Inferring Models of Bacterial Dynamics toward Point Sources

Experiments have shown that bacteria can be sensitive to small variations in chemoattractant (CA) concentrations. Motivated by these findings, our focus here is on a regime rarely studied in experiments: bacteria tracking point CA sources (such as food patches or even prey). In tracking point sources, the CA detected by bacteria may show very large spatiotemporal fluctuations which vary with distance from the source. We present a general statistical model to describe how bacteria locate point sources of food on the basis of stochastic event detection, rather than CA gradient information. We show how all model parameters can be directly inferred from single cell tracking data even in the limit of high detection noise. Once parameterized, our model recapitulates bacterial behavior around point sources such as the “volcano effect”. In addition, while the search by bacteria for point sources such as prey may appear random, our model identifies key statistical signatures of a targeted search for a point source given any arbitrary source configuration.     

Immunoelectron microscopic localization of estrogen receptor with monoclonal estrophilin antibodies

The recent production of a series of monoclonal estrophilin (estrogen receptor) antibodies recognizing estrogen receptor derived from a wide variety of animals and target tissues permits the development of immunoelectron microscopic techniques for identifying estrogen receptor. We have determined suitable conditions for the ultrastructural localization of estrogen receptor in tissue sections. Localization of receptor was observed in the euchromatin, but not in the marginated heterochromatin or nucleoli of epithelial and stromal nuclei of human endometrium. Competition studies indicate that only estrogen receptor specifically inhibits nuclear staining. The absence of any specific cytoplasmic localization at the electron-microscopic level is consistent with earlier light-microscopic observations and suggests that the majority of the cellular pool of estrophilin exists in the nucleus of hormone-responsive cells.     

Awareness modifies the skill-learning benefits of sleep

Behind every skilled movement lies months of practice. However, practice alone is not responsible for the acquisition of all skill; performance can improve between, not just within, practice sessions. An important principle shaping these offline improvements may be an individual's awareness of learning a new skill. New skills, such as a sequence of finger movements, can be learned unintentionally (with little awareness for the sequence, implicit learning) or intentionally (explicit learning). We measured skill in an implicit and explicit sequence-learning task before and after a 12 hr interval. This interval either did (8 p.m. to 8 a.m.) or did not (8 a.m. to 8 p.m.) include a period of sleep. Following explicit sequence learning, offline skill improvements were only observed when the 12 hr interval included sleep. This overnight improvement was correlated with the amount of NREM sleep. The same improvement could also be observed in the evening (with an interval from 8 p.m. to 8 p.m.), so it was not coupled to retesting at a particular time of day and cannot therefore be attributed to circadian factors. In contrast, in the implicit learning task, offline learning was observed regardless of whether the 12 hr interval did or did not contain a period of sleep. However, these improvements were not observed with only a 15 min interval between sessions. Therefore, the practice available within each session cannot account for these skill improvements. Instead, sufficient time is necessary for offline learning to occur. These results show a behavioral dissociation, based upon an individual's awareness for having learned a sequence of finger movements. Offline learning is sleep dependent for explicit skills but time dependent for implicit skills.     

Growth and survivorship of dipterocarp seedlings: differences in shade persistence create a special case of dispersal limitation

A series of growth experiments and observations on natural populations have been carried out on dipterocarp species of contrasting ecology growing in artificial gaps and the forest understorey. These studies have demonstrated that although differences exist between species in photosynthetic and growth responses to the high-light environment, competition for light in canopy gaps is highly asymmetrical and tends to reinforce any pre-existing dominance hierarchy. We propose that differences in seedling persistence in forest canopy shade are highly influenced by species-specific biotic and abiotic interactions. Our experiments suggest that as seedlings, dipterocarp species trade off traits which enhance persistence and growth in shade against those that enhance their ability to exploit gaps. Less competitive species survive for progressively longer periods of time after a gregarious fruiting event. This leads to significant shifts with time in the number of species present in the seedling bank and hence in the importance of interspecific competition in determining which species dominates regrowth in gaps. We propose that this special case of dispersal limitation is more likely to account for coexistence of dipterocarp species than differences in growth responses to gaps of different size, with stochastic and environmental variables interacting to determine species distribution and abundance.     

Litter of the hemiparasite<Emphasis Type="Italic"> Bartsia alpina</Emphasis> enhances plant growth: evidence for a functional role in nutrient cycling

Hemiparasitic angiosperms concentrate nutrients in their leaves and also produce high quality litter, which can decompose faster and release more nutrients than that of surrounding species. The impact of these litters on plant growth may be particularly important in nutrient-poor communities where hemiparisites can be abundant, such as the sub-Arctic. We tested the hypothesis that plant growth is enhanced by the litter of the hemiparasite Bartsia alpina, in comparison with litter of co-occurring dwarf shrub species, using a pot based bioassay approach. Growth of Betula nana and Poa alpina was up to 51% and 41% greater, respectively, in the presence of Bartsia alpina litter than when grown with dwarf shrub litter (Vaccinium uliginosum, Betula nana and Empetrum nigrum subsp. hermaphroditum). The nutrient concentrations of Betula nana plants grown with Bartsia alpina litter were almost double those of plants grown with dwarf shrub litter, and a significantly greater proportion of biomass was allocated to shoots rather than roots, strongly suggesting that nutrient availability was higher where Bartsia alpina litter was present. The presence of litter from dwarf shrubs, or the moss Hylocomium splendens, did not reduce the positive effect of Bartsia alpina litter on plant growth. E. nigrum litter did not appear to affect plant growth substantially differently from litter of other dwarf shrub species, despite earlier reports of its allelopathic action. The enhanced nutrient uptake and growth of plants in the presence of Bartsia alpina (and potentially other hemiparasitic species) litter could have important implications for communities in which it occurs, including enhanced survival of seedlings of co-occurring species and increased resource patchiness.     

A biomimetic pH-responsive polymer directs endosomal release and intracellular delivery of an endocytosed antibody complex

Poly(propylacrylic acid) (PPAAc) is a synthetic pH-responsive polymer that has been shown to disrupt cell membranes at low pH values typical of the endosome, but not at physiological pH, suggesting its use as an endosomal-releasing agent [Murthy et al. J. Controlled Release 61, 137-43]. We have constructed an antibody-targeted biotherapeutic model to investigate whether PPAAc can enhance intracellular trafficking of proteins to the cytoplasm. A ternary complex composed of a biotinylated anti-CD3 antibody, streptavidin, and biotinylated PPAAc was fluorescently labeled, and its intracellular fate was analyzed by confocal microscopy, flow cytometry, and quantitative western blotting of cell fractionates. The 64.1 anti-CD3 antibody was previously shown to direct receptor-mediated endocytosis in the Jurkat T-cell lymphoma cell line and was rapidly trafficked from the endosome to the lysosomal compartment. The antibody-streptavidin complex was also rapidly internalized to the endosomal/lysosomal compartment and retained there, as evidenced by punctate regions of fluorescence observed by confocal fluorescence microscopy. In samples containing the ternary complex of antibody, streptavidin, and PPAAc-biotin, diffuse fluorescence in the cytoplasm was observed, indicating that PPAAc enhanced translocation to the cytoplasm. This was confirmed by western blotting analysis of the isolated cytoplasm. Flow cytometry results demonstrated that neither streptavidin nor PPAAc caused nonspecific uptake of the complex, nor did they inhibit antibody-mediated endocytosis. The striking enhancement of protein delivery to the cytoplasm by complexed PPAAc suggests that this polymer could provide a new delivery agent for therapeutic, vaccine, and diagnostics development.     

Plant eats plant: sap-feeding witchweeds and other parasitic angiosperms

About one in every hundred species of flowering plant is parasitic and obtain some or all of their carbon, nutrients and water from the sap of their hosts. They possess unique morphological and metabolic adaptations but are more than just botanical curiosities.     

FV-162 is a novel,orally bioavailable,irreversible proteasome inhibitor with improved pharmacokinetics displaying preclinical efficacy with continuous daily dosing

Approved proteasome inhibitors have advanced the treatment of multiple myeloma but are associated with serious toxicities, poor pharmacokinetics, and most with the inconvenience of intravenous administration. We therefore sought to identify novel orally bioavailable proteasome inhibitors with a continuous daily dosing schedule and improved therapeutic window using a unique drug discovery platform. We employed a fluorine-based medicinal chemistry technology to synthesize 14 novel analogs of epoxyketone-based proteasome inhibitors and screened them for their stability, ability to inhibit the chymotrypsin-like proteasome, and antimyeloma activity in vitro. The tolerability, pharmacokinetics, pharmacodynamic activity, and antimyeloma efficacy of our lead candidate were examined in NOD/SCID mice. We identified a tripeptide epoxyketone, FV-162, as a metabolically stable, potent proteasome inhibitor cytotoxic to human myeloma cell lines and primary myeloma cells. FV-162 had limited toxicity and was well tolerated on a continuous daily dosing schedule. Compared with the benchmark oral irreversible proteasome inhibitor, ONX-0192, FV-162 had a lower peak plasma concentration and longer half-life, resulting in a larger area under the curve (AUC). Oral FV-162 treatment induced rapid, irreversible inhibition of chymotrypsin-like proteasome activity in murine red blood cells and inhibited tumor growth in a myeloma xenograft model. Our data suggest that oral FV-162 with continuous daily dosing schedule displays a favorable safety, efficacy, and pharmacokinetic profile in vivo, identifying it as a promising lead for clinical evaluation in myeloma therapy.The ubiquitin–proteasome system is responsible for the regulation and degradation of the majority of the intracellular proteins in eukaryotic cells.¹ The 26S proteasome is a multi-subunit protein complex that mediates the proteolytic degradation and turnover of damaged, misfolded or excess proteins that have been polyubiquitylated in the cytoplasm and nucleus.^{1, 2} The 26S proteasome consists of a 20S core particle, capped by 19S regulatory particles.^{3, 4} The 19S particle participates in the recognition, processing, unfolding, and translocation of ubiquitylated protein substrates into the 20S core.⁵ Substrates are then degraded inside the chamber of the barrel-like 20S core particle, where the active sites of multiple β1, β2, and β5 subunits catalyze caspase-like (C-L), trypsin-like (T-L), or chymotrypsin-like (CT-L) proteolysis, respectively.^{6, 7} Inhibition of the 26S proteasome activity leads to disruption of the cell cycle and induction of apoptosis.⁸Cancer cells have an increased dependency on the integrity of the ubiquitin–proteasome system machinery compared with normal cells in preclinical studies. This finding is predominantly evident in hematological malignancies, identifying the 26S proteasome as a promising anticancer therapeutic target.^{9, 10, 11, 12} In particular, cells derived from multiple myeloma are notably sensitive to proteasome inhibition, at least in part, owing to their characteristically high rates of immunoglobulin protein biosynthesis and increased proteasome activity.^{13, 14, 15} The continuous activity of the proteasome in myeloma cells makes them particularly susceptible to prolonged inhibition.¹⁶Bortezomib, the first proteasome inhibitor approved for clinical use, is a dipeptide boronic acid that reversibly binds to the active site of the β5 and β1 subunit to competitively inhibit proteasome function.^{9, 10, 17} By inhibiting the proteasome, bortezomib acts through multiple cellular pathways that ultimately result in cell cycle arrest and apoptosis.¹⁸ Bortezomib is currently approved for the treatment of newly diagnosed, relapsed or refractory multiple myeloma and mantle cell lymphoma.¹⁸ Carfilzomib was subsequently developed as a second-generation inhibitor that belongs to the epoxyketone class and irreversibly binds to the active site of the β5 subunit of the proteasome. Carfilzomib is structurally and mechanistically distinct from bortezomib and overcomes bortezomib resistance in multiple myeloma cell lines and in primary multiple myeloma cells from patients.^{17, 19} Carfilzomib is currently also approved for relapsed and refractory multiple myeloma. ONX-0912 (also known as oprozomib) is another epoxyketone class oral proteasome inhibitor that is an analog of carfilzomib.^{20, 21} Similar to carfilzomib, ONX-0912 promotes cell death in primary myeloma cells from patients who relapsed after treatment with bortezomib.²⁰ ONX-0912 has advanced into phase I/II trials in hematological and solid malignancies.^{22, 23, 24}Despite their clinical efficacy, treatment with proteasome inhibitors is associated with a number of toxicities, including neuropathy, thrombocytopenia, and cardiotoxicity.^{25, 26, 27} The toxicity of currently available proteasome inhibitors necessitates administering the drugs in intermittent dosing schedules, typically biweekly. Although intermittent dosing permits proteasome activity in normal tissues during dose holidays, it has been shown to be sub-optimal for therapy in malignant cells.¹⁶ Moreover, infrequent administration at relatively high exposures may give rise to undesirable and potentially unnecessary toxicities in normal cells. Potentially, by moderating exposures, an optimized oral proteasome inhibitor with continuous daily dosing could be developed that exploits the high proteasome dependency in malignant cells while sparing normal cells.In the present study, we report the development of FV-162, a novel, metabolically stable and orally bioavailable epoxyketone-based proteasome inhibitor. FV-162 displays potent anticancer activity and maintains a wide differential activity between malignant and normal cells despite a continuous daily dosing schedule in multiple myeloma cell lines, primary patient cells, and animal models. Overall, our results show that FV-162 inhibits the proteasome, displays metabolic stability, and has a favorable toxicity profile.     

Structure and dynamics of the estrogen receptor

To evaluate the structure and function of estrogen receptor (ER) in various mammalian systems, the cytosolic forms of receptor from calf uterus and from MCF-7 human breast cancer cells have been purified to virtual homogeneity by sequential selective adsorption to estradiol-Sepharose and heparin-Sepharose. In both cases, the purified steroid-receptor complex appears to exist as an activated 5S homo- or heterodimer of mol. wt 65,000 (4S) steroid-binding subunits. Purified ER has high affinity for DNA and serves as a substrate for phosphorylation by a purified rat brain kinase. Several monoclonal antibodies prepared against affinity-purified MCF-7 cytosol ER have been used to localize receptor by an indirect immunoperoxidase technique in fixed, frozen sections of human breast tumors, human uterus, rabbit uterus and in other mammalian reproductive tissues and cancers, as well as in fixed MCF-7 cell cultures and in paraffin-embedded sections of breast tumors and human endometrium. In all cases, we have observed only nuclear localization of immunoreactive receptor in tissues and whole cells, even under conditions in which virtually all of the receptor is found in a low-salt extract (cytosol) of the target cells. Treatment of cells or tissues in vivo or in vitro with estradiol alters the intensity but not the distribution of specific staining for ER. By immunoelectron microscopy, receptor was localized in the euchromatin, but not in the marginated heterochromatin or nucleoli of MCF-7 nuclei and epithelial and stromal nuclei of postmenopausal human endometrium. These observations suggest that the majority of the unoccupied receptor may actually reside in the nucleus, rather than in the cytoplasm as previously thought. Thus, hormone action may involve binding of the steroid directly to receptor loosely associated with nuclear components, followed by conversion of the steroid-receptor complex to an activated form which becomes more tightly associated with chromatin.