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OBJECTIVE To evaluate the efficacy of doxorubicin for treatment of histiocytic sarcoma (HS) in dogs, whether administered as the sole treatment or as an adjunct to surgery or radiation therapy. ANIMALS 31 client-owned dogs with localized or disseminated HS examined between 2003 and 2017. PROCEDURES Medical records were reviewed retrospectively, and data were collected. The Kaplan-Meier method was used to estimate time-to-progression from the date of first doxorubicin administration and survival time from initial diagnosis. Factors that could be associated with poorer outcomes with doxorubicin treatment were analyzed with log-rank tests. RESULTS The objective response rate (ORR) was 26%. When stratified by disease status, dogs with localized and disseminated forms experienced 43% and 21% ORRs, respectively. Median time to progression after initiating doxorubicin treatment (n = 30 dogs) was 42 days. Median survival time from initial diagnosis to death (n = 29 dogs) was 169 days. Complete responses were obtained in only 2 dogs that had localized disease and received multimodality therapy. CLINICAL RELEVANCE Benefits of doxorubicin administration in canine HS are modest, with a limited ORR and delay in tumor progression, and are comparable to effects attained with other single-agent regimens.

The HIV-1 Envelope (Env) glycoprotein facilitates host cell fusion through a complex series of receptor-induced structural changes. Although significant progress has been made in understanding the structures of various Env conformations and transition intermediates that occur within the millisecond timescale, faster transitions in the microsecond timescale have not yet been observed. In this study, we employed time-resolved, temperature-jump small angle X- ray scattering to monitor structural rearrangements in an HIV-1 Env ectodomain construct with microsecond precision. We detected a transition correlated with Env opening that occurs in the hundreds of microseconds range and another more rapid transition that preceded this opening. Model fitting indicated that the early rapid transition involved an order-to-disorder transition in the trimer apex loop contacts, suggesting that conventional conformation-locking design strategies that target the allosteric machinery may be ineffective in preventing this movement. Utilizing this information, we engineered an envelope that locks the apex loop contacts to the adjacent protomer. This modification resulted in significant angle-of-approach shifts in the interaction of a neutralizing antibody. Our findings imply that blocking the intermediate state could be crucial for inducing antibodies with the appropriate bound state orientation through vaccination.

Hv1 proteins form a voltage-gated proton channel that is inhibited by extracellular Zn2+. De La Rosa et al find that extracellular Zn2+ is octahedrally coordinated by specific protein atoms and that Zn2+ occupancy modulates voltage sensor activation in Hv1 by long-range conformational coupling to a conserved intracellular electrostatic network., The voltage sensor (VS) domain in Hv1 proton channels mediates a voltage-dependent and H+-selective “aqueous” conductance (GAQ) that is potently modulated by extracellular Zn2+. Although two conserved His residues are required for Zn2+ effects on GAQ gating, the atomic structure of the Zn2+ coordination site and mechanism by which extracellular Zn2+ stabilizes a closed-state conformation remain unknown. Here we use His mutagenesis to identify residues that increase Zn2+ potency and are therefore likely to participate in first solvation shell interactions with Zn2+. Experimental Zn2+-mapping data were then used to constrain the structure of a new resting-state Hv1 model (Hv1 F). Molecular dynamics (MD) simulations show how protein and water atoms directly contribute to octahedral Zn2+ coordination spheres in Zn2+-bound and -unbound Hv1 F models. During MD simulations, we observed correlated movements of Zn2+-interacting side chains and residues in a highly conserved intracellular Coulombic network (ICN) that contains highly conserved Arg “gating charges” in S4 as well as acidic “counter-charges” in S2 and S3 and is known to control VS activation, suggesting that occupancy of the extracellular Zn2+ site is conformationally coupled to reorganization of the ICN. To test this hypothesis, we neutralized an ICN Glu residue (E153) and show that in addition to shifting GAQ activation to more negative voltages, E153A also decreases Zn2+ potency. We speculate that extracellular gating-modifier toxins and other ligands may use a generally similar long-range conformational coupling mechanism to modulate VS activation in related ion channel proteins.

δ-lysin, secreted by a Gram-positive bacterium Staphylococcus aureus, is a 26-residue membrane active peptide that shares many common features with antimicrobial peptides (AMPs). However, it possesses a few unique features that differentiate itself from typical AMPs. In particular, δ-lysin has zero net charge, even though it has many charged residues, and it preferentially lyses eukaryotic cells over bacterial cells. Here, we present the results of coarse-grained molecular dynamics simulations of δ-lysin interacting with a zwitterionic membrane over a wide range of peptide concentrations. When the peptides concentration is low, spontaneous dimerization of peptides is observed on the membrane surface, but deep insertion of peptides or pore formation was not observed. However, the calculated free energy of peptide insertion suggests that a small fraction of peptides is likely to be present inside the membrane at the peptide concentrations typically seen in dye efflux experiments. When the simulations with multiple peptides are carried out with a single pre-inserted transmembrane peptide, spontaneous pore formation occurs with a peptide-to-lipid ratio (P/L) as low as P/L = 1:42. Inter-peptide salt bridges among the transmembrane peptides seem to play a role in creating compact pores with very low level of hydration. More importantly, the transmembrane peptides making up the pore are constantly pushed to the opposite side of the membrane when the mass imbalance between the two sides of membrane is significant. Thus, the pore is very dynamic, allowing multiple peptides to translocate across the membrane simultaneously.

The Hv1 proton channel is evidently unique among voltage sensor domain proteins in mediating an intrinsic ‘aqueous’ H+ conductance (GAQ). Mutation of a highly conserved ‘gating charge’ residue in the S4 helix (R1H) confers a resting-state H+ ‘shuttle’ conductance (GSH) in VGCs and Ci VSP, and we now report that R1H is sufficient to reconstitute GSH in Hv1 without abrogating GAQ. Second-site mutations in S3 (D185A/H) and S4 (N4R) experimentally separate GSH and GAQ gating, which report thermodynamically distinct initial and final steps, respectively, in the Hv1 activation pathway. The effects of Hv1 mutations on GSH and GAQ are used to constrain the positions of key side chains in resting- and activated-state VS model structures, providing new insights into the structural basis of VS activation and H+ transfer mechanisms in Hv1. DOI: http://dx.doi.org/10.7554/eLife.18017.001

Voltage sensor (VS) activation likely involves an outward movement of the S4 helix from its resting state conformation, but high resolution resting-state VS structures are rare and experimental data is needed to spatially constrain model structures. Zn2+ potently (EC50 ≈ 1 uM, pHO 6.5) attenuates H+ currents in the human voltage-gated proton channel (hHv1) by shifting the apparent POPEN-V relation toward more positive potentials (Cherny, et al. 1999). Zn2+ is likely to be coordinated by two extracellularly-accessible His residues, H140 (S2 helix) and H193 (S3-S4 linker) in the resting conformation of the Hv1 VS domain (Ramsey et al., 2006). The intermediate potency (EC50 20-70 uM) of Zn2+ in single H140A or H193A mutant channels suggests that additional residues may also participate resting-state Zn2+ coordination. Candidate residues include the acidic side chains of E119 (S1) and D123 (S1-S2 linker), which may be near H140 and H193 in the mouse Hv1-based voltage sensor chimeric protein mHv1cc (Takeshita et al., 2014). To identify Zn2+-coordinating residues in hHv1, we mutated candidate acidic residues to His in the background of either H140A or H193A and measured the potency of Zn2+ to attenuate voltage-gated H+ currents. We find that E119H, but not D123H or D130H, is sufficient to reconstitute potent (low micromolar EC50) block of current by Zn2+ when combined with either H193A or H140A, indicating that E119 is likely to coordinate Zn2+ in conjunction with H140 and H193 in WT hHv1. Our experimental data limit the distances of E119, H140 and H193 side chains from the coordinated Zn2+ ion and therefore strongly constrain the positions of these groups, allowing us to generate refined resting-state hHv1 VS model structures.

The structure of an Hv1-based chimeric voltage sensor protein (mHv1cc) was recently solved by X-ray crystallography. Although mHv1cc mediates voltage-gated H+ currents when expressed in mammalian cells, the crystallized protein likely represents a closed, possibly resting, conformation. However, the mHv1cc structure is apparently incompatible with our experimental measurements of the resting-state H+ ‘shuttle’ conductance (GSH) in hHv1 R205H. In order to explore possible conformations of the Hv1 voltage sensor (VS) domain in its resting state, we created an Hv1 VS domain homology model (Hv1 D) based on a previously reported resting-state Rosetta model structure of the Shaker K+ channel VS domain (Pathak, et al. 2007) and subjected the Hv1 model to all-atom MD simulations. We subsequently created a model of the Hv1 D R205H point mutant that mediates GSH. In contrast to mHv1cc and a resting-state model of the Ciona Hv1 VS domain (Chamberlin, et al. 2013), the central crevice in our new model is not obviously occluded by hydrophobic side chains. A prominent feature of Hv1 D R205H is that the imidazole ring of the introduced histidine appears to be simultaneously accessible to intra- and extra-cellular aqueous vestiblues, and therefore appears competent for Grotthuss-type proton conduction. Hv1 D represents the first VS domain resting-state model structure that satisfies the rigorous structural constraints imposed by experimental data and thus serves as a template for understanding the structural basis of VS activation in a variety of VS domain-containing proteins.