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.     

Studies on Calf Diarrhoea in Mozambique: Prevalence of Bacterial Pathogens

The prevalence of diarrhoea in calves was investigated in 8 dairy farms in Mozambique at 4 occasions during 2 consecutive years. A total of 1241 calves up to 6 months of age were reared in the farms, and 63 (5%) of them had signs of diarrhoea. Two farms had an overall higher prevalence (13% and 21%) of diarrhoea. Faecal samples were collected from all diarrhoeal calves (n = 63) and from 330 healthy calves and analysed for Salmonella species, Campylobacter jejuni and enterotoxigenic Escherichia coli (ETEC). Salmonella spp. was isolated in only 2% of all calves. Campylobacter was isolated in 11% of all calves, irrespective of health condition, and was more frequent (25%) in one of the 2 diarrhoeal farms (p = 0.001). 80% of the isolates were identified as C. jejuni. No ETEC strains were detected among the 55 tested strains from diarrhoeal calves, but 22/55 (40%) strains from diarrhoeal calves and 14/88 (16%) strains from healthy calves carried the K99 adhesin (p = 0.001). 6,757 E. coli isolates were typed with a biochemical fingerprinting method (the PhenePlate?) giving the same E. coli diversity in healthy and diarrhoeal calves. Thus it was concluded: i) the overall prevalence of diarrhoea was low, but 2 farms had a higher prevalence that could be due to an outbreak situation, ii) Salmonella did not seem to be associated with diarrhoea, iii) Campylobacter jejuni was common at one of the 2 diarrhoeal farms and iv) ETEC strains were not found, but K99 antigen was more prevalent in E. coli strains from diarrhoeal calves than from healthy, as well as more prevalent in one diarrhoeal farm.     

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.     

Advanced age promotes colonic dysfunction and gut‐derived lung infection after stroke

Bacterial infection a leading cause of death among patients with stroke, with elderly patients often presenting with more debilitating outcomes. The findings from our retrospective study, supported by previous clinical reports, showed that increasing age is an early predictor for developing fatal infectious complications after stroke. However, exactly how and why older individuals are more susceptible to infection after stroke remains unclear. Using a mouse model of transient ischaemic stroke, we demonstrate that older mice (>12 months) present with greater spontaneous bacterial lung infections compared to their younger counterparts (7–10 weeks) after stroke. Importantly, we provide evidence that older poststroke mice exhibited elevated intestinal inflammation and disruption in gut barriers critical in maintaining colonic integrity following stroke, including reduced expression of mucin and tight junction proteins. In addition, our data support the notion that the localized pro‐inflammatory microenvironment driven by increased tumour necrosis factor‐α production in the colon of older mice facilitates the translocation and dissemination of orally inoculated bacteria to the lung following stroke onset. Therefore, findings of this study demonstrate that exacerbated dysfunction of the intestinal barrier in advanced age promotes translocation of gut‐derived bacteria and contributes to the increased risk to poststroke bacterial infection.     

Inferring Diffusion Dynamics from FCS in Heterogeneous Nuclear Environments

Fluorescence correlation spectroscopy (FCS) is a noninvasive technique that probes the diffusion dynamics of proteins down to single-molecule sensitivity in living cells. Critical mechanistic insight is often drawn from FCS experiments by fitting the resulting time-intensity correlation function, G(t), to known diffusion models. When simple models fail, the complex diffusion dynamics of proteins within heterogeneous cellular environments can be fit to anomalous diffusion models with adjustable anomalous exponents. Here, we take a different approach. We use the maximum entropy method to show—first using synthetic data—that a model for proteins diffusing while stochastically binding/unbinding to various affinity sites in living cells gives rise to a G(t) that could otherwise be equally well fit using anomalous diffusion models. We explain the mechanistic insight derived from our method. In particular, using real FCS data, we describe how the effects of cell crowding and binding to affinity sites manifest themselves in the behavior of G(t). Our focus is on the diffusive behavior of an engineered protein in 1) the heterochromatin region of the cell’s nucleus as well as 2) in the cell’s cytoplasm and 3) in solution. The protein consists of the basic region-leucine zipper (BZip) domain of the CCAAT/enhancer-binding protein (C/EBP) fused to fluorescent proteins.     

A preliminary investigation into the relationship between functional movement screen scores and athletic physical performance in female team sport athletes

There is little research investigating relationships between the Functional Movement Screen (FMS) and athletic performance in female athletes. This study analyzed the relationships between FMS (deep squat; hurdle step [HS]; in-line lunge [ILL]; shoulder mobility; active straight-leg raise [ASLR]; trunk stability push-up; rotary stability) scores, and performance tests (bilateral and unilateral sit-and-reach [flexibility]; 20-m sprint [linear speed]; 505 with turns from each leg; modified T-test with movement to left and right [change-of-direction speed]; bilateral and unilateral vertical and standing broad jumps; lateral jumps [leg power]). Nine healthy female recreational team sport athletes (age = 22.67 ± 5.12 years; height = 1.66 ± 0.05 m; body mass = 64.22 ± 4.44 kilograms) were screened in the FMS and completed the afore-mentioned tests. Percentage between-leg differences in unilateral sit-and-reach, 505 turns and the jumps, and difference between the T-test conditions, were also calculated. Spearman''s correlations (p ≤ 0.05) examined relationships between the FMS and performance tests. Stepwise multiple regressions (p ≤ 0.05) were conducted for the performance tests to determine FMS predictors. Unilateral sit-and-reach positive correlated with the left-leg ASLR (r = 0.704-0.725). However, higher-scoring HS, ILL, and ASLR related to poorer 505 and T-test performance (r = 0.722-0.829). A higher-scored left-leg ASLR related to a poorer unilateral vertical and standing broad jump, which were the only significant relationships for jump performance. Predictive data tended to confirm the correlations. The results suggest limitations in using the FMS to identify movement deficiencies that could negatively impact athletic performance in female team sport athletes.     

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.