Brain Imaging Investigation of the Memory-Enhancing Effect of Emotion

Emotional events tend to be better remembered than non-emotional events^1,2. One goal of cognitive and affective neuroscientists is to understand the neural mechanisms underlying this enhancing effect of emotion on memory. A method that has proven particularly influential in the investigation of the memory-enhancing effect of emotion is the so-called subsequent memory paradigm (SMP). This method was originally used to investigate the neural correlates of non-emotional memories³, and more recently we and others also applied it successfully to studies of emotional memory (reviewed in^{4, 5-7}). Here, we describe a protocol that allows investigation of the neural correlates of the memory-enhancing effect of emotion using the SMP in conjunction with event-related functional magnetic resonance imaging (fMRI). An important feature of the SMP is that it allows separation of brain activity specifically associated with memory from more general activity associated with perception. Moreover, in the context of investigating the impact of emotional stimuli, SMP allows identification of brain regions whose activity is susceptible to emotional modulation of both general/perceptual and memory-specific processing. This protocol can be used in healthy subjects^8-15, as well as in clinical patients where there are alterations in the neural correlates of emotion perception and biases in remembering emotional events, such as those suffering from depression and post-traumatic stress disorder (PTSD)^{16, 17}. Download video file.^{(95M, mov)}     

Prediction and prenosological diagnostics of heart diseases based on energy characteristics of acupuncture points and fuzzy logic

Many theories of reflexology use ancient concepts which do not coincide with the modern medical terminology of anatomy, physiology and biophysics. This substantially reduces the trust of physicians in reflexology methods. During this research, several mathematical models for the interaction of the internal and biological active points of meridian structures have been proposed. The analysis of these models allows the specification of a list of heart diseases for which reflex diagnostics and reflex therapy methods are most effective and also allows increasing the effectiveness of these procedures. It is shown that good results for the prediction and early diagnosis of diseases from the reaction energy of biologically active points (acupuncture points) are obtained using fuzzy logic decision making.     

How old are you—Evaluation of age reading methods for the invasive round goby (Neogobius melanostomus,Pallas 1814)

In the Baltic, the first observation of the round goby (Neogobius melanostomus, Pallas 1814) was made in 1990. Within the past decade the species became invasive and spread rapidly throughout the Baltic Sea. Studies about the fishes potential impacts on resident species promote the need for an increasing knowledge of their basic stock structures such as growth rates, longevity and mortality, which all rely on accurate estimates of age. Former studies on the round goby have used several different age reading techniques. In this study, we compared three standard otolith preparation methods for ageing and present the best procedure for the invasive round goby. The results showed significant differences in age estimates of the same fish between the different preparation methods and between readers. The estimation of the first annulus, the first year, was the most problematic. The overall agreement was lowest when reading the whole otoliths while the best performance was achieved with sectioned and stained preparation method. Depending on method used the growth estimates also differed. The results question comparability between previous studies and highlight the importance of harmonised aging procedures for the round goby for obtaining correct estimates of population parameters such as growth rate, age at maturity, and longevity.     

Human amylin proteotoxicity impairs protein biosynthesis,and alters major cellular signaling pathways in the heart,brain and liver of humanized diabetic rat model in vivo

Introduction

Chronic hypersecretion of the 37 amino acid amylin is common in type 2 diabetics (T2D). Recent studies implicate human amylin aggregates cause proteotoxicity (cell death induced by misfolded proteins) in both the brain and the heart.

Objectives

Identify systemic mechanisms/markers by which human amylin associated with cardiac and brain defects might be identified.

Methods

We investigated the metabolic consequences of amyloidogenic and cytotoxic amylin oligomers in heart, brain, liver, and plasma using non-targeted metabolomics analysis in a rat model expressing pancreatic human amylin (HIP model).

Results

Four metabolites were significantly different in three or more of the four compartments (heart, brain, liver, and plasma) in HIP rats. When compared to a T2D rat model, HIP hearts uniquely had significant DECREASES in five amino acids (lysine, alanine, tyrosine, phenylalanine, serine), with phenylalanine decreased across all four tissues investigated, including plasma. In contrast, significantly INCREASED circulating phenylalanine is reported in diabetics in multiple recent studies.

Conclusion

DECREASED phenylalanine may serve as a unique marker of cardiac and brain dysfunction due to hyperamylinemia that can be differentiated from alterations in T2D in the plasma. While the deficiency in phenylalanine was seen across tissues including plasma and could be monitored, reduced tyrosine was seen only in the brain. The 50 % reduction in phenylalanine and tyrosine in HIP brains is significant given their role in supporting brain chemistry as a precursor for catecholamines (dopamine, norepinephrine, epinephrine), which may contribute to the increased morbidity and mortality in diabetics at a multi-system level beyond the effects on glucose metabolism.

     

Comparison of two methods for one-step in-straw thawing and direct transfer of cattle blastocysts

The aim of this experiment was to evaluate the feasibility of dilution of the cryoprotectant by a sucrose solution in the straw followed by direct transfer without any selection of the embryos. A comparison was made between the method described by Renard et al. (7) and a method slightly modified from that published by Leibo (8). The feasibility is proved by a mean pregnancy rate of 41.4%. Although the difference was not statistically significant, the method described by Renard et al. (7) led to a higher pregnancy rate (44.7%) than that of Leibo (8) (38.5%).     

Co-occurrence of denitrification and nitrogen fixation in a meromictic lake, Lake Cadagno (Switzerland)

The nitrogen cycling of Lake Cadagno was investigated by using a combination of biogeochemical and molecular ecological techniques. In the upper oxic freshwater zone inorganic nitrogen concentrations were low (up to ～3.4 μM nitrate at the base of the oxic zone), while in the lower anoxic zone there were high concentrations of ammonium (up to 40 μM). Between these zones, a narrow zone was characterized by no measurable inorganic nitrogen, but high microbial biomass (up to 4 × 10⁷ cells ml^?1). Incubation experiments with ¹⁵N‐nitrite revealed nitrogen loss occurring in the chemocline through denitrification (～3 nM N h^?1). At the same depth, incubations experiments with ¹⁵N₂‐ and ¹³C_DIC‐labelled bicarbonate, indicated substantial N₂ fixation (31.7–42.1 pM h^?1) and inorganic carbon assimilation (40–85 nM h^?1). Catalysed reporter deposition fluorescence in situ hybridization (CARD‐FISH) and sequencing of 16S rRNA genes showed that the microbial community at the chemocline was dominated by the phototrophic green sulfur bacterium Chlorobium clathratiforme. Phylogenetic analyses of the nifH genes expressed as mRNA revealed a high diversity of N₂ fixers, with the highest expression levels right at the chemocline. The majority of N₂ fixers were related to Chlorobium tepidum/C. phaeobacteroides. By using Halogen In Situ Hybridization‐Secondary Ion Mass Spectroscopy (HISH‐SIMS), we could for the first time directly link Chlorobium to N₂ fixation in the environment. Moreover, our results show that N₂ fixation could partly compensate for the N loss and that both processes occur at the same locale at the same time as suggested for the ancient Ocean.     

Mechanism of Activation and Functional Role of Protein Kinase C�� in Human Platelets

The novel class of protein kinase C (nPKC) isoform η is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCη using pharmacological and gene knock-out approaches. nPKCη was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y₁ receptor antagonist, or YM-254890, a G_q blocker, abolished 2MeSADP-induced phosphorylation of nPKCη. Similarly, ADP failed to activate nPKCη in platelets isolated from P2Y₁ and G_q knock-out mice. However, pretreatment of platelets with P2Y₁₂ receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCη phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCη was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin α_IIbβ₃ mediated outside-in signaling. Moreover, in the presence of SC-57101, a α_IIbβ₃ receptor antagonist, nPKCη dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cγ, a catalytic subunit of serine/threonine phosphatase, α_IIbβ₃ failed to dephosphorylate nPKCη. Thus, we conclude that ADP activates nPKCη via P2Y₁ receptor and is subsequently dephosphorylated by PP1γ phosphatase activated by α_IIbβ₃ integrin. In addition, pretreatment of platelets with η-RACK antagonistic peptides, a specific inhibitor of nPKCη, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCη positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.Platelets are the key cellular components in maintaining hemostasis (1). Vascular injury exposes subendothelial collagen that activates platelets to change shape, secrete contents of granules, generate thromboxane, and finally aggregate via activated α_IIbβ₃ integrin, to prevent further bleeding (2, 3). ADP is a physiological agonist of platelets secreted from dense granules and is involved in feedback activation of platelets and hemostatic plug stabilization (4). It activates two distinct G-protein-coupled receptors (GPCRs) on platelets, P2Y₁ and P2Y₁₂, which couple to G_q and G_i, respectively (5–8). G_q activates phospholipase Cβ (PLCβ), which leads to diacyl glycerol (DAG)² generation and calcium mobilization (9, 10). On the other hand, G_i is involved in inhibition of cAMP levels and PI 3-kinase activation (4, 6). Synergistic activation of G_q and G_i proteins leads to the activation of the fibrinogen receptor integrin α_IIbβ₃. Fibrinogen bound to activated integrin α_IIbβ₃ further initiates feed back signaling (outside-in signaling) in platelets that contributes to the formation of a stable platelet plug (11).Protein kinase Cs (PKCs) are serine/threonine kinases known to regulate various platelet functional responses such as dense granule secretion and integrin α_IIbβ₃ activation (12, 13). Based on their structure and cofactor requirements, PKCs are divided in to three classes: classical (cofactors: DAG, Ca²⁺), novel (cofactors: DAG) and atypical (cofactors: PIP₃) PKC isoforms (14). All the members of the novel class of PKC isoforms (nPKC), viz. nPKC isoforms δ, θ, η, and ε, are expressed in platelets (15), and they require DAG for activation. Among all the nPKCs, PKCδ (15, 16) and PKCθ (17–19) are fairly studied in platelets. Whereas nPKCδ is reported to regulate protease-activated receptor (PAR)-mediated dense granule secretion (15, 20), nPKCθ is activated by outside-in signaling and contributes to platelet spreading on fibrinogen (18). On the other hand, the mechanism of activation and functional role of nPKCη is not addressed as yet.PKCs are cytoplasmic enzymes. The enzyme activity of PKCs is modulated via three mechanisms (14, 21): 1) cofactor binding: upon cell stimulus, cytoplasmic PKCs mobilize to membrane, bind cofactors such as DAG, Ca²⁺, or PIP3, release autoinhibition, and attain an active conformation exposing catalytic domain of the enzyme. 2) phosphorylations: 3-phosphoinositide-dependent kinase 1 (PDK1) on the membrane phosphorylates conserved threonine residues on activation loop of catalytic domain; this is followed by autophosphorylations of serine/threonine residues on turn motif and hydrophobic region. These series of phosphorylations maintain an active conformation of the enzyme. 3) RACK binding: PKCs in active conformation bind receptors for activated C kinases (RACKs) and are lead to various subcellular locations to access the substrates (22, 23). Although various leading laboratories have elucidated the activation of PKCs, the mechanism of down-regulation of PKCs is not completely understood.The premise of dynamic cell signaling, which involves protein phosphorylations by kinases and dephosphorylations by phosphatases has gained immense attention over recent years. PP1, PP2A, PP2B, PHLPP are a few of the serine/threonine phosphatases reported to date. Among them PP1 and PP2 phosphatases are known to regulate various platelet functional responses (24, 25). Furthermore, PP1c, is the catalytic unit of PP1 known to constitutively associate with α_IIb and is activated upon integrin engagement with fibrinogen and subsequent outside-in signaling (26). Among various PP1 isoforms, recently PP1γ is shown to positively regulate platelet functional responses (27). Thus, in this study we investigated if the above-mentioned phosphatases are involved in down-regulation of nPKCη. Furthermore, reports from other cell systems suggest that nPKCη regulates ERK/JNK pathways (28). In platelets ERK is known to regulate agonist induced thromboxane generation (29, 30). Thus, we also investigated if nPKCη regulates ERK phosphorylation and thereby agonist-induced platelet functional responses.In this study, we evaluated the activation of nPKCη downstream of ADP receptors and its inactivation by an integrin-associated phosphatase PP1γ. We also studied if nPKCη regulates functional responses in platelets and found that this isoform regulates ADP-induced thromboxane generation, but not fibrinogen receptor activation in platelets.