Structural Basis for the Function of the C-Terminal Proton Release Pathway in the Calcium Pump

The calcium pump (sarco/endoplasmic reticulum Ca²⁺-ATPase, SERCA) plays a major role in calcium homeostasis in muscle cells by clearing cytosolic Ca²⁺ during muscle relaxation. Active Ca²⁺ transport by SERCA involves the structural transition from a low-Ca²⁺ affinity E2 state toward a high-Ca²⁺ affinity E1 state of the pump. This structural transition is accompanied by the countertransport of protons to stabilize the negative charge and maintain the structural integrity of the transport sites and partially compensate for the positive charges of the two Ca²⁺ ions passing through the membrane. X-ray crystallography studies have suggested that a hydrated pore located at the C-terminal domain of SERCA serves as a conduit for proton countertransport, but the existence and function of this pathway have not yet been fully characterized. We used atomistic simulations to demonstrate that in the protonated E2 state and the absence of initially bound water molecules, the C-terminal pore becomes hydrated in the nanosecond timescale. Hydration of the C-terminal pore is accompanied by the formation of water wires that connect the transport sites with the cytosol. Water wires are known as ubiquitous proton-transport devices in biological systems, thus supporting the notion that the C-terminal domain serves as a conduit for proton release. Additional simulations showed that the release of a single proton from the transport sites induces bending of transmembrane helix M5 and the interaction between residues Arg762 and Ser915. These structural changes create a physical barrier against full hydration of the pore and prevent the formation of hydrogen-bonded water wires once proton transport has occurred through this pore. Together, these findings support the notion that the C-terminal proton release pathway is a functional element of SERCA and also provide a mechanistic model for its operation in the catalytic cycle of the pump.     

Map management for robust long-term visual localization of an autonomous shuttle in changing conditions

Multimedia Tools and Applications - Changes in appearance present a tremendous problem for the visual localization of an autonomous vehicle in outdoor environments. Data association between the...     

Quantitative Proteomic Approach Reveals Altered Metabolic Pathways in Response to the Inhibition of Lysine Deacetylases in A549 Cells under Normoxia and Hypoxia

Growing evidence is showing that acetylation plays an essential role in cancer, but studies on the impact of KDAC inhibition (KDACi) on the metabolic profile are still in their infancy. Here, we analyzed, by using an iTRAQ-based quantitative proteomics approach, the changes in the proteome of KRAS-mutated non-small cell lung cancer (NSCLC) A549 cells in response to trichostatin-A (TSA) and nicotinamide (NAM) under normoxia and hypoxia. Part of this response was further validated by molecular and biochemical analyses and correlated with the proliferation rates, apoptotic cell death, and activation of ROS scavenging mechanisms in opposition to the ROS production. Despite the differences among the KDAC inhibitors, up-regulation of glycolysis, TCA cycle, oxidative phosphorylation and fatty acid synthesis emerged as a common metabolic response underlying KDACi. We also observed that some of the KDACi effects at metabolic levels are enhanced under hypoxia. Furthermore, we used a drug repositioning machine learning approach to list candidate metabolic therapeutic agents for KRAS mutated NSCLC. Together, these results allow us to better understand the metabolic regulations underlying KDACi in NSCLC, taking into account the microenvironment of tumors related to hypoxia, and bring new insights for the future rational design of new therapies.     

Functional Dysregulations in CA1 Hippocampal Networks of a 3-Hit Mouse Model of Schizophrenia

For a better translation from treatment designs of schizophrenia to clinical efficiency, there is a crucial need to refine preclinical animal models. In order to consider the multifactorial nature of the disorder, a new mouse model associating three factors (genetic susceptibility—partial deletion of the MAP6 gene, early-life stress—maternal separation, and pharmacological treatment—chronic Δ-9-tetrahydrocannabinol during adolescence) has recently been described. While this model depicts a schizophrenia-like phenotype, the neurobiological correlates remain unknown. Synaptic transmission and functional plasticity of the CA1 hippocampal region of male and female 3-hit mice were therefore investigated using electrophysiological recordings on the hippocampus slice. While basal excitatory transmission remained unaffected, NMDA receptor (NMDAr)-mediated long-term potentiation (LTP) triggered by theta-burst (TBS) but not by high-frequency (HFS) stimulation was impaired in 3-hit mice. Isolated NMDAr activation was not affected or even increased in female 3-hit mice, revealing a sexual dimorphism. Considering that the regulation of LTP is more prone to inhibitory tone if triggered by TBS than by HFS, the weaker potentiation in 3-hit mice suggests a deficiency of intrinsic GABA regulatory mechanisms. Indeed, NMDAr activation was increased by GABA_A receptor blockade in wild-type but not in 3-hit mice. This electrophysiological study highlights dysregulations of functional properties and plasticity in hippocampal networks of 3-hit mice, one of the mechanisms suspected to contribute to the pathophysiology of schizophrenia. It also shows differences between males and females, supporting the sexual dimorphism observed in the disorder. Combined with the previously reported study, the present data reinforce the face validity of the 3-hit model that will help to consider new therapeutic strategies for psychosis.     

Implementing Qubits with Superconducting Integrated Circuits

Superconducting qubits are solid state electrical circuits fabricated using techniques borrowed from conventional integrated circuits. They are based on the Josephson tunnel junction, the only non-dissipative, strongly non-linear circuit element available at low temperature. In contrast to microscopic entities such as spins or atoms, they tend to be well coupled to other circuits, which make them appealling from the point of view of readout and gate implementation. Very recently, new designs of superconducting qubits based on multi-junction circuits have solved the problem of isolation from unwanted extrinsic electromagnetic perturbations. We discuss in this review how qubit decoherence is affected by the intrinsic noise of the junction and what can be done to improve it. PACS: 03.67.-a, 03.65.Yz, 85.25.-j, 85.35.Gv     

Inhibition of intratracheal lung cancer development by systemic delivery of E1A

Amplification or overexpression of HER-2/neu in human lung cancer has been correlated with poor prognosis and chemoresistance. We have previously reported that the adenovirus type 5 early region 1A (E1A) gene product can suppress HER-2/neu-mediated transformation phenotypes through inhibition of HER-2/neu expression. To find an efficient way to treat HER-2/neu-overexpressing lung cancer with E1A, a replication-deficient adenovirus containing the E1A gene, Ad.E1A(+), was used to transduce E1A into HER-2/neu-overexpressing and low expressing human lung cancer cell lines. Tumour cell growth in vitro and colony formation in soft agarose were greatly inhibited by Ad.E1A(+) transduction in HER-2/neu-overexpressing lung cancer cell lines. In HER-2/neu low expressing cell lines, E1A could not inhibit cell growth in vitro but could reduce the colony formation ability in soft agarose, indicating different effects of E1A in these two types of cancer cells. To test the therapeutic efficacy of E1A to lung cancer by systemic delivery in vivo, tumor-bearing mice were established by intratracheal injection of lung cancer cells and treated by i.v. tail injections of Ad.E1A(+). As a result, Ad.E1A(+) suppressed HER-2/neu overexpression and inhibited intratracheal lung cancer growth. However, no significant tumor suppression effect of Ad.E1A(+) was observed in mice bearing HER-2/neu low expressing cell line when the same therapeutic procedure was followed. Thus, we conclude that systemic delivery of Ad.E1A(+) can efficiently achieve therapeutic effect in HER-2/neu-overexpressing lung cancer in vivo.     

Baclofen: intestinal absorption mechanism in mice

Systemic lupus erythematosus (SLE) is a multisystem organ disease, and involvement of the gastrointestinal system is relatively rare. We describe a 13-year-old girl who presented initially with abdominal pain, diarrhea, edema, and hypoalbuminemia. She was diagnosed with protein losing enteropathy (PLE) based on the significant increase of alpha 1-antitrypsin clearance in the stool. Two weeks after admission she developed clinical and serological findings that fulfilled the ACR criteria for SLE. Over 22 cases of lupus associated PLE have now been reported, but only 3 in children. Children with PLE should be evaluated for SLE. In addition, PLE should be suspected as a possible cause of unexplained edema and/or hypoalbuminemia in SLE.     

Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.     

The influence of semantic encoding on recognition memory in Alzheimer's disease

Within the framework of the distinction between episodic and semantic memory, it has been argued that these two memory systems are organised in a hierarchical way. The hierarchical hypothesis assumes that episodic memory is a specific subsystem of semantic memory and therefore implies that episodic memory cannot exist without semantic memory. If this hypothesis is correct, it should be expected that patients with impaired semantic memory also have impaired episodic memory. In the present study, two experiments investigated the influence of semantic encoding on recognition memory performance in a population of 20 patients with Alzheimer's disease and 18 normal controls. Both experiments assessed recognition memory for semantically-related items. In Experiment 2, but not in Experiment 1, subjects were explicitly instructed to make a semantic association between the items. Alzheimer's disease patients were impaired, compared to the normal controls, on the recognition memory performance of both experiments. The ability to make a semantic association between two items was significantly and positively correlated with the subjects' performance on the recognition tasks. A further analysis showed that patients who were impaired on the semantic association task did significantly worse on the recognition task of Experiment 2 than normal controls and patients who were unimpaired on the semantic association task. These findings are discussed in the context of memory deficits in Alzheimer's disease, and are interpreted as supporting the view that episodic memory for an item is affected by the level of semantic awareness of that same item.