Semisynthetic derivatives of the fungal metabolite eupenifeldin via targeting the tropolone hydroxy groups.

Eupenifeldin (1) is a fungal secondary metabolite possessing bis-tropolone moieties that demonstrates nanomolar cytotoxic activity against a number of cancer cell types. As a potential anticancer lead, this meroterpenoid was used to access 29 semisynthetic analogues via functionalization of the reactive hydroxy groups of the bis-tropolones. A series of ester (2-6), carbonate (7-8), sulfonate (9-16), carbamate (17-20), and ether (21-30) analogues of 1 were generated via 22 reactions. Most of these compounds were disubstituted, produced via functionalization of both of the tropolonic hydroxy moieties, although three mono-functionalized analogues (6, 8, and 24) and one tri-functionalized analogue (3) were also obtained. The cytotoxic activities of 1-30 were evaluated against human melanoma and ovarian cancer cell lines (i.e., MDA-MB-435 and OVCAR3, respectively). Ester and carbonate analogues of 1 (i.e., 2-8) maintained cytotoxicity at the nanomolar level, and the greatest improvement in aqueous solubility came from the monosuccinate analogue (6), which was acylated on the secondary hydroxy at the 11 position.

Pro197Ser and the new Trp574Leu mutations together with enhanced metabolism contribute to cross-resistance to ALS inhibiting herbicides in Sinapis alba.

White mustard, (Sinapis alba), a problematic broadleaf weed in many Mediterranean countries in arable fields has been detected as resistant to tribenuron-methyl in Tunisia. Greenhouse and laboratory studies were conducted to characterize Target-Site Resistance (TSR) and the Non-Target Site Resistance (NTSR) mechanisms in two suspected white mustard biotypes. Herbicide dose-response experiments confirmed that the two S. alba biotypes were resistant to four dissimilar acetolactate synthase (ALS)-pinhibiting herbicide chemistries indicating the presence of cross-resistance mechanisms. The highest resistance factor (>144) was attributed to tribenuron-methyl herbicide and both R populations survived up to 64-fold the recommended field dose (18.7 g ai ha-1). In this study, the metabolism experiments with malathion (a cytochrome P450 inhibitor) showed that malathion reduced resistance to tribenuron-methyl and imazamox in both populations, indicating that P450 may be involved in the resistance. Sequence analysis of the ALS gene detected target site mutations in the two R biotypes, with amino acid substitutions Trp574Leu, the first report for the species, and Pro197Ser. Molecular docking analysis showed that ALSPro197Ser enzyme cannot properly bind to tribenuron-methyl's aromatic ring due to a reduction in the number of hydrogen bonds, while imazamox can still bind. However, Trp574Leu can weaken the binding affinity between the mutated ALS enzyme and both herbicides with the loss of crucial interactions. This investigation provides substantial evidence for the risk of evolving multiple resistance in S. alba to auxin herbicides while deciphering the TSR and NTSR mechanisms conferring cross resistance to ALS inhibitors.

Inhibition of suplatast tosilate on EPSC in the single paratracheal ganglion neurons attached with presynaptic boutons.

Using single neurons of rat paratracheal ganglia (PTG) attached with presynaptic boutons, the effects of suplatast tosilate on excitatory postsynaptic currents (EPSCs) were investigated with nystatin-perforated patch-clamp recording technique. We found that suplatast concentration dependently inhibited the EPSC amplitude and its frequency in single PTG neurons attached with presynaptic boutons. EPSC frequency was higher sensitive to suplatast than EPSC amplitude. IC50 for EPSC frequency was 1.1 × 10-5 M, being similar to that for the effect on histamine release from mast cells and lower than that for the inhibitory effect on cytokine production. Suplatast also inhibited the EPSCs potentiated by bradykinin (BK), but it did not affect the potentiation itself by BK. Thus suplatast inhibited the EPSC of PTG neurons attached with presynaptic boutons at both the presynaptic and postsynaptic sites.NEW & NOTEWORTHY In this study, using single neurons of rat paratracheal ganglia (PTG) attached with presynaptic boutons, the effects of suplatast tosilate on excitatory postsynaptic currents (EPSCs) were investigated with patch-clamp recording technique. We found that suplatast concentration dependently inhibited the EPSC amplitude and its frequency in single PTG neurons attached with presynaptic boutons. Thus suplatast inhibited the function of PTG neurons at both of presynaptic and postsynaptic sites.

Comparative proteomic and physiological analyses reveal tribenuron-methyl phytotoxicity and nontarget-site resistance mechanisms in Brassica napus.

The application of herbicides is the most effective strategy for weed control and the development of herbicide-resistant crops will facilitate the weed management. The acetolactate synthase-inhibiting herbicide, tribenuron-methyl (TBM), is broadly used for weed control. However, its application in rapeseed field is restricted since rapeseed is sensitive to TBM. Herein, an integrated study of cytological, physiological and proteomic analysis of the TBM-resistant rapeseed mutant M342 and its wild-type (WT) plants was conducted. After TBM spraying, M342 showed improved tolerance to TBM, and proteins implicated in non-target-site resistance (NTSR) to herbicides had a significantly higher level in M342 as compared with the WT. Differentially accumulated proteins (DAPs) between these two genotypes were enriched in glutathione metabolism and oxidoreduction coenzyme metabolic process, which protected the mutant from oxidative stress triggered by TBM. Important DAPs related to stress or defence response were up-accumulated in M342 regardless of the TBM treatment, which might serve as the constitutive part of NTSR to TBM. These results provide new clues for further exploration of the NTSR mechanism in plants and establish a theoretical basis for the development of herbicide-resistant crops.

Computer-Aided Design and Synthesis of (Functionalized quinazoline)-(α-substituted coumarin)-arylsulfonate Conjugates against Chikungunya Virus.

Chikungunya virus (CHIKV) has repeatedly spread via the bite of an infected mosquito and affected more than 100 countries. The disease poses threats to public health and the economy in the infected locations. Many efforts have been devoted to identifying compounds that could inhibit CHIKV. Unfortunately, successful clinical candidates have not been found yet. Computations through the simulating recognition process were performed on complexation of the nsP3 protein of CHIKV with the structures of triply conjugated drug lead candidates. The outcomes provided the aid on rational design of functionalized quinazoline-(α-substituted coumarin)-arylsulfonate compounds to inhibit CHIKV in Vero cells. The molecular docking studies showed a void space around the ß carbon atom of coumarin when a substituent was attached at the α position. The formed vacancy offered a good chance for a Michael addition to take place owing to steric and electronic effects. The best conjugate containing a quinazolinone moiety exhibited potency with EC50 = 6.46 µM, low toxicity with CC50 = 59.7 µM, and the selective index (SI) = 9.24. Furthermore, the corresponding 4-anilinoquinazoline derivative improved the anti-CHIKV potency to EC50 = 3.84 µM, CC50 = 72.3 µM, and SI = 18.8. The conjugate with 4-anilinoquinazoline exhibited stronger binding affinity towards the macro domain than that with quinazolinone via hydrophobic and hydrogen bond interactions.

Discovery of pyrazole N-aryl sulfonate: A novel and highly potent cyclooxygenase-2 (COX-2) selective inhibitors.

Based on a new pyrazole sulfonate synthetic method, a novel class of molecules with a basic structure of pyrazole N-aryl sulfonate have been designed and synthesized. The interest in conducting intensive research stems from quite evident anti-inflammatory effects exhibited by the compounds in preliminary animal experiments. A series of compounds were synthesized by different substitutions of the R1, R2, and R3 groups. Within the series, 4-iodophenyl 5-methyl-3-(p-tolyl)-1H-pyrazole-1-sulfonate and phenyl 5-methyl-3-(4-(trifluoromethyl) phenyl)-1H-pyrazole-1-sulfonate exhibited excellent anti-inflammatory activity (% inhibition of auricular edemas = 27.0 and 35.9, respectively); the in vivo analgesic activity of phenyl 5-methyl-3-(p-tolyl)-1H-pyrazole-1-sulfonate and 2-chlorophenyl 5-methyl-3-(p-tolyl)-1H-pyrazole-1-sulfonate was confirmed to be effective (inhibition ratio of writhing = 50.7% and 48.5% separately), and compounds phenyl 5-methyl-3-(p-tolyl)-1H-pyrazole-1-sulfonate , 4-iodophenyl 5-methyl-3-(p-tolyl)-1H-pyrazole-1-sulfonate and 2-chlorophenyl 5-methyl-3-(p-tolyl)-1H-pyrazole-1-sulfonate were identified as selective COX-2 inhibitors (SI = 455, 10,497 and >189 severally). In Acute Oral Toxicity assays conducted in vivo, the lethal dose 50 (LD50) of 4-iodophenyl 5-methyl-3-(p-tolyl)-1H-pyrazole-1-sulfonate and 2-chlorophenyl 5-methyl-3-(p-tolyl)-1H-pyrazole-1-sulfonate to mice was >2000 mg/kg BW.

Biological effects of the free and embedded metribuzin and tribenuron-methyl herbicides on various cultivated weed species.

The present study addresses the herbicidal activity and biological effects of the metribuzin (MET) and tribenuron-methyl (TBM) herbicides used to control various weed species (Amaranthus retroflexus, Sinapis arvensis, and Leucanthemum maximum). The effects of the free herbicides and the herbicides embedded in granules of degradable polymer poly-3-hydroxybutyrate [P(3HB)] blended with birch wood flour were compared. Metribuzin, regardless of the form, caused 100% mortality of the three weeds by day 21. The herbicidal activity of tribenuron-methyl was lower than that of metribuzin, but the embedded TBM was superior to the free herbicide in the length and strength of its action on the weeds. Both metribuzin forms dramatically decreased the main parameters of fluorescence: maximum quantum yield of photosystem-II [Y(II)max], maximum quantum yield of non-photochemical quenching [Y(NPQ)max], and maximum rate of non-cyclic electron transport [ETRmax] and concentrations of chlorophyll a and b. The effect of the embedded TBM on the photosynthetic activity of the weeds was lower in the first two weeks of the growth of herbicide-treated plants but lasted longer than the effect of the free TBM and increased over time. Embedding of metribuzin in the matrix of degradable blend did not decrease its herbicidal activity.

How exposure to ALS-inhibiting gametocide tribenuron-methyl induces male sterility in rapeseed.

BACKGROUND: Acetolactate synthase (ALS)-inhibiting herbicide tribenuron-methyl (TBM) is an efficient gametocide that can cause rapeseed (Brassica napus L.) to become male sterile and outcrossing. To find the reason the TBM treatment leads to male sterility, an integrated study using cytological, physiological, and transcriptomic methods was conducted. RESULTS: Some temporary symptoms, including the discoloration of young leaves and a short halt of raceme elongation, were observed in the rapeseed plants exposed to TBM at an application rate of 1 µg per plant. Both chloroplasts in young leaves and plastids in anthers were deformed. TBM also reduced the leaf photosynthetic rate and the contents of chlorophyll, soluble sugar and pyruvate. Both the tapetal cells and uni-nucleate microspores in the treated plants showed large autophagic vacuoles, and the tissue degenerated quickly. A transcriptomic comparison with the control identified 200 upregulated and 163 downregulated differential expression genes in the small flower buds of the TBM treatment. The genes encoding functionally important proteins, including glucan endo-1,3-beta-glucosidase A6, QUARTET3 (QRT3), ARABIDOPSIS ANTHER 7 (ATA7), non-specific lipid-transfer protein LTP11 and LTP12, histone-lysine N-methyltransferase ATXR6, spermidine coumaroyl-CoA acyltransferase (SCT), and photosystem II reaction centre protein psbB, were downregulated by TBM exposure. Some important genes encoding autophagy-related protein ATG8a and metabolic detoxification related proteins, including DTX1, DTX6, DTX35, cytosolic sulfotransferase SOT12, and six members of glutathione S-transferase, were upregulated. In addition, several genes related to hormone stimulus, such as 1-aminocyclopropane-1-carboxylate synthase 8 (ACS8), ethylene-responsive factor ERF1A, ERF1, ERF71, CRF6, and RAP2-3, were also upregulated. The transcriptional regulation is in accordance with the functional abnormalities of pollen wall formation, lipid metabolism, chloroplast structure, ethylene generation, cell cycle, and tissue autophagy. CONCLUSION: The results suggested that except for ALS, the metabolic pathways related to lipid metabolism, pollen exine formation, photosynthesis and hormone response are associated with male sterility induced by TBM. The results provide new insight into the molecular mechanisms of inducing male sterility by sulfonylurea.

PCR-based identification of point mutation mediating acetolactate synthase-inhibiting herbicide resistance in weed wild mustard (Sinapis arvensis).

Acetolactate synthase (ALS)-inhibiting herbicides have been widely used for effective management and control of wild mustard (Sinapis arvensis) biotypes in Iran. The resistance of the ALS inhibitor to weeds is attributed to either target site alteration or enhanced herbicide degradation. Molecular and genetic characterization of the resistance mechanism is relevant to the evolution and management of herbicide resistance. The aims of this research were (a) to characterize the mechanism molecular suspected to Granstar (tribenuron methyl) and Atlantis (Mesosulfuron + Iodosulfuron) resistance in S. arvensis biotypes in the greenhouse and laboratory (b) to investigate the organization of the target-site loci in field selected S. arvensis populations and (c) instantly recognize the mutations that cause resistance to ALS inhibitors. Eighty resistant populations of S. arvensis were carefully collected from fields repeatedly treated with Granstar and Atlantis. The resistance level and pattern of the population were determined through a greenhouse dose-response experiment by applying the above-mentioned herbicides. Extraction of genomic DNA was carried out for PCR and ALS gene analysis. Our results showed that by greenhouse experiment across 80 biotypes suspected to resistance collected in the fields of whole Kermanshah Province, 30 biotypes (37.5%) conferred S. arvensis resistance species reported in the farm. Among 30 biotypes screened in a greenhouse experiment, six biotypes (20%), No. 9, 14, 17, 19, 23 and 28 revealed a mutation in the ALS gene that was detected by PCR-based method. Biotype No. 9 in the position 376 (Asp376-Gly, GAC to GGC), biotypes 14 and 19 in the position 197 (Pro197-Ala, CCT to GCT), biotypes 17, 23 and 28 in the position 574 (Trp574-Leu, TGG to TTG) and biotype No. 23 in the position 122 (Thr-122-Ala, ACA to GCA) showed herbicide resistance. The specific mutation in the position of 122 of the ALS gene in S. arvensis is the first report. Other biotypes showed resistance in the greenhouse but didn't indicate any mutation by PCR-based method. Most of the resistance to Granstar and Atlantis are genetic and are induced by mutations in the ALS gene. The resistance to herbicides may contain a non-mutagenic and non-genetic origin. The reason of herbicide resistance as non-target-site in some of the biotypes may relate to the activity of the herbicide-metabolizing enzyme(s) or transporter proteins that will naturally lead to an increase in herbicide degradation or compartmentation away from its active site.

Th2 and Th17 Induce Dry Skin in a Mouse Model of Arthritis.

Skin dryness is a characteristic of rheumatoid arthritis (RA) model mice. However, the mechanism underlying the induction of dry skin by RA is unclear. We hypothesized that T helper (Th)2 and Th17 cells mediate this process. A mouse model of DBA/1JJmsSlc collagen-induced arthritis was treated with Th2 or Th17 cell inhibitor, and transepidermal water loss (TEWL) and the expression of markers associated with allergic reaction and inflammation were evaluated. TEWL and plasma levels of thymic stromal lymphopoietin, interleukin (IL)-6 and -17, and tumor necrosis factor (TNF)-α were increased in the arthritis mouse model compared to that in control mice. Administration of Th2 cell inhibitor abolished the increase in TEWL, IL-6, and TNF-α levels, whereas Th17 cell inhibitor reversed TEWL and decreased IL-17 level. Th2 and Th17 cells contribute to the induction of dry skin, but via distinct mechanisms.

Target-site and non-target-site-based resistance to tribenuron-methyl in multiply-resistant Myosoton aquaticum L.

Myosoton aquaticum L., a widespread and competitive winter weed of wheat in China, has evolved resistance to many classes of herbicides. In one M. aquaticum population (AH03), collected from Anhui Province, where tribenuron-methyl and florasulam had been used to control this weed resistance to both herbicides had evolved. Compared with the sensitive population, HN03(S), the resistant (R) population, AH03, was highly resistant to tribenuron-methyl, flucarbazone-Na and pyroxsulam, moderately resistant to pyrithiobac­sodium, and florasulam, and had low resistance to diflufenican. AH03 was still controlled by imazethapyr, 2,4-D butylate, fluroxypyr-meptyl, and isoproturon. Pretreatment with the P450 inhibitor malathion reduced the GR50 value of tribenuron-methyl by 43% in the R population, and by 25% in the S population. This indicates that P450-mediated enhanced metabolism is one likely mechanism for tribenuron-methyl resistance in M. aquaticum. Glutathione-S-transferase (GST) activity could be induced by tribenuron-methyl in both the R and S populations. However, both the basal and induced GST activity of the R population was lower than that of the S population. The in vitro ALS assay confirmed that the ALS from the R plants showed a high resistance (52.93-fold) to tribenuron-methyl. ALS gene sequencing revealed a Pro197Ala substitution in the R plants. Based on the ALS gene sequence analysis, molecular markers were also developed to identify the specific Pro197Ala mutation. This population of M. aquaticum has multiple resistance and target-site (ALS Pro197Ala) and non-target-site resistance mechanisms contribute to tribenuron-methyl resistance.

Resistance evolution and mechanisms to ALS-inhibiting herbicides in Capsella bursa-pastoris populations from China.

Capsella bursa-pastoris is a serious broadleaf weed in winter wheat fields in China. It has evolved high levels of resistance to acetolactate synthase (ALS) inhibiting herbicides and has caused substantial losses of wheat yield in recent years. We monitored the herbicide resistance of Capsella bursa-pastoris collected from 18 regions of Shandong Province in 2009, 2013 and 2017, respectively. Compared with the 2009 populations, the number of populations resistant to florasulam had increased in 2013 and 2017. Resistance to tribenuron-methyl increased in 2013, but decreased in 2017. The 2009 and 2013 populations developed resistance only to tribenuron-methyl, but some 2017 populations developed cross-resistance to imazethapyr and florasulam as well. Mutations in ALS (Pro-197-Thr/Ser/His/Arg/Leu/Gln) were identified in the 2009 and 2013 populations; however, two ALS mutations (Pro197 and/or Trp574) were identified in 2017 plants. Meanwhile, plants containing both point mutations (Pro197 + Trp574) were identified in the 2017 populations. This study demonstrated that target site gene mutations were the main reason for Capsella bursa-pastoris resistance to ALS-inhibiting herbicides. Although target-site mutation is the reason for resistance to ALS-inhibiting herbicides in Capsella bursa-pastoris, the resistance patterns and mutations identified have changed over time.

Non-target site-based resistance to tribenuron-methyl and essential involved genes in Myosoton aquaticum (L.).

BACKGROUND: Water chickweed (Myosoton aquaticum (L.)) is a dicot broadleaf weed that is widespread in winter fields in China, and has evolved serious resistance to acetolactate synthase (ALS) inhibiting herbicides. RESULTS: We identified a M. aquaticum population exhibiting moderate (6.15-fold) resistance to tribenuron-methyl (TM). Target-site ALS gene sequencing revealed no known resistance mutations in these plants, and the in vitro ALS activity assays showed no differences in enzyme sensitivity between susceptible and resistant populations; however, resistance was reversed by pretreatment with the cytochrome P450 (CYP) monooxygenase inhibitor malathion. An RNA sequencing transcriptome analysis was performed to identify candidate genes involved in metabolic resistance, and the unigenes obtained by de novo transcriptome assembly were annotated across seven databases. In total, 34 differentially expressed genes selected by digital gene expression analysis were validated by quantitative real-time (qRT)-PCR. Ten consistently overexpressed contigs, including four for CYP, four for ATP-binding cassette (ABC) transporter, and two for peroxidase were further validated by qRT-PCR using additional plants from resistant and susceptible populations. Three CYP genes (with homology to CYP734A1, CYP76C1, and CYP86B1) and one ABC transporter gene (with homology to ABCC10) were highly expressed in all resistant plants. CONCLUSION: The mechanism of TM resistance in M. aquaticum is controlled by NTSR rather than TSR. Four genes, CYP734A1, CYP76C1, CYP86B1, and ABCC10 could play essential role in metabolic resistance to TM and justify further functional studies. To our knowledge, this is the first large-scale transcriptome analysis of genes associated with NTSR in M. aquaticum using the Illumina platform. Our data provide resource for M. aquaticum biology, and will facilitate the study of herbicide resistance mechanism at the molecular level in this species as well as in other weeds.

Direct Potentiation of Capsaicin Current by Histamine and Its Effect by Suplatast on Rat Trigeminal Ganglia Neurons.

In this study, we investigated the effect of histamine on capsaicin-induced current and its influence by suplatast in rat trigeminal ganglia neurons using a patch-clamp technique. We found that histamine directly potentiated capsaicin-induced currents in rat sensory neurons, and suplatast had little effect on this potentiation. Since it has been known that suplatast suppresses histamine release from mast cells, it is possible that suplatast inhibits the activation of nociceptive fibers in the pathological condition via prevention of histamine-induced potentiation of the transient receptor potential vanilloid 1 receptor-mediated currents.

A sialosyl sulfonate as a potent inhibitor of influenza virus replication.

A new direction for influenza virus sialidase inhibitor development was identified using a sulfonate congener of 2-deoxy-2-ß-H N-acetylneuraminic acid. Sialosyl sulfonates can be synthesised efficiently in four steps from N-acetylneuraminic acid via a microwave assisted decarboxylation. The presence of the sulfonate group significantly increases inhibition of influenza virus sialidase and viral infection when compared to the carboxylate congener, and also to the benchmark sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, Neu5Ac2en.

Trp574 substitution in the acetolactate synthase of Sinapis arvensis confers cross-resistance to tribenuron and imazamox.

Rate-response experiments with nine putative resistant wild mustard (Sinapis arvensis) populations from Greece showed cross-resistance to tribenuron and imazamox. The calculated GR50 values [herbicide rate (gaiha-1) required for 50% reduction of fresh weight] of the nine resistant (R) populations ranged from 51.8 to 555.6gaitribenuronha-1 and from 66.3 to 900.4gaiimazamoxha-1. Regarding the susceptible population, GR50 value was not estimated for tribenuron as its lower treatment reduced fresh weight by >95%, whereas the respective value for imazamox was 0.5gaiha-1. Gene sequencing of als revealed that a point mutation at Trp574 position, leading to amino acid substitution by Leu in the ALS enzyme was present and the likely cause of resistance. The in vitro activity of the ALS enzyme indicated I50 values (herbicide concentration required for 50% reduction of the ALS activity) ranging from 19.11 to 217.45µM for tribenuron, whereas the respective value for the S population was 1.17µM. All populations were susceptible to MCPA at the recommended rate. These results strongly support that cross-resistance of 9 S. arvensis populations was due a point mutation of the als gene, which resulted in a less sensitive ALS enzyme.

Herbicidal activity of slow-release herbicide formulations in wheat stands infested by weeds.

The present study reports the herbicidal activity of metribuzin and tribenuron-methyl embedded in the degradable matrix of natural poly-3-hydroxybutyrate [P(3HB)/MET and P(3HB)/TBM]. The developed formulations were constructed as films and microgranules, which were tested against the weeds such as white sweet clover Melilotus albus and lamb's quarters Chenopodium album in the presence of soft spring wheat (Triticum aestivum, cv. Altaiskaya 70) as the subject crop for investigation. The activity was measured in laboratory scale experiments by determining the density and weight of the vegetative organs of weeds. The study was also aimed at testing the effect of the experimental formulation on the growth of wheat crop as dependent on the method of herbicide delivery. The experimental MET and TBM formulations showed pronounced herbicidal activity against the weed species used in the study. The effectiveness of the experimental formulations in inhibiting weed growth was comparable to and, sometimes, higher than that of the commercial formulations (positive control). The amount of the biomass of the wheat treated with the experimental herbicide formulations was significantly greater than that of the wheat treated with commercial formulations.

Target-site and non-target-site based resistance to the herbicide tribenuron-methyl in flixweed (Descurainia sophia L.).

BACKGROUND: Flixweed (Descurainia sophia L.) is a troublesome and widespread broadleaf weed in winter fields in China, and has evolved high level resistance to acetolactate synthase (ALS)-inhibiting sulfonylurea herbicide tribenuron-methyl. RESULTS: We identified a resistant flixweed population (N11) exhibiting 116.3-fold resistance to tribenuron-methyl relative to the susceptible population (SD8). Target-site ALS gene mutation Pro-197-Thr was identified in resistant plants. Moreover, the resistance can be reversed to 28.7-fold by the cytochrome P450 inhibitor malathion. The RNA-Sequencing was employed to identify candidate genes involved in non-target-site metabolic resistance in this population. Total 26 differentially expressed contigs were identified and eight of them (four P450s, one ABC transporter, three glycosyltransferase) verified by qRT-PCR. Consistent over-expression of the two contigs homology to CYP96A13 and ABCC1 transporter, respectively, were further qRT-PCR validated using additional plants from the resistant and susceptible populations. CONCLUSIONS: Tribenuron-methyl resistance in flixweed is controlled by target-site ALS mutation and non-target-site based mechanisms. Two genes, CYP96A13 and ABCC1 transporter, could play an important role in metabolic resistance to tribenuron-methyl in the resistant flixweed population and justify further functional studies.

Suplatast tosilate alleviates nasal symptoms through the suppression of nuclear factor of activated T-cells-mediated IL-9 gene expression in toluene-2,4-diisocyanate-sensitized rats.

Histamine H1 receptor (H1R) gene is upregulated in patients with pollinosis; its expression level is highly correlated with the nasal symptom severity. Antihistamines are widely used as allergy treatments because they inhibit histamine signaling by blocking H1R or suppressing H1R signaling as inverse agonists. However, long-term treatment with antihistamines does not completely resolve toluene-2,4-diisocyanate (TDI)-induced nasal symptoms, although it can decrease H1R gene expression to the basal level, suggesting additional signaling is responsible for the pathogenesis of the allergic symptoms. Here, we show that treatment with suplatast tosilate in combination with antihistamines markedly alleviates nasal symptoms in TDI-sensitized rats. Suplatast suppressed TDI-induced upregulation of IL-9 gene expression. Suplatast also suppressed ionomycin/phorbol-12-myristate-13-acetate-induced upregulation of IL-2 gene expression in Jurkat cells, in which calcineurin (CN)/nuclear factor of activated T-cells (NFAT) signaling is known to be involved. Immunoblot analysis demonstrated that suplatast inhibited binding of NFAT to DNA. Furthermore, suplatast suppressed ionomycin-induced IL-9 mRNA upregulation in RBL-2H3 cells, in which CN/NFAT signaling is also involved. These data suggest that suplatast suppressed NFAT-mediated IL-9 gene expression in TDI-sensitized rats and this might be the underlying mechanism of the therapeutic effects of combined therapy of suplatast with antihistamine.

Simultaneous inhibition of T helper 2 and T regulatory cell differentiation by small molecules enhances Bacillus Calmette-Guerin vaccine efficacy against tuberculosis.

Tuberculosis affects nine million individuals and kills almost two million people every year. The only vaccine available, Bacillus Calmette-Guerin (BCG), has been used since its inception in 1921. Although BCG induces host-protective T helper 1 (Th1) cell immune responses, which play a central role in host protection, its efficacy is unsatisfactory, suggesting that additional methods to enhance protective immune responses are needed. Recently we have shown that simultaneous inhibition of Th2 cells and Tregs by using the pharmacological inhibitors suplatast tosylate and D4476, respectively, dramatically enhances Mycobacterium tuberculosis clearance and induces superior Th1 responses. Here we show that treatment with these two drugs during BCG vaccination dramatically improves vaccine efficacy. Furthermore, we demonstrate that these drugs induce a shift in the development of T cell memory, favoring central memory T (Tcm) cell responses over effector memory T (Tem) cell responses. Collectively, our findings provide evidence that simultaneous inhibition of Th2 cells and Tregs during BCG vaccination promotes vaccine efficacy.