COEXPRESSION OF CYTOCHROME P4502A6 AND HUMAN NADPH-P450 OXIDOREDUCTASE IN THE BACULOVIRUS SYSTEM

ABSTRACT: Heterologous expression using baculovirus vectors has become a popular method for the production of catalytically active cytochrome P450s (CYPs). We have systematically optimized the multiplicity of infection (MOI) for a coinfection approach for the coexpression of CYP2A6 (viral vector designated v2A6) and NADPH-P450 oxidoreductase (OR; viral vector designated vOR) using Sf9 insect cells. A 3000-fold range of MOI was examined in stationary culture and stirred suspension culture. Surprisingly, our results indicate that the best CYP2A6 catalytic activity (850-1300 pmol/ min/mg total lysate protein as measured by coumarin 7-hydroxylase activity) was obtained only when using a low MOI of v2A6 (1.5-3 ؋ 10 ؊2 ) and a vOR of 10-to 20-fold less. This activity was ϳ7-to 11-fold higher than the best activity obtained when infecting cells with v2A6 alone. At this level of coinfection, the P450 content ranged from 180 to 250 pmol/mg total lysate protein, and the NADPH cytochrome c reductase activity ranged from 350 to 520 nmol/min/mg total lysate protein. Increasing the MOI of both viruses to 50-fold higher resulted in lower overall activity with the optimum (250 pmol/min/mg total lysate protein) being seen earlier postinfection (60 vs. 72 hr). Increasing the MOI of vOR to levels comparable with those of v2A6, decreased coumarin 7-hydroxylase activity 14-fold. These results suggest that the best CYP2A6 catalytic activity depends on properly posttranslationally modified proteins accumulating in a right ratio as a result of primary, secondary, and possibly tertiary infection of both viruses. These results also suggest that high OR expression results in degradation of P450. CYPs 1 are a multienzyme, membrane-bound system that metabolizes many drugs and other xenobiotics (1). The catalytic activity of CYP enzymes requires the presence of NADPH-CYP OR. In addition, cytochrome b 5 stimulates catalytic activity for some CYP forms (2). Several efficient systems for the heterologous expression of mammalian CYP enzymes have been developed, including bacterial, yeast, and mammalian and baculovirus/insect cell-based systems (reviewed in ref. 2). Of the systems available, the baculovirus system has distinct advantages for the production of high levels of active, native CYP enzyme. Although efficient mammalian CYP expression is possible in bacterial systems, modification of the amino acid sequence is usually required for high level expression (2). Three distinct approaches have been taken for production of catalytically active CYP enzymes using baculovirus system. Expression of the CYP enzyme alone and then reconstitution with OR using purified P450s or total cell lysate (3-5), coexpression of the CYP and OR using a single virus (6), and coinfection with independent viruses containing CYP or OR (7). Each approach has unique advantages and disadvantages. Purification/reconstitution provides flexibility in controlling the CYP to OR ratio, but is time-and labor-intensive because of the need for column purifications. The single virus approach is more time-and labor-efficient (simple cell lysate can be used), but, because of the small number of promoters characterized for baculovirus expression, the ability to control the CYP to OR ratio is limited The human CYP2A6 is the only enzyme known to date responsible for coumarin 7-hydroxylase activity in human liver (9, 10). CYP2A6 also metabolically activates certain carcinogens and promutagens (for review, see refs. 1, 9, and 10). It has the highest activity for activation of the mutagen N-nitrosodiethylamine among all human P450s studied (9, 11). It also activates tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal, n-nitrosonornicotine (12, 13), and the mycotoxin aflatoxin B1 In this study, we describe a systematic characterization of a coinfection approach using CYP2A6 and human OR. To obtain an overall phenomena for coexpression of P450 and OR proteins in the baculovirus system, we examined a wide range of MOI for both v2A6 and vOR, with different ratios for coinfection to determine the best ratio for optimal coumarin 7-hydroxylase catalytic activity in insect cell lysate. Materials and Methods Rabbit anti-rat 2A1 (16) and rabbit anti-rat OR (17) antibodies were described in the previous studies. Recombinant baculoviruses v2A6 and vOR This study was supported in part by SBIR Contract N43-ES-31001 from the National Institute of Environmental Health Sciences. 1 Abbreviations used are: CYP, cytochrome P450; OR, NADPH-P450 oxidoreductase; P450, cytochrome P450; MOI, multiplicity of infection (the number of virus used per cell); v2A6, a recombinant baculovirus containing the human CYP2A6 cDNA; vOR, a recombinant baculovirus containing the human OR cDNA; Sf9, Spodoptera frugiperda; pfu, plaque-forming units (measurement of infectivity of a virus); AcMNPV, Autographa californica nuclear polyhedrosis virus. Send reprint requests to: Dr. Liping Chen

Constraints on cosmic strings using data from the first Advanced LIGO observing run

Cosmic strings are topological defects which can be formed in grand unified theory scale phase transitions in the early universe. They are also predicted to form in the context of string theory. The main mechanism for a network of Nambu-Goto cosmic strings to lose energy is through the production of loops and the subsequent emission of gravitational waves, thus offering an experimental signature for the existence of cosmic strings. Here we report on the analysis conducted to specifically search for gravitational-wave bursts from cosmic string loops in the data of Advanced LIGO 2015-2016 observing run (O1). No evidence of such signals was found in the data, and as a result we set upper limits on the cosmic string parameters for three recent loop distribution models. In this paper, we initially derive constraints on the string tension Gμ and the intercommutation probability, using not only the burst analysis performed on the O1 data set but also results from the previously published LIGO stochastic O1 analysis, pulsar timing arrays, cosmic microwave background and big-bang nucleosynthesis experiments. We show that these data sets are complementary in that they probe gravitational waves produced by cosmic string loops during very different epochs. Finally, we show that the data sets exclude large parts of the parameter space of the three loop distribution models we consider

Directional Limits on Persistent Gravitational Waves from Advanced LIGO’s First Observing Run

We employ gravitational-wave radiometry to map the stochastic gravitational wave background expected from a variety of contributing mechanisms and test the assumption of isotropy using data from the Advanced Laser Interferometer Gravitational Wave Observatory’s (aLIGO) first observing run. We also search for persistent gravitational waves from point sources with only minimal assumptions over the 20–1726 Hz frequency band. Finding no evidence of gravitational waves from either point sources or a stochastic background, we set limits at 90% confidence. For broadband point sources, we report upper limits on the gravitational wave energy flux per unit frequency in the range Fα;ΘðfÞ < ð0.1–56Þ × 10−8 erg cm−2 s−1 Hz−1ðf=25 HzÞα−1 depending on the sky location Θ and the spectral power index α. For extended sources, we report upper limits on the fractional gravitational wave energy density required to close the Universe of Ωðf; ΘÞ < ð0.39–7.6Þ × 10−8 sr−1ðf=25 HzÞα depending on Θ and α. Directed searches for narrowband gravitational waves from astrophysically interesting objects (Scorpius X-1, Supernova 1987 A, and the Galactic Center) yield median frequency-dependent limits on strain amplitude of h0 < ð6.7; 5.5; and 7.0Þ × 10−25, respectively, at the most sensitive detector frequencies between 130–175 Hz. This represents a mean improvement of a factor of 2 across the band compared to previous searches of this kind for these sky locations, considering the different quantities of strain constrained in each case

Full band all-sky search for periodic gravitational waves in the O1 LIGO data

We report on a new all-sky search for periodic gravitational waves in the frequency band 475–2000 Hz and with a frequency time derivative in the range of ½−1.0; þ0.1 × 10−8 Hz=s. Potential signals could be produced by a nearby spinning and slightly nonaxisymmetric isolated neutron star in our Galaxy. This search uses the data from Advanced LIGO’s first observational run O1. No gravitational-wave signals were observed, and upper limits were placed on their strengths. For completeness, results from the separately published low-frequency search 20–475 Hz are included as well. Our lowest upper limit on worst-case (linearly polarized) strain amplitude h0 is ∼4 × 10−25 near 170 Hz, while at the high end of our frequency range, we achieve a worst-case upper limit of 1.3 × 10−24. For a circularly polarized source (most favorable orientation), the smallest upper limit obtained is ∼1.5 × 10−25

Prospects for observing and localizing gravitational-wave transients with Advanced LIGO, Advanced Virgo and KAGRA

We present possible observing scenarios for the Advanced LIGO, Advanced Virgo and KAGRA gravitational-wave detectors over the next decade, with the intention of providing information to the astronomy community to facilitate planning for multi-messenger astronomy with gravitational waves. We estimate the sensitivity of the network to transient gravitational-wave signals, and study the capability of the network to determine the sky location of the source. We report our findings for gravitational-wave transients, with particular focus on gravitational-wave signals from the inspiral of binary neutron star systems, which are the most promising targets for multi-messenger astronomy. The ability to localize the sources of the detected signals depends on the geographical distribution of the detectors and their relative sensitivity, and 90% credible regions can be as large as thousands of square degrees when only two sensitive detectors are operational. Determining the sky position of a significant fraction of detected signals to areas of 5– 20 deg2 requires at least three detectors of sensitivity within a factor of ∼2 of each other and with a broad frequency bandwidth. When all detectors, including KAGRA and the third LIGO detector in India, reach design sensitivity, a significant fraction of gravitational-wave signals will be localized to a few square degrees by gravitational-wave observations alone

Social work students as community partners in a family intervention programme

Summary: This paper describes the findings from the evaluation of a UK initiative which engaged social work students as community partners within an educational based family intervention programme. Fourteen social work students in the first year of a BA (Hons) were placed in the programme to meet the volunteering requirements of their 'Community Project'. By engaging with the community-based family programme at an early stage in their education, students experienced the benefits of interventions, focussing on sustainability, citizenship and parent participation. We describe the approach and discuss the evaluation outcomes to illustrate the potential of utilising co-learning with families in social work education. In taking up the role of community partners, students observed first-hand the value of incorporating horizontal relationships into their learning and experienced direct knowledge exchange with service users at an early stage in their training. Findings: The findings suggest that this experience enabled students to connect with conceptualisations of macro alongside micro practice in their professional development as social workers. Further, connecting critical social theories with the direct experience of families promoted the learners own empowerment and conscientization. Application: This provided a means of embedding social work values and aspirations towards social justice in their future practice orientation

Small Change: Economics and the British coin-tree

This is the accepted manuscript for the following article: Ceri Houlbrook, “Small Change: Economics and the British coin-tree”, Post Medieval Archaeology, Vol. 49(1), June 2015. The final published version can be found at: http://www.tandfonline.com/doi/full/10.1179/0079423615Z.00000000074 © Society for Post-Medieval Archaeology 2015Throughout the c.2000 year period coins have been circulated in Britain, they have also been ritually employed, most notably as votive deposits. Focusing specifically on the understudied custom of the British coin-tree, whereby coins are ritually embedded into the barks of trees, this paper considers the coin’s role and applicability as a deposit. It aims to demonstrate that our understanding of the coin’s past, present, and future ritual employment is not only aided by a consideration of economics and the coin’s secular function; it would be utterly incomplete without it.Peer reviewedFinal Accepted Versio

Incidence and survival of oesophageal and gastric cancer in England between 1998 and 2007, a population-based study

BACKGROUND: Major changes in the incidence of oesophageal and gastric cancers have been reported internationally. This study describes recent trends in incidence and survival of subgroups of oesophageal and gastric cancer in England between 1998 and 2007 and considers the implications for cancer services and policy. METHODS: Data on 133,804 English patients diagnosed with oesophageal and gastric cancer between 1998 and 2007 were extracted from the National Cancer Data Repository. Using information on anatomical site and tumour morphology, data were divided into six groups; upper and middle oesophagus, lower oesophagus, oesophagus with an unspecified anatomical site, cardia, non-cardia stomach, and stomach with an unspecified anatomical site. Age-standardised incidence rates (per 100,000 European standard population) were calculated for each group by year of diagnosis and by socioeconomic deprivation. Survival was estimated using the Kaplan-Meier method. RESULTS: The majority of oesophageal cancers were in the lower third of the oesophagus (58%). Stomach with an unspecified anatomical site was the largest gastric cancer group (53%). The incidence of lower oesophageal cancer increased between 1998 and 2002 and remained stable thereafter. The incidence of cancer of the cardia, non-cardia stomach, and stomach with an unspecified anatomical site declined over the 10 year period. Both lower oesophageal and cardia cancers had a much higher incidence in males compared with females (M:F 4:1). The incidence was also higher in the most deprived quintiles for all six cancer groups. Survival was poor in all sub-groups with 1 year survival ranging from 14.8-40.8% and 5 year survival ranging from 3.7-15.6%. CONCLUSIONS: An increased focus on prevention and early diagnosis, especially in deprived areas and in males, is required to improve outcomes for these cancers. Improved recording of tumour site, stage and morphology and the evaluation of focused early diagnosis programmes are also needed. The poor long-term survival reinforces the need for early detection and multidisciplinary care

Combined Point-of-Care Nucleic Acid and Antibody Testing for SARS-CoV-2 following Emergence of D614G Spike Variant

Rapid COVID-19 diagnosis in the hospital is essential, although this is complicated by 30%-50% of nose/throat swabs being negative by SARS-CoV-2 nucleic acid amplification testing (NAAT). Furthermore, the D614G spike mutant dominates the pandemic and it is unclear how serological tests designed to detect anti-spike antibodies perform against this variant. We assess the diagnostic accuracy of combined rapid antibody point of care (POC) and nucleic acid assays for suspected COVID-19 disease due to either wild-type or the D614G spike mutant SARS-CoV-2. The overall detection rate for COVID-19 is 79.2% (95% CI 57.8-92.9) by rapid NAAT alone. The combined point of care antibody test and rapid NAAT is not affected by D614G and results in very high sensitivity for COVID-19 diagnosis with very high specificity

GWTC-1: A Gravitational-Wave Transient Catalog of Compact Binary Mergers Observed by LIGO and Virgo during the First and Second Observing Runs

We present the results from three gravitational-wave searches for coalescing compact binaries with component masses above 1  M⊙ during the first and second observing runs of the advanced gravitational-wave detector network. During the first observing run (O1), from September 12, 2015 to January 19, 2016, gravitational waves from three binary black hole mergers were detected. The second observing run (O2), which ran from November 30, 2016 to August 25, 2017, saw the first detection of gravitational waves from a binary neutron star inspiral, in addition to the observation of gravitational waves from a total of seven binary black hole mergers, four of which we report here for the first time: GW170729, GW170809, GW170818, and GW170823. For all significant gravitational-wave events, we provide estimates of the source properties. The detected binary black holes have total masses between 18.6−0.7+3.2  M⊙ and 84.4−11.1+15.8  M⊙ and range in distance between 320−110+120 and 2840−1360+1400  Mpc. No neutron star-black hole mergers were detected. In addition to highly significant gravitational-wave events, we also provide a list of marginal event candidates with an estimated false-alarm rate less than 1 per 30 days. From these results over the first two observing runs, which include approximately one gravitational-wave detection per 15 days of data searched, we infer merger rates at the 90% confidence intervals of 110−3840  Gpc−3 y−1 for binary neutron stars and 9.7−101  Gpc−3 y−1 for binary black holes assuming fixed population distributions and determine a neutron star-black hole merger rate 90% upper limit of 610  Gpc−3 y−1