Loss of Acid Sensing Ion Channel-1a and Bicarbonate Administration Attenuate the Severity of Traumatic Brain Injury

Traumatic brain injury (TBI) is a common cause of morbidity and mortality in people of all ages. Following the acute mechanical insult, TBI evolves over the ensuing minutes and days. Understanding the secondary factors that contribute to TBI might suggest therapeutic strategies to reduce the long-term consequences of brain trauma. To assess secondary factors that contribute to TBI, we studied a lateral fluid percussion injury (FPI) model in mice. Following FPI, the brain cortex became acidic, consistent with data from humans following brain trauma. Administering HCO₃ ⁻ after FPI prevented the acidosis and reduced the extent of neurodegeneration. Because acidosis can activate acid sensing ion channels (ASICs), we also studied ASIC1a^−/− mice and found reduced neurodegeneration after FPI. Both HCO₃ ⁻ administration and loss of ASIC1a also reduced functional deficits caused by FPI. These results suggest that FPI induces cerebral acidosis that activates ASIC channels and contributes to secondary injury in TBI. They also suggest a therapeutic strategy to attenuate the adverse consequences of TBI.     

The Social Life of Infants in the Context of Infectious Disease Transmission; Social Contacts and Mixing Patterns of the Very Young

Insight into how humans interact helps further understanding of the transmission of infectious diseases. For diseases such as pertussis, infants are at particular risk for severe outcomes. To understand the contact pattern of infants, especially those too young to be vaccinated, we sent contact diaries to a representative sample of 1000 mothers in the United Kingdom. We received 115 responses with a total of 758 recorded contacts. The average number of daily contacts for an infant was 6.68 overall and 5.7 for those aged ≤10 weeks. Of the latter, 2.1 (37%) contacts were with non-household members and were >15 minutes duration, suggesting that a cocooning programme may miss a substantial proportion of exposures leading to disease transmission. The least contact was between adolescents and infants. Thus the impact of adolescent (pertussis) vaccination on infants would likely be limited, unless it reduces transmission to other age groups whose contact with infants is greater.     

Brainstem Neurons Survive the Identical Ischemic Stress That Kills Higher Neurons: Insight to the Persistent Vegetative State

Global ischemia caused by heart attack, pulmonary failure, near-drowning or traumatic brain injury often damages the higher brain but not the brainstem, leading to a ‘persistent vegetative state’ where the patient is awake but not aware. Approximately 30,000 U.S. patients are held captive in this condition but not a single research study has addressed how the lower brain is preferentially protected in these people. In the higher brain, ischemia elicits a profound anoxic depolarization (AD) causing neuronal dysfunction and vasoconstriction within minutes. Might brainstem nuclei generate less damaging AD and so be more resilient? Here we compared resistance to acute injury induced from simulated ischemia by ‘higher’ hippocampal and striatal neurons versus brainstem neurons in live slices from rat and mouse. Light transmittance (LT) imaging in response to 10 minutes of oxygen/glucose deprivation (OGD) revealed immediate and acutely damaging AD propagating through gray matter of neocortex, hippocampus, striatum, thalamus and cerebellar cortex. In adjacent brainstem nuclei, OGD-evoked AD caused little tissue injury. Whole-cell patch recordings from hippocampal and striatal neurons under OGD revealed sudden membrane potential loss that did not recover. In contrast brainstem neurons from locus ceruleus and mesencephalic nucleus as well as from sensory and motor nuclei only slowly depolarized and then repolarized post-OGD. Two-photon microscopy confirmed non-recoverable swelling and dendritic beading of hippocampal neurons during OGD, while mesencephalic neurons in midbrain appeared uninjured. All of the above responses were mimicked by bath exposure to 100 µM ouabain which inhibits the Na⁺/K⁺ pump or to 1–10 nM palytoxin which converts the pump into an open cationic channel.Therefore during ischemia the Na⁺/K⁺ pump of higher neurons fails quickly and extensively compared to naturally resilient hypothalamic and brainstem neurons. The selective survival of lower brain regions that maintain vital functions will support the persistent vegetative state.     

Impact of Bottom Trawling on Deep-Sea Sediment Properties along the Flanks of a Submarine Canyon

The offshore displacement of commercial bottom trawling has raised concerns about the impact of this destructive fishing practice on the deep seafloor, which is in general characterized by lower resilience than shallow water regions. This study focuses on the flanks of La Fonera (or Palamós) submarine canyon in the Northwestern Mediterranean, where an intensive bottom trawl fishery has been active during several decades in the 400–800 m depth range. To explore the degree of alteration of surface sediments (0–50 cm depth) caused by this industrial activity, fishing grounds and control (untrawled) sites were sampled along the canyon flanks with an interface multicorer. Sediment cores were analyzed to obtain vertical profiles of sediment grain-size, dry bulk density, organic carbon content and concentration of the radionuclide ²¹⁰Pb. At control sites, surface sediments presented sedimentological characteristics typical of slope depositional systems, including a topmost unit of unconsolidated and bioturbated material overlying sediments progressively compacted with depth, with consistently high ²¹⁰Pb inventories and exponential decaying profiles of ²¹⁰Pb concentrations. Sediment accumulation rates at these untrawled sites ranged from 0.3 to 1.0 cm y⁻¹. Sediment properties at most trawled sites departed from control sites and the sampled cores were characterized by denser sediments with lower ²¹⁰Pb surface concentrations and inventories that indicate widespread erosion of recent sediments caused by trawling gears. Other alterations of the physical sediment properties, including thorough mixing or grain-size sorting, as well as organic carbon impoverishment, were also visible at trawled sites. This work contributes to the growing realization of the capacity of bottom trawling to alter the physical properties of surface sediments and affect the seafloor integrity over large spatial scales of the deep-sea.     

Synthesis of Two 2′-Deoxyuridine Analogs Substituted in Position 5 with 2-Atom Tethered Nitroxide Radicals

Abstract

The synthesis of two novel spin labeled 2′-deoxyuridine analogs is described. The nucleic acid building block is substituted in position 5 with a short methylamino tether, which bears either a six- or five-membered nitroxide ring.     

Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein

Hepatitis C virus (HCV) co-opts hepatic lipid pathways to facilitate its pathogenesis. The virus alters cellular lipid biosynthesis and trafficking, and causes an accumulation of lipid droplets (LDs) that gives rise to hepatic steatosis. Little is known about how these changes are controlled at the molecular level, and how they are related to the underlying metabolic states of the infected cell. The HCV core protein has previously been shown to independently induce alterations in hepatic lipid homeostasis. Herein, we demonstrate, using coherent anti-Stokes Raman scattering (CARS) microscopy, that expression of domain 2 of the HCV core protein (D2) fused to GFP is sufficient to induce an accumulation of larger lipid droplets (LDs) in the perinuclear region. Additionally, we performed fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotides [NAD(P)H], a key coenzyme in cellular metabolic processes, to monitor changes in the cofactor’s abundance and conformational state in D2-GFP transfected cells. When expressed in Huh-7 human hepatoma cells, we observed that the D2-GFP induced accumulation of LDs correlated with an increase in total NAD(P)H fluorescence and an increase in the ratio of free to bound NAD(P)H. This is consistent with an approximate 10 fold increase in cellular NAD(P)H levels. Furthermore, the lifetimes of bound and free NAD(P)H were both significantly reduced – indicating viral protein-induced alterations in the cofactors’ binding and microenvironment. Interestingly, the D2-expressing cells showed a more diffuse localization of NAD(P)H fluorescence signal, consistent with an accumulation of the co-factor outside the mitochondria. These observations suggest that HCV causes a shift of metabolic control away from the use of the coenzyme in mitochondrial electron transport and towards glycolysis, lipid biosynthesis, and building of new biomass. Overall, our findings demonstrate that HCV induced alterations in hepatic metabolism is tightly linked to alterations in NAD(P)H functional states.     

The Role of Protein Denaturation Energetics and Molecular Chaperones in the Aggregation and Mistargeting of Mutants Causing Primary Hyperoxaluria Type I

Primary hyperoxaluria type I (PH1) is a conformational disease which result in the loss of alanine:glyoxylate aminotransferase (AGT) function. The study of AGT has important implications for protein folding and trafficking because PH1 mutants may cause protein aggregation and mitochondrial mistargeting. We herein describe a multidisciplinary study aimed to understand the molecular basis of protein aggregation and mistargeting in PH1 by studying twelve AGT variants. Expression studies in cell cultures reveal strong protein folding defects in PH1 causing mutants leading to enhanced aggregation, and in two cases, mitochondrial mistargeting. Immunoprecipitation studies in a cell-free system reveal that most mutants enhance the interactions with Hsc70 chaperones along their folding process, while in vitro binding experiments show no changes in the interaction of folded AGT dimers with the peroxisomal receptor Pex5p. Thermal denaturation studies by calorimetry support that PH1 causing mutants often kinetically destabilize the folded apo-protein through significant changes in the denaturation free energy barrier, whereas coenzyme binding overcomes this destabilization. Modeling of the mutations on a 1.9 Å crystal structure suggests that PH1 causing mutants perturb locally the native structure. Our work support that a misbalance between denaturation energetics and interactions with chaperones underlie aggregation and mistargeting in PH1, suggesting that native state stabilizers and protein homeostasis modulators are potential drugs to restore the complex and delicate balance of AGT protein homeostasis in PH1.     

The cAMP Signaling Pathway and Direct Protein Kinase A Phosphorylation Regulate Polycystin-2 (TRPP2) Channel Function

Polycystin-2 (PC2) is a TRP-type, Ca²⁺-permeable non-selective cation channel that plays an important role in Ca²⁺ signaling in renal and non-renal cells. The effect(s) of the cAMP pathway and kinase mediated phosphorylation of PC2 seem to be relevant to PC2 trafficking and its interaction with polycystin-1. However, the role of PC2 phosphorylation in channel function is still poorly defined. Here we reconstituted apical membranes of term human syncytiotrophoblast (hST), containing endogenous PC2 (PC2_hst), and in vitro translated channel protein (PC2_iv). Addition of the catalytic subunit of PKA increased by 566% the spontaneous PC2_hst channel activity in the presence of ATP. Interestingly, 8-Br-cAMP also stimulated spontaneous PC2_hst channel activity in the absence of the exogenous kinase. Either stimulation was inhibited by addition of alkaline phosphatase, which in turn, was reversed by the phosphatase inhibitor vanadate. Neither maneuver modified the single channel conductance but instead increased channel mean open time. PKA directly phosphorylated PC2, which increased the mean open time but not the single channel conductance of the channel. PKA phosphorylation did not modify either R742X truncated or S829A-mutant PC2_iv channel function. The data indicate that the cAMP pathway regulates PC2-mediated cation transport in the hST. The relevant PKA site for PC2 channel regulation centers on a single residue serine 829, in the carboxyl terminus.