Activation of Autophagic Flux against Xenoestrogen Bisphenol-A-induced Hippocampal Neurodegeneration via AMP kinase (AMPK)/Mammalian Target of Rapamycin (mTOR) Pathways

The human health hazards related to persisting use of bisphenol-A (BPA) are well documented. BPA-induced neurotoxicity occurs with the generation of oxidative stress, neurodegeneration, and cognitive dysfunctions. However, the cellular and molecular mechanism(s) of the effects of BPA on autophagy and association with oxidative stress and apoptosis are still elusive. We observed that BPA exposure during the early postnatal period enhanced the expression and the levels of autophagy genes/proteins. BPA treatment in the presence of bafilomycin A₁ increased the levels of LC3-II and SQSTM1 and also potentiated GFP-LC3 puncta index in GFP-LC3-transfected hippocampal neural stem cell-derived neurons. BPA-induced generation of reactive oxygen species and apoptosis were mitigated by a pharmacological activator of autophagy (rapamycin). Pharmacological (wortmannin and bafilomycin A₁) and genetic (beclin siRNA) inhibition of autophagy aggravated BPA neurotoxicity. Activation of autophagy against BPA resulted in intracellular energy sensor AMP kinase (AMPK) activation, increased phosphorylation of raptor and acetyl-CoA carboxylase, and decreased phosphorylation of ULK1 (Ser-757), and silencing of AMPK exacerbated BPA neurotoxicity. Conversely, BPA exposure down-regulated the mammalian target of rapamycin (mTOR) pathway by phosphorylation of raptor as a transient cell''s compensatory mechanism to preserve cellular energy pool. Moreover, silencing of mTOR enhanced autophagy, which further alleviated BPA-induced reactive oxygen species generation and apoptosis. BPA-mediated neurotoxicity also resulted in mitochondrial loss, bioenergetic deficits, and increased PARKIN mitochondrial translocation, suggesting enhanced mitophagy. These results suggest implication of autophagy against BPA-mediated neurodegeneration through involvement of AMPK and mTOR pathways. Hence, autophagy, which arbitrates cell survival and demise during stress conditions, requires further assessment to be established as a biomarker of xenoestrogen exposure.     

Blastomere ablation and the developmental origin of identified monoamine-containing neurons in the leech

Ablation of different identifiable blastomeres of the early embryo of the leech Helobdella triserialis was found to lead to the absence of different sets of segmentally iterated monoamine-containing neurons in subsequent development. Thus the ablation of one of the paired N ectoteloblasts leads to the absence of one member of each of the three bilateral pairs of serotonin-containing neurons (one of which is the Retzius cell) from each segmental ganglion. The ablation of one of the paired OP blastomeres (precursors of the paired O and P ectoteloblasts) leads to the absence of one member of each of the two bilateral pairs of lateral dopamine-containing neurons that lie in the body wall of each segment. And the ablation of one of the paired Q ectoteloblast leads to the absence of one member of the bilateral pair of medial dopamine-containing neurons that lie in the body wall of each segment. These results suggest that each of these sets of monoamine-containing neurons is derived from a particular blastomere. Upon ablation of that blastomere the set does not develop from any other source.     

Cleavage of the signaling mucin Msb2 by the aspartyl protease Yps1 is required for MAPK activation in yeast

Signaling mucins are cell adhesion molecules that activate RAS/RHO guanosine triphosphatases and their effector mitogen-activated protein kinase (MAPK) pathways. We found that the Saccharomyces cerevisiae mucin Msb2p, which functions at the head of the Cdc42p-dependent MAPK pathway that controls filamentous growth, is processed into secreted and cell-associated forms. Cleavage of the extracellular inhibitory domain of Msb2p by the aspartyl protease Yps1p generated the active form of the protein by a mechanism incorporating cellular nutritional status. Activated Msb2p functioned through the tetraspan protein Sho1p to induce MAPK activation as well as cell polarization, which involved the Cdc42p guanine nucleotide exchange factor Cdc24p. We postulate that cleavage-dependent activation is a general feature of signaling mucins, which brings to light a novel regulatory aspect of this class of signaling adhesion molecule.     

Evaluation of the eutomer of 4-{3-(4-chlorophenyl)-3-hydroxypyrrolidin-1-yl}-1-(4-fluorophenyl)butan-1-one, {(+)-SYA 09}, a pyrrolidine analog of haloperidol

Enantiomeric separation of the racemic 4-{3-(4-chlorophenyl)-3-hydroxypyrrolidin-1-yl}-1-(4-fluorophenyl)butan-1-one, a pyrrolidine analog of haloperidol, {(+/-)-SYA 09}, and subsequent binding studies revealed that most of the binding affinity at dopamine and serotonin receptors resides in the (+)-isomer {(+)-SYA 09} or the eutomer. Further pharmacological evaluation of the eutomer revealed that it has a higher affinity for the dopamine D4 (DAD4) receptor subtype (Ki = 3.6 nM) than for the DAD2 subtype (Ki = 51.1 nM) with a ratio of 14.2 (D2Ki/D4Ki ratio = 14.2). In an animal model of antipsychotic efficacy, the (+)-SYA 09 was efficacious with an ED50 value of 1.6 mg/kg, i.p., and at twice this value, (+)-SYA 09 did not induce significant catalepsy in rats.     

Proportions and variability in the Indian lorises: Bone weight observations onLoris tardigradus lydekkerianus andNycticebus coucang

Thirty uniformly prepared, disarticulated complete skeletons of adultLoris tardigradus lydekkerianus andNycticebus coucang have been weighed for preliminary study of the weight relations between different major skeletal parts. In these forms, combined relative weight of the precaudal vertebrae, ribs and sternum surpasses the averages for the rest of the skeletal parts. The weight of the skull equals one-fifth of the total skeletal weight inLoris andNycticebus. The weight of the skeletons of the limbs (including the shoulder girdle and the hip bones) contributes less than half of the total skeletal weight in lorises. The bones of the upper limbs are lighter than those of the lower limbs in the 2 series examined.Loris andNycticebus are distinguished by having the proportionately heaviest axial skeleton and skull and the lowest relative weights of the bones of the 4 limbs. The combined relative skeletal weight of the upper and lower limbs ranges between 45.34 and 50.01 inLoris andNycticebus. The relative weight of the skeletons of the hands and feet are almost similar to the corresponding weights in the 2 series.As far as the weights of the different skeletal parts are concerned, there is no relative asymmetry in the 2 series ofLoris andNycticebus examined.     

Docosahexaenoic acid-induced unfolded protein response, cell cycle arrest, and apoptosis in vascular smooth muscle cells are triggered by Ca²⁺-dependent induction of oxidative stress

Proliferation of vascular smooth muscle cells is a characteristic of pathological vascular remodeling and represents a significant therapeutic challenge in several cardiovascular diseases. Docosahexaenoic acid (DHA), a member of the n-3 polyunsaturated fatty acids, was shown to inhibit proliferation of numerous cell types, implicating several different mechanisms. In this study we examined the molecular events underlying the inhibitory effects of DHA on proliferation of primary human smooth muscle cells isolated from small pulmonary artery (hPASMCs). DHA concentration-dependently inhibited hPASMC proliferation, induced G1 cell cycle arrest, and decreased cyclin D1 protein expression. DHA activated the unfolded protein response (UPR), evidenced by increased mRNA expression of HSPA5, increased phosphorylation of eukaryotic initiation factor 2α, and splicing of X-box binding protein 1. DHA altered cellular lipid composition and led to increased reactive oxygen species (ROS) production. DHA-induced ROS were dependent on both intracellular Ca(2+) release and entry of extracellular Ca(2+). Overall cellular ROS and mitochondrial ROS were decreased by RU360, a specific inhibitor of mitochondrial Ca(2+) uptake. DHA-induced mitochondrial dysfunction was evidenced by decreased mitochondrial membrane potential and decreased cellular ATP content. DHA triggered apoptosis as found by increased numbers of cleaved caspase-3- and TUNEL-positive cells. The free radical scavenger Tempol counteracted DHA-induced ROS, cell cycle arrest, induction of UPR, and apoptosis. We conclude that Ca(2+)-dependent oxidative stress is the central and initial event responsible for induction of UPR, cell cycle arrest, and apoptosis in DHA-treated hPASMCs.     

Protective impacts of household-based tuberculosis contact tracing are robust across endemic incidence levels and community contact patterns

There is an emerging consensus that achieving global tuberculosis control targets will require more proactive case finding approaches than are currently used in high-incidence settings. Household contact tracing (HHCT), for which households of newly diagnosed cases are actively screened for additional infected individuals is a potentially efficient approach to finding new cases of tuberculosis, however randomized trials assessing the population-level effects of such interventions in settings with sustained community transmission have shown mixed results. One potential explanation for this is that household transmission is responsible for a variable proportion of population-level tuberculosis burden between settings. For example, transmission is more likely to occur in households in settings with a lower tuberculosis burden and where individuals mix preferentially in local areas, compared with settings with higher disease burden and more dispersed mixing. To better understand the relationship between endemic incidence levels, social mixing, and the impact of HHCT, we developed a spatially explicit model of coupled household and community transmission. We found that the impact of HHCT was robust across settings of varied incidence and community contact patterns. In contrast, we found that the effects of community contact tracing interventions were sensitive to community contact patterns. Our results suggest that the protective benefits of HHCT are robust and the benefits of this intervention are likely to be maintained across epidemiological settings.     

Oxidative damage of albumin in advanced liver disease

Albumin has a number of biological functions and the serum albumin level is related to prognosis in advanced liver disease. Oxidative stress is believed to play an important role in the pathogenesis of liver failure. The aim of the present study was to characterize oxidative modification of albumin in patients with various degrees of liver failure and to investigate implications for its binding function. Patients with liver cirrhosis (n=10), acute-on-chronic liver failure (n=8) and healthy controls (n=15) were included in the study. Three fractions of albumin were separated by HPLC according to the redox state of cysteine-34 and detected by fluorescence as well as UV absorption. Carbonyl groups were measured as a marker of oxidative modification in plasma proteins and, by western blotting, on albumin. Progressive oxidative modification of albumin was found with increasing severity of liver failure indicated by an increased content of carbonyl groups and oxidation of cysteine-34. Fluorescence properties of albumin were altered by oxidation and, in patients with acute-on-chronic liver failure, by high plasma levels of bilirubin. This alteration of albumin fluorescence by bilirubin provides evidence for a preferred binding of bilirubin to the fully reduced form of albumin.     

Single-Stranded siRNAs Activate RNAi in Animals

The therapeutic utility of siRNAs is limited by the requirement for complex formulations to deliver them to tissues. If potent single-stranded RNAs could be identified, they would provide a simpler path to pharmacological agents. Here, we describe single-stranded siRNAs (ss-siRNAs) that silence gene expression in animals absent lipid formulation. Effective ss-siRNAs were identified by iterative design by determining structure-activity relationships correlating chemically modified single strands and Argonaute 2 (AGO2) activities, potency in cells, nuclease stability, and pharmacokinetics. We find that the passenger strand is not necessary for potent gene silencing. The guide-strand activity requires AGO2, demonstrating action through the RNAi pathway. ss-siRNA action requires a 5' phosphate to achieve activity in?vivo, and we developed a metabolically stable 5'-(E)-vinylphosphonate (5'-VP) with conformation and sterioelectronic properties similar to the natural phosphate. Identification of potent ss-siRNAs offers an additional option for RNAi therapeutics and an alternate perspective on RNAi mechanism.