Firefly Luciferase Mutant with Enhanced Activity and Thermostability

The luciferase isolated from the firefly Photinus pyralis (Ppy) catalyzes a two-step reaction that results in the oxidation of d-luciferin accompanied by emission of yellow-green light with a peak at 560 nm. Among many applications, Ppy luciferase has been used extensively as a reporter gene in living cells and organisms. However, some biological applications are limited by the low stability of the luciferase and limited intracellular luciferin concentration. To address these challenges, efforts to protein engineer Ppy luciferase have resulted in a number of mutants with improved properties such as thermostability, pH tolerance, and catalytic turn over. In this work, we combined amino acid mutations that were shown to enhance the enzyme\u27s thermostability (Mutant E) with those reported to enhance catalytic activity (LGR). The resulting mutant (YY5) contained eight amino acid changes from the wild-type luciferase and exhibited both improved thermostability and brighter luminescence at low luciferin concentrations. Therefore, YY5 may be useful for reporter gene applications

Gas-Phase Synthesis for Label-Free Biosensors: Zinc-Oxide Nanowires Functionalized with Gold Nanoparticles

Metal oxide semiconductor nanowires have important applications in label-free biosensing due to their ease of fabrication and ultralow detection limits. Typically, chemical functionalization of the oxide surface is necessary for specific biological analyte detection. We instead demonstrate the use of gas-phase synthesis of gold nanoparticles (Au NPs) to decorate zinc oxide nanowire (ZnO NW) devices for biosensing applications. Uniform ZnO NW devices were fabricated using a vapor-solid-liquid method in a chemical vapor deposition (CVD) furnace. Magnetron-sputtering of a Au target combined with a quadrupole mass filter for cluster size selection was used to deposit Au NPs on the ZnO NWs. Without additional functionalization, we electrically detect DNA binding on the nanowire at sub-nanomolar concentrations and visualize individual DNA strands using atomic force microscopy (AFM). By attaching a DNA aptamer for streptavidin to the biosensor, we detect both streptavidin and the complementary DNA strand at sub-nanomolar concentrations. Au NP decoration also enables sub-nanomolar DNA detection in passivated ZnO NWs that are resilient to dissolution in aqueous solutions. This novel method of biosensor functionalization can be applied to many semiconductor materials for highly sensitive and label-free detection of a wide range of biomolecules

Beyond directed evolution: Darwinian selection as a tool for synthetic biology

Synthetic biology is an engineering approach that seeks to design and construct new biological parts, devices and systems, as well as to re-design existing components. However, rationally designed synthetic circuits may not work as expected due to the context-dependence of biological parts. Darwinian selection, the main mechanism through which evolution works, is a major force in creating biodiversity and may be a powerful tool for synthetic biology. This article reviews selection-based techniques and proposes strict Darwinian selection as an alternative approach for the identification and characterization of parts. Additionally, a strategy for fine-tuning of relatively complex circuits by coupling them to a master standard circuit is discussed

Selection of silk-binding peptides by phage display

Peptides that bind to silkworm-derived silk fibroin fiber were selected from a phage-displayed random peptide library. The selected silk-binding peptides contained a consensus sequence QSWS which is important for silk-binding as confirmed by binding assays using phage and synthetic peptides. With further optimization, we anticipate that the silk-binding peptides will be useful for functionalization of silk for biomaterial applications

Optimisation of the Schizosaccharomyces pombe urg1 expression system

The ability to study protein function in vivo often relies on systems that regulate the presence and absence of the protein of interest. Two limitations for previously described transcriptional control systems that are used to regulate protein expression in fission yeast are: the time taken for inducing conditions to initiate transcription and the ability to achieve very low basal transcription in the "OFF-state". In previous work, we described a Cre recombination-mediated system that allows the rapid and efficient regulation of any gene of interest by the urg1 promoter, which has a dynamic range of approximately 75-fold and which is induced within 30-60 minutes of uracil addition. In this report we describe easy-to-use and versatile modules that can be exploited to significantly tune down P urg1 "OFF-levels" while maintaining an equivalent dynamic range. We also provide plasmids and tools for combining P urg1 transcriptional control with the auxin degron tag to help maintain a null-like phenotype. We demonstrate the utility of this system by improved regulation of HO-dependent site-specific DSB formation, by the regulation Rtf1-dependent replication fork arrest and by controlling Rhp18(Rad18)-dependent post replication repair

Association between social relationship and glycemic control among older Japanese: JAGES cross-sectional study

AIM: The present study examined whether social support, informal socializing and social participation are associated with glycemic control in older people. METHODS: Data for this population-based cross-sectional study was obtained from the Japan Gerontological Evaluation Study (JAGES) 2010 linked to the annual health check-up data in Japan. We analyzed 9,554 individuals aged ≥65 years without the certification of needed long-term care. Multivariate logistic regression models were used to assess the effect of social support, informal socializing and social participations on glycemic control. The outcome measure was HbA1c ≥8.4%. RESULTS: 1.3% of the participants had a level of HbA1c over 8.4%. Better glycemic control was significantly associated with meeting with friends one to four times per month (odds ratio [OR] 0.51, 95% confidence interval [CI]0.30-0.89, compared to meeting with friends a few times per year or less) and participation in sports groups (OR 0.50, 95% CI 0.26-0.97) even after adjusting for other variables. Meeting with friends more than twice per week, receiving social support, and being married were not associated with better control of diabetes. CONCLUSIONS: Meeting with friends occasionally is associated with better glycemic control among older people

Design principles for riboswitch function

Scientific and technological advances that enable the tuning of integrated regulatory components to match network and system requirements are critical to reliably control the function of biological systems. RNA provides a promising building block for the construction of tunable regulatory components based on its rich regulatory capacity and our current understanding of the sequence–function relationship. One prominent example of RNA-based regulatory components is riboswitches, genetic elements that mediate ligand control of gene expression through diverse regulatory mechanisms. While characterization of natural and synthetic riboswitches has revealed that riboswitch function can be modulated through sequence alteration, no quantitative frameworks exist to investigate or guide riboswitch tuning. Here, we combined mathematical modeling and experimental approaches to investigate the relationship between riboswitch function and performance. Model results demonstrated that the competition between reversible and irreversible rate constants dictates performance for different regulatory mechanisms. We also found that practical system restrictions, such as an upper limit on ligand concentration, can significantly alter the requirements for riboswitch performance, necessitating alternative tuning strategies. Previous experimental data for natural and synthetic riboswitches as well as experiments conducted in this work support model predictions. From our results, we developed a set of general design principles for synthetic riboswitches. Our results also provide a foundation from which to investigate how natural riboswitches are tuned to meet systems-level regulatory demands

SCF Ensures Meiotic Chromosome Segregation Through a Resolution of Meiotic Recombination Intermediates

The SCF (Skp1-Cul1-F-box) complex contributes to a variety of cellular events including meiotic cell cycle control, but its function during meiosis is not understood well. Here we describe a novel function of SCF/Skp1 in meiotic recombination and subsequent chromosome segregation. The skp1 temperature-sensitive mutant exhibited abnormal distribution of spindle microtubules in meiosis II, which turned out to originate from abnormal bending of the spindle in meiosis I. Bent spindles were reported in mitosis of this mutant, but it remained unknown how SCF could affect spindle morphology. We found that the meiotic bent spindle in skp1 cells was due to a hypertension generated by chromosome entanglement. The spindle bending was suppressed by inhibiting double strand break (DSB) formation, indicating that the entanglement was generated by the meiotic recombination machinery. Consistently, Rhp51/Rad51-Rad22/Rad52 foci persisted until meiosis I in skp1 cells, proving accumulation of recombination intermediates. Intriguingly bent spindles were also observed in the mutant of Fbh1, an F-box protein containing the DNA helicase domain, which is involved in meiotic recombination. Genetic evidence suggested its cooperation with SCF/Skp1. Thus, SCF/Skp1 together with Fbh1 is likely to function in the resolution of meiotic recombination intermediates, thereby ensuring proper chromosome segregation

Chiasmata Promote Monopolar Attachment of Sister Chromatids and Their Co-Segregation toward the Proper Pole during Meiosis I

The chiasma is a structure that forms between a pair of homologous chromosomes by crossover recombination and physically links the homologous chromosomes during meiosis. Chiasmata are essential for the attachment of the homologous chromosomes to opposite spindle poles (bipolar attachment) and their subsequent segregation to the opposite poles during meiosis I. However, the overall function of chiasmata during meiosis is not fully understood. Here, we show that chiasmata also play a crucial role in the attachment of sister chromatids to the same spindle pole and in their co-segregation during meiosis I in fission yeast. Analysis of cells lacking chiasmata and the cohesin protector Sgo1 showed that loss of chiasmata causes frequent bipolar attachment of sister chromatids during anaphase. Furthermore, high time-resolution analysis of centromere dynamics in various types of chiasmate and achiasmate cells, including those lacking the DNA replication checkpoint factor Mrc1 or the meiotic centromere protein Moa1, showed the following three outcomes: (i) during the pre-anaphase stage, the bipolar attachment of sister chromatids occurs irrespective of chiasma formation; (ii) the chiasma contributes to the elimination of the pre-anaphase bipolar attachment; and (iii) when the bipolar attachment remains during anaphase, the chiasmata generate a bias toward the proper pole during poleward chromosome pulling that results in appropriate chromosome segregation. Based on these results, we propose that chiasmata play a pivotal role in the selection of proper attachments and provide a backup mechanism that promotes correct chromosome segregation when improper attachments remain during anaphase I