Repeated polyketide synthase modules involved in the biosynthesis of a heptaene macrolide by Streptomyces sp. FR-008

Genes for biosynthesis of a Streptomyces sp. FR-008 heptaene macrolide antibiotic with antifungal and mosquito larvicidal activity were cloned in Escherichia coli using heterologous DNA probes. The cloned genes were implicated in heptaene biosynthiesis by gene replacement. The FR-008 antibiotic contains a 38-membered, poiyketide-derived macrolide ring. Southern hybridization using probes encoding domains of the type i modular erythromycin polyketide synthase (PKS) showed that the Streptomyces sp. FR-008 PKS gene cluster contains repeated sequences spanning c. 105 kb of contiguous DNA; assuming c. 5 kb for each PKS module, this is in striking agreement with the expectation for the 21-step condensation process required for synthesis of the FR-008 carbon chain. The methods developed for transformation and gene replacement in Streptomyces sp. FR-008 make it possible to genetically manipulate polyene macrolide production, and may later lead to the biosynthesis of novel polyene macrolides.     

The effect of intrinsic curvature on supercoiling: Predictions of elasticity theory

Elasticity theory of naturally curved rods is employed to study the effects of intrinsic curvature on the properties of the equilibrium conformations of supercoiled DNA. The results stand in sharp contrast to those obtained when the molecule is viewed as being straight in its relaxed form. Starting from very fundamental principles of the theory, we show that the torsion of an open segment with a curved duplex axis can vary when the temperature, and along with it, the intrinsic twist is changed. Conversely, an imposed helicity, such as might be associated with binding to a histone, can change the intrinsic twist. It is also shown that another consequence of the presence of naturally curved sequences is that the twist density will, in general, vary with position along the chain in all equilibrium states. Then portions of the molecule will be more or less susceptible to interaction with other agents sensitive to such a variation. Finally, some closed equilibrium global structures uniquely associated with intrinsic curvature are discussed. © 1993 John Wiley & Sons, Inc.     

Tropical dry woodland loss occurs disproportionately in areas of highest conservation value

Tropical and subtropical dry woodlands are rich in biodiversity and carbon. Yet, many of these woodlands are under high deforestation pressure and remain weakly protected. Here, we assessed how deforestation dynamics relate to areas of woodland protection and to conservation priorities across the world's tropical dry woodlands. Specifically, we characterized different types of deforestation frontier from 2000 to 2020 and compared them to protected areas (PAs), Indigenous Peoples' lands and conservation areas for biodiversity, carbon and water. We found that global conservation priorities were always overrepresented in tropical dry woodlands compared to the rest of the globe (between 4% and 96% more than expected, depending on the type of conservation priority). Moreover, about 41% of all dry woodlands were characterized as deforestation frontiers, and these frontiers have been falling disproportionately in areas with important regional (i.e. tropical dry woodland) conservation assets. While deforestation frontiers were identified within all tropical dry woodland classes of woodland protection, they were lower than the average within protected areas coinciding with Indigenous Peoples' lands (23%), and within other PAs (28%). However, within PAs, deforestation frontiers have also been disproportionately affecting regional conservation assets. Many emerging deforestation frontiers were identified outside but close to PAs, highlighting a growing threat that the conserved areas of dry woodland will become isolated. Understanding how deforestation frontiers coincide with major types of current woodland protection can help target context-specific conservation policies and interventions to tropical dry woodland conservation assets (e.g. PAs in which deforestation is rampant require stronger enforcement, inactive deforestation frontiers could benefit from restoration). Our analyses also identify recurring patterns that can be used to test the transferability of governance approaches and promote learning across social–ecological contexts.     

Intratumoral interleukin-2 immunotherapy: Activation of tumor-infiltrating and splenic lymphocytes in vivo

Direct intratumoral injection of interleukin-2 (IL-2) was evaluated in a murine model. Balb/c mice received 5 × 10⁴ Line 1 alveolar carcinoma cells (L1C2) by subcutaneous injection. On the third day following tumor implantation, mice received injections of IL-2 (5 × 10³–5 × 10⁴ units) or diluent twice daily, either by i. p. or intratumoral injection, 5 days/week for 3 weeks. Intratumoral injection of 5 × 10⁴ units IL-2 significantly reduced tumor volume (P <0.05 versus control), increased median survival time (P = 0.0001), and resulted in a 23.5% cure rate (P = 0.008). There were no long-term survivors in the other treatment groups. Both tumor-infiltrating lymphocytes (TIL) and splenic lymphocytes isolated directly from IL-2-treated mice demonstrated enhanced cytolytic activity compared to diluent-treated controls. To determine whether non-T-cell-mediated antitumor responses were active in our model, intratumoral immunotherapy was evaluated in athymic Balb/cnu/nu mice. In order to decrease the recruitment of lymphocyte precursors, nude mice were splenectomized and received cyclophosphamide prior to tumor injection and IL-2 therapy. Intratumoral IL-2 immunotherapy also significantly decreased tumor volume in these immunodeficient mice (P <0.02), but did not lead to long-term survival. We conclude that both TIL and splenic lymphocytes are activated in vivo in response to intratumoral IL-2 immunotherapy, suggesting that intratumoral therapy with IL-2 activates both local and systemic antitumor responses.Supported by the Tobacco-Related Disease Research Program of the University of California, the Cancer Research Coordinating Committee, the Jonsson Cancer Center Foundation, and Veterans Administration Medical Research Funds     

A data-driven framework to model the organism–environment system

Organisms modify their development and function in response to the environment. At the same time, the environment is modified by the activities of the organism. Despite the ubiquity of such dynamical interactions in nature, it remains challenging to develop models that accurately represent them, and that can be fitted using data. These features are desirable when modeling phenomena such as phenotypic plasticity, to generate quantitative predictions of how the system will respond to environmental signals of different magnitude or at different times, for example, during ontogeny. Here, we explain a modeling framework that represents the organism and environment as a single coupled dynamical system in terms of inputs and outputs. Inputs are external signals, and outputs are measurements of the system in time. The framework uses time-series data of inputs and outputs to fit a nonlinear black-box model that allows to predict how the system will respond to novel input signals. The framework has three key properties: it captures the dynamical nature of the organism–environment system, it can be fitted with data, and it can be applied without detailed knowledge of the system. We study phenotypic plasticity using in silico experiments and demonstrate that the framework predicts the response to novel environmental signals. The framework allows us to model plasticity as a dynamical property that changes in time during ontogeny, reflecting the well-known fact that organisms are more or less plastic at different developmental stages.     

The agony of choice: Impact of the host animal species on the enzyme-linked immunosorbent assay performance for host cell protein quantification

Host cell proteins (HCPs) are inevitable process-related impurities in biotherapeutics commonly monitored by enzyme-linked immunosorbent assays (ELISAs). Of particular importance for their reliable detection are the anti-HCP polyclonal antibodies (pAbs), supposed to detect a broad range of HCPs. The present study focuses on the identification of suitable host animal species for the development of high-performance CHO-HCP ELISAs, assuming the generation of pAbs with adequate coverage and specificity. Hence, antibodies derived from immunization of sheep, goats, donkeys, rabbits, and chickens were compared concerning their amount of HCP-specific antibodies, coverage, and performance in a sandwich ELISA. Immunization of sheep, goats, donkeys, and rabbits met all test criteria, whereas the antibodies from chickens cannot be recommended based on the results of this study. Additionally, a mixture of antibodies from the five host species was prepared to assess if coverage and ELISA performance can be improved by a multispecies approach. Comparable results were obtained for the single- and multispecies ELISAs in different in-process samples, indicating no substantial improvement for the latter in ELISA performance while raising ethical and financial concerns.     

A Mycobacterium leprae-specific gene encoding an immunologically recognized 45 kDa protein

By screening a Mycobacterium leprae lambda gt11 expression library with a serum from an Ethiopian lepromatous leprosy (LL) patient a clone was isolated (LL4) belonging to hybridization group III of a panel of previously isolated M. leprae clones. Members of this hybridization group encode a serologically recognized 45 kDa protein. The complete DNA sequences of the partially overlapping clones LL4 and L1 (hybridization group III) are presented and these revealed the presence of an open reading frame (ORF) predicting a protein with a molecular size of 42 448 Da. Southern hybridizations on total genomic DNA of M. Ieprae, Mycobacterium tuberculosis and eight atypical mycobacteria showed that the LL4 DNA fragment is specific for M. Ieprae DNA even under low-stringency conditions. The M. Ieprae specificity of LL4 DNA was further confirmed by the polymerase chain reaction using four different sets of primers. Western blotting analyses showed that the M. Ieprae 45 kDa protein is frequently recognized by antibodies from leprosy patients and that this recognition is specific since no antibodies could be detected in sera of tuberculosis patients. T-cell proliferation assays also demonstrated T-cell recognition by leprosy patients and healthy contacts of the M. Ieprae 45 kDa protein. The specificity of the LL4 DNA region and the 45 kDa antigen that is encoded by hybridization group III could provide unique tools for the development of M. Ieprae-specific immunological and DNA reagents.     

Poleward increase in feeding efficiency of leafminer Stigmella lapponica (Lepidoptera: Nepticulidae) in a latitudinal gradient crossing a boreal forest zone

Damage to plant communities imposed by insect herbivores generally decreases from low to high latitudes. This decrease is routinely attributed to declines in herbivore abundance and/or diversity, whereas latitudinal changes in per capita food consumption remain virtually unknown. Here, we tested the hypothesis that the lifetime food consumption by a herbivore individual decreases from low to high latitudes due to a temperature-driven decrease in metabolic expenses. From 2016 to 2019, we explored latitudinal changes in multiple characteristics of linear (gallery) mines made by larvae of the pygmy moth, Stigmella lapponica, in leaves of downy birch, Betula pubescens. The mined leaves were larger than intact leaves at the southern end of our latitudinal gradient (at 60°N) but smaller than intact leaves at its northern end (at 69°N), suggesting that female oviposition preference changes with latitude. No latitudinal changes were observed in larval size, mine length or area, and in per capita food consumption, but the larval feeding efficiency (quantified as the ratio between larval size and mine size) increased with latitude. Consequently, S. lapponica larvae consumed less foliar biomass at higher latitudes than at lower latitudes to reach the same size. Based on space-for-time substitution, we suggest that climate warming will increase metabolic expenses of insect herbivores with uncertain consequences for plant–herbivore interactions.