Transmission of Human and Macaque Plasmodium spp. to Ex-Captive Orangutans in Kalimantan, Indonesia

We identified 4 discrete Plasmodium spp. sequences from the blood of orangutans, including 1 of P. vivax, which has implications for human residents and orangutan rehabilitation programs

Regional ion channel gene expression heterogeneity and ventricular fibrillation dynamics in human hearts

RATIONALE: Structural differences between ventricular regions may not be the sole determinant of local ventricular fibrillation (VF) dynamics and molecular remodeling may play a role. OBJECTIVES: To define regional ion channel expression in myopathic hearts compared to normal hearts, and correlate expression to regional VF dynamics. METHODS AND RESULTS: High throughput real-time RT-PCR was used to quantify the expression patterns of 84 ion-channel, calcium cycling, connexin and related gene transcripts from sites in the LV, septum, and RV in 8 patients undergoing transplantation. An additional eight non-diseased donor human hearts served as controls. To relate local ion channel expression change to VF dynamics localized VF mapping was performed on the explanted myopathic hearts right adjacent to sampled regions. Compared to non-diseased ventricles, significant differences (p<0.05) were identified in the expression of 23 genes in the myopathic LV and 32 genes in the myopathic RV. Within the myopathic hearts significant regional (LV vs septum vs RV) expression differences were observed for 13 subunits: Nav1.1, Cx43, Ca3.1, Cavalpha2delta2, Cavbeta2, HCN2, Na/K ATPase-1, CASQ1, CASQ2, RYR2, Kir2.3, Kir3.4, SUR2 (p<0.05). In a subset of genes we demonstrated differences in protein expression between control and myopathic hearts, which were concordant with the mRNA expression profiles for these genes. Variability in the expression of Cx43, hERG, Na(+)/K(+) ATPase ss1 and Kir2.1 correlated to variability in local VF dynamics (p<0.001). To better understand the contribution of multiple ion channel changes on VF frequency, simulations of a human myocyte model were conducted. These simulations demonstrated the complex nature by which VF dynamics are regulated when multi-channel changes are occurring simultaneously, compared to known linear relationships. CONCLUSIONS: Ion channel expression profile in myopathic human hearts is significantly altered compared to normal hearts. Multi-channel ion changes influence VF dynamic in a complex manner not predicted by known single channel linear relationships

Structural determinants regulating surface expression and function of Kv-related ion channels

To date, the mechanisms and structural determinants which contribute to the regulation of ion channel trafficking, surface expression and function have only been limitedly explored. Through biophysical and molecular characterization of the hyperpolarization-activated cyclic-nucleotide gated channel 2 (HCN2), we have identified a four-amino acid motif (EEYP) in the B-helix of the cyclic-nucleotide binding domain (CNBD) that strongly promotes channel export from the endoplasmic reticulum (ER) and cell surface expression but does not contribute to the inhibition of channel opening. We further demonstrate that this motif augments a step in the trafficking pathway and/or the efficiency of correct folding and assembly. The role of post-translational modifications, specifically N-linked glycosylation, has also been investigated in two different HCN isoforms. All four mammalian HCN channel isoforms have been shown to undergo N-linked glycosylation in the brain. HCN channels have further been suggested to require N-glycosylation for function, a provocative finding that would make them unique in the voltage-gated potassium channel superfamily. Here, we show that both the HCN1 and HCN2 isoforms are also predominantly N-glycosylated in the embryonic heart, where they are found in significant amounts and where HCN-mediated currents are known to regulate beating frequency. Surprisingly, we find that N-glycosylation is not required for HCN2 function, although its cell surface expression is highly dependent on the presence of N-glycans. Comparatively, disruption of N-glycosylation only modestly impacts cell surface expression of HCN1 and leaves permeation and gating functions almost unchanged. The evolutionary significance of this isoforms specific regulation is also examined. Finally, the role of palmitoylation in the regulation of Kv4 channels is examined. Using acylbiotin exchange (ABE) chemistry we are able demonstrate that Kv4.2 is present as a palmitoylated protein in both rat cortical neurons and COS-7 cells. Through mutational analysis of the twelve intracellular cysteine residues within Kv4.2, we were able to localize the site of palmitoylation to the intracellular COOH-terminus. Palmitoylation of Kv4.2 does not contribute to the regulation of activation and inactivation gating parameters. Rather, inhibition of palmitoylation through either mutation of COOH-terminal cysteine residues or the pharmacological agent 2-bromopalmitate results in significant reductions in overall current density measurements.Medicine, Faculty ofCellular and Physiological Sciences, Department ofGraduat

Mechanical circulatory support in the heart failure population.

PURPOSE OF REVIEW Use of durable left ventricular assist devices (LVADs) has increased considerably in recent years because of the insufficient supply of donor hearts for cardiac transplantation and improvement in outcomes from refinements in technology. This review examines clinical utility of these devices and summarizes the most recent evidence supporting the use of LVAD therapy. RECENT FINDINGS There continues to be significant advancements made in LVAD technology, which has resulted in improvements in the rates of adverse events and overall patient quality of life. Specifically, less invasive and improved surgical techniques have resulted in fewer incidence of pump thrombosis and stringent blood pressure management have been shown to significantly decrease stroke rates. SUMMARY The continued advances in LVAD therapy have resulted in significant improvement in overall survival; however, complication rates remain relatively high. Future work will focus on improvements in adverse outcomes and ultimately the possibility that LVADs will be a viable alternative to transplantation in patients with end-stage heart failure

Opening of ciHCNs is facilitated by a rise in intracellular cAMP.

<p>A. Current traces for ciHCNa (above) and ciHCNb (below), elicited by the staggered voltage protocols shown above each set of current traces. “Tail” currents were elicited at −30 mV for ciHCNa and +30 mV for ciHCNb, and an example for each isoform is highlighted by a solid black arrow. B. Mean activation curves determined from single order Boltzmann fits of plots of normalized tail currents versus voltage from individual experiments and oocytes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047590#pone.0047590-Proenza1" target="_blank">[35]</a>, under different conditions. The gray and black lines represent the mean curves obtained from control experiments and from experiments carried out in the presence of 10 mM 8-bromo cAMP, respectively. Number of experiments (representing one oocyte per experiment), without and with cAMP, were 14 and 11, respectively, for ciHCNa, and 8 and 8, respectively, for ciHCNb. C. Plot of mean V½ values ± s.e.m. determined by fitting the tail current data, obtained from individual experiments in oocytes expressing ciHCNa or ciHCNb in the absence (open squares) or presence of 8-Br-cAMP (filled squares), with a Boltzmann equation. *indicates significant difference (p<0.05, two-tailed unpaired t-test) between values obtained in control solution as compared to those obtained in a solution containing 8-Br-cAMP. The numbers of oocytes used per group are as for ‘B’.</p

Phylogenetic pattern and N-linked glycosylation of a subset of <i>Ciona</i> HCNs.

<p>A. An alignment of the pore region produced by ClustalX and generated by GeneDoc. Red boxes indicates known and putative HCN N-linked glycosylation site and the yellow line indicates the division between the presence and absence of this functional site. B. An HCN cladogram generated by aligning the sequences shown as described in the text. “X” indicates the predicted emergence point of N-linked glycosylation in the HCN family. Sequences other than those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047590#pone-0047590-g001" target="_blank">Figure 1</a>, and other than those for <i>Oikopleura dioica</i> and <i>Branchiostoma floridae</i> (lancelet), are: gi: 260829977; <i>Strongylocentrotus purpuratus</i> (sea urchin) gi:47551101; <i>Panulirus interruptus</i>, splice variant I (lobster) gi:115334851; <i>Apis mellifera</i> (bee) gi:33355927; <i>Drosophila melanogaster</i>, isoform B, gi:84795752; <i>Aedes aegypti</i> (mosquito) gi:108875949. C. Western blot of membrane fractions from oocytes injected with cRNA of ciHCNa, ciHCNa-N380Q or ciHCNb, untreated (−) or treated (+) with PNGaseF and probed with anti-V5 antibody. Actin, robed with an anti-actin antibody, was used as a loading control. ciHCNa, but not ciHCNa-N380Q or ciHCNb, is shifted to a lower molecular mass in the presence of PNGaseF. Molecular weights, indicated by the short black bars to the right of the blots, are 130 kDa (top), 100 kDa (upper middle) and 70 kDa (lower middle) and 35 kDa (bottom). D. Western blot of whole cell lysates from CHO cells transfected with 1.5 ug cDNA of ciHCNa, ciHCNa-N380Q or ciHCNb, either treated (+) or untreated (−) with PNGaseF and probed for using an anti-V5 antibody. Molecular weights, indicated by the short black bars to the right of the blots, are 95 kDa (top), 72 kDa (middle) and 34 kDa (bottom). Predicted mass of <i>Ciona</i> HCN channels without post-translational modification, is 78.8 kDa and 93.3 kDa for ciHCNa and ciHCNb, respectively.</p

The time course of I_h activation and deactivation is different between ciHCNa and ciHCNb.

<p>A. (Left) Sample current traces elicited by a hyperpolarization of the membrane potential from a holding potential of −10 mV to −90 (ciHCNa) or −70 mV (ciHCNb). The slow portion of the current traces obtained by hyperpolarization to −90 or −70 mV (I_h) was fit with a double (ciHCNa) or single (ciHCNb) exponential function. The sample fits of these traces are shown in dark gray, along with a plot of the residuals of the fits in light gray at the top of each current trace. (Right) Plots of Tau values, which were obtained from fitting with exponential functions, versus test voltage. B. Plot of the ratio of amplitudes of fast component versus the amplitudes of both the fast and slow component of the double exponential fit for the current traces, obtained from oocytes expressing ciHCNa as described in ‘A’, versus test voltage. For both ‘A’ and ‘B’, the values for “n” refer to the number of oocytes used. For all plots, the values represent the mean ± s.e.m.</p

Variable opening by hyperpolarization and cesium block of current produced by the two <i>Ciona</i> HCNs.

<p>A. Black current traces elicited by a single 6 second hyperpolarizing pulse to −70 mV, from a holding potential of −10 mV, followed by pulse back to −30 mV, in an oocyte containing ciHCNa (above) or ciHCNb (below), bathed an extracellular solution containing 96 mM potassium. The voltage protocol utilized is shown between the two sets of current traces. The black vertical lines to the left indicate the instantaneous (I_inst) and the slowly-activating (I_h) components of the current trace. Gray current traces were produced by the same voltage protocol, following incubation with the same extracellular solution, with the addition of 5 mM Cs+, for three minutes while clamped at −10 mV. The dotted lines represent the zero current level. Capacitive transients elicited upon current activation have been removed for clarity. B. Plots of amplitudes of I_inst and I_h before and during perfusion with Cs+, as indicated. The number of oocytes that were used in each group are shown above each bar in brackets. Values represent mean ± s.e.m.; those in black and gray represent with and without Cs+. *indicates a significant difference induced by Cs+ (p<0.05 in a two-tailed paired t-test).</p