Diverse energetic effects of charge reversal mutations of poxvirus topoisomerase IB

A key aspect of the reaction mechanism of type IB topoisomerases is the controlled unwinding of DNA supercoils while the enzyme is transiently bound to one strand of the DNA duplex via a phosphotyrosyl linkage. In this complex, the mobile segment of the bound DNA downstream from the site of cleavage must rotate around the helical axis, requiring that interactions with the enzyme must break and re-form multiple times during the course of removing supercoils. A crystal structure of variola virus type IB topoisomerase (vTopo) bound to DNA shows several positively charged side chains that interact with the downstream mobile and upstream rigid segments, suggesting that these groups may play a role in catalysis, including the processive unwinding of supercoils. We have mutated three such residues, R67, K35, and K271, to Ala and Glu and determined the energetic effects of these mutations at each point along the reaction coordinate of vTopo. R67 interacts with a phosphate group in the rigid DNA segment across from the site of DNA strand cleavage. The ~30-fold damaging effects of the R67A and R67E mutations were primarily on the phosphoryl transfer step, with little effect on enzyme-DNA binding, or the processivity of supercoil unwinding. Removal of the K35 interaction shows mutational effects similar to those of R67, even though this residue interacts with the mobile segment 3 bp from the cleavage site. The two mutations of K271, which interacts with the mobile region even further from the site of covalent linkage, show significant effects not only on phosphoryl transfer but also on downstream DNA strand positioning. Moreover, supercoil unwinding measurements indicate that the K271A and K271E mutations increase the average number of supercoils that are removed during the lifetime of the covalent complex, enhancing the processivity of supercoil unwinding. These measurements support the proposal that the processivity of supercoil unwinding can be regulated by electrostatic interactions between the enzyme and the mobile DNA phosphate backbone.     

Influence of polyploidy on insect herbivores of native and invasive genotypes of Solidago gigantea (Asteraceae)

Herbivores are sensitive to the genetic structure of plant populations, as genetics underlies plant phenotype and host quality. Polyploidy is a widespread feature of angiosperm genomes, yet few studies have examined how polyploidy influences herbivores. Introduction to new ranges, with consequent changes in selective regimes, can lead to evolution of changes in plant defensive characteristics and also affect herbivores. Here, we examine how insect herbivores respond to polyploidy in Solidago gigantea, using plants derived from both the native range (USA) and introduced range (Europe). S. gigantea has three cytotypes in the US, with two of these present in Europe. We performed bioassays with generalist (Spodoptera exigua) and specialist (Trirhabda virgata) leaf-feeding insects. Insects were reared on detached leaves (Spodoptera) or potted host plants (Trirhabda) and mortality and mass were measured. Trirhabda larvae showed little variation in survival or pupal mass attributable to either cytotype or plant origin. Spodoptera larvae were more sensitive to both cytotype and plant origin: they grew best on European tetraploids and poorly on US diploids (high mortality) and US tetraploids (low larval mass). These results show that both cytotype and plant origin influence insect herbivores, but that generalist and specialist insects may respond differently.Key words: polyploidy, cytotype, Solidago gigantea, insect herbivore, herbivory, invasive plant, introduced plantPolyploidy, or the possession of more than two sets of homologous chromosomes, is a fundamental force in angiosperm evolution.^1,2 Many plant species or species complexes consist of multiple cytotypes that may occur sympatrically;³ this is an important source of genetic structure in plant populations that is often overlooked.⁴ Possession of multiple genomes may confer advantages to polyploid plants such as increased heterozygosity, a decreased probability of inbreeding depression, or a greater gene pool available for selection; these traits contribute to the widespread success of polyploids and may make them prone to invasiveness.^5,6 In a recent article,⁷ we examined the functional consequences of polyploidy for different cytotypes of Solidago gigantea Ait. (Asteraceae), collected from both its native range (North America) and its introduced range (Europe). In this addendum, we show how cytotype and continent of origin influence interactions of S. gigantea with insect herbivores. Interactions with herbivores are expected to vary with cytotype because of phenotypic changes associated with polyploidy, but this area has received little study (reviewed in refs. ^8–11). Plant origin, from either the native range or an introduced range, should also influence herbivores. Plants may escape from their specialist natural enemies in the introduced range, thereby experiencing reduced herbivore pressure from an insect community dominated by generalists.^12,13 Given sufficient time, plants from the introduced range may evolve to decrease investment in anti-herbivore defenses, particularly those effective against specialists.¹⁴ While a growing body of research has addressed whether plant defenses against herbivory are lower in the introduced range,^12,15,16 few of these studies have also examined the influence of cytotype.¹⁷Three cytotypes of S. gigantea can be found in its native range in North America (diploid, tetraploid and hexaploid, 2n = 18, 36 and 54 respectively). These are morphologically indistinguishable and not generally treated as separate species.¹⁸ In Europe, where S. gigantea was introduced in the mid 18^th century,¹⁹ tetraploids are the dominant cytotype but diploids also occur. S. gigantea supports a diverse array of insect herbivores in its native range, but has few natural enemies in its introduced range.²⁰ We report here on experiments using both a generalist and a specialist leaf-chewing insect. The generalist, Spodoptera exigua (Lepidoptera: Noctuidae) is widely distributed and highly polyphagous, while the specialist Trirhabda virgata (Coleoptera: Chrysomelidae) feeds only on closely-related species within the genus Solidago. T. virgata is an outbreak insect that can be a major defoliator of S. gigantea and related species in North America.²¹ We grew plants originating from 10 populations in the US and 20 populations in Europe in common gardens at the University of Wisconsin-Milwaukee Field Station in Saukville, Wisconsin. There were five plant origin-cytotype combinations: three cytotypes from the US and two from Europe. Insects were reared on detached leaves from a single plant (Spodoptera) or on potted host plants (Trirhabda), for a set period of 21 d (Spodoptera) or until pupation (Trirhabda). We recorded insect survival and mass at the end of 21 d (Spodoptera) or at pupation (Trirhabda) (reviewed in ref. ²²).Overall survival was much better for the specialist Trirhabda than for the generalist Spodoptera (91% vs. 72%). Spodoptera larvae are not generally found on S. gigantea in the field, and while they are able to complete development, we found that this plant was not an ideal host. Spodoptera larvae were more sensitive to differences among cytotype and plant origin than were Trirhabda larvae. Percent survival was particularly poor for Spodoptera larvae reared on diploids from the US, where slightly more than half of the caterpillars survived for 21 days (Fig. 1). Trirhabda pupal mass was remarkably consistent across the five ploidy-plant origin combinations. In contrast, Spodoptera larvae responded to both cytotype and continent of origin. Surviving Spodoptera larvae did particularly well on tetraploid plants from the introduced range (Europe), and particularly poorly on tetraploids from the US (Fig. 1). We have previously reported that Spodoptera grow better on plants from Europe;²² our current results reveal that this difference is due exclusively to better growth on tetraploid plants. However, our results also show that both diploids and tetraploids from the US were poor hosts for Spodoptera: diploids because they caused high mortality and tetraploids because they resulted in poor growth. These results indicate that plants from the introduced range have reduced defenses against herbivores, even when accounting for polyploidy.Open in a separate windowFigure 1Mass ± se of S. exigua (A) and T. virgata (B) larvae reared on host plants of different cytotypes of Solidago gigantea originating from the US (native range) or europe (introduced range). Means in A followed by different letters are significantly different at p < 0.05 (ANOVA followed by multiple Student''s t-tests with Bonferroni correction). There were no significant differences in (B). Sample sizes for (A and B) shown in

Combined ipilimumab and nivolumab first‐line and after BRAF‐targeted therapy in advanced melanoma

The combination of ipilimumab and nivolumab is a highly active systemic therapy for metastatic melanoma but can cause significant toxicity. We explore the safety and efficacy of this treatment in routine clinical practice, particularly in the setting of serine/threonine‐protein kinase B‐Raf (BRAF)‐targeted therapy. Consecutive patients with unresectable stage IIIC/IV melanoma commenced on ipilimumab and nivolumab across 10 tertiary melanoma institutions in Australia were identified retrospectively. Data collected included demographics, response and survival outcomes. A total of 152 patients were included for analysis, 39% were treatment‐naïve and 22% failed first‐line BRAF/MEK inhibitors. Treatment‐related adverse events occurred in 67% of patients, grade 3–5 in 38%. The overall objective response rate was 41%, 57% in treatment‐naïve and 21% in BRAF/MEK failure patients. Median progression‐free survival was 4.0 months (95% CI, 3.0–6.0) in the whole cohort, 11.0 months (95% CI, 6.0‐NR) in treatment‐naïve and 2.0 months (95% CI, 1.4–4.6) in BRAF/MEK failure patients. The combination of ipilimumab and nivolumab can be used safely and effectively in a real‐world population. While first‐line efficacy appears comparable to trial populations, BRAF‐mutant patients failing prior BRAF/MEK inhibitors show less response.     

Toward an understanding of broad-scale patterns of the habitat suitability of fountain grass (Cenchrus setaceus (Forssk.) Morrone,Poaceae)

Plant Ecology - Understanding the factors contributing to the introduction and spread of invasive species is crucial to help develop management strategies to control and eradicate them in sensitive...     

Dopamine- and cAMP-regulated Phosphoprotein of 32-kDa (DARPP-32)-dependent Activation of Extracellular Signal-regulated Kinase (ERK) and Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling in Experimental Parkinsonism

Dyskinesia, a motor complication caused by prolonged administration of the antiparkinsonian drug l-3,4-dihydroxyphenylalanine (l-DOPA), is accompanied by activation of cAMP signaling and hyperphosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). Here, we show that the abnormal phosphorylation of DARPP-32 occurs specifically in medium spiny neurons (MSNs) expressing dopamine D1 receptors (D1R). Using mice in which DARPP-32 is selectively deleted in D1R-expressing MSNs, we demonstrate that this protein is required for l-DOPA-induced activation of the extracellular signal-regulated protein kinases 1 and 2 and the mammalian target of rapamycin complex 1 (mTORC1) pathways, which are implicated in dyskinesia. We also show that mutation of the phosphorylation site for cAMP-dependent protein kinase on DARPP-32 attenuates l-DOPA-induced dyskinesia and reduces the concomitant activations of ERK and mTORC1 signaling. These studies demonstrate that, in D1R-expressing MSNs, l-DOPA-induced activation of ERK and mTORC1 requires DARPP-32 and indicates the importance of the cAMP/DARPP-32 signaling cascade in dyskinesia.     

Mitochondrial superoxide anion radicals mediate induction of apoptosis in cardiac myoblasts exposed to chronic hypoxia

Both reactive oxygen species (ROS) and ATP depletion may be significant in hypoxia-induced damage and death, either collectively or independently, with high energy requiring, metabolically active cells being the most susceptible to damage.We investigated the kinetics and effects of ROS production in cardiac myoblasts, H9C2 cells, under 2%, 10% and 21% O₂ in the presence or absence of apocynin, rotenone and carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone.H9C2 cells showed significant loss of viability within 30 min of culture at 2% oxygen which was not due to apoptosis, but was associated with an increase in protein oxidation. However, after 4 h, apoptosis induction was observed at 2% oxygen and also to a lesser extent at 10% oxygen; this was dependent on the levels of mitochondrial superoxide anion radicals determined using dihydroethidine. Hypoxia-induced ROS production and cell death could be rescued by the mitochondrial complex I inhibitor, rotenone, despite further depletion of ATP.In conclusion, a change to superoxide anion radical steady state level was not detectable after 30 min but was evident after 4 h of mild or severe hypoxia. Superoxide anion radicals from the mitochondrion and not ATP depletion is the major cause of apoptotic cell death in cardiac myoblasts under chronic, severe hypoxia.