Statistics of the model of TbCof in complex with G-actin.

<p>Statistics of the model of TbCof in complex with G-actin.</p

Interactions between TbCof and G-actin revealed by ITC.

<p>The negative peaks indicate an exothermic reaction. The area under each peak represents the heat released after an injection of TbCof into G-actin solution (upper panel). Binding isotherms were obtained by plotting the peak areas against the molar ratio of TbCof to G-actin (lower panel). The lines represent the best-fit curves obtained from least-squares regression analyses assuming a one-site binding model. A. ITC of TbCof-G-actin in the presence of ADP. B. ITC of TbCof-G-actin in the presence of ATP.</p

NMR solution structure of TbCof.

<p>A. The lowest-energy conformation was used for the cartoon representation of TbCof, showing a central β sheet surrounded by α helices. The key secondary structure elements, both termini, and the F-loop are labeled; B. Superposition of backbone traces of the 20 lowest-energy NMR structures of TbCof. C. Electrostatic surface diagram of the lowest-energy conformation of TbCof is shown from two different orientations 180 degrees apart (red, negative; blue, positive; white, neutral).</p

Structural and Functional Insight into ADF/Cofilin from <em>Trypanosoma brucei</em>

<div><p>The ADF/cofilin family has been characterized as a group of actin-binding proteins critical for controlling the assembly of actin within the cells. In this study, the solution structure of the ADF/cofilin from <em>Trypanosoma brucei</em> (TbCof) was determined by NMR spectroscopy. TbCof adopts the conserved ADF/cofilin fold with a central β-sheet composed of six β-strands surrounded by five α-helices. Isothermal titration calorimetry experiments denoted a submicromolar affinity between TbCof and G-actin, and the affinity between TbCof and ADP-G-actin was five times higher than that between TbCof and ATP-G-actin at low ionic strength. The results obtained from electron microscopy and actin filament sedimentation assays showed that TbCof depolymerized but did not co-sediment with actin filaments and its ability of F-actin depolymerization was pH independent. Similar to actin, TbCof was distributed throughout the cytoplasm. All our data indicate a structurally and functionally conserved ADF/cofilin from <em>Trypanosoma brucei</em>.</p> </div

Sequence alignment of TbCof with other ADF/cofilin members.

<p>The alignment was prepared using ClustalW2 and ESPript 2.2. Identical residues are boxed in red. The accession numbers of the proteins used for the alignment are listed as follows: Swiss-Prot, Q2QKR1, ADF/cofilin from <i>Leishmania donovani</i> (LdCof); Swiss-Prot, B9Q2C8, ADF from <i>Toxoplasma gondii</i> (TgADF); Swiss-Prot, P86292, ADF1 from <i>Plasmodium falciparum</i> (PfADF1); Swiss-Prot, Q4YT54, ADF2 from <i>Plasmodium berghei</i> (PbADF2); Swiss-Prot, P23528, cofilin from <i>Homo sapiens</i> (HuCof)<i>;</i> Swiss-Prot, Q39250, ADF1 from <i>Arabidopsis Thaliana</i> (AtADF1); Swiss-Prot, Q03048, cofilin from <i>Saccharomyces cerevisiae</i> (ScCof). The secondary structure elements of TbCof are labeled on the top of the alignment.</p

Structural comparison of TbCof with other ADF/cofilin family members.

<p>A. ADF/cofilin from <i>Trypanosoma brucei</i>; B. cofilin from <i>Saccharomyces cerevisiae</i> (PDB ID: 1COF); C. ADF1 from <i>Plasmodium falciparum</i> (PfADF1) (PDB ID: 2XF1); D. ADF2 from <i>Plasmodium berghei</i> (PbADF2) (PDB ID: 2XFA). These ADF/cofilin family members all share the classical fold except for a short helical turn in the loop between β6 and the C-terminal helix from residue D119 to L123 in TbCof and the shorter C-terminal region in PfADF1. The key secondary structure elements are labeled.</p

Model of TbCof (cyan) in complex with G-actin (magenta).

<p>The G-actin binding site contains 3 regions: a, the N-terminal extension that interacts with actin subdomain 1; b, the long kinked helix α3 that binds to the cleft between actin subdomains 1 and 3; c, the region before the C-terminal α-helix that interacts with actin subdomain 3. F-actin binding is mediated by additional regions consisting of the F-loop between β4-β5 (with label d) and the C-terminal α-helix (with label e). The F-actin binding site is circled by a dashed line.</p

Effect of TbCof on F-actin examined by electron microscopy.

<p>F-actin (5 µM) was incubated without (A) or with 0.05 µM TbCof (B), negatively stained with uranyl acetate, and observed by electron microscopy. Actin alone maintains long filaments. While only short filaments are observed in the presence of TbCof (B). The scale bars represent 100 nm.</p

Localization of TbCof in the procylic form <i>T. brucei</i>.

<p>Cells overexpressing TbCof with an HA₃-tag at the C-terminus were treated with tetracycline (1 µg/ml) for 2 days. (A) The overexpressed TbCof-HA₃ examined by western blot using an HA probe. (B) Cells overexpressing TbCof-HA₃ stained with an HA probe and DAPI, and examined with a fluorescence microscope. TbCof is localized to the cytoplasm throughout the cell cycle.</p

Table1_Efficacy of pharmacologic therapies in patients with acute heart failure: A network meta-analysis.DOCX

Background: A network meta-analysis (NMA) of the current recommended drugs for the treatment of acute heart failure (AHF), was performed to compare the relative efficacy.Methods: We used PubMed, EMBASE, Cochrane Clinical Trials Register, and Web of Science systems to search studies of randomized controlled trials (RCT) for the treatment of AHF recommended by the guidelines and expert consensus until 1 December 2020. The primary outcome was all-cause mortality within 30 days. The secondary outcomes included 30-days all-cause rehospitalization, rates of HF-related rehospitalization, rates of adverse events, and rates of serious adverse events. A Bayesian NMA based on random effects model was performed.Results: After screening 14,888 citations, 23 RCTs (17,097 patients) were included, focusing on nesiritide, placebo, serelaxin, rhANP, omecamtiv mecarbil, tezosentan, KW-3902, conivaptan, tolvaptan, TRV027, chlorothiazide, metolazone, ularitide, relaxin, and rolofylline. Omecamtiv mecarbil had significantly lower all-cause mortality rates than the placebo (odds ratio 0.04, 0.01–0.22), rhANP (odds ratio 0.03, 0–0.40), serelaxin (odds ratio 0.05, 0.01–0.38), tezosentan (odds ratio 0.04, 0–0.22), tolvaptan (odds ratio 0.04, 0.01–0.30), and TRV027 (odds ratio 0.03, 0–0.36). No drug was superior to the other drugs for the secondary outcomes and safety outcomes.Conclusion: No drug was superior to the other drugs for the secondary outcomes and safety outcomes. Current drugs for AHF show similar efficacy and safety.</p