The effects of Fusilade (Fluazifop-<Emphasis Type="Italic">p</Emphasis>-butyl) on germination,mitotic frequency and α-amylase activity of lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> Medik.) seeds

In this study, seed germination percentages, effects on phases of mitosis and α-amylase enzyme activity of lentil seeds treated with four different concentrations (0.25, 0.5, 1 and 1.5%) of Fusilade (Fluazifop-p-butyl) were determined. Median EC (effective concentration) values were calculated according to seed germination percentages after treatment for 72 h. Germination percentages of primary lentil roots decreased with increasing Fusilade concentrations. Cytological observations showed that the mitotic frequency in root meristematic cells were decreased parallel to the increase in concentrations and all Fusilade concentrations applied decreased the activity of α-amylase enzyme in lentil seeds. The obtained results indicate that the herbicide Fusilade had the ability to cause reduction in seed germination, mitotic frequency and also α-amylase activity of lentil seeds.     

SNX-25a,a novel Hsp90 inhibitor,inhibited human cancer growth more potently than 17-AAG

17-Allylamino-17-demethoxygeldanamycin (17-AAG), a typical Hsp90 inhibitor derived from geldanamycin (GA), has entered Phase III clinical trials for cancer therapy. However, it has several significant limitations such as poor solubility, limited bioavailability and unacceptable hepatotoxicity. In this study, the anticancer activity and mechanism of SNX-25a, a novel Hsp90 inhibitor, was investigated comparing with that of 17-AAG. We showed that SNX-25a triggered growth inhibition more sensitively than 17-AAG against many human cancer cells, including K562, SW-620, A375, Hep-2, MCF-7, HepG2, HeLa, and A549 cell lines, especially at low concentrations (<1 μM). It showed low cytotoxicity in L-02, HDF and MRC5 normal human cells. Compared with 17-AAG, SNX-25a was more potent in arresting the cell cycle at G2 phase, and displayed more potent effects on human cancer cell apoptosis and Hsp90 client proteins. It also exhibited a stronger binding affinity to Hsp90 than 17-AAG using molecular docking. Considering the superiority effects on Hsp90 affinity, cell growth, cell cycle, apoptosis, and Hsp90 client proteins, SNX-25a is supposed as a potential anticancer agent that needs to be explored in detail.     

Cathepsin S Causes Inflammatory Pain via Biased Agonism of PAR2 and TRPV4

Serine proteases such as trypsin and mast cell tryptase cleave protease-activated receptor-2 (PAR₂) at R³⁶↓S³⁷ and reveal a tethered ligand that excites nociceptors, causing neurogenic inflammation and pain. Whether proteases that cleave PAR₂ at distinct sites are biased agonists that also induce inflammation and pain is unexplored. Cathepsin S (Cat-S) is a lysosomal cysteine protease of antigen-presenting cells that is secreted during inflammation and which retains activity at extracellular pH. We observed that Cat-S cleaved PAR₂ at E⁵⁶↓T⁵⁷, which removed the canonical tethered ligand and prevented trypsin activation. In HEK and KNRK cell lines and in nociceptive neurons of mouse dorsal root ganglia, Cat-S and a decapeptide mimicking the Cat-S-revealed tethered ligand-stimulated PAR₂ coupling to Gαs and formation of cAMP. In contrast to trypsin, Cat-S did not mobilize intracellular Ca²⁺, activate ERK1/2, recruit β-arrestins, or induce PAR₂ endocytosis. Cat-S caused PAR₂-dependent activation of transient receptor potential vanilloid 4 (TRPV4) in Xenopus laevis oocytes, HEK cells and nociceptive neurons, and stimulated neuronal hyperexcitability by adenylyl cyclase and protein kinase A-dependent mechanisms. Intraplantar injection of Cat-S caused inflammation and hyperalgesia in mice that was attenuated by PAR₂ or TRPV4 deletion and adenylyl cyclase inhibition. Cat-S and PAR₂ antagonists suppressed formalin-induced inflammation and pain, which implicates endogenous Cat-S and PAR₂ in inflammatory pain. Our results identify Cat-S as a biased agonist of PAR₂ that causes PAR₂- and TRPV4-dependent inflammation and pain. They expand the role of PAR₂ as a mediator of protease-driven inflammatory pain.     

Use of Four Next-Generation Sequencing Platforms to Determine HIV-1 Coreceptor Tropism

HIV-1 coreceptor tropism assays are required to rule out the presence of CXCR4-tropic (non-R5) viruses prior treatment with CCR5 antagonists. Phenotypic (e.g., Trofile™, Monogram Biosciences) and genotypic (e.g., population sequencing linked to bioinformatic algorithms) assays are the most widely used. Although several next-generation sequencing (NGS) platforms are available, to date all published deep sequencing HIV-1 tropism studies have used the 454™ Life Sciences/Roche platform. In this study, HIV-1 co-receptor usage was predicted for twelve patients scheduled to start a maraviroc-based antiretroviral regimen. The V3 region of the HIV-1 env gene was sequenced using four NGS platforms: 454™, PacBio® RS (Pacific Biosciences), Illumina®, and Ion Torrent™ (Life Technologies). Cross-platform variation was evaluated, including number of reads, read length and error rates. HIV-1 tropism was inferred using Geno2Pheno, Web PSSM, and the 11/24/25 rule and compared with Trofile™ and virologic response to antiretroviral therapy. Error rates related to insertions/deletions (indels) and nucleotide substitutions introduced by the four NGS platforms were low compared to the actual HIV-1 sequence variation. Each platform detected all major virus variants within the HIV-1 population with similar frequencies. Identification of non-R5 viruses was comparable among the four platforms, with minor differences attributable to the algorithms used to infer HIV-1 tropism. All NGS platforms showed similar concordance with virologic response to the maraviroc-based regimen (75% to 80% range depending on the algorithm used), compared to Trofile (80%) and population sequencing (70%). In conclusion, all four NGS platforms were able to detect minority non-R5 variants at comparable levels suggesting that any NGS-based method can be used to predict HIV-1 coreceptor usage.     

Wnt signaling regulates acetylcholine receptor translocation and synaptic plasticity in the adult nervous system

The adult nervous system is plastic, allowing us to learn, remember, and forget. Experience-dependent plasticity occurs at synapses--the specialized points of contact between neurons where signaling occurs. However, the mechanisms that regulate the strength of synaptic signaling are not well understood. Here, we define a Wnt-signaling pathway that modifies synaptic strength in the adult nervous system by regulating the translocation of one class of acetylcholine receptors (AChRs) to synapses. In Caenorhabditis elegans, we show that mutations in CWN-2 (Wnt ligand), LIN-17 (Frizzled), CAM-1 (Ror receptor tyrosine kinase), or the downstream effector DSH-1 (disheveled) result in similar subsynaptic accumulations of ACR-16/α7 AChRs, a consequent reduction in synaptic current, and predictable behavioral defects. Photoconversion experiments revealed defective translocation of ACR-16/α7 to synapses in Wnt-signaling mutants. Using optogenetic nerve stimulation, we demonstrate activity-dependent synaptic plasticity and its dependence on ACR-16/α7 translocation mediated by Wnt signaling via LIN-17/CAM-1 heteromeric receptors.     

Assessing recruitment of lung diffusing capacity in exercising guinea pigs with a rebreathing technique.

Noninvasive techniques for assessing cardiopulmonary function in small animals are limited. We previously developed a rebreathing technique for measuring lung volume, pulmonary blood flow, diffusing capacity for carbon monoxide (Dl(CO)) and its components, membrane diffusing capacity (Dm(CO)) and pulmonary capillary blood volume (Vc), and septal volume, in conscious nonsedated guinea pigs at rest. Now we have extended this technique to study guinea pigs during voluntary treadmill exercise with a sealed respiratory mask attached to a body vest and a test gas mixture containing 0.5% SF(6) or Ne, 0.3% CO, and 0.8% C(2)H(2) in 40% or 98% O(2). From rest to exercise, O(2) uptake increased from 12.7 to 25.5 ml x min(-1) x kg(-1) while pulmonary blood flow increased from 123 to 239 ml/kg. The measured Dl(CO), Dm(CO), and Vc increased linearly with respect to pulmonary blood flow as expected from alveolar microvascular recruitment; body mass-specific relationships were consistent with those in healthy human subjects and dogs studied with a similar technique. The results show that 1) cardiopulmonary interactions from rest to exercise can be measured noninvasively in guinea pigs, 2) guinea pigs exhibit patterns of exercise response and alveolar microvascular recruitment similar to those of larger species, and 3) the rebreathing technique is widely applicable to human ( approximately 70 kg), dog (20-30 kg), and guinea pig (1-1.5 kg). In theory, this technique can be extended to even smaller animals provided that species-specific technical hurdles can be overcome.     

Discovery of dual Axl/VEGF-R2 inhibitors as potential anti-angiogenic and anti-metastatic drugs for cancer chemotherapy

Axl tyrosine kinase has been shown to be involved in multiple pathways contributing to tumor development, angiogenesis, and metastasis. High Axl expression has been observed in many human tumors where it appears to confer aggressive tumor behavior. Here we present several series of dual Axl-VEGF-R2 kinase inhibitors based on extensive optimization of an acyl diaminotriazole. It was hypothesized that dual inhibition of these two receptor tyrosine kinases may have a synergistic affect in inhibiting tumor angiogenesis and metastasis. One of these molecules, R916562 showed comparable activity to Sunitinib in two mouse tumor xenograft models and a mouse corneal micropocket model.     

Temporal variation and succession in an algal-dominated high intertidal assemblage

We determined whether temporal variation and succession were similar among sites with similar species composition by sampling unmanipulated and cleared plots in a high intertidal assemblage dominated by Endocladia muricata and Mastocarpus papillatus. Sampling was done for 6 years at six sites spanning over 4° of latitude in California. Ten 1×2-m permanent plots were chosen in the central portion of the assemblage at each site. Four of these served as unmanipulated controls, three were cleared (scraped and burned) in the spring of 1985, and three were cleared in the fall of 1985. The cover of sessile and density of motile species were determined by subsampling within the plots from 1985 until 1991. Recovery of the clearings was determined by their similarity to the controls. The algae E. muricata, M. papillatus, and Fucus gardneri, and the barnacle Balanus glandula, were the most abundant sessile organisms in the control plots, although the latter never exceeded 12% cover at any site. The grazing gastropods Littorina scutulata/plena, various limpets, and Tegula funebralis were the most common mobile organisms. The species composition of the common species remained constant in the control plots over the study period and there were few large changes in relative abundance. Significant seasonal variation was detected in 11 species but variation was commonly site-specific.Ephemeral algae were abundant during early succession at only two of the six sites, and barnacle cover was low (<15% cover) at four sites and moderate (15-50% cover) at the remaining two throughout succession. Recovery rate varied considerably among sites and between times of clearing (1-10%/month). Correlations between ephemeral algae and grazer abundance, and between these variables and recovery rate were not significant. The effects of grazers on recovery rate were only evident at one site where they appeared to reduce an initially high cover of ephemeral algae and delay the establishment of perennials. Some of the largest differences in recovery rate were between clearing times, associated with differences in the phenology of the dominant perennial algae. In spite of these differences, most plots recovered by the end of the study period.These results indicate that the assemblages in the control plots at each site were relatively stable and, while successional pathways and processes varied, the assemblage at most sites still recovered. Current models, based largely on biological interactions, that attempt to explain within assemblage structure and succession were not broadly applicable.