Calcium oxalate is the most abundant insoluble mineral found in plants and its crystals have been reported in more than 200 plant families. In the barrel medic Medicago truncatula Gaertn., these crystals accumulate predominantly in a sheath surrounding secondary veins of leaves. Mutants of M. truncatula with decreased levels of calcium oxalate crystals were used to assess the defensive role of this mineral against insects. Caterpillar larvae of the beet armyworm Spodoptera exigua Hü bner show a clear feeding preference for tissue from calcium oxalate-defective (cod) mutant lines cod5 and cod6 in choice test comparisons with wild-type M. truncatula. Compared to their performance on mutant lines, larvae feeding on wild-type plants with abundant calcium oxalate crystals suffer significantly reduced growth and increased mortality. Induction of woundresponsive genes appears to be normal in cod5 and cod6, indicating that these lines are not deficient in induced insect defenses. Electron micrographs of insect mouthparts indicate that the prismatic crystals in M. truncatula leaves act as physical abrasives during feeding. Food utilization measurements show that, after consumption, calcium oxalate also interferes with the conversion of plant material into insect biomass during digestion. In contrast to their detrimental effects on a chewing insect, calcium oxalate crystals do not negatively affect the performance of the pea aphid Acyrthosiphon pisum Harris, a sap-feeding insect with piercing-sucking mouthparts. The results confirm a long-held hypothesis for the defensive function of these crystals and point to the potential value of genes controlling crystal formation and localization in crop plants.

Jasmonates, such as jasmonic acid (JA), are plant-signaling compounds that trigger induced resistance against certain pathogens and a broad range of arthropod herbivores. One goal of this study was to determine the effects of JA-dependent defenses in tomato on root-knot nematodes. Another was to determine if the artificial induction of these defenses could enhance nematode control on plants that carry Mi-1.2, a nematode resistance gene that is present in many tomato cultivars. At moderate soil temperatures, Mi-1.2 can effectively suppress reproduction of most isolates of the common root-knot nematode species Meloidogyne javanica, M. incognita, and M. arenaria. Mi-mediated resistance has its limitations, however. Mi-1.2 is reported to lose its effectiveness at soil temperatures above 28 degrees C, and certain virulent nematode isolates can overcome resistance even at moderate soil temperatures. This study used a foliar application of JA to activate induced resistance in two near-isogenic lines of tomato (Lycopersicon esculentum) with and without Mi-1.2, and evaluated the effects of induced resistance at moderate soil temperatures on one avirulent nematode isolate (M. javanica isolate VW4) and two virulent isolates (M. javanica isolate VW5 and M. incognita isolate 557R). In addition, the effects of induced resistance on avirulent nematode performance were examined at a high temperature (32 degrees C). The results indicate that JA application induces a systemic defense response that reduces avirulent nematode reproduction on susceptible tomato plants. Furthermore, JA-dependent defenses proved to be heat-stable, whereas the effects of Mi-mediated resistance were reduced but not eliminated at 32 degrees C. JA treatment enhanced Mi-mediated resistance at high temperature, but did not suppress either of the virulent nematode isolates tested.

Patients with type 2 diabetes (T2D) have decreased butyrate-producing bacteria. We hypothesized that supplementation with butyrate-producing bacteria may exert beneficial effects on T2D. The current study investigated the effects of well-characterized butyrate-producing bacteria Clostridium butyricum CGMCC0313.1 (CB0313.1) on hyperglycemia and associated metabolic dysfunction in two diabetic mouse models. CB0313.1 was administered daily by oral gavage to leptindb/db mice for 5 weeks starting from 3 weeks of age, and to HF diabetic mice induced by high fat diet (HFD) plus streptozotocin (STZ) in C57BL/6J mice for 13 weeks starting from 4 weeks of age. CB0313.1 improved diabetic markers (fasting glucose, glucose tolerance, insulin tolerance, GLP-1 and insulin secretion), and decreased blood lipids and inflammatory tone. Furthermore, CB0313.1 reversed hypohepatias and reduced glucose output. We also found that CB0313.1 modulated gut microbiota composition, characterized by a decreased ratio of Firmicutes to Bacteroidetes, reduced Allobaculum bacteria that were abundant in HF diabetic mice and increased butyrate-producing bacteria. Changes in gut microbiota following CB0313.1 treatment were associated with enhanced peroxisome proliferator–activated receptor-γ (PPARγ), insulin signaling molecules and mitochondrial function markers. Together, our study suggests that CB0313.1 may act as a beneficial probiotic for the prevention and treatment of hyperglycemia and associated metabolic dysfunction.

We examined the effects of three forms of host plant resistance in tomato, Lycopersicon esculentum, on the potato aphid, Macrosiphum euphorbiae. Mi-1.2, a resistance gene (R-gene) in tomato that deters aphid feeding, reduced the population growth of both potato aphid isolates tested, although it appeared to have a greater impact on isolate WU11 than on isolate WU12. The results suggest that there may be quantitative differences in virulence between these two aphid isolates. We also examined two distinct forms of acquired resistance in tomato, jasmonic acid (JA)-dependent and salicylic acid (SA)-dependent induced defenses. Exogenous foliar application of JA triggered expression of a JA-inducible proteinase inhibitor in tomato cultivars with and without Mi-1.2, although the effects of treatment on aphid performance differed between these cultivars. JA-treatment reduced aphid population growth on a susceptible tomato cultivar that lacks Mi-1.2, but did not significantly enhance or inhibit aphid control on a near-isogenic resistant tomato cultivar that carries this gene. Foliar application of an SA analog, benzothiadiazole (BTH), was used to induce SA-dependent defenses. BTH treatment reduced the population growth of both aphid isolates on a susceptible tomato cultivar, and also enhanced aphid control on a resistant cultivar. The results indicate that both SA- and JA-dependent acquired resistance in tomato have a direct negative effect on a phloem-feeding insect. Furthermore, this study demonstrates that acquired resistance and R-gene-mediated resistance can interact for enhanced suppression of insect herbivores.

We report here that disruption of function of the v-3 FATTY ACID DESATURASE7 (FAD7) enhances plant defenses against aphids. The suppressor of prosystemin-mediated responses2 (spr2) mutation in tomato (Solanum lycopersicum), which eliminates the function of FAD7, reduces the settling behavior, survival, and fecundity of the potato aphid (Macrosiphum euphorbiae). Likewise, the antisense suppression of LeFAD7 expression in wild-type tomato plants reduces aphid infestations. Aphid resistance in the spr2 mutant is associated with enhanced levels of salicylic acid (SA) and mRNA encoding the pathogenesis-related protein P4. Introduction of the Naphthalene/salicylate hydroxylase transgene, which suppresses SA accumulation, restores wild-type levels of aphid susceptibility to spr2. Resistance in spr2 is also lost when we utilize virus-induced gene silencing to suppress the expression of NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1), a positive regulator of many SAdependent defenses. These results indicate that FAD7 suppresses defenses against aphids that are mediated through SA and NPR1. Although loss of function of FAD7 also inhibits the synthesis of jasmonate (JA), the effects of this desaturase on aphid resistance are not dependent on JA; other mutants impaired in JA synthesis (acx1) or perception (jai1-1) show wild-type levels of aphid susceptibility, and spr2 retains aphid resistance when treated with methyl jasmonate. Thus, FAD7 may influence JAdependent defenses against chewing insects and SA-dependent defenses against aphids through independent effects on JA synthesis and SA signaling. The Arabidopsis (Arabidopsis thaliana) mutants Atfad7-2 and Atfad7-1fad8 also show enhanced resistance to the green peach aphid (Myzus persicae) compared with wild-type controls, indicating that FAD7 influences plantaphid interactions in at least two plant families.

Recent evidence indicates that indigenous Clostridium species induce colonic regulatory T cells (Tregs), and gut lymphocytes are able to migrate to pancreatic islets in an inflammatory environment. Thus, we speculate that supplementation with the well-characterized probiotics Clostridium butyricum CGMCC0313.1 (CB0313.1) may induce pancreatic Tregs and consequently inhibit the diabetes incidence in non-obese diabetic (NOD) mice. CB0313.1 was administered daily to female NOD mice from 3 to 45 weeks of age. The control group received an equal volume of sterile water. Fasting glucose was measured twice a week. Pyrosequencing of the gut microbiota and flow cytometry of mesenteric lymph node (MLN), pancreatic lymph node (PLN), pancreatic and splenic immune cells were performed to investigate the effect of CB0313.1 treatment. Early oral administration of CB0313.1 mitigated insulitis, delayed the onset of diabetes, and improved energy metabolic dysfunction. Protection may involve increased Tregs, rebalanced Th1/Th2/Th17 cells and changes to a less proinflammatory immunological milieu in the gut, PLN, and pancreas. An increase of α4β7+ (the gut homing receptor) Tregs in the PLN suggests that the mechanism may involve increased migration of gut-primed Tregs to the pancreas. Furthermore, 16S rRNA gene sequencing revealed that CB0313.1 enhanced the Firmicutes/Bacteroidetes ratio, enriched Clostridium-subgroups and butyrate-producing bacteria subgroups. Our results provide the basis for future clinical investigations in preventing type 1 diabetes by oral CB0313.1 administration.

BMS-790052, a first-in-class hepatitis C virus (HCV) replication complex inhibitor, targeting nonstructural protein 5A (NS5A), displays picomolar to nanomolar potency against genotypes 1 to 5. This exceptional potency translated into robust anti-HCV activity in clinical studies with HCV genotype 1-infected subjects. To date, all BMS-790052-associated resistance mutations have mapped to the N-terminal region of NS5A. To further characterize the antiviral activity of BMS-790052, HCV replicon elimination and colony formation assays were performed. Replicon was cleared from genotype 1a and 1b replicon cells in a time-and dose-dependent manner. Elimination of the genotype 1a replicon required longer treatment durations and higher concentrations of BMS-790052 than those for the genotype1b replicon. Single amino acid substitutions that conferred relatively low levels of resistance were observed at early time points and at low doses. Higher doses and longer treatment durations yielded mutations that conferred greater levels of resistance, including linked amino acid substitutions. Replicon cells that survived inhibitor treatment remained fully sensitivity to pegylated alpha interferon (pegIFN-␣) and other HCV inhibitors. Moreover, genotype 1a replicon elimination was markedly enhanced when pegIFN-␣ and BMS-790052 were combined. Resistant variants observed in this study were very similar to those observed in a multiple ascending dose (MAD) monotherapy trial of BMS-790052, validating replicon elimination studies as a model to predict clinical resistance. Insights gained from the in vitro anti-HCV activity and resistance profiles of BMS-790052 will be used to help guide the clinical development of this novel HCV inhibitor. H epatitis C virus (HCV), a member of the Flaviviridae family of RNA viruses, is a major cause of liver disease worldwide (1). The ϳ9.6-kb HCV genome encodes a polyprotein that is processed into structural proteins (core, E1, and E2), a small ion channel protein (p7), and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) required for polyprotein processing and RNA replication (2). Until very recently, standard-of-care therapy for HCV-infected individuals consisted of a combination of pegylated interferon (pegIFN) and ribavirin (RBV) (18). Because of complications with side effects and incomplete antiviral efficacy, only ϳ50% of individuals infected with HCV genotype 1 achieved a sustained viral response upon treatment (18). Today, an increasing number of small-molecule inhibitors targeting specific viral proteins are in various stages of development, and two drugs that target the HCV NS3 protease, telaprevir and boceprevir, have been approved for clinical use for HCV genotype 1-infected patient treatment in combination with pegIFN and RBV. Collectively referred to as directly acting antiviral agents (DAA), these virus-specific inhibitors hold the promise of improving or even replacing IFN-based HCV therapy (9). Many of the DAA in development are directed against the viral enzymatic activities...

A comparison of the daclatasvir (DCV [BMS-790052]) resistance barrier on authentic or hybrid replicons containing NS5A from hepatitis C virus (HCV) genotypes 1 to 6 (GT-1 to -6) was completed using a replicon elimination assay. The data indicated that genotype 1b (GT-1b) has the highest relative resistance barrier and genotype 2a (GT-2a M31) has the lowest. The rank order of resistance barriers to DCV was 1b > 4a > 5a > 6a Х 1a > 2a JFH > 3a > 2a M31. Importantly, DCV in combination with a protease inhibitor (PI) eliminated GT-2a M31 replicon RNA at a clinically relevant concentration. Previously, we reported the antiviral activity and resistance profiles of DCV on HCV genotypes 1 to 4 evaluated in the replicon system. Here, we report the antiviral activity and resistance profiles of DCV against hybrid replicons with NS5A sequences derived from HCV GT-5a and GT-6a clinical isolates. DCV was effective against both GT-5a and -6a hybrid replicon cell lines (50% effective concentrations [EC 50 s] ranging from 3 to 7 pM for GT-5a, and 74 pM for GT-6a). Resistance selection identified amino acid substitutions in the N-terminal domain of NS5A. For GT-5a, L31F and L31V, alone or in combination with K56R, were the major resistance variants (EC 50 s ranging from 2 to 40 nM). In GT-6a, Q24H, L31M, P32L/S, and T58A/S were identified as resistance variants (EC 50 s ranging from 2 to 250 nM). The in vitro data suggest that DCV has the potential to be an effective agent for HCV genotypes 1 to 6 when used in combination therapy. D aclatasvir (DCV [BMS-790052]) is a cross-genotypic NS5Ainhibitor with picomolar to low nanomolar potency in the replicon system (1, 2). The antiviral activity of DCV in vitro translated into clinical efficacy, with hepatitis C virus (HCV) RNA declines of ϳ3 to 4 log 10 observed in genotype 1a (GT-1a)-infected subjects treated once daily (QD) with 60 mg of DCV in a 14-day multiple ascending dose (MAD) monotherapy study (3, 4). Moreover, DCV was effective against GT-1b and -1a in combinations that include either pegylated interferon and ribavirin (PEG-IFN-RBV) or other direct-acting anti-HCV agents (DAAs) (5-8).There are large populations of viral quasispecies preexisting in infected individuals, and variants that confer resistance to antiviral agents can be rapidly enriched and/or selected during antiviral treatment (9-11). Since DCV resistance variants show no crossresistance to other DAAs, DCV should rapidly suppress wild-type virus and variants resistant to other DAAs, thereby enhancing the effectiveness of other DAAs in combination therapies (2, 3). This effect is predicted to lead to higher rates of sustained viral response (SVR) and/or shorten the duration of treatment necessary to achieve SVR. Recent clinical results with DCV plus asunaprevir (ASV ) in patients infected with GT-1b and with DCV plus sofosbuvir (SOF ) in patients infected with GT-1, -2, and 3 demonstrate the effectiveness of DCV in interferon-free DAA combination therapies (6,12).Prior studies using the in vitro replicon system in...