We have investigated the physical binding of pyrene and benzo[a]pyrene derivatives to denatured DNA. These compounds exhibit a red shift in their absorbance spectra of 9 nm when bound to denatured calf thymus DNA, compared to a shift of 10 nm when binding occurs to native DNA. Fluorescence from the hydrocarbons is severely quenched when bound to both native and denatured DNA. Increasing sodium ion concentration decreases binding of neutral polycyclic aromatic hydrocarbons to native DNA and increases binding to denatured DNA. The direct relationship between binding to denatured DNA and salt concentration appears to be a general property of neutral polycyclic aromatic hydrocarbons. Absorption measurements at 260 nm were used to determine the duplex content of denatured DNA. When calculated on the basis of duplex binding sites, equilibrium constants for binding of 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydro-benzo[a]pyrene to denatured DNA are an order of magnitude larger than for binding to native DNA. The effect of salt on the binding constant was used to calculate the sodium ion release per bound ligand, which was 0.36 for both native and denatured DNA. Increasing salt concentration increases the duplex content of denatured DNA, and it appears that physical binding of polycyclic aromatic hydrocarbons consists of intercalation into these sites.

Human induced pluripotent stem cells (iPSCs) promise to revolutionize research and therapy of liver diseases by providing a source of hepatocytes for autologous cell therapy and disease modeling. However, despite progress in advancing the differentiation of iPSCs into hepatocytes (iPSC-Heps) in vitro1–3, cells that replicate the ability of human primary adult hepatocytes (aHeps) to proliferate extensively in vivo have not been reported. This deficiency has hampered efforts to recreate human liver diseases in mice, and has cast doubt on the potential of iPSC-Heps for liver cell therapy. The reason is that extensive post-transplant expansion is needed to establish and sustain a therapeutically effective liver cell mass in patients, a lesson learned from clinical trials of aHep transplantation4. As a solution to this problem, we report generation of human fibroblast-derived hepatocytes that can repopulate mouse livers. Unlike current protocols for deriving hepatocytes from human fibroblasts, ours did not generate iPSCs, but shortcut reprogramming to pluripotency to generate an induced multipotent progenitor cell (iMPC) state from which endoderm progenitor cells (iMPC-EPCs) and subsequently hepatocytes (iMPC-Heps) could be efficiently differentiated. For this, we identified small molecules that aided endoderm and hepatocyte differentiation without compromising proliferation. After transplantation into an immune-deficient mouse model of human liver failure, iMPC-Heps proliferated extensively and acquired levels of hepatocyte function similar to aHeps. Unfractionated iMPC-Heps did not form tumors, most likely because they never entered a pluripotent state. To our knowledge, this is the first demonstration of significant liver repopulation of mice with human hepatocytes generated in vitro, which removes a long-standing roadblock on the path to autologous liver cell therapy.

To examine the suitability of synthetic peptides as DNA-binding and -compacting agents for receptor-mediated gene delivery, we have synthesized and characterized a series of branched oligocationic peptides that differ in the number and type (lysine, arginine, ornithine) of cationic amino acids in the DNA-binding moiety. The peptides were designed as branched molecules to provide a coupling site via a spacer for the attachment of effectors at a flexible distance from the DNA-binding moiety. This design provides torsional flexibility in the peptide backbone of the DNA-binding moiety to maximize cation-DNA phosphate interactions and also minimizes the potential for interference by the effector with DNA binding. The branched peptides bind DNA with affinities that increase with the number of cationic groups. The peptides compact DNA into microparticulate structures as judged by an ethidium bromide displacement assay, dynamic light scattering, and electron microscopy. In general, differences in DNA binding and compaction owing to variation in the cationic side chain were modest, with the rank order being arginyl > lysyl approximately ornithyl. Incorporation of tryptophans into the DNA-binding moiety had no major effect on apparent binding affinity but clearly reduced the DNA-compacting potency of the peptides. Compared with polylysine, the peptides and their DNA complexes are weak activators of the complement system. Complement activation by an octaarginyl peptide was stronger than that induced by an octalysyl peptide. The microparticulate peptide-DNA complexes are suitable for receptor-mediated gene delivery as evidenced by transferrinfection of K562 cells in the presence of chloroquine. The results obtained in gene delivery in vitro suggest that a minimum chain length of six to eight cationic amino acids is required to compact DNA into structures active in receptor-mediated gene delivery.

Solid organ transplantation in human immunodeficiency virus (HIV)-infected individuals requiring con) is complicated by significant drug interactions. To assist in appropriate clinical management, we describe the pharmacokinetics and dosing modifications in 35 patients (20 kidney, 13 liver and two kidney-liver HIV-infected subjects with end-stage kidney or liver disease), on both IS and NNRTIs, PIs, and combined NNRTIs + PIs, in studies done at weeks 2-4 and/or 12 weeks after transplantation or after a change in IS or ARV drug regimen (n = 97 studies). CsA, SrL and FK concentrations were measured in whole blood by LC/MS. HIV-infected transplant recipients using PIs with IS had marked increases in CsA, FK or SrL trough levels compared to those on NNRTIs alone or to patients not on ARVs, necessitating either a reduction in dose or an increase in dosing interval. Subjects on efavirenz (EFV) and CsA required much higher doses of CsA than those using any other ARV. Changes in antiretroviral therapy should be carefully managed to avoid insufficient immunosuppression or toxicity due to drug interactions.

Cruzain is an essential cysteine protease of Trypanosoma cruzi and a therapeutic target for Chagas' disease. Eight dogs were infected with T. cruzi; three were treated with an inhibitor of cruzain, K777, for 14 days. Treatment with K777 abrogated myocardial damage by T. cruzi, as documented by histopathological lesion scores and serum troponin I levels.