Dimer-assisted mechanism of (un)saturated fatty acid decarboxylation for alkene production.

The enzymatic decarboxylation of fatty acids (FAs) represents an advance toward the development of biological routes to produce drop-in hydrocarbons. The current mechanism for the P450-catalyzed decarboxylation has been largely established from the bacterial cytochrome P450 OleTJE. Herein, we describe OleTPRN, a poly-unsaturated alkene-producing decarboxylase that outrivals the functional properties of the model enzyme and exploits a distinct molecular mechanism for substrate binding and chemoselectivity. In addition to the high conversion rates into alkenes from a broad range of saturated FAs without dependence on high salt concentrations, OleTPRN can also efficiently produce alkenes from unsaturated (oleic and linoleic) acids, the most abundant FAs found in nature. OleTPRN performs carbon-carbon cleavage by a catalytic itinerary that involves hydrogen-atom transfer by the heme-ferryl intermediate Compound I and features a hydrophobic cradle at the distal region of the substrate-binding pocket, not found in OleTJE, which is proposed to play a role in the productive binding of long-chain FAs and favors the rapid release of products from the metabolism of short-chain FAs. Moreover, it is shown that the dimeric configuration of OleTPRN is involved in the stabilization of the A-A' helical motif, a second-coordination sphere of the substrate, which contributes to the proper accommodation of the aliphatic tail in the distal and medial active-site pocket. These findings provide an alternative molecular mechanism for alkene production by P450 peroxygenases, creating new opportunities for biological production of renewable hydrocarbons.

AAV-mediated neuronal expression of an scFv antibody selective for Aß oligomers protects synapses and rescues memory in Alzheimer models.

The accumulation of soluble oligomers of the amyloid-ß peptide (AßOs) in the brain has been implicated in synapse failure and memory impairment in Alzheimer's disease. Here, we initially show that treatment with NUsc1, a single-chain variable-fragment antibody (scFv) that selectively targets a subpopulation of AßOs and shows minimal reactivity to Aß monomers and fibrils, prevents the inhibition of long-term potentiation in hippocampal slices and memory impairment induced by AßOs in mice. As a therapeutic approach for intracerebral antibody delivery, we developed an adeno-associated virus vector to drive neuronal expression of NUsc1 (AAV-NUsc1) within the brain. Transduction by AAV-NUsc1 induced NUsc1 expression and secretion in adult human brain slices and inhibited AßO binding to neurons and AßO-induced loss of dendritic spines in primary rat hippocampal cultures. Treatment of mice with AAV-NUsc1 prevented memory impairment induced by AßOs and, remarkably, reversed memory deficits in aged APPswe/PS1ΔE9 Alzheimer's disease model mice. These results support the feasibility of immunotherapy using viral vector-mediated gene delivery of NUsc1 or other AßO-specific single-chain antibodies as a potential therapeutic approach in Alzheimer's disease.

Paralytic activity of lysophosphatidylcholine from saliva of the waterbug Belostoma anurum.

Lysophosphatidylcholine (LPC) is a major bioactive lipid that is enzymatically generated by phospholipase A(2) (PLA(2)). Previously, we showed that LPC is present in the saliva of the blood-sucking hemipteran Rhodnius prolixus and modulates cell-signaling pathways involved in vascular biology, which aids blood feeding. Here, we show that the saliva of the predator insect Belostoma anurum contains a large number of lipids with LPC accounting for 25% of the total phospholipids. A PLA(2) enzyme likely to be involved in LPC generation was characterized. The activity of this enzyme is 5-fold higher in Belostoma saliva than in other studied hemipterans, suggesting a close association with the predator feeding habits of this insect. Belostoma employs extra-oral digestion, which allows for ingestion of larger prey than itself, including small vertebrates such as amphibians and fish. Therefore, prey immobilization during digestion is essential, and we show here that Belostoma saliva and B. anurum saliva purified LPC have paralytic activity in zebrafish. This is the first evidence that lysophospholipids might play an important role in prey immobilization, in addition to contributing to blood feeding, and might have been an evolutionary acquisition that occurred long before the appearance of hematophagy in this animal group.

Evolution of EPSPS double mutation imparting glyphosate resistance in wild poinsettia (Euphorbia heterophylla L.).

The evolution of glyphosate resistance (GR) in weeds is an increasing problem. Glyphosate has been used intensively on wild poinsettia (Euphorbia heterophylla L.) populations for at least 20 years in GR crops within South America. We investigated the GR mechanisms in a wild poinsettia population from a soybean field in southern Brazil. The GR population required higher glyphosate doses to achieve 50% control (LD50) and 50% dry mass reduction (MR50) compared to a glyphosate susceptible (GS) population. The ratio between the LD50 and MR50 of GR and GS resulted in resistance factors (RF) of 6.9-fold and 6.1-fold, respectively. Shikimate accumulated 6.7 times more in GS than in GR when leaf-discs were incubated with increasing glyphosate concentrations. No differences were found between GR and GS regarding non-target-site mechanisms. Neither population metabolized glyphosate to significant levels following treatment with 850 g ha-1 glyphosate. Similar levels of 14C-glyphosate uptake and translocation were observed between the two populations. No differences in EPSPS expression were found between GS and GR. Two target site mutations were found in all EPSPS alleles of homozygous resistant plants: Thr102Ile + Pro106Thr (TIPT-mutation). Heterozygous individuals harbored both alleles, wild-type and TIPT. Half of GR individuals were heterozygous, suggesting that resistance is still evolving in the population. A genotyping assay was developed based on the Pro106Thr mutation, demonstrating high efficiency to identify homozygous, heterozygous or wild-type EPSPS sequences across different plants. This is the first report of glyphosate-resistant wild-poinsettia harboring an EPSPS double mutation (TIPT) in the same plant.

Adenovirus-Mediated Transduction of Insulin-Like Growth Factor 1 Protects Hippocampal Neurons from the Toxicity of Aß Oligomers and Prevents Memory Loss in an Alzheimer Mouse Model.

Alzheimer's disease (AD) is the main cause of dementia in the elderly. Although activation of brain insulin signaling has been shown to be neuroprotective, to preserve memory in AD models, and appears beneficial in patients, the role of insulin-like growth factor 1 (IGF1) remains incompletely understood. We found reduced active/inactive IGF1 ratio and increased IGF1R expression in postmortem hippocampal tissue from AD patients, suggesting impaired brain IGF1 signaling in AD. Active/inactive IGF-1 ratio was also reduced in the brains of mouse models of AD. We next investigated the possible protective role of IGF1 in AD models. We used a recombinant adenoviral vector, RAd-IGF1, to drive the expression of IGF1 in primary hippocampal neuronal cultures prior to exposure to AßOs, toxins that accumulate in AD brains and have been implicated in early synapse dysfunction and memory impairment. Cultures transduced with RAd-IGF1 showed decreased binding of AßOs to neurons and were protected against AßO-induced neuronal oxidative stress and loss of dendritic spines. Significantly, in vivo transduction with RAd-IGF1 blocked memory impairment caused by intracerebroventricular (i.c.v.) infusion of AßOs in mice. Our results demonstrate altered active IGF1 and IGF1R levels in AD hippocampi, and suggest that boosting brain expression of IGF1 may comprise an approach to prevent neuronal damage and memory loss in AD.

Zika virus replicates in adult human brain tissue and impairs synapses and memory in mice.

Neurological complications affecting the central nervous system have been reported in adult patients infected by Zika virus (ZIKV) but the underlying mechanisms remain unknown. Here, we report that ZIKV replicates in human and mouse adult brain tissue, targeting mature neurons. ZIKV preferentially targets memory-related brain regions, inhibits hippocampal long-term potentiation and induces memory impairment in adult mice. TNF-α upregulation, microgliosis and upregulation of complement system proteins, C1q and C3, are induced by ZIKV infection. Microglia are found to engulf hippocampal presynaptic terminals during acute infection. Neutralization of TNF-α signaling, blockage of microglial activation or of C1q/C3 prevent synapse and memory impairment in ZIKV-infected mice. Results suggest that ZIKV induces synapse and memory dysfunction via aberrant activation of TNF-α, microglia and complement. Our findings establish a mechanism by which ZIKV affects the adult brain, and point to the need of evaluating cognitive deficits as a potential comorbidity in ZIKV-infected adults.

Interesterified fat or palm oil as substitutes for partially hydrogenated fat in maternal diet can predispose obesity in adult male offspring.

BACKGROUND & AIMS: Palm oil (PO) and interesterified fat (IF) have been used to replace partially hydrogenated fat (PHF), which is rich in trans isomers, in processed foods. The purpose of this study was to investigate whether normolipidic diets containing PHF, IF, or PO consumed during pregnancy and lactation affect total body adiposity and adipose tissue morphology of adult offspring mice. METHODS: Four groups of female C57BL/6 mice were fed, during pregnancy and lactation, a control diet (control group, CG), a PHF diet (trans group, TG), a PO diet (PG group), or an IF diet (IG group). After weaning (at 21 days), male pups received the control diet for 70 days. Food intake and body weight were monitored in all groups throughout the experimental period. At 3 months of age, mice were sacrificed and the inguinal (IWAT), epididymal (EWAT), retroperitoneal (RPWAT), and mesenteric (MWAT) adipose fat pads were removed and weighed. Adiposity was quantified by micro computed tomography (micro-CT), and adipocyte areas and cell number were analyzed by histology. RESULTS: PG and IG offspring gained more weight than CG and TG groups (p < 0.01) during the first 10 weeks after weaning, resulting in higher final body weights (p < 0.05). IG mice and PG mice had respectively heavier EWAT and IWAT than TG and CG mice. Micro-CT scanning revealed that the total volumes of internal, external, and total fat depots were greater in IG animals, as compared to the other groups. Larger adipocyte areas were observed in EWAT and IWAT in IG and TG, respectively, in comparison to CG and PG mice. PG mice showed increased adipocyte numbers in IWAT. CONCLUSIONS: Maternal intake of IF and/or PO during pregnancy and lactation predisposes the offspring to the development of obesity in adult life in mice.

Interesterified fat or palm oil as substitutes for partially hydrogenated fat during the perinatal period produces changes in the brain fatty acids profile and increases leukocyte-endothelial interactions in the cerebral microcirculation from the male offspring in adult life.

We investigated whether maternal intake of normolipidic diets with distinct fatty acid (FA) compositions alters the lipidic profile and influences the inflammatory status of the adult offsprings׳ brains. C57BL/6 female mice during pregnancy and lactation received diets containing either soybean oil (CG), partially hydrogenated vegetable fat rich in trans-fatty acids (TG), palm oil (PG), or interesterified fat (IG). After weaning, male offspring from all groups received control diet. The FA profile was measured in the offspring׳s brains at post-natal days 21 and 90. Brain functional capillary density as well as leukocyte-endothelial interactions in the cerebral post-capillary venules was assessed by intravital fluorescence microscopy at post-natal day 90. Inflammation signaling was evaluated through toll-like receptor 4 (TLR4) content in brain of the adult offspring. In the 21-day old offspring, the brains of the TG showed higher levels of trans FA and reduced levels of linoleic acid (LA) and total n-6 polyunsaturated fatty acids (PUFA). At post-natal day 90, TG and IG groups showed reduced levels of eicosapentaenoic acid (EPA) and total n-3 PUFA tended to be lower compared to CG. The offspring׳s brains exhibited an altered microcirculation with increased leukocyte rolling in groups TG, PG and IG and in TG group increased leukocyte adhesion. The TLR4 content of TG, IG and PG groups only tended to increase (23%; 20% and 35%, respectively). Maternal consumption of trans FA, palm oil or interesterified fat during pregnancy and lactation can trigger the initial steps of inflammatory pathways in the brain of offspring in adulthood.