Insect herbivores should follow plants escaping their relatives

Numerous observational studies have documented conspecific negative density-dependence that is consistent with the Janzen–Connell Hypothesis (JCH) of diversity maintenance. However, there have been few experimental tests of a central prediction of the JCH: that removing host-specific enemies should lead to greater increases in per capita recruitment in areas of higher host density or lower relative phylogenetic diversity. Using spatially randomized plots of high and low host biomass in a temperate grassland biodiversity experiment, we treated developing seedheads of six prairie perennials to factorial applications of fungicide and insecticide. We measured predispersal seed production, seed viability, and seedling biomass. Results were highly species-specific and idiosyncratic. Effects of insect seed predators and fungal pathogens on predispersal responses varied with neither conspecific biomass nor phylogenetic diversity, suggesting that—at least at the predispersal stage and for the insect and fungal seed predators we were able to exclude—the JCH is not sufficient to contribute to negative conspecific density-dependence for these dominant prairie species.     

Hypercapnia prevents neovascularization via nitrative stress

Neovascularization after an ischemic insult is a beneficial attempt to salvage the injured tissue. Yet, despite the production of angiogenic factors within ischemic tissues, compensatory growth of new vessels fails to provide adequate vascularization. Thus, we hypothesized that local factors counter efficient revascularization. Whereas ischemia is often considered to be synonymous with an oxygen deficit, it is also associated with a concomitant local elevation of carbon dioxide (CO2). Although studies suggest that hypercapnia impacts tissue neovascularization, its significance relative to the abundantly described effects of hypoxia and its underlying mechanisms have yet to be elucidated. Therefore, we investigated the effects of hypercapnia on blood vessel growth in models of developmental and ischemic neovascularization. Acute and prolonged CO2 exposure inhibited developmental neovascularization of the rodent retina, as well as revascularization of the ischemic retina. Hypercapnia induced early increases in endothelial nitric oxide synthase and nitrative stress, associated with astrocyte impairment and endothelial cell death, as well as downregulation of the proangiogenic prostaglandin E2 receptor EP3. These results establish a previously unexplored means by which hypercapnia hinders efficient neovascularization, a mechanism that may contribute to ischemic tissue injury.     

Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors

A key challenge for the academic and biopharmaceutical communities is the rapid and scalable production of recombinant proteins for supporting downstream applications ranging from therapeutic trials to structural genomics efforts. Here, we describe a novel system for the production of recombinant mammalian proteins, including immune receptors, cytokines and antibodies, in a human cell line culture system, often requiring <3 weeks to achieve stable, high-level expression: Daedalus. The inclusion of minimized ubiquitous chromatin opening elements in the transduction vectors is key for preventing genomic silencing and maintaining the stability of decigram levels of expression. This system can bypass the tedious and time-consuming steps of conventional protein production methods by employing the secretion pathway of serum-free adapted human suspension cell lines, such as 293 Freestyle. Using optimized lentiviral vectors, yields of 20-100 mg/l of correctly folded and post-translationally modified, endotoxin-free protein of up to ~70 kDa in size, can be achieved in conventional, small-scale (100 ml) culture. At these yields, most proteins can be purified using a single size-exclusion chromatography step, immediately appropriate for use in structural, biophysical or therapeutic applications.     

The use of chimeric Venezuelan equine encephalitis viruses as an approach for the molecular identification of natural virulence determinants

Venezuelan equine encephalitis (VEE) virus antigenic subtypes and varieties are considered either epidemic/epizootic or enzootic. In addition to epidemiological differences between the epidemic and enzootic viruses, several in vitro and in vivo laboratory markers distinguishing the viruses have been identified, including differential plaque size, sensitivity to interferon (IFN), and virulence for guinea pigs. These observations have been shown to be useful predictors of natural, equine virulence and epizootic potential. Chimeric viruses containing variety IAB (epizootic) nonstructural genes with variety IE (enzootic) structural genes (VE/IAB-IE) or IE nonstructural genes and IAB structural genes (IE/IAB) were constructed to systematically analyze and map viral phenotype and virulence determinants. Plaque size analysis showed that both chimeric viruses produced a mean plaque diameter that was intermediate between those of the parental strains. Additionally, both chimeric viruses showed intermediate levels of virus replication and virulence for guinea pigs compared to the parental strains. However, IE/IAB produced a slightly higher viremia and an average survival time 2 days shorter than the VE/IAB-IE virus. Finally, IFN sensitivity assays revealed that only one chimera, VE/IAB-IE, was intermediate between the two parental types. The second chimera, containing the IE nonstructural genes, was at least five times more sensitive to IFN than the IE parental virus and greater than 50 times more sensitive than the IAB parent. These results implicate viral components in both the structural and nonstructural portions of the genome in contributing to the epizootic phenotype and indicate the potential for epidemic emergence from the IE enzootic VEE viruses.     

Prmt2 regulates the lipopolysaccharide-induced responses in lungs and macrophages

Precise control of the LPS stimulation in the lung modulates inflammation and airway hyperresponsiveness involving the well-known TLR4/NF-κB pathway. As a consequence, the expression and secretion of proinflammatory cytokines is tightly regulated with the recruitment of neutrophils. Changes in the LPS-induced responses have been observed in the Prmt2-Col6a1 monosomic model, suggesting the presence of dosage-sensitive genes controlling LPS pathway in the mouse. In this article, we report that the Prmt2 regulates the LPS-induced lung responses in lungs and macrophages. We demonstrate that Prmt2 gene dosage influences the lung airway hyperresponsiveness, the recruitment of neutrophils, and the expression of proinflammatory cytokines, such as IL-6 and TNF-α. In addition, Prmt2 loss of function also altered the nuclear accumulation of NF-κB in stimulated macrophages. Prmt2 should be considered as a new member of the NF-κB pathway controlling LPS-induced inflammatory and lung responses in a dosage-dependent manner, certainly through regulating nuclear accumulation of NF-κB as shown already in fibroblasts.     

Characterization of PTZ-induced seizure susceptibility in a down syndrome mouse model that overexpresses CSTB

Down syndrome (DS) is a complex genetic syndrome characterized by intellectual disability, dysmorphism and variable additional physiological traits. Current research progress has begun to decipher the neural mechanisms underlying cognitive impairment, leading to new therapeutic perspectives. Pentylenetetrazol (PTZ) has recently been found to have positive effects on learning and memory capacities of a DS mouse model and is foreseen to treat DS patients. But PTZ is also known to be a convulsant drug at higher dose and DS persons are more prone to epileptic seizures than the general population. This raises concerns over what long-term effects of treatment might be in the DS population. The cause of increased propensity for epilepsy in the DS population and which Hsa21 gene(s) are implicated remain unknown. Among Hsa21 candidate genes in epilepsy, CSTB, coding for the cystein protease inhibitor cystatin B, is involved in progressive myoclonus epilepsy and ataxia in both mice and human. Thus we aim to evaluate the effect of an increase in Cstb gene dosage on spontaneous epileptic activity and susceptibility to PTZ-induced seizure. To this end we generated a new mouse model trisomic for Cstb by homologous recombination. We verified that increasing copy number of Cstb from Trisomy (Ts) to Tetrasomy (Tt) was driving overexpression of the gene in the brain, we checked transgenic animals for presence of locomotor activity and electroencephalogram (EEG) abnormalities characteristic of myoclonic epilepsy and we tested if those animals were prone to PTZ-induced seizure. Overall, the results of the analysis shows that an increase in Cstb does not induce any spontaneous epileptic activity and neither increase or decrease the propensity of Ts and Tt mice to myoclonic seizures suggesting that Ctsb dosage should not interfere with PTZ-treatment.     

Isolation and characterization of EstC, a new cold-active esterase from Streptomyces coelicolor A3(2)

The genome sequence of Streptomyces coelicolor A3(2) contains more than 50 genes coding for putative lipolytic enzymes. Many studies have shown the capacity of this actinomycete to store important reserves of intracellular triacylglycerols in nutrient depletion situations. In the present study, we used genome mining of S. coelicolor to identify genes coding for putative, non-secreted esterases/lipases. Two genes were cloned and successfully overexpressed in E. coli as His-tagged fusion proteins. One of the recombinant enzymes, EstC, showed interesting cold-active esterase activity with a strong potential for the production of valuable esters. The purified enzyme displayed optimal activity at 35°C and was cold-active with retention of 25% relative activity at 10°C. Its optimal pH was 8.5–9 but the enzyme kept more than 75% of its maximal activity between pH 7.5 and 10. EstC also showed remarkable tolerance over a wide range of pH values, retaining almost full residual activity between pH 6–11. The enzyme was active toward short-chain p-nitrophenyl esters (C2–C12), displaying optimal activity with the valerate (C5) ester (k _cat/K _m = 737±77 s⁻¹ mM⁻¹). The enzyme was also very active toward short chain triglycerides such as triacetin (C2:0) and tributyrin (C4:0), in addition to showing good primary alcohol and organic solvent tolerance, suggesting it could function as an interesting candidate for organic synthesis of short-chain esters such as flavors.     

Development of a novel noncompetitive antagonist of IL-1 receptor

IL-1 is a major proinflammatory cytokine which interacts with the IL-1 receptor I (IL-1RI) complex, composed of IL-1RI and IL-1R accessory protein subunits. Currently available strategies to counter pathological IL-1 signaling rely on a recombinant IL-1 receptor antagonist, which directly competes with IL-1 for its binding site. Presently, there are no small antagonists of the IL-1RI complex. Given this void, we derived 15 peptides from loops of IL-1R accessory protein, which are putative interactive sites with the IL-1RI subunit. In this study, we substantiate the merits of one of these peptides, rytvela (we termed "101.10"), as an inhibitor of IL-1R and describe its properties consistent with those of an allosteric negative modulator. 101.10 (IC(50) approximately 1 nM) blocked human thymocyte proliferation in vitro, and demonstrated robust in vivo effects in models of hyperthermia and inflammatory bowel disease as well as topically in contact dermatitis, superior to corticosteroids and IL-1ra; 101.10 did not bind to IL-1RI deficient cells and was ineffective in vivo in IL-1RI knockout mice. Importantly, characterization of 101.10, revealed noncompetitive antagonist actions and functional selectivity by blocking certain IL-1R pathways while not affecting others. Findings describe the discovery of a potent and specific small (peptide) antagonist of IL-1RI, with properties in line with an allosteric negative modulator.     

Reaction between the anesthetic agent propofol and the free radical DPPH in semiaqueous media: kinetics and characterization of the products

The reaction of the free radical diphenylpicrylhydrazyl (DPPH ) with the anesthetic agent 2,6-diisopropylphenol (propofol, PPF) was investigated in buffered hydroalcoholic media. The kinetics was followed using a stopped-flow system. DPPH was reduced to the hydrazine analogue DPPH-H with a measured stoichiometry (DPPH /PPF) of 2. The main product of the reaction, 3,5,3',5'-tetraisopropyl-(4,4')-diphenoquinone (PPFDQ) was isolated by chromatography and its structure was fully characterized. The reaction mechanism was inferred from the stoichiometry, kinetics, and product identification. The first step, which primarily determines the kinetics, is the reaction of DPPH with PPF to produce DPPH-H and the PPF radical. The rate constant was found to be 31.8, 207, and 908 M(-1) s(-1) at pH 6.4, 7.4, and 8.4, respectively. The pH dependence is indicative of a higher reactivity of the phenolate form of PPF. Then, PPF radicals combine to form dipropofol, which is quickly oxidized to PPFDQ by the remaining DPPH . This reaction scheme is corroborated by numerical simulations of the kinetics. In the course of this study we also disclosed an unexpected effect, the photochemical degradation of PPFDQ. The need to compare antioxidants on a kinetics basis is again emphasized. In our hands, PPF presents a significantly weaker reactivity than Trolox.