Ileal bile acid transporter inhibition in Cyp2c70 KO mice ameliorates cholestatic liver injury.

Cyp2c70 is the liver enzyme in rodents responsible for synthesis of the primary 6-hydroxylated muricholate bile acid (BA) species. Cyp2c70 KO mice are devoid of protective, hydrophilic muricholic acids, leading to a more human-like BA composition and subsequent cholestatic liver injury. Pharmacological inhibition of the ileal BA transporter (IBAT) has been shown to be therapeutic in cholestatic models. Here, we aimed to determine if IBAT inhibition with SC-435 is protective in Cyp2c70 KO mice. As compared to WT mice, we found male and female Cyp2c70 KO mice exhibited increased levels of serum liver injury markers, and our evaluation of liver histology revealed increased hepatic inflammation, macrophage infiltration, and biliary cell proliferation. We demonstrate serum and histologic markers of liver damage were markedly reduced with SC-435 treatment. Additionally, we show hepatic gene expression in pathways related to immune cell activation and inflammation were significantly upregulated in Cyp2c70 KO mice and reduced to levels indistinguishable from WT with IBAT inhibition. In Cyp2c70 KO mice, the liver BA content was significantly increased, enriched in chenodeoxycholic acid, and more hydrophobic, exhibiting a hydrophobicity index value and red blood cell lysis properties similar to human liver BAs. Furthermore, we determined IBAT inhibition reduced the total hepatic BA levels but did not affect overall hydrophobicity of the liver BAs. These findings suggest that there may be a threshold in the liver for pathological accretion of hydrophobic BAs and reducing hepatic BA accumulation can be sufficient to alleviate liver injury, independent of BA pool hydrophobicity.

Coarse-grained simulations of hemolytic peptide δ-lysin interacting with a POPC bilayer.

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.

A molecular dynamics study of cell-penetrating peptide transportan-10 (TP10): Binding, folding and insertion to transmembrane state in zwitterionic membrane.

Transportan 10 (TP10) is a 21-residue, cationic, α-helical cell-penetrating peptide that can be used as a delivery vector for various bioactive molecules. Based on recent confocal microscopy studies, it is believed that TP10 can translocate across neutral lipid membrane passively, possibly as a monomer, without the formation of permanent pore. Here, we performed extensive molecular dynamics (MD) simulations of TP10W (Y3W variant of TP10) to find the microscopic details of binding, folding and insertion of TP10W to transmembrane state in POPC bilayer. Binding study with CHARMM36 force field showed that TP10W initially binds to the membrane surface in unstructured configuration, but it spontaneously folds into α-helical conformation under the lipid head groups. Further insertion of TP10W, changing from a surface bound state to a vertically oriented transmembrane state, was investigated via umbrella simulations. The resulting free energy profile shows a relatively small barrier between two states, suggesting a possible translocation pathway as a monomer. In fact, unbiased simulation of transmembrane TP10W revealed how a charged Lys side chain can move from one leaflet to the other without a significant free energy cost. Finally, we compared the results of TP10W simulations with those of point mutated variants (TP10W-K12A18 and TP10W-K19L) to understand the effect of charge distribution on the peptide. It was observed that such a conservative mutation can cause noticeable changes in the conformations of both surface bound and transmembrane states. The results of present study will be discussed in relation to the experimentally observed activities of TP10W against neutral membrane.

Microsecond dynamics control the HIV-1 Envelope conformation.

The HIV-1 Envelope (Env) glycoprotein facilitates host cell fusion through a complex series of receptor-induced structural changes. Although remarkable progress has been made in understanding the structures of various Env conformations, microsecond timescale dynamics have not been studied experimentally. Here, we used time-resolved, temperature-jump small-angle x-ray scattering to monitor structural rearrangements in an HIV-1 Env SOSIP ectodomain construct with microsecond precision. In two distinct Env variants, we detected a transition that correlated with known Env structure rearrangements with a time constant in the hundreds of microseconds range. A previously unknown structural transition was also observed, which occurred with a time constant below 10 µs, and involved an order-to-disorder transition in the trimer apex. Using this information, we engineered an Env SOSIP construct that locks the trimer in the prefusion closed state by connecting adjacent protomers via disulfides. Our findings show that the microsecond timescale structural dynamics play an essential role in controlling the Env conformation with impacts on vaccine design.

Microsecond dynamics control the HIV-1 envelope conformation.

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.

Doxorubicin for treatment of histiocytic sarcoma in dogs: 31 cases (2003-2017).

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.

Coupling between an electrostatic network and the Zn2+ binding site modulates Hv1 activation.

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.

Antibody-mediated immune suppression by antigen modulation is antigen-specific.

Alloantibodies developing after exposure to red blood cell (RBC) alloantigens can complicate pregnancy and transfusion therapy. The only method currently available to actively inhibit RBC alloantibody formation is administration of antigen-specific antibodies, a phenomenon termed antibody-mediated immune suppression (AMIS). A well-known example of AMIS is RhD immune globulin prophylaxis to prevent anti-D formation in RhD- individuals. However, whether AMIS is specific or impacts alloimmunization to other antigens on the same RBC remains unclear. To evaluate the specificity of AMIS, we passively immunized antigen-negative recipients with anti-KEL or anti-hen egg lysozyme (HEL) antibodies, followed by transfusion of murine RBC expressing both the HEL-ovalbumin-Duffy (HOD) and human KEL antigens (HOD × KEL RBC). Significant immunoglobulin G deposition on transfused HOD × KEL RBC occurred in all passively immunized recipients. Complement deposition and antigen modulation of the KEL antigen occurred on transfused RBC only in anti-KEL-treated recipients, whereas HEL antigen levels decreased only in the presence of anti-HEL antibodies. Western blot analysis confirmed the specificity of antigen loss, which was not attributable to RBC endocytosis and appears distinct for the 2 antigens. Specifically, removal of KEL was attenuated by clodronate treatment, whereas loss of HEL was unaffected by clodronate in vivo but sensitive to protease treatment in vitro. Antigen-specific modulation correlated with antigen-specific AMIS, with anti-KEL treated recipients forming antibodies to the HOD antigen and anti-HEL-treated recipients developing antibodies to the KEL antigen. Together, these results demonstrate that passively administered antibodies can selectively inhibit the immune response to a specific antigen.

Marginal Zone B Cells Induce Alloantibody Formation Following RBC Transfusion.

Red blood cell (RBC) alloimmunization represents a significant immunological challenge for some patients. While a variety of immune constituents likely contribute to the initiation and orchestration of alloantibodies to RBC antigens, identification of key immune factors that initiate alloantibody formation may aid in the development of a therapeutic modality to minimize or prevent this process. To define the immune factors that may be important in driving alloimmunization to an RBC antigen, we determined the specific immune compartment and distinct cells that may initially engage transfused RBCs and facilitate subsequent alloimmunization. Our findings demonstrate that the splenic compartment is essential for formation of anti-KEL antibodies following KEL RBC transfusion. Within the spleen, transfused KEL RBCs are found within the marginal sinus, where they appear to specifically co-localize with marginal zone (MZ) B cells. Consistent with this, removal of MZ B cells completely prevented alloantibody formation following KEL RBC transfusion. While MZ B cells can mediate a variety of key downstream immune pathways, depletion of follicular B cells or CD4 T cells failed to similarly impact the anti-KEL antibody response, suggesting that MZ B cells may play a key role in the development of anti-KEL IgM and IgG following KEL RBC transfusion. These findings highlight a key contributor to KEL RBC-induced antibody formation, wherein MZ B cells facilitate antibody formation following RBC transfusion.

Proton currents constrain structural models of voltage sensor activation.

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.