Lake restoration by biomanipulation using piscivore and Daphnia stocking; results of the biomanipulation in Japan

Biomanipulation has been employed in numerous locations throughout the world as a means for reducing phytoplankton biomass; however, it has not been employed very often in Japan. A common approach involves the introduction of piscivorous fish to reduce the abundance of planktivorous fish. In our study, to first apply biomanipulation, we stocked Lake Shirakaba (a high-altitude, protected area in a park) in central Japan with rainbow trout fingerlings and cladoceran Daphnia (Daphnia galeata) in 2000. A “pre-biomanipulation” data set (1997–1999) and “a post-biomanipulation” data set (2000–2006) allowed us to evaluate the lake's response to biomanipulation. After the biomanipulation, zoo-planktivorous pond smelt disappeared and a large population of Daphnia had been established, which substantially reduced the number of the previously dominant small cladocerans and rotifers. Water transparency increased from about 2 m (before biomanipulation) to more than 4 m (after biomanipulation). Reductions in algal biomass and increased transparency led to expansion of the submerged macrophyte Elodea nuttallii. Total phosphorus concentrations declined as well over this time period. Based on these results, we concluded that biomanipulation using piscivore and Daphnia stocking succeeded in improving lake water quality by reducing algal abundance and providing favorable conditions for the establishment of rooted plants.     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies     

Parthenogenetic development of dwarf surf dam,Mulinia lateralis,oocytes treated with polar body suppressing agents

Summary

Parthenogenesis following oocyte activation has been observed in a number of marine invertebrates, but the fate of parthenogenesis in bivalve mollusc embryos is unclear. We used the dwarf surf clam, Mulinia lateralis, to examine parthenogenetic development of KC1-activated oocytes using the polar body suppressing agents caffeine and heat or cytochalasin B. Development was followed by epifluorescence microscopy and flow-cytometric analysis using the DNA-specific fluorochrome DAPI. All agents suppressed polar body formation to some degree, putatively increasing the ploidy level and retaining a meiotic centrosome in the zygote; but the zygotes failed to develop normally. Failure of the zygotes to develop suggests that the meiotic centrosome is incapable of participating in mitosis in bivalves.     

Gravel dredging alters diversity and structure of riverine fish assemblages

1. Human activities affect fish assemblages in a variety of ways. Large‐scale and long‐term disturbances such as in‐stream dredging and mining alter habitat and hydrodynamic characteristics within rivers which can, in turn, alter fish distribution. Habitat heterogeneity is decreased as the natural riffle–pool–run sequences are lost to continuous pools and, as a consequence, lotic species are displaced by lentic species, while generalist and invasive species displace native habitat specialists. Sediment and organic detritus accumulate in deep, dredged reaches and behind dams, disrupting nutrient flow and destroying critical habitat for habitat specialist species. 2. We used standard ecological metrics such as species richness and diversity, as well as stable isotope analysis of δ¹³C and δ¹⁵N, to quantify the differences in fish assemblages sampled by benthic trawls among dredged and undredged sites in the Allegheny River, Pennsylvania, U.S.A. 3. Using mixed‐effects models, we found that total catch, species richness and diversity were negatively correlated with depth (P < 0.05), while species richness, diversity and proportion of species in lithophilic (‘rock‐loving’) reproductive guilds were lower at dredged than at undredged sites (P < 0.05). 4. Principal components analysis and manova revealed that taxa such as darters in brood hider and substratum chooser reproductive guilds were predominantly associated with undredged sites along principal component axis 1 (PC1 and manova P < 0.05), while nest spawners such as catfish and open substratum spawners including suckers were more associated with dredged sites along PC2 (P < 0.05). 5. Stable isotope analysis of δ¹³C and δ¹⁵N revealed shifts from reliance on shallow water and benthic‐derived nutrients at undredged sites to reliance on phytoplankton and terrestrial detritus at deep‐water dredged sites. Relative trophic positions were also lower at dredged sites for many species; loss of benthic nutrient pathways associated with depth and dredging history is hypothesised. 6. The combination of ecological metrics and stable isotope analysis thus shows how anthropogenic habitat loss caused by gravel dredging can decrease benthic fish abundance and diversity, and that species in substratum‐specific reproductive guilds are at particular risk. The effects of dredging also manifest by altering resource use and nutrient pathways within food webs. Management and conservation decisions should therefore consider the protection of relatively shallow areas with suitable substratum for spawning for the protection of native fishes.     

Isolation of Possible Intermediate of Chlorogenic Acid Biosynthesis in Sweet Potato Root Tissue

Thienodolin, a new plant growth-regulating substance, was isolated from the fermentation broth of a streptomycete strain identified as Streptomyces albogriseolus.

The active principle was extracted with ethyl acetate and purified by silica gel column chromatography and preparative HPLC. The substance showed growth promoting activity with 1.2 × 10^?6–1.2 × 10^?5 M treatment to rice seedlings, and inhibitory activity with 4.0 × 10^?5 M treatment.     

Role of Surface Substances on Germinated Spores of Ceratosystis fimbriata,Black Rot Fungus,in Host-parasite Interaction

Surface substances were isolated by sonication from the germinated spores of various strains of Ceratocystis fimbriata and characterized in relation to host-parasite specificity. The substances from the sweet potato strain, compatible with sweet potato, potently inhibited the spore agglutination of various strains by spore-agglutinating factor from sweet potato roots, while the substances from incompatible strains, that is, coffee, taro, and almond strains, weakly inhibited this agglutination. The substances from the sweet potato strain increased ethylene production from sweet potato roots infected by all strains tested, sweet potato, coffee, taro, and almond strains, which was possibly an index of pathogenicity. On the other hand, the substances from incompatible strains, coffee, taro, and almond strains, suppressed the ethylene production from the tissue infected by all four strains except the substances from almond strains on almond strain. Heat and trypsin treatments inactivated the spore agglutination inhibitory activity of the surface substances. Coincidently, these treatments extinguished the effect of the surface substances on pathogenicity of C. fimbriata on sweet potato roots.     

Ameloblastin in Hertwig’s Epithelial Root Sheath Regulates Tooth Root Formation and Development

Tooth root formation begins after the completion of crown morphogenesis. At the end edge of the tooth crown, inner and outer enamel epithelia form Hertwig’s epithelial root sheath (HERS). HERS extends along with dental follicular tissue for root formation. Ameloblastin (AMBN) is an enamel matrix protein secreted by ameloblasts and HERS derived cells. A number of enamel proteins are eliminated in root formation, except for AMBN. AMBN may be related to tooth root formation; however, its role in this process remains unclear. In this study, we found AMBN in the basal portion of HERS of lower first molar in mice, but not at the tip. We designed and synthesized small interfering RNA (siRNA) targeting AMBN based on the mouse sequence. When AMBN siRNA was injected into a prospective mandibular first molar of postnatal day 10 mice, the root became shorter 10 days later. Furthermore, HERS in these mice revealed a multilayered appearance and 5-bromo-2′-deoxyuridine (BrdU) positive cells increased in the outer layers. In vitro experiments, when cells were compared with and without transiently expressing AMBN mRNA, expression of growth suppressor genes such as p21^Cip1 and p27^Kip1 was enhanced without AMBN and BrdU incorporation increased. Thus, AMBN may regulate differentiation state of HERS derived cells. Moreover, our results suggest that the expression of AMBN in HERS functions as a trigger for normal root formation.     

IL-17 Induction by ArtinM is Due to Stimulation of IL-23 and IL-1 Release and/or Interaction with CD3 in CD4+ T Cells

ArtinM is a D-mannose-binding lectin extracted from the seeds of Artocarpus heterophyllus that interacts with TLR2 N-glycans and activates antigen-presenting cells (APCs), as manifested by IL-12 production. In vivo ArtinM administration induces Th1 immunity and confers protection against infection with several intracellular pathogens. In the murine model of Candida albicans infection, it was verified that, in addition to Th1, ArtinM induces Th17 immunity manifested by high IL-17 levels in the treated animals. Herein, we investigated the mechanisms accounting for the ArtinM-induced IL-17 production. We found that ArtinM stimulates the IL-17 production by spleen cells in BALB/c or C57BL/6 mice, a response that was significantly reduced in the absence of IL-23, MyD88, or IL-1R. Furthermore, we showed that ArtinM directly induced the IL-23 mRNA expression and the IL-1 production by macrophages. Consistently, in cell suspensions depleted of macrophages, the IL-17 production stimulated by ArtinM was reduced by 53% and the exogenous IL-23 acted synergistically with ArtinM in promoting IL-17 production by spleen cell suspensions. We verified that the absence of IL-23, IL-1R, or MyD88 inhibited, but did not block, the IL-17 production by ArtinM-stimulated spleen cells. Therefore, we investigated whether ArtinM exerts a direct effect on CD4⁺ T cells in promoting IL-17 production. Indeed, spleen cell suspensions depleted of CD4⁺ T cells responded to ArtinM with very low levels of IL-17 release. Likewise, isolated CD4⁺ T cells under ArtinM stimulus augmented the expression of TGF-β mRNA and released high levels of IL-17. Considering the observed synergism between IL-23 and ArtinM, we used cells from IL-23 KO mice to assess the direct effect of lectin on CD4⁺ T cells. We verified that ArtinM increased the IL-17 production significantly, a response that was inhibited when the CD4⁺ T cells were pre-incubated with anti-CD3 antibody. In conclusion, ArtinM stimulates the production of IL-17 by CD4⁺ T cells in two major ways: (I) through the induction of IL-23 and IL-1 by APCs and (II) through the direct interaction with CD3 on the CD4⁺ T cells. This study contributes to elucidation of mechanisms accounting for the property of ArtinM in inducing Th17 immunity and opens new perspectives in designing strategies for modulating immunity by using carbohydrate recognition agents.