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141.
Jared Lynn Dopp Samuel Michael Rothstein Thomas Joseph Mansell Nigel Forest Reuel 《Biotechnology and bioengineering》2019,116(3):667-676
In this study, we present a minimal template design and accompanying methods to produce assayable quantities of custom sequence proteins within 24 hr from receipt of inexpensive gene fragments from a DNA synthesis vendor. This is done without the conventional steps of plasmid cloning or cell-based amplification and expression. Instead the linear template is PCR amplified, circularized, and isothermally amplified using a rolling circle polymerase. The resulting template can be used directly with cost-optimized, scalably-manufactured Escherichia coli extract and minimal supplement reagents to perform cell-free protein synthesis (CFPS) of the template protein. We demonstrate the utility of this template design and 24 hr process with seven fluorescent proteins (sfGFP, mVenus, mCherry, and four GFP variants), three enzymes (chloramphenicol acetyltransferase, a chitinase catalytic domain, and native subtilisin), a capture protein (anti-GFP nanobody), and 2 antimicrobial peptides (BP100 and CA(1–7)M(2–9)). We detected each of these directly from the CFPS reaction using colorimetric, fluorogenic, and growth assays. Of especial note, the GFP variant sequences were found from genomic screening data and had not been expressed or characterized before, thus demonstrating the utility of this approach for phenotype characterization of sequenced libraries. We also demonstrate that the rolling circle amplified version of the linear template exhibits expression similar to that of a complete plasmid when expressing sfGFP in the CFPS reaction. We evaluate the cost of this approach to be $61/mg sfGFP for a 4 hr reaction. We also detail limitations of this approach and strategies to overcome these, namely proteins with posttranslational modifications. 相似文献
142.
David J. Curnick Aaron B. Carlisle Matthew J. Gollock Robert J. Schallert Nigel E. Hussey 《Journal of fish biology》2019,94(4):680-685
Stable-isotope analyses (δ13C, δ15N and δ34S) of multiple tissues (fin, muscle, red blood cells and plasma), revealed ontogenetic shifts in resource use by grey reef sharks Carcharhinus amblyrhynchos and resource partitioning with silvertip sharks Carcharhinus albimarginatus within the British Indian Ocean Territory marine protected area (MPA). Resource partitioning varied temporally, with C. albimarginatus feeding on more pelagic prey during October to January, potentially attributable to an influx of pelagic prey from outside the MPA at that time. Reef sharks may therefore be affected by processes outside an MPA, even if the sharks do not leave the MPA. 相似文献
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144.
Valrie Bgay Branko Cirovic Alison J. Barker Robert Klopfleisch Daniel W. Hart Nigel C. Bennett Gary R. Lewin 《Open biology》2022,12(4)
Naked mole-rats (NM-R; Heterocephalus glaber) live in multi-generational colonies with a social hierarchy, and show low cancer incidence and long life-spans. Here we asked if an immune component might underlie such extreme physiology. The largest lymphoid organ is the spleen, which plays an essential role in responding to immunological insults and may participate in combating cancer and slowing ageing. We investigated the anatomy, molecular composition and function of the NM-R spleen using RNA-sequencing and histological analysis in healthy NM-Rs. Spleen size in healthy NM-Rs showed considerable inter-individual variability, with some animals displaying enlarged spleens. In all healthy NM-Rs, the spleen is a major site of adult haematopoiesis under normal physiological conditions. However, myeloid-to-lymphoid cell ratio is increased and splenic marginal zone showed markedly altered morphology when compared to other rodents. Healthy NM-Rs with enlarged spleens showed potentially better anti-microbial profiles and were much more likely to have a high rank within the colony. We propose that the anatomical plasticity of the spleen might be regulated by social interaction and gives immunological advantage to increase the lifespan of higher-ranked animals. 相似文献
145.
Nguyen T. Hien Nigel W. Kerby Gordon C. Machray Peter Rowell William D.P. Stewart 《FEMS microbiology letters》1988,56(3):337-341
Abstract The nucleoside analogue ganciclovir has clinical efficacy in the treatment of serious infections with human cytomegalovirus (CMV) in AIDS patients. The mechanism of action of the drug against CMV is different from that described for herpes simplex viruses (HSVs) as the crucial formation of the monophosphate derivative appears to be carried out by cellular rather than virus-coded enzymes. Adenovirus infections also induce the expression of cellular genes including kinase activity and a novel DNA polymerase and the results reported here show that these viruses are sensitive to ganciclovir. The 50% effective dose (ED50 ) range for known serotypes and one clinical isolate was 4.5−33 μM. By comparison with the sensitivity of CMV in vitro and the known clinical response of infections with this virus to ganciclovir, our results suggest that this drug or its analogous may form the basis of chemotherapy for adenovirus infections. 相似文献
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147.
Keller Larkin M Deng WM Holder K Tworoger M Clegg N Ruohola-Baker H 《Development genes and evolution》1999,209(5):301-311
During Drosophila oogenesis the body axes are determined by signaling between the oocyte and the somatic follicle cells that surround the egg
chamber. A key event in the establishment of oocyte anterior-posterior polarity is the differential patterning of the follicle
cell epithelium along the anterior-posterior axis. Both the Notch and epithelial growth factor (EGF) receptor pathways are
required for this patterning. To understand how these pathways act in the process we have analyzed markers for anterior and
posterior follicle cells accompanying constitutive activation of the EGF receptor, loss of Notch function, and ectopic expression
of Delta. We find that a constitutively active EGF receptor can induce posterior fate in anterior but not in lateral follicle
cells, showing that the EGF receptor pathway can act only on predetermined terminal cells. Furthermore, Notch function is
required at both termini for appropriate expression of anterior and posterior markers, while loss of both the EGF receptor
and Notch pathways mimic the Notch loss-of-function phenotype. Ectopic expression of the Notch ligand, Delta, disturbs EGF
receptor dependent posterior follicle cell differentiation and anterior-posterior polarity of the oocyte. Our data are consistent
with a model in which the Notch pathway is required for early follicle cell differentiation at both termini, but is then repressed
at the posterior for proper determination of the posterior follicle cells by the EGF receptor pathway.
Received: 5 November 1998 / Accepted: 14 December 1998 相似文献
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149.
Van Klink JW Baek SH Barlow AJ Ishii H Nakanishi K Berova N Perry NB Weavers RT 《Chirality》2004,16(8):549-558
Several Anisotome diterpene derivatives were synthesized in an attempt to obtain a crystalline compound for X-ray analysis. Although we were unable to obtain a suitable crystal, the absolute configuration of the irregular diterpene skeleton was determined using two other techniques: a circular dichroism (CD) protocol based on a tetraarylporphyrin molecular tweezer that allowed prediction of the absolute stereochemistry on a microscale level, and a method employing differences in NMR shifts from derivatization of the naturally occurring acid 1 with enantiomers of a phenylglycine methyl ester (PGME) chiral anisotropic reagent. The excellent agreement between the CD and NMR methods led to the assignment of a 2S-absolute configuration for anisotomenoic acid 1. 相似文献
150.