Prenatal Stress Down-Regulates Reelin Expression by Methylation of Its Promoter and Induces Adult Behavioral Impairments in Rats

Prenatal stress causes predisposition to cognitive and emotional disturbances and is a risk factor towards the development of neuropsychiatric conditions like depression, bipolar disorders and schizophrenia. The extracellular protein Reelin, expressed by Cajal-Retzius cells during cortical development, plays critical roles on cortical lamination and synaptic maturation, and its deregulation has been associated with maladaptive conditions. In the present study, we address the effect of prenatal restraint stress (PNS) upon Reelin expression and signaling in pregnant rats during the last 10 days of pregnancy. Animals from one group, including control and PNS exposed fetuses, were sacrificed and analyzed using immunohistochemical, biochemical, cell biology and molecular biology approaches. We scored changes in the expression of Reelin, its signaling pathway and in the methylation of its promoter. A second group included control and PNS exposed animals maintained until young adulthood for behavioral studies. Using the optical dissector, we show decreased numbers of Reelin-positive neurons in cortical layer I of PNS exposed animals. In addition, neurons from PNS exposed animals display decreased Reelin expression that is paralleled by changes in components of the Reelin-signaling cascade, both in vivo and in vitro. Furthermore, PNS induced changes in the DNA methylation levels of the Reelin promoter in culture and in histological samples. PNS adult rats display excessive spontaneous locomotor activity, high anxiety levels and problems of learning and memory consolidation. No significant visuo-spatial memory impairment was detected on the Morris water maze. These results highlight the effects of prenatal stress on the Cajal-Retzius neuronal population, and the persistence of behavioral consequences using this treatment in adults, thereby supporting a relevant role of PNS in the genesis of neuropsychiatric diseases. We also propose an in vitro model that can yield new insights on the molecular mechanisms behind the effects of prenatal stress.     

Atrial Fibrillation Complicated by Heart Failure Induces Distinct Remodeling of Calcium Cycling Proteins

Atrial fibrillation (AF) and heart failure (HF) are two of the most common cardiovascular diseases. They often coexist and account for significant morbidity and mortality. Alterations in cellular Ca²⁺ homeostasis play a critical role in AF initiation and maintenance. This study was designed to specifically elucidate AF-associated remodeling of atrial Ca²⁺ cycling in the presence of mild HF. AF was induced in domestic pigs by atrial burst pacing. The animals underwent electrophysiologic and echocardiographic examinations. Ca²⁺ handling proteins were analyzed in right atrial tissue obtained from pigs with AF (day 7; n = 5) and compared to sinus rhythm (SR) controls (n = 5). During AF, animals exhibited reduction of left ventricular ejection fraction (from 73% to 58%) and prolonged atrial refractory periods. AF and HF were associated with suppression of protein kinase A (PKA)_RII (-62%) and Ca²⁺-calmodulin-dependent kinase II (CaMKII) δ by 37%, without changes in CaMKIIδ autophosphorylation. We further detected downregulation of L-type calcium channel (LTCC) subunit α₂ (-75%), sarcoplasmic reticulum Ca²⁺-ATPase (Serca) 2a (-29%), phosphorylated phospholamban (Ser16, -92%; Thr17, -70%), and phospho-ryanodine receptor 2 (RyR2) (Ser2808, -62%). Na⁺-Ca²⁺ exchanger (NCX) levels were upregulated (+473%), whereas expression of Ser2814-phosphorylated RyR2 and LTCCα_1c subunits was not significantly altered. In conclusion, AF produced distinct arrhythmogenic remodeling of Ca²⁺ handling in the presence of tachycardia-induced mild HF that is different from AF without structural alterations. The changes may provide a starting point for personalized approaches to AF treatment.     

Introgression of mitochondrial DNA among lineages in a hybridogenetic ant

We report a remarkable pattern of incongruence between nuclear and mitochondrial variations in a social insect, the desert ant Cataglyphis hispanica. This species reproduces by social hybridogenesis. In all populations, two distinct genetic lineages coexist; non-reproductive workers develop from hybrid crosses between the lineages, whereas reproductive offspring (males and new queens) are typically produced asexually by parthenogenesis. Genetic analyses based on nuclear markers revealed that the two lineages remain highly differentiated despite constant hybridization for worker production. Here, we show that, in contrast with nuclear DNA, mitochondrial DNA (mtDNA) does not recover the two lineages as monophyletic. Rather, mitochondrial haplotypes cluster according to their geographical origin. We argue that this cytonuclear incongruence stems from introgression of mtDNA among lineages, and review the mechanisms likely to explain this pattern under social hybridogenesis.     

An industrial scale process for the enzymatic removal of steryl glucosides from biodiesel

Background

Biodiesels produced from transesterification of vegetable oils have a major quality problem due to the presence of precipitates, which need to be removed to avoid clogging of filters and engine failures. These precipitates have been reported to be mostly composed of steryl glucosides (SGs), but so far industrial cost-effective methods to remove these compounds are not available. Here we describe a novel method for the efficient removal of SGs from biodiesel, based on the hydrolytic activity of a thermostable β-glycosidase obtained from Thermococcus litoralis.

Results

A steryl glucosidase (SGase) enzyme from T. litoralis was produced and purified from Escherichia coli cultures expressing a synthetic gene, and used to treat soybean-derived biodiesel. Several optimization steps allowed for the selection of optimal reaction conditions to finally provide a simple and efficient process for the removal of SGs from crude biodiesel. The resulting biodiesel displayed filterability properties similar to distilled biodiesel according to the total contamination (TC), the cold soak filtration test (CSFT), filter blocking tendency (FBT), and cold soak filter blocking tendency (CSFBT) tests. The process was successfully scaled up to a 20 ton reactor, confirming its adaptability to industrial settings.

Conclusions

The results presented in this work provide a novel path for the removal of steryl glucosides from biodiesel using a cost-effective, environmentally friendly and scalable enzymatic process, contributing to the adoption of this renewable fuel.

     

Impaired Mitochondrial Energy Production Causes Light-Induced Photoreceptor Degeneration Independent of Oxidative Stress

Two insults often underlie a variety of eye diseases including glaucoma, optic atrophy, and retinal degeneration—defects in mitochondrial function and aberrant Rhodopsin trafficking. Although mitochondrial defects are often associated with oxidative stress, they have not been linked to Rhodopsin trafficking. In an unbiased forward genetic screen designed to isolate mutations that cause photoreceptor degeneration, we identified mutations in a nuclear-encoded mitochondrial gene, ppr, a homolog of human LRPPRC. We found that ppr is required for protection against light-induced degeneration. Its function is essential to maintain membrane depolarization of the photoreceptors upon repetitive light exposure, and an impaired phototransduction cascade in ppr mutants results in excessive Rhodopsin1 endocytosis. Moreover, loss of ppr results in a reduction in mitochondrial RNAs, reduced electron transport chain activity, and reduced ATP levels. Oxidative stress, however, is not induced. We propose that the reduced ATP level in ppr mutants underlies the phototransduction defect, leading to increased Rhodopsin1 endocytosis during light exposure, causing photoreceptor degeneration independent of oxidative stress. This hypothesis is bolstered by characterization of two other genes isolated in the screen, pyruvate dehydrogenase and citrate synthase. Their loss also causes a light-induced degeneration, excessive Rhodopsin1 endocytosis and reduced ATP without concurrent oxidative stress, unlike many other mutations in mitochondrial genes that are associated with elevated oxidative stress and light-independent photoreceptor demise.     

A Voltage-Gated Calcium Channel Regulates Lysosomal Fusion with Endosomes and Autophagosomes and Is Required for Neuronal Homeostasis

Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis.     

Sustainability of Evidence-Based Practices for HIV Prevention among Female Sex Workers in Mexico

Objective

This study examined service provider perceptions of requirements for successful sustainment of an efficacious intervention for preventing HIV/AIDS and STIs in female sex workers (FSWs) in Mexico.

Methods

Semi-structured interviews were conducted with 77 leaders and counselors from 12 community-based reproductive health clinics located throughout Mexico participating in a large hybrid effectiveness-implementation randomized controlled trial to scale-up the use of Mujer Segura, a psychoeducational intervention designed to promote condom use and enhance safer sex negotiation skills among FSWs.

Results

Five sets of requirements for sustainment were identified: 1) characteristics of the provider, including competence in delivering the intervention, need for continued technical support and assistance from outside experts, and satisfaction with addressing the needs of this population; 2) characteristics of the clients (i.e., FSWs), including client need and demand for services and incentives for participation; 3) characteristics of the organization, including its mission, benefits, and operations; 4) characteristics of the outer setting, including financial support and relationship with the community-based organization’s central offices, and transportation and security in areas where FSWs live and work; and 5) outcomes associated with the intervention itself, including a reduction of risk through education and increased outreach through referrals from FSWs who received the intervention.

Conclusions

Although the requirements for successful sustainment of interventions like Mujer Segura are consistent with the factors identified in many models of implementation, the results illustrate the importance of local context in assigning priority to these model elements and suggest that the five categories are not discrete entities but interconnected.     

Effects of Partial Replacement of Corn with Glycerin on Ruminal Fermentation in a Dual-Flow Continuous Culture System

The objective of this study was to evaluate the effects of partially replacing dry ground corn with glycerin on ruminal fermentation using a dual-flow continuous culture system. Six fermenters (1,223 ± 21 ml) were used in a replicated 3x3 Latin square arrangement with three periods of 10 d each, with 7 d for diet adaptation and 3 d for sample collections. All diets contained 75% concentrate and three dietary glycerin levels (0, 15, and 30% on dry matter basis), totaling six replicates per treatment. Fermenters were fed 72 g of dry matter/d equally divided in two meals/d, at 0800 and 2000 h. Solid and liquid dilution rates were adjusted daily to 5.5 and 11%/h, respectively. On d 8, 9, and 10, samples of 500 ml of solid and liquid digesta effluent were mixed, homogenized, and stored at -20°C. Subsamples of 10 ml were collected and preserved with 0.2 mL of a 50% H₂SO₄ solution for later determination of NH₃-N and volatile fatty acids. Microbial biomass was isolated from fermenters for chemical analysis at the end of each experimental period. Data were analyzed using the MIXED procedure in SAS with α = 0.05. Glycerin levels did not affect apparent digestibility of DM (P _Lin. = 0.13; P _Quad. = 0.40), OM (P _Lin. = 0.72; P _Quad. = 0.15), NDF (P _Lin. = 0.38; P _Quad. = 0.50) and ADF (P _Lin. = 0.91; P _Quad. = 0.18). Also, glycerin inclusion did not affect true digestibility of DM (P _Lin. = 0.35; P _Quad. = 0.48), and OM (P _Lin. = 0.08; P _Quad. = 0.19). Concentrations of propionate (P < 0.01) and total volatile fatty acids (P < 0.01) increased linearly and concentrations of acetate (P < 0.01), butyrate (P = 0.01), iso-valerate (P < 0.01), and total branched-chain volatile fatty acids, as well as the acetate: propionate ratio (P < 0.01) decreased with glycerin inclusion. Linear increases on NH₃-N concentration in digesta effluent (P < 0.01) and on NH₃-N flow (P < 0.01) were observed due to glycerin inclusion in the diets. Crude protein digestibility (P = 0.04) and microbial N flow (P = 0.04) were greater in the control treatment compared with the other treatments and responded quadratically with glycerin inclusion. Furthermore, the inclusion of glycerin linearly decreased (P = 0.02) non-ammonia N flow. Glycerin levels did not affect the flows of total N (P _Lin. = 0.79; P _Quad. = 0.35), and dietary N (P _Lin. = 0.99; P _Quad. = 0.07), as well as microbial efficiency (P _Lin. = 0.09; P _Quad. = 0.07). These results suggest that partially replacing dry ground corn with glycerin may change ruminal fermentation, by increasing total volatile fatty acids, and propionate concentration without affecting microbial efficiency, which may improve glucogenic potential of beef cattle diets.