Cloning of spiramycin biosynthetic genes and their use in constructing Streptomyces ambofaciens mutants defective in spiramycin biosynthesis

Several cosmid clones from Streptomyces ambofaciens containing the spiramycin resistance gene srmB were introduced into S. fradiae PM73, a mutant defective in tylosin synthesis, resulting in tylosin synthesis. The DNA responsible for this complementation was localized to a 10.5-kilobase EcoRI fragment. A 32-kilobase DNA segment which included the srmB spiramycin resistance gene and DNA which complemented the defect in strain PM73 were mutagenized in vivo with Tn10 carrying the gene for Nmr (which is expressed in Streptomyces spp.) or in vitro by insertional mutagenesis with a drug resistance gene (Nmr) cassette. When these mutagenized DNA segments were crossed into the S. ambofaciens chromosome, three mutant classes blocked in spiramycin synthesis were obtained. One mutant accumulated two precursors of spiramycin, platenolide I and platenolide II. Two mutants, when cofermented with the platenolide-accumulating mutant, produced spiramycin. Tylactone supplementation of these two mutants resulted in the synthesis of a group of compounds exhibiting antibiotic activity. Two other mutants failed to coferment with any of the other mutants or to respond to tylactone supplementation.     

Effects of Hierarchical Roost Removal on Northern Long-Eared Bat (Myotis septentrionalis) Maternity Colonies

Forest roosting bats use a variety of ephemeral roosts such as snags and declining live trees. Although conservation of summer maternity habitat is considered critical for forest-roosting bats, bat response to roost loss still is poorly understood. To address this, we monitored 3 northern long-eared bat (Myotis septentrionalis) maternity colonies on Fort Knox Military Reservation, Kentucky, USA, before and after targeted roost removal during the dormant season when bats were hibernating in caves. We used 2 treatments: removal of a single highly used (primary) roost and removal of 24% of less used (secondary) roosts, and an un-manipulated control. Neither treatment altered the number of roosts used by individual bats, but secondary roost removal doubled the distances moved between sequentially used roosts. However, overall space use by and location of colonies was similar pre- and post-treatment. Patterns of roost use before and after removal treatments also were similar but bats maintained closer social connections after our treatments. Roost height, diameter at breast height, percent canopy openness, and roost species composition were similar pre- and post-treatment. We detected differences in the distribution of roosts among decay stages and crown classes pre- and post-roost removal, but this may have been a result of temperature differences between treatment years. Our results suggest that loss of a primary roost or ≤ 20% of secondary roosts in the dormant season may not cause northern long-eared bats to abandon roosting areas or substantially alter some roosting behaviors in the following active season when tree-roosts are used. Critically, tolerance limits to roost loss may be dependent upon local forest conditions, and continued research on this topic will be necessary for conservation of the northern long-eared bat across its range.     

Persistent infection of rhesus macaques with a molecular clone of human immunodeficiency virus type 2: evidence of minimal genetic drift and low pathogenetic effects.

In an attempt to generate a suitable animal model to study the infectivity and possible pathogenicity of human immunodeficiency viruses, we intravenously inoculated juvenile rhesus macaques and African green monkeys with a molecularly cloned virus, human immunodeficiency virus type 2 HIV-2sbl/isy, as well as with the uncloned HIV-2nih-z virus. Infection was monitored by virus recovery from the peripheral blood cells and by seroconversion against HIV-2 antigens measured by Western immunoblot, radioimmunoprecipitation, and enzyme-linked immunosorbent assay. We successfully infected two out of two macaques with the molecularly cloned virus and one macaque out of two with the HIV-2nih-z. No evidence of infection was seen in the African green monkeys with either virus. We followed the infected animals for 2 years. The animals remained healthy, although we observed intermittent lymphadenopathy and a transient decrease in the absolute number of circulating CD4+ T lymphocytes in both animals infected with the molecularly cloned virus. Virus isolation from the peripheral blood cells of the infected animals was successful only within the first few months after inoculation. Evidence of persistent infection was provided by the detection of proviral DNA by polymerase chain reaction analysis of the blood cells of the inoculated animals and by the stability of antiviral antibody titers. To evaluate the genetic drift of the proviral DNA, we molecularly cloned viruses which were reisolated 1 and 5 months postinoculation from one of these animals. Comparison of the DNA sequences of the envelope genes of both these isolates indicated that a low degree of variation (0.2%) in the envelope protein had occurred in vivo during the 5-month period. These data suggest that the use of HIV-2sbl/isy in rhesus macaques may represent a good animal model system to study prevention of viral infection. In particular, molecularly cloned virus can be manipulated for functional studies of viral genes in the pathogenesis of acquired immune deficiency syndrome and provides a reproducible source of virus for vaccine studies.     

Synthesis and biological evaluation of benzazepine oxazolidinone antibacterials

Novel benzazepine oxazolidinone antibacterials were synthesized and evaluated against clinically relevant susceptible and resistant organisms. The effect of ring nitrogen position and N-substitution on antibacterial activity is examined.     

Rational Design of a Fibroblast Growth Factor 21-Based Clinical Candidate,LY2405319

Fibroblast growth factor 21 is a novel hormonal regulator with the potential to treat a broad variety of metabolic abnormalities, such as type 2 diabetes, obesity, hepatic steatosis, and cardiovascular disease. Human recombinant wild type FGF21 (FGF21) has been shown to ameliorate metabolic disorders in rodents and non-human primates. However, development of FGF21 as a drug is challenging and requires re-engineering of its amino acid sequence to improve protein expression and formulation stability. Here we report the design and characterization of a novel FGF21 variant, LY2405319. To enable the development of a potential drug product with a once-daily dosing profile, in a preserved, multi-use formulation, an additional disulfide bond was introduced in FGF21 through Leu118Cys and Ala134Cys mutations. FGF21 was further optimized by deleting the four N-terminal amino acids, His-Pro-Ile-Pro (HPIP), which was subject to proteolytic cleavage. In addition, to eliminate an O-linked glycosylation site in yeast a Ser167Ala mutation was introduced, thus allowing large-scale, homogenous protein production in Pichia pastoris. Altogether re-engineering of FGF21 led to significant improvements in its biopharmaceutical properties. The impact of these changes was assessed in a panel of in vitro and in vivo assays, which confirmed that biological properties of LY2405319 were essentially identical to FGF21. Specifically, subcutaneous administration of LY2405319 in ob/ob and diet-induced obese (DIO) mice over 7–14 days resulted in a 25–50% lowering of plasma glucose coupled with a 10–30% reduction in body weight. Thus, LY2405319 exhibited all the biopharmaceutical and biological properties required for initiation of a clinical program designed to test the hypothesis that administration of exogenous FGF21 would result in effects on disease-related metabolic parameters in humans.     

Serotonergic component of SCH 23390: in vitro and in vivo binding analyses

A series of benzazepines related to SCH 23390 were tested for binding to the 5HT-2 receptor. The compounds tested inhibited the binding of 3H-ketanserin with KI values generally greater than those observed for the D-1 receptor, but less than those for the D-2 receptor. When this serotonergic activity was correlated to the D-1 activity, the resulting coefficient was 0.84, indicating a strong correlation between the two activities. Conversely, the 5HT-2 activity did not show a good correlation with the D-2 activity. To further test the significance of the 5HT-2 binding of the SCH 23390, in vivo binding studies were performed using 125I-SCH 38840 in the frontal cortex, an area containing both D-1 and 5HT-2 receptors. The in vivo binding of 125I-SCH 38840 to frontal cortex exhibited peak levels one hour following subcutaneous administration, similar to the time course previously observed in striatum. The binding was both D-1 and tissue specific. Competition studies with selected standards demonstrated that inhibition of the binding to frontal cortex, in contrast to the inhibition observed in the striatum, exhibited a Hill coefficient less than unity, implying interaction at more than one receptor subtype. When SCH 23390 and ketanserin were administered simultaneously, the inhibition of the in vivo binding of 125I-SCH 38840 to striatum was not different than that observed with SCH 23390, alone. However, the inhibition of binding to frontal cortex was significantly greater than that demonstrated with either SCH 23390 or ketanserin, alone, suggesting that 125I-SCH 38840 was binding to both D-1 and 5HT-2 receptors, in vivo.     

Integration of the Rosetta suite with the python software stack via reproducible packaging and core programming interfaces for distributed simulation

The Rosetta software suite for macromolecular modeling is a powerful computational toolbox for protein design, structure prediction, and protein structure analysis. The development of novel Rosetta‐based scientific tools requires two orthogonal skill sets: deep domain‐specific expertise in protein biochemistry and technical expertise in development, deployment, and analysis of molecular simulations. Furthermore, the computational demands of molecular simulation necessitate large scale cluster‐based or distributed solutions for nearly all scientifically relevant tasks. To reduce the technical barriers to entry for new development, we integrated Rosetta with modern, widely adopted computational infrastructure. This allows simplified deployment in large‐scale cluster and cloud computing environments, and effective reuse of common libraries for simulation execution and data analysis. To achieve this, we integrated Rosetta with the Conda package manager; this simplifies installation into existing computational environments and packaging as docker images for cloud deployment. Then, we developed programming interfaces to integrate Rosetta with the PyData stack for analysis and distributed computing, including the popular tools Jupyter, Pandas, and Dask. We demonstrate the utility of these components by generating a library of a thousand de novo disulfide‐rich miniproteins in a hybrid simulation that included cluster‐based design and interactive notebook‐based analyses. Our new tools enable users, who would otherwise not have access to the necessary computational infrastructure, to perform state‐of‐the‐art molecular simulation and design with Rosetta.     

Determination of Effective Transport Coefficients for Bacterial Migration in Sand Columns

A well-characterized experimental system was designed to evaluate the effect of porous media on macroscopic transport coefficients which are used to characterize the migration of bacterial populations. Bacterial density profiles of Pseudomonas putida PRS2000 were determined in the presence and absence of a chemical attractant (3-chlorobenzoate) gradient within sand columns having a narrow distribution of particle diameters. These experimental profiles were compared with theoretical predictions to evaluate the macroscopic transport coefficients. The effective random motility coefficient, used to quantify migration due to a random process in a porous medium, decreased nearly 20-fold as grain size in the columns decreased from 800 to 80 (mu)m. The effective random motility coefficient (mu)(infeff) was related to the random motility coefficient (mu), measured in a bulk aqueous system, according to (mu)(infeff) = ((epsilon)/(tau))(mu) with porosity (epsilon) and tortuosity (tau). Over the times and distances examined in these experiments, bacterial density profiles were unaffected by the presence of an attractant gradient. Theoretical profiles with the aqueous phase value of the chemotactic sensitivity coefficient (used to quantify migration due to a directed process) were consistent with this result and suggested that any chemotactic effect on bacterial migration was below the detection limits of our assay.     

The induction of taxol production in the endophytic fungus—Periconia sp from Torreya grandifolia

A Periconia sp was isolated from Torreya grandifolia (a relative of yew that does not synthesize taxol) near Huangshan National Park in the People’s Republic of China. This fungus, not previously known as a tree endophyte, was isolated from the inner bark of a small lower limb. When freshly isolated from the tree and placed in a semi-synthetic medium, the fungus produced readily detectable quantities of the anticancer drug taxol. Other taxol-producing endophytes were also isolated from this source. The production of taxol by Periconia sp was demonstrated unequivocally via spectroscopic and immunological methods. However, successive transfers of the fungus in semi-synthetic medium resulted in gradual attenuation until low production occurred even though fungal growth was relatively unaffected. Several compounds, known previously as activators of microbial metabolism, including serinol, p-hydroxybenzoic acid, and a mixture of phenolic acids, were capable of fully or partially restoring taxol production to otherwise taxol-attenuated cultures. The compound with the most impressive ability to activate taxol production was benzoic acid at 0.01 mM. Benzoic acid was not a taxol precursor. Received 19 December 1997/ Accepted in revised form 19 February 1998