Recombinant environmental libraries provide access to microbial diversity for drug discovery from natural products

To further explore possible avenues for accessing microbial biodiversity for drug discovery from natural products, we constructed and screened a 5,000-clone "shotgun" environmental DNA library by using an Escherichia coli-Streptomyces lividans shuttle cosmid vector and DNA inserts from microbes derived directly (without cultivation) from soil. The library was analyzed by several means to assess diversity, genetic content, and expression of heterologous genes in both expression hosts. We found that the phylogenetic content of the DNA library was extremely diverse, representing mostly microorganisms that have not been described previously. The library was screened by PCR for sequences similar to parts of type I polyketide synthase genes and tested for the expression of new molecules by screening of live colonies and cell extracts. The results revealed new polyketide synthase genes in at least eight clones. In addition, at least five additional clones were confirmed by high-pressure liquid chromatography analysis and/or biological activity to produce heterologous molecules. These data reinforce the idea that exploiting previously unknown or uncultivated microorganisms for the discovery of novel natural products has potential value and, most importantly, suggest a strategy for developing this technology into a realistic and effective drug discovery tool.     

Transfer of severe experimental autoimmune encephalomyelitis by IL-12- and IL-18-potentiated T cells is estrogen sensitive

The aim of this study was to evaluate the roles of IL-18 and IL-12 in potentiating the encephalitogenic activity of T cell lines specific for myelin oligodendrocyte glycoprotein (MOG(35-55)). MOG-specific T cells stimulated with anti-CD3 and anti-CD28 in the presence of IL-12 or IL-18 alone transferred only mild experimental autoimmune encephalomyelitis (EAE) into a low percentage of recipients. However, T cells cocultured with both cytokines transferred aggressive clinical and histological EAE into all recipients. Coculture of T cells with IL-12 enhanced the secretion of IFN-gamma, but not TNF-alpha, whereas coculture with IL-18 enhanced the secretion of TNF-alpha, but not INF-gamma. However, coculture with both IL-18 and IL-12 induced high levels of both TNF-alpha and IFN-gamma. Additionally, IL-12 selectively enhanced mRNA expression of CCR5, whereas IL-18 selectively enhanced the expression of CCR4 and CCR7, and CCR4 and CCR5 were coexpressed on the surface of T cells cocultured with IL-12 and IL-18. Finally, estrogen treatment, previously found to inhibit both TNF-alpha and IFN-gamma production, completely abrogated all signs of passive EAE. These data demonstrate that optimal potentiation of encephalitogenic activity can be achieved by conditioning MOG-specific T cells with the combination of IL-12 and IL-18, which, respectively, induce the secretion of IFN-gamma/CCR5 and TNF-alpha/CCR4/CCR7, and that estrogen treatment, which is known to inhibit both proinflammatory cytokines, can completely ablate this aggressive form of passive EAE.     

CD30 expression identifies the predominant proliferating T lymphocyte population in human alloimmune responses

CD30 is an inducible member of the TNFR superfamily that is expressed on activated T and B cells and some lymphoid malignancies. We have previously shown that human CD30(+) T cells elicited with allogeneic APC are a major source of IFN-gamma and IL-5 production. In the present study we have used alloantigen, as well as anti-CD3 plus anti-CD28 mAb stimulation, to further characterize human CD30(+) T cells with respect to function and the expression of other activation-dependent cell surface molecules, including the related TNFR family members OX-40 and 4-1BB (CD137). Our results indicate that human CD30(+) T cells are a subset of activated T cells that also express CD25 and CD45RO. Moreover, we observed that allogeneic APC consistently induced a greater proportion of CD30(+) cells within the activated T cell population than did stimulation with plate-bound anti-CD3 plus anti-CD28 mAb or stimulation with soluble anti-CD3 plus anti-CD28 and autologous APC. The enhanced induction of CD30 expression by alloantigen was not common to other inducible TNFR family members because anti-CD3 plus anti-CD28 mAbs were far more effective in inducing expression of 4-1BB and OX-40. Furthermore, CD30 expression marked the predominant proliferating T cell population induced by alloantigen as determined by CFSE staining and flow cytometry. These results indicate that CD30, but not 4-1BB or OX-40, is preferentially induced by alloantigen, suggesting that CD30 may be important in human alloimmune responses.     

Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

Background  

The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene.     

Neurocognitive Function in HIV Infected Patients on Antiretroviral Therapy

Objective

To describe factors associated with neurocognitive (NC) function in HIV-positive patients on stable combination antiretroviral therapy.

Design

We undertook a cross-sectional analysis assessing NC data obtained at baseline in patients entering the Protease-Inhibitor-Monotherapy-Versus-Ongoing-Triple therapy (PIVOT) trial.

Main outcome measure

NC testing comprised of 5 domains. Raw results were z-transformed using standard and demographically adjusted normative datasets (ND). Global z-scores (NPZ-5) were derived from averaging the 5 domains and percentage of subjects with test scores >1 standard deviation (SD) below population means in at least two domains (abnormal Frascati score) calculated. Patient characteristics associated with NC results were assessed using multivariable linear regression.

Results

Of the 587 patients in PIVOT, 557 had full NC results and were included. 77% were male, 68% Caucasian and 28% of Black ethnicity. Mean (SD) baseline and nadir CD4+ lymphocyte counts were 553(217) and 177(117) cells/µL, respectively, and HIV RNA was <50 copies/mL in all. Median (IQR) NPZ-5 score was −0.5 (−1.2/−0) overall, and −0.3 (−0.7/0.1) and −1.4 (−2/−0.8) in subjects of Caucasian and Black ethnicity, respectively. Abnormal Frascati scores using the standard-ND were observed in 51%, 38%, and 81%, respectively, of subjects overall, Caucasian and Black ethnicity (p<0.001), but in 62% and 69% of Caucasian and Black subjects using demographically adjusted-ND (p = 0.20). In the multivariate analysis, only Black ethnicity was associated with poorer NPZ-5 scores (P<0.001).

Conclusions

In this large group of HIV-infected subjects with viral load suppression, ethnicity but not HIV-disease factors is closely associated with NC results. The prevalence of abnormal results is highly dependent on control datasets utilised.

Trial registry

ClinicalTrials.gov, {"type":"clinical-trial","attrs":{"text":"NCT01230580","term_id":"NCT01230580"}}NCT01230580     

Modern evolution of a single-copy gene: the immunoglobulin C kappa locus in wild mice

The immunoglobulin kappa light-chain constant region gene (C kappa) has been cloned and sequenced from five wild mouse species. Analysis of these data has permitted an assessment of single-copy gene evolution during a limited time period as defined by the genus Mus. Sequence conservation was found to be as high (or higher) in the 5' and enhancer regions as in the coding region. The pattern of substitutions throughout these genes suggests that parallel evolution has occurred frequently and that substitutions at replacement sites have not decreased significantly, owing to saturation during this period of approximately 10 Myr. Phylogenetic relationships have been determined among these wild species as well as among members of the genus Rattus.      

Middle-age male mice have increased severity of experimental autoimmune encephalomyelitis and are unresponsive to testosterone therapy

Treatment with sex hormones is known to protect against experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, little is known about how age affects the course of EAE or response to hormone treatment. This study demonstrates striking differences between middle-age vs young C57BL/6 male mice in the clinical course of EAE and response to both testosterone (T4) and estrogen (E2) hormone therapy. Unlike young males that developed an acute phase of EAE followed by a partial remission, middle-age males suffered severe chronic and unremitting EAE that was likely influenced by alterations in the distribution and function of splenic immunocytes and a significant reduction in suppressive activity of CD4+CD25+ regulatory T cells in the spleen and spinal cord. Middle-age males had reduced numbers of splenic CD4+ T cells that were generally hypoproliferative, but enhanced numbers of splenic macrophages and MHC class II-expressing cells, and increased secretion of the proinflammatory factors IFN-gamma and MCP-1. Surprisingly, middle-age males were unresponsive to the EAE-protective effects of T4 and had only a transient benefit from E2 treatment; young males were almost completely protected by both hormone treatments. T4 treatment of young males inhibited proliferation of myelin oligodendrocyte glycoprotein 35-55-specific T cells and secretion of TNF-alpha and IFN-gamma. The effects of T4 in vivo and in vitro were reversed by the androgen receptor antagonist, flutamide, indicating that the regulatory effects of T4 were mediated through the androgen receptor. These data are the first to define age-dependent differences in EAE expression and response to hormone therapy.     

The flat-plate plant-microbial fuel cell: the effect of a new design on internal resistances

Due to a growing world population and increasing welfare, energy demand worldwide is increasing. To meet the increasing energy demand in a sustainable way, new technologies are needed. The Plant-Microbial Fuel Cell (P-MFC) is a technology that could produce sustainable bio-electricity and help meeting the increasing energy demand. Power output of the P-MFC, however, needs to be increased to make it attractive as a renewable and sustainable energy source. To increase power output of the P-MFC internal resistances need to be reduced. With a flat-plate P-MFC design we tried to minimize internal resistances compared to the previously used tubular P-MFC design. With the flat-plate design current and power density per geometric planting area were increased (from 0.15 A/m² to 1.6 A/m² and from 0.22 W/m² to and 0.44 W/m²)as were current and power output per volume (from 7.5 A/m³ to 122 A/m³ and from 1.3 W/m³ to 5.8 W/m³). Internal resistances times volume were decreased, even though internal resistances times membrane surface area were not. Since the membrane in the flat-plate design is placed vertically, membrane surface area per geometric planting area is increased, which allows for lower internal resistances times volume while not decreasing internal resistances times membrane surface area. Anode was split into three different sections on different depths of the system, allowing to calculate internal resistances on different depths. Most electricity was produced where internal resistances were lowest and where most roots were present; in the top section of the system. By measuring electricity production on different depths in the system, electricity production could be linked to root growth. This link offers opportunities for material-reduction in new designs. Concurrent reduction in material use and increase in power output brings the P-MFC a step closer to usable energy density and economic feasibility.     

Genetically Modified Bacterial Strains and Novel Bacterial Artificial Chromosome Shuttle Vectors for Constructing Environmental Libraries and Detecting Heterologous Natural Products in Multiple Expression Hosts

The enormous diversity of uncultured microorganisms in soil and other environments provides a potentially rich source of novel natural products, which is critically important for drug discovery efforts. Our investigators reported previously on the creation and screening of an Escherichia coli library containing soil DNA cloned and expressed in a bacterial artificial chromosome (BAC) vector. In that initial study, our group identified novel enzyme activities and a family of antibacterial small molecules encoded by soil DNA cloned and expressed in E. coli. To continue our pilot study of the utility and feasibility of this approach to natural product drug discovery, we have expanded our technology to include Streptomyces lividans and Pseudomonas putida as additional hosts with different expression capabilities, and herein we describe the tools we developed for transferring environmental libraries into all three expression hosts and screening for novel activities. These tools include derivatives of S. lividans that contain complete and unmarked deletions of the act and red endogenous pigment gene clusters, a derivative of P. putida that can accept environmental DNA vectors and integrate the heterologous DNA into the chromosome, and new BAC shuttle vectors for transferring large fragments of environmental DNA from E. coli to both S. lividans and P. putida by high-throughput conjugation. Finally, we used these tools to confirm that the three hosts have different expression capabilities for some known gene clusters.