We have studied the three-dimensional arrangement of ventricular muscle cells and the associated extracellular connective tissue matrix in dog hearts. Four hearts were potassium-arrested, excised, and perfusion-fixed at zero transmural pressure. Full-thickness segments were cut from the right and left ventricular walls at a series of precisely located sites. Morphology was visualized macroscopically and with scanning electron microscopy in 1) transmural planes of section and 2) planes tangential to the epicardial surface. The appearance of all specimens was consistent with an ordered laminar arrangement of myocytes with extensive cleavage planes between muscle layers. These planes ran radially from endocardium toward epicardium in transmural section and coincided with the local muscle fiber orientation in tangential section. Stereological techniques were used to quantify aspects of this organization. There was no consistent variation in the cellular organization of muscle layers (48.4 +/- 20.4 microns thick and 4 +/- 2 myocytes across) transmurally or in different ventricular regions (23 sites in 6 segments), but there was significant transmural variation in the coupling between adjacent layers. The number of branches between layers decreased twofold from subepicardium to midwall, whereas the length distribution of perimysial collagen fibers connecting muscle layers was greatest in the midwall. We conclude that ventricular myocardium is not a uniformly branching continuum but a laminar hierarchy in which it is possible to identify three axes of material symmetry at any point.

Process intensification in biomanufacturing has attracted a great deal of interest in recent years. Manufacturing platform improvements leading to higher cell density and bioreactor productivity have been pursued. Here we evaluated a variety of intensified mammalian cell culture processes for producing monoclonal antibodies. Cell culture operational modes including fed-batch (normal seeding density or high seeding density with N-1 perfusion), perfusion, and concentrated fed-batch (CFB) were assessed using the same media set with the same Chinese Hamster Ovary (CHO) cell line. Limited media modification was done to quickly fit the media set to different operational modes. Perfusion and CFB processes were developed using an alternating tangential flow filtration device. Independent of the operational modes, comparable cell specific productivity (fed-batch: 29.4 pg/cell/day; fed-batch with N-1 perfusion: 32.0 pg/cell/day; perfusion: 31.0 pg/cell/day; CFB: 20.1 - 45.1 pg/cell/day) was reached with similar media conditions. Continuous media exchange enabled much higher bioreactor productivity in the perfusion (up to 2.29 g/L/day) and CFB processes (up to 2.04 g/L/day), compared with that in the fed-batch processes (ranging from 0.39 to 0.49 g/L/day), largely due to the higher cell density maintained. Furthermore, media cost per gram of antibody produced from perfusion was found to be highly comparable with that from fed-batch; and the media cost for CFB was the highest due to the short batch duration. Our experimental data supports the argument that media cost for perfusion process could be even lower than that in a fed-batch process, as long as sufficient bioreactor productivity is achieved. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:867-878, 2017.

Mcl-1, an antiapoptotic member of the Bcl-2 family of proteins, is a validated and attractive target for cancer therapy. Overexpression of Mcl-1 in many cancers results in disease progression and resistance to current chemotherapeutics. Utilizing high-throughput screening, compound 1 was identified as a selective Mcl-1 inhibitor and its binding to the BH3 binding groove of Mcl-1 was confirmed by several different, but complementary, biochemical and biophysical assays. Guided by structure-based drug design and supported by NMR experiments, comprehensive SAR studies were undertaken and a potent and selective inhibitor, compound 21, was designed which binds to Mcl-1 with a Ki of 180 nM. Biological characterization of 21 showed that it disrupts the interaction of endogenous Mcl-1 and biotinylated Noxa-BH3 peptide, causes cell death through a Bak/Bax-dependent mechanism, and selectively sensitizes Eμ-myc lymphomas overexpressing Mcl-1, but not Eμ-myc lymphoma cells overexpressing Bcl-2. Treatment of human leukemic cell lines with compound 21 resulted in cell death through activation of caspase-3 and induction of apoptosis.

Some investigators have suggested that the marked activity of flavone acetic acid (FAA) against advanced solid tumors in mice results from an indirect effect. This study indicates that the critical effect of FAA is irreversible inhibition of tumor blood flow. Perfusion of sc Colon 38 tumors, assessed with H33342 as a fluorescent stain for functional blood vessels, was reduced to 50% of controls within 3 hours of an ip injection of 1.2 mmol of FAA/kg and was completely inhibited by 24 hours. A double-label fluorescence technique demonstrated a significant decrease in blood flow in both sc Colon 38 and im EMT-6/Ak tumors as early as 15 minutes after iv treatment with 1.2 mmol of FAA/kg, with progressively enlarging zones of perfusion failure. The rate of cell death in totally ischemic EMT-6 tumors was shown to be sufficiently rapid to represent a major component of the observed antitumor effect of FAA if the flavonoid acts via inhibition of blood flow. Further, avascular EMT-6/Ak multicellular spheroids growing in the mouse peritoneum are relatively resistant to killing by FAA administered iv or ip, despite extensive infiltration with host immune cells. These results indicate that inhibition of tumor blood flow by FAA is a necessary component of its antitumor activity against solid tumors.

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