and biological evaluation of substituted 4-(thiophen-2-ylmethyl)-2H-phthalazin-1-ones as potent PARP-1 inhibitors.

We have developed a series of substituted 4-(thiophen-2-ylmethyl)-2H-phthalazin-1-ones as potent PARP-1 inhibitors. Preliminary biological evaluation indicated that most compounds possessed inhibitory potencies comparable to, or higher than AZD-2281. Among these compounds, 18q appeared to be the most notable one, which displayed an 8-fold improvement in enzymatic activity compared to AZD-2281. These efforts lay the foundation for our further investigation.

An efficient synthesis of enantiopure (R)-heteroarylpyrimidine analogs.

An efficient synthesis of enantiopure (R)-heteroarylpyrimidine analogs is described here, which involves introduction of a chiral group, formation and separation of diasteroisomers and final transformation of an amide to an ester. The absolute configuration of the enantiopure HAPs is confirmed by X-ray analysis of their intermediates.

Topical application of FTY720 and cyclosporin A prolong corneal graft survival in mice.

PURPOSE: To investigate the effects of topical FTY720 and cyclosporin A (CsA) on allogeneic corneal transplantation in mice. METHODS: A total of 75 BALB/c mice received corneal grafts from C57BL/6 donors. Recipients were treated with 0.1%, 0.3%, or 0.5% FTY720 ophthalmic gel or 1% CsA eye-drops after the graft (controls received no treatment). The number of cluster of differentiation (CD)4+ T cells and CD4+CD25+forkhead box P3 (Foxp3)+ regulatory (Treg) cell phenotypes were measured by flow cytometry. Cytokine mRNA expression in corneal grafts was analyzed by real-time quantitative PCR. CD4 + T cells and cytokines in corneal samples were identified by immunohistochemical staining. RESULTS: Corneal graft survival was prolonged by treatment with topical 0.5% FTY720 (mean survival time [MST], 24.1±1.6 days) or 1% CsA eye-drops (MST 25.0±1.9 days) compared with controls (MST, 13.4±0.5 days; n=9, both p<0.01). Topical 0.5% FTY720 treatment significantly increased the percentages of CD4 + T (p<0.05) and Treg cells (p<0.01; n=5) in the cervical lymph nodes compared with controls. Transforming growth factor-ß1 (TGF-ß1) mRNA transcription in corneal grafts after topical 0.5% FTY720 increased (p<0.05, n=3), while interleukin-2 (IL-2) and interferon-γ (IFN-γ) mRNA expression in corneal grafts treated with 1% CsA decreased (p<0.01, p<0.05, respectively). These cytokine results were paralleled by similar immunohistochemical staining. Topical 0.5% FTY720 and 1% CsA treatment reduced the infiltration of CD4+ Tcells in the grafts. CONCLUSIONS: Topical 0.5% FTY720 and 1% CsA can effectively prolong allogeneic corneal graft survival in mice. Treatment with topical 0.5% FTY720 increases the percentage of CD4+ T cells and the percentage of Treg cells in cervical lymph nodes. The 0.5% FTY720 increased TGF-ß1 mRNA expression and decreases infiltration of CD4+ T cells in corneal grafts, while topical 1% CsA down-regulated the expression of IL-2 and IFN-γ.

A Functional Screening Identifies a New Organic Selenium Compound Targeting Cancer Stem Cells: Role of c-Myc Transcription Activity Inhibition in Liver Cancer.

Cancer stem cells (CSCs) are reported to play essential roles in chemoresistance and metastasis. Pathways regulating CSC self-renewal and proliferation, such as Hedgehog, Notch, Wnt/ß-catenin, TGF-ß, and Myc, may be potential therapeutic targets. Here, a functional screening from the focused library with 365 compounds is performed by a step-by-step strategy. Among these candidate molecules, phenyl-2-pyrimidinyl ketone 4-allyl-3-amino selenourea (CU27) is chosen for further identification because it proves to be the most effective compound over others on CSC inhibition. Through ingenuity pathway analysis, it is shown CU27 may inhibit CSC through a well-known stemness-related transcription factor c-Myc. Gene set enrichment analysis, dual-luciferase reporter assays, expression levels of typical c-Myc targets, molecular docking, surface plasmon resonance, immunoprecipitation, and chromatin immunoprecipitation are conducted. These results together suggest CU27 binds c-Myc bHLH/LZ domains, inhibits c-Myc-Max complex formation, and prevents its occupancy on target gene promoters. In mouse models, CU27 significantly sensitizes sorafenib-resistant tumor to sorafenib, reduces the primary tumor size, and inhibits CSC generation, showing a dramatic anti-metastasis potential. Taken together, CU27 exerts inhibitory effects on CSC and CSC-associated traits in hepatocellular carcinoma (HCC) via c-Myc transcription activity inhibition. CU27 may be a promising therapeutic to treat sorafenib-resistant HCC.

[The observation of the effects of a micromolecular compound J2 on rat corneal allograft using atomic force microscopy].

OBJECTIVE: To study the cell membrane of corneal endothelium with a micromolecular compound J2 in corneal allograft of rat using atomic force microscopy (AFM). METHODS: Cohort study. Subjects were divided into two groups: group A (n = 15): experimental group; group B (n = 15): placebo control group. At the fifth, tenth, fifteen, twentieth, twenty-fifth day after penetrating keratoplasty, the donor implant was separated from receipt bed, one part of which was stained by HE and the others fixed into AFM sample. Amplitude and height images were obtained in the tapping mode with a scan rate of 2 Hz and an integral gain of 0.3 to 0.5. Statistical analysis was performed using single-factor analysis of variance and P value was calculated. RESULTS: The average transplant survival time in group A was (33.12 ± 6.80) d, and those in group B was (18.87 ± 4.19) d. There were significant difference between two group (F = 47.7449, P = 0.00). There were obvious differences on ultrastructure measured by AFM between two groups. At the fifth day after penetrating keratoplasty, regular hexagonal structure of corneal endothelium was observed by AFM in both two group. The diameter of corneal endothelium was about 15 µm, uneven microstructure of cell could be found. The time being, different changes were arose in two group: a clear microstructure could be found in group A, however the microstructure of cell could not be recognized in group B. One way analysis of variance showed that significant differences on parameters (Ra, Rp and Rv) were found between two groups (P < 0.05). At the fifth day after penetrating keratoplasty, group A: Ra (97.64 ± 31.58) nm, Rp (297.79 ± 25.19) nm, Rv (545.55 ± 25.83) nm; group B: Ra (112.61 ± 34.29) nm, Rp (265.06 ± 24.17) nm, Rv (544.41 ± 21.78) nm (Fa = 30.9416, P = 0.0000; Fp = 263.6018, P = 0.0000; Pv = 1.2013, P = 0.2735). At the tenth day after penetrating keratoplasty, group A: Ra (102.98 ± 32.98) nm, Rp (711.38 ± 21.94) nm, Rv (639.89 ± 22.58) nm; group B: Ra (222.85 ± 31.28) nm, Rp (111.22 ± 20.35) nm, Rv (746.49 ± 23.17) nm (Fa = 2086.4535, P = 0.0000; Fp = 53768.4676, P = 0.0000; Pv = 3257.3178, P = 0.0000). At the fifteenth day after penetrating keratoplasty, group A: Ra (87.44 ± 34.97) nm, Rp (344.18 ± 21.09) nm, Rv (482.61 ± 22.27) nm; group B: Ra (197.64 ± 35.72) nm, Rp (510.76 ± 24.98) nm, Rv (545.62 ± 23.17) nm (Fa = 1458.1057, P = 0.0000; Fp = 7788.6963, P = 0.0000; Pv = 1153.2860, P = 0.0000). At the twentieth day after penetrating keratoplasty, group A: Ra (85.85 ± 32.53) nm, Rp (348.69 ± 21.26) nm, Rv (367.65 ± 23.12) nm; group B: Ra (201.36 ± 34.12) nm, Rp (788.58 ± 20.34) nm, Rv (563.33 ± 21.01) nm (Fa = 1801.1215, P = 0.0000; Fp = 67 057.9516, P = 0.0000; Fv = 11 770.2195, P = 0.0000). At the twenty-fifth day after penetrating keratoplasty, group A: Ra (104.97 ± 32.47) nm, Rp (395.05 ± 20.38) nm, Rv (396.17 ± 21.59) nm; group B: Ra (43.85 ± 31.28) nm, Rp (249.88 ± 20.79) nm, Rv (154.88 ± 22.37) nm (Fa = 551.4134, P = 0.0000; Fp = 7458.9255, P = 0.0000; Pv = 18 070.5189, P = 0.0000). CONCLUSIONS: The morphology and ultrastructure of corneal endothelium in group A with J2 were different from group B by observation with AFM. J2 was an effect micromolecular in prevention of corneal allograft rejection.

CRISPR-Cas13a-Based Detection for Bovine Viral Diarrhea Virus.

Bovine Viral Diarrhea Virus (BVDV) is the main pathogen of bovine viral diarrhea disease (BVD), which leads to enormous economic losses in the cattle industry. A sensitive and specific detection for BVDV is advantageous to the control of BVDV. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have been used for detecting virus RNA. In this study, the expression and purification of LwCas13a protein was optimized and the RNase activity of LwCas13a in vitro was verified. CRISPR-LwCas13a system could detect BVDV virus and BVDV RNA with high specificity and simplicity. The detection limit of the LwCas13a system was 103 pM, and there were no cross-reactions with HEK293T and MDBK. In summary, a sensitive, specific, and simple nucleic acid detection method based on CRISPR-Cas13a was developed for BVDV. This method provides a new detection strategy for early diagnosis of BVDV.

[On-line coupling of microcolumn electrophoresis and UV-Vis spectrophotometry].

In the present paper, a laboratory-made high-performance electrophoresis microcolumn unit was prepared for UV-Vis spectrophotometer. X-ray diffraction was used in the preparation of electrophoretic microcolumns. And an analytical technique of microcolumn electrophoresis coupled with UV-Vis spectrophotometry was introduced. Uniform quartz microncrystals were prepared by hydrothermal synthesis. Their crystalline phase and morphology were identified by X-ray diffraction and scanning electron microscope, respectively. The quartz microncrystals were packed into a 2-mm i. d. fused-silica tube to prepare the electrophoretic microcolumn. With 1.5 mmol x L(-1) disodium phosphate buffer solution (pH 11.5) containing 25% (phi) methanol and 10% (phi) acetonitrile, tryptophan, phenylalanine and tyrosine were on-line separated on line and detected by microcolumn electrophoresis coupled with UV-Vis spectrophotometry without derivatization. The limits of detection were 0.037, 0.20 and 0.20 micromol x L(-1), respectively. The separation efficiency of tryptophan was 4.5 x 10(4) plates/m. The sample capacity of the electrophoretic microcolumn achieved 35 microL. It was found that the electrophoretic microcolumn packed with quartz microncrystals was able to limit Joule heat, increase sample capacity and enhance detection sensitivity. The laboratory-made electrophoretic microcolumn could be a high-performance separation unit for conventional UV-Vis spectrophotometer. The on-line coupling of microcolumn electrophoresis and UV-Vis spectrophotometry could separate and determine samples with complicated matrices, reduce zone broadening and enhance separation efficiency, so expand the analytical function of spectrophotometer in the trace analysis of mixed components with overlapped spectra.

Identification and Profiling of Pituitary microRNAs of Sheep during Anestrus and Estrus Stages.

MicroRNAs (miRNAs) are a class of small non-coding RNAs, molecules of 21 to 25 nucleotides in length, that regulate gene expression by binding to their target mRNA and play a significant role in animal development. The expression and role of miRNAs in regulating sheep estrus, however, remain elusive. Transcriptome analysis is helpful to understand the biological roles of miRNAs in the pituitary gland of sheep. A sheep's pituitary gland has a significant difference between estrus and anestrus states. Here, we investigate the expression profiles of sheep anterior pituitary microRNAs (miRNAs) in two states, estrus and anestrus, using Illumina HiSeq-technology. This study identified a total of 199 miRNAs and 25 differentially expressed miRNAs in the estrus and anestrus pituitary gland in sheep. Reverse transcription quantitative-PCR (RT-qPCR) analysis shows six differentially (p < 0.05) expressed miRNAs, that are miR-143, miR-199a, miR-181a, miR-200a, miR-218, and miR-221 in both estrus and anestrus states. miRNAs containing estrus-related terms and pathways regulation are enriched using enrichment analysis from gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Moreover, we also envisioned a miRNA-mRNA interaction network to understand the function of miRNAs involved in the pituitary gland regulatory network. In conclusion, miRNA expression profiles in sheep pituitary gland in the anestrus and estrus deliver a theoretical basis for the study of pituitary gland biology in sheep.

1-Oxo-3-substitute-isothiochroman-4-carboxylic acid compounds: synthesis and biological activities of FAS inhibition.

A new series 1-oxo-3-substitute-isothiochroman-4-carboxylic acid compounds have been designed and synthesized. Screening of these molecules for FAS inhibition in vitro has indicated that compounds 2c and 2d showed more effective FAS inhibition activities and higher therapeutic index than C75.

Novel fatty acid synthase (FAS) inhibitors: design, synthesis, biological evaluation, and molecular docking studies.

Several novel series of C75 derivatives were synthesized and evaluated for their FAS inhibitory activities. The results showed compound 4-methylene-2-octyl-5-oxo-tetrahydro-thiophene-3-carboxylic acid (1c) had more effective FAS inhibitory (IC(50) was 2.56 microM and T.I. was 9.26) and potent anti-tumor activities on HL60 and Hela cells in vitro (IC(50) were 5.38 microM and 46.10 microM, respectively).

Determination of amino acids in tobacco samples by capillary electrophoresis/indirect absorbance detection with isolation of the electrolysis compartment and p-Aminobenzoic acid as a background electrolyte.

A fast, convenient and sensitive method of capillary zone electrophoresis (CZE) and indirect UV detection was proposed for the determination of 16 amino acids. p-Aminobenzoic acid (PAB) was selected as a background electrolyte (BGE). An isolated cell included a BGE buffer part and an electrode buffer one, which were jointed with a glass frit. The isolated cell can prevent PAB from the electrode reaction and improve the stability of the detection baseline. The separation conditions of amino acids were investigated, such as different BGEs, BGE concentration, buffer pH and electroosmotic flow (EOF) modifiers. Under the selected separation conditions, 14 amino acid peaks could be separated in 12 min. The detection limits of the amino acids were in the range of 1.7 - 4.5 micromol/L. The isolated cell is suitable for reagents reacting on the electrodes in capillary electrophoresis. The proposed method has been successfully applied to the determination of the amino acids in tobacco samples.

The sphingosine-1-phosphate receptor agonist FTY720 and its phosphorylated form affect the function of CD4+CD25+ T cells in vitro.

The sphingosine-1-phosphate receptor agonist FTY720 and FTY720-P have a wide variety of fundamental functions. Many studies have demonstrated that CD4+CD25+ regulatory T (Treg) cells engage in the maintenance of immunological self-tolerance by actively suppressing self-reactive lymphocytes. Although FTY720 has also recently shown to possess an additional effect that increases the functional activity of Treg cells, the mechanism leading to the enhanced Treg activity after FTY720 treatment is still not clear. We isolated Treg cells, which were co-cultured with FTY720 or FTY720-P. The proliferation of co-cultured Treg cells was detected by the cell counting kit-8. The changes of the phenotype CD25+ and forkhead box P3 (Foxp3)+ of co-cultured Treg cells were measured by flow cytometry. The levels of IL-10 and TGF-ß1 in the supernatants were detected by Elisa. Cytokine mRNA expressions in co-cultured Treg cells were analyzed by real-time quantitative PCR. Mixed lymphocyte reaction assay examined the suppressive function. We found that neither FTY720 nor FTY720-P affected the proliferation of co-cultured Treg cells. The percentages of CD25+ and Foxp3+ were enhanced in the high-dose FTY720-P group. The levels of TGF-ß1 in the supernatants were enhanced in the high-dose FTY720 group. Medium and high-dose FTY720-P also enhanced the levels of TGF-ß1. TGF-ß1 and Foxp3 mRNA expression were upregulated in the high-dose FTY720-P group. The proliferation of effector T (Teff) cells was suppressed significantly in the medium and high-dose FTY720-P group at a Treg/Teff cell ratio of 1:1. At a ratio of 1:1, the proliferation of Teff cells was also suppressed in the high-dose FTY720 group. It can be concluded that high-dose FTY720-P can enhance the immune function of co-cultured Treg cells, and that medium-dose FTY720-P and high-dose FTY720 could partly enhance the function. The reason may be attributed to enhanced levels of TGF-ß1 and Foxp3.

Electrophoretic separation with 2-mm inner diameter fused-silica microcolumn packed with quartz microncrystals and its application.

The feasibility of a microcolumn electrophoresis technique was investigated with a 100mm length, 2mm I.D. fused-silica microcolumn packed with uniform quartz microncrystals prepared by hydrothermal synthesis. To evaluate the separation technique, tryptophan, phenylalanine and tyrosine were primarily separated by the microcolumn electrophoresis and detected at 216 nm without derivatization by an ordinary spectrophotometer. The separation conditions of the amino acids were optimized. With 1.5 mmol/L disodium phosphate buffer solution (pH 11.5) containing 25% (v/v) methanol and 10% (v/v) acetonitrile, the three amino acids were separated and the separation efficiency of tryptophan was 4.5x10(4)plates/m. The limits of detection were 0.035, 0.22 and 0.20 micromol/L, respectively. The sample capacity of the electrophoretic microcolumn achieved 35 microL. The proposed method was used to determine these amino acids in compound amino acid injection samples without derivatization. For the simplicity and portability of the microcolumn electrophoresis, it is studied as one of the high-performance separation techniques for an in situ and real-time electrokinetic flow analysis system. For its high detection sensitivity and large sample capacity, it can be developed for preparative electrophoresis.

Determination of polyphenol components in herbal medicines by micellar electrokinetic capillary chromatography with Tween 20.

A simple and convenient method of micellar electrokinetic capillary chromatography (MEKC) using polyoxyethylene sorbitan monolaurate (Tween 20) to form single micelle and methanol as a buffer additive was introduced for the simultaneous determination of five polyphenols, including scopoletin, rutin, esculetin, chlorogenic acid and caffeic acid. A running buffer solution of pH 9.3, 20 mmol/L sodium tetraborate containing 64 mmol/L Tween 20 and 9% (v/v) methanol was adopted in the separation. Because rutin and esculetin were difficult to be separated by capillary zone electrophoresis (CZE) and SDS-based MEKC, Tween 20-based MEKC was adopted and the polyphenols were separated satisfactorily. The proposed method was used to determine the polyphenol components in the herbal medicine of Cortex fraxini. The separation mechanism of Tween 20-based MEKC for the polyphenols was discussed preliminarily.

[Determination of amino acids in tea samples by capillary electrophoresis with partition cell and indirect ultraviolet detection].

An improved capillary electrophoretic (CE) separation and indirect ultraviolet (in-UV) detection system was proposed for the amino acid analysis in tea samples with a home-made partition cell and a background electrolyte (BGE) of p-aminobenzoic acid (PAB). PAB improved the separation efficiency and detection limits of the amino acids. The partition cell prevented PAB from chemical reaction at the electrode, reduced baseline noise and kept electric current inside the cell. The separation parameters of the amino acids, such as different BG-Es, BGE concentration, buffer pH and electroosmotic flow (EOF) modifiers, were investigated. The CE separation was carried out with the running buffer solution of pH 11. 2, 10 mmol/L PAB containing 0. 014 mmol/L cetyltrimethylamonium bromide (CTAB), an applied voltage of - 15 kV and a detection wavelength of 254 nm. Sixteen amino acids were separated within 14 min under the selected conditions. The linear ranges of the amino acids were 0. 02 - 0. 60 mmol/L except for theanine (0. 02 - 3. 80 mmol/L) and gamma-aminobutyric acid (0. 02 - 2. 00 mmol/L). The recoveries were in the range from 83. 0% to 106%. The relative standard deviations of peak area were less than 5% (n = 5) and the detection limits were in the range of 1. 7 -4. 5 gammamol/L. The method is fast, convenient and sensitive, and has been applied to the determination of 11 amino acids in tea samples satisfactorily.

cDNA microarray analysis of the expression profiles of Trichophyton rubrum in response to novel synthetic fatty acid synthase inhibitor PHS11A.

Trichophyton rubrum (T. rubrum) is a major pathogen responsible for dermatophytosis. Because of potential relapse of disease with current antifungal therapy protocols, there is a need for additional and/or alternative antifungal agents for the treatment of disease caused by T. rubrum. We synthesized a potent fungal fatty acid synthase inhibitor, PHS11A, based on the structure of fungal fatty acid synthase. The antifungal activities of PHS11A were tested against 38 clinical isolates of T. rubrum and compared with those of ketoconazole and terbinafine, the MIC(50) and MIC(90) of PHS11A on the isolates were 2 and 4 microg/ml, respectively. We evaluated the transcriptional response of T. rubrum hyphae exposed to PHS11A using 11,232-spot cDNA microarrays. PHS11A exposure increased transcription of fatty acid synthases (FASs) genes FAS1 and FAS2. PHS11A also affected transcription of some genes involved in lipid metabolism, cAMP and MAPK pathways, and multidrug resistance. Quantitative real-time PCR was performed for selected genes to verify the microarray results.