Mechanism and Kinetics of Copper Complexes Binding to the Influenza A M2 S31N and S31N/G34E Channels

Copper(II) is known to bind in the influenza virus His37 cluster in the homotetrameric M2 proton channel and block the proton current needed for uncoating. Copper complexes based on iminodiacetate also block the M2 proton channel and show reduced cytotoxicity and zebrafish-embryo toxicity. In voltage-clamp oocyte studies using the ubiquitous amantadine-insensitive M2 S31N variant, the current block showed fast and slow phases, in contrast to the single phase found for amantadine block of wild-type M2. Here, we evaluate the mechanism of block by copper adamantyl iminodiacitate and copper cyclooctyl iminodiacitate complexes and address whether the complexes can coordinate with one or more of the His37 imidazoles. The current traces were fitted to parametrized master equations. The energetics of binding and the rate constants suggest that the first step is copper complex binding within the channel, and the slow step in the current block is the formation of a Cu-histidine coordination complex. Solution-phase isothermal titration calorimetry and density functional theory (DFT) calculations indicate that imidazole binds to the copper complexes. Structural optimization using DFT reveals that the complexes fit inside the channel and project the Cu(II) toward the His37 cluster, allowing one imidazole to form a coordination complex with Cu(II). Electrophysiology and DFT studies also show that the complexes block the G34E amantadine-resistant mutant despite some crowding in the binding site by the glutamates.     

Highly Stable Plasmonic Nanostructures on a Nickel-Sputtered Glass and Polymeric Optical Fiber Sensors

Plasmonics - This study shows development of highly sensitive and stable localized surface plasmon resonance (LSPR)-active U-bent glass and polymeric optical fiber (GOF and POF) sensor probes by a...     

Conditional and domain‐specific inactivation of the Tsc2 gene in neural progenitor cells

Tuberous sclerosis complex (TSC) is a genetic disease characterized by multiorgan benign tumors as well as neurological manifestations. Epilepsy and autism are two of the more prevalent neurological complications and are usually severe. TSC is caused by mutations in either the TSC1 (encodes hamartin) or the TSC2 (encodes tuberin) genes with TSC2 mutations being associated with worse outcomes. Tuberin contains a highly conserved GTPase‐activating protein (GAP) domain that indirectly inhibits mammalian target of rapamycin complex 1 (mTORC1). mTORC1 dysregulation is currently thought to cause much of the pathogenesis in TSC but mTORC1‐independent mechanisms may also contribute. We generated a novel conditional allele of Tsc2 by flanking exons 36 and 37 with loxP sites. Mice homozygous for this knock‐in Tsc2 allele are viable and fertile with normal appearing growth and development. Exposure to Cre recombinase then creates an in‐frame deletion involving critical residues of the GAP domain. Homozygous conditional mutant mice generated using Emx1^Cre have increased cortical mTORC1 signaling, severe developmental brain anomalies, seizures, and die within 3 weeks. We found that the normal levels of the mutant Tsc2 mRNA, though GAP‐deficient tuberin protein, appear unstable and rapidly degraded. This novel animal model will allow further study of tuberin function including the requirement of the GAP domain for protein stability. genesis 51:284–292. © 2013 Wiley Periodicals, Inc.     

Multi-organ Abnormalities and mTORC1 Activation in Zebrafish Model of Multiple Acyl-CoA Dehydrogenase Deficiency

Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is a severe mitochondrial disorder featuring multi-organ dysfunction. Mutations in either the ETFA, ETFB, and ETFDH genes can cause MADD but very little is known about disease specific mechanisms due to a paucity of animal models. We report a novel zebrafish mutant dark xavier (dxa^vu463) that has an inactivating mutation in the etfa gene. dxa^vu463 recapitulates numerous pathological and biochemical features seen in patients with MADD including brain, liver, and kidney disease. Similar to children with MADD, homozygote mutant dxa^vu463 zebrafish have a spectrum of phenotypes ranging from moderate to severe. Interestingly, excessive maternal feeding significantly exacerbated the phenotype. Homozygous mutant dxa^vu463 zebrafish have swollen and hyperplastic neural progenitor cells, hepatocytes and kidney tubule cells as well as elevations in triacylglycerol, cerebroside sulfate and cholesterol levels. Their mitochondria were also greatly enlarged, lacked normal cristae, and were dysfunctional. We also found increased signaling of the mechanistic target of rapamycin complex 1 (mTORC1) with enlarged cell size and proliferation. Treatment with rapamycin partially reversed these abnormalities. Our results indicate that etfa gene function is remarkably conserved in zebrafish as compared to humans with highly similar pathological, biochemical abnormalities to those reported in children with MADD. Altered mTORC1 signaling and maternal nutritional status may play critical roles in MADD disease progression and suggest novel treatment approaches that may ameliorate disease severity.     

Views of Swedish Commissioning Parents Relating to the Exploitation Discourse in Using Transnational Surrogacy

Transnational surrogacy, when people travel abroad for reproduction with the help of a surrogate mother, is a heavily debated phenomenon. One of the most salient discourses on surrogacy is the one affirming that Westerners, in their quest for having a child, exploit poor women in countries such as India. As surrogacy within the Swedish health care system is not permitted, Swedish commissioning parents have used transnational surrogacy, and the majority has turned to India. This interview study aimed to explore how commissioning parents negotiate the present discourses on surrogacy. Findings from the study suggest that the commissioning parents’ views on using surrogacy are influenced by competing discourses on surrogacy represented by media and surrogacy agencies. The use of this reproductive method resulted, then, in some ambiguity. Although commissioning parents defy the exploitation discourse by referring to what they have learnt about the surrogate mother’s life situation and by pointing at the significant benefits for her, they still had a request for regulation of surrogacy in Sweden, to better protect all parties involved. This study, then, gives a complex view on surrogacy, where the commissioning parents simultaneously argue against the exploitation discourse but at the same time are uncertain if the surrogate mothers are well protected in the surrogacy arrangements. Their responses to the situation endorse the need for regulation both in Sweden and India.     

Cystogenesis and elongated primary cilia in Tsc1-deficient distal convoluted tubules

Tuberous sclerosis complex (TSC) is a multiorgan hamartomatous disease caused by loss of function mutations of either the TSC1 or TSC2 genes. Neurological symptoms of TSC predominate in younger patients, but renal pathologies are a serious aspect of the disease in older children and adults. To study TSC pathogenesis in the kidney, we inactivated the mouse Tsc1 gene in the distal convoluted tubules (DCT). At young ages, Tsc1 conditional knockout (CKO) mice have enlarged kidneys and mild cystogenesis with increased mammalian target of rapamycin complex (mTORC)1 but decreased mTORC2 signaling. Treatment with the mTORC1 inhibitor rapamycin reduces kidney size and cystogenesis. Rapamycin withdrawal led to massive cystogenesis involving both distal as well as proximal tubules. To assess the contribution of decreased mTORC2 signaling in kidney pathogenesis, we also generated Rictor CKO mice. These animals did not have any detectable kidney pathology. Finally, we examined primary cilia in the DCT. Cilia were longer in Tsc1 CKO mice, and rapamycin treatment returned cilia length to normal. Rictor CKO mice had normal cilia in the DCT. Overall, our findings suggest that loss of the Tsc1 gene in the DCT is sufficient for renal cystogenesis. This cytogenesis appears to be mTORC1 but not mTORC2 dependent. Intriguingly, the mechanism may be cell autonomous as well as non-cell autonomous and possibly involves the length and function of primary cilia.     

Glycogen and triglyceride utilization in relation to muscle metabolic characteristics in men performing heavy-resistance exercise

Nine bodybuilders performed heavy-resistance exercise activating the quadriceps femoris muscle. Intermittent 30-s exhaustive exercise bouts comprising 6-12 repetitions were interspersed with 60-s periods for 30 min. Venous blood samples were taken repeatedly during and after exercise for analyses of plasma free fatty acid (FFA) and glycerol concentration. Muscle biopsies were obtained from the vastus lateralis muscle before and after exercise and assayed for glycogen, glycerol-3-phosphate, lactate and triglyceride (TG) content. The activities of citrate synthase (CS), lactate dehydrogenase, hexokinase (HK), myokinase, creatine kinase and 3-hydroxyacyl-CoA dehydrogenase (HAD), were analysed. Histochemical staining procedures were used to assess fibre type composition, fibre area and capillary density. TG content before and after exercise averaged (SD) 23.9 (13.3) and 16.7 (6.4) mmol kg-1 dry wt. The basal triglyceride content varied sixfold among individuals and the higher the levels the greater was the change during exercise. The glycogen content decreased (P less than 0.001) from 690 (82) to 495 (95) mmol kg-1 dry wt. and lactate and glycerol-3-phosphate increased (P less than 0.001) to 79.5 (5.5) and 14.5 (7.3) mmol kg-1 dry wt., respectively, after exercise. The HK and HAD/CS content respectively correlated with glycogen or TG content at rest and with changes in these metabolites during exercise. FFA and glycerol concentrations increased slightly (P less than 0.001) during exercise. Lipolysis may, therefore, provide energy during heavy-resistance exercise of relatively short duration. Also, storage and utilization of intramuscular substrates appear to be influenced by the metabolic profile of muscle.     

Improving consensus structure by eliminating averaging artifacts

Background  

Common structural biology methods (i.e., NMR and molecular dynamics) often produce ensembles of molecular structures. Consequently, averaging of 3D coordinates of molecular structures (proteins and RNA) is a frequent approach to obtain a consensus structure that is representative of the ensemble. However, when the structures are averaged, artifacts can result in unrealistic local geometries, including unphysical bond lengths and angles.