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Home Safety
A short list of safety check points to make your home safer.
I. Lock all doors and windows.
1. Make a security check list and post in a prominent place.
I. Lock all doors and windows.
II. Leave a light on if you will not be back until after dark.
III. Verify that any window air-conditioning units are secured to the window frame.
IV. Verify that motion sensor lights are turned on.
V. Verify that you have set a "Charlie Bar" or commercial locking device in your sliding doors.
VI. Set alarm.
2. Account for all of the keys to your house.
3. If you use an emergency key hider, make sure it is not placed in an obvious location and that it is not easily identifiable.

subset: commoncrawl


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Looking for GED® testing in Hubertus WI? The examination is only offered in person and not online. Typically to be able to sit for the GED® exam the test taker must be older than 17 years old. The testing fee in the state of Wisconsin typically varies from $40 to $90.
The examination consists of five topics: reading, writing, mathematics, science and social studies. Each section of the test gives a score between 200 and 800. Most states require test takers to receive a score of 410 on each of the five tests and an average of 450 for the GED® test to pass.
Typically GED® testing sites in Hubertus WI are offered at:
- Hubertus Community Colleges
- Hubertus ISD
- Hubertus Literacy Centers
- Hubertus High Schools


text:
HOME DEPOT – Technical Analysis – 2016-09-29
Page 1: Index – Company- technical measures and spreads
Theoretical exposure Asset Manager – Trader
Daily and Weekly Absolute and Relative charts
Page 2: Read mode and disclaimer
This document has been prepared solely for informational purposes, and is intended only fo use be the designated recipient(s). This information was obtained from sources we believe to be reliable, but its accuracy is not guaranteed. All information is subject to change without notice. This does not constitute a solicitation or offer to buy securities or sell secutities or any other instruments, or a recommandation with respect to any security or instrument mentioned herein. This is not a confirmation of terms of any transaction. No representations are made herein with respect to availability, pricing or performance. Additional information available on request. This document should only be read by those persons to whom it is addressed and is not intended to be relied upon by any person without subsequent written confirmation of its contents. If you have received this e-mail message in error, please destroy it and delete it from your computer, Any form of reproduction, dissemination, copying, disclosure, modification, distribution and/or publication of this e-maim message is strictly prohibited.


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Between the Pipes
I then realized that my comment was so long (long-winded??) that I should probably just post it on my own blog. Without further ado, here goes:
I completely agree that the instructor has always been the stitching that holds the small pieces of every course together, both in terms of content and delivery. As you noted in your post, in the f2f classroom, the instructor joins multiple media herself by switching from handouts to overhead slides to the textbook. This "multimedia" has been around for so long that we hardly consider it media at all any more; as such, it has -- perhaps for the best -- become transparent, leaving the instructor and the students to focus on the important part of the lesson, namely the content.
Online education, being such a relatively new field dealing with such relatively new media, has brought the media of instruction back to its opaque state. With online multimedia staring us so squarely in the face, it's easy for both instructors and students to get lost in the "how" at the expense of the "why." Of course, the "why" must always win out (although in reality it often doesn't). However, I don't think it's possible to leave the "how" completely up to the individual students in the online environment any more than an instructor would leave the "how" up to the students in the f2f classroom.
I think online media may offer a wider array of options than are available in the f2f classroom. After all, a f2f classroom could get pretty chaotic if an instructor told the class to read an assignment either from the textbook, from a photocopy she was passing around or from a slide she was showing. Out of necessity, the f2f instructor needs to choose one medium for that reading and ask everyone to use it, even though some students might prefer one form over the other if given the choice. No such confusion need occur in the online classroom. One group of students could choose to complete a project by communicating through wikis; another group could choose to use a blog; still another could choose to use a forum. However, the instructor still needs to give direction, especially when dealing with students who are less familiar with what technology is out there. The online instructor who tells a group to "complete this project" without providing any suggestions for which media to use may find that the group, when faced with boundless -- perhaps overwhelming -- options, chooses nothing at all. I think this will certainly become less and less of an issue as online media become more and more transparent; this will happen naturally over time as generations of children raised with this technology come of age. Just as the written word was once a revolutionary form of media that completely transformed the nature of knowledge transfer but has now become such a standard that we don't even give its use a second thought, today's "revolutionary" online multimedia will one day be second nature to everyone. In the meantime, though, too many options and too little direction may cause more problems than not (see here for an interesting example).


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Machine Learning for Estimating Rock Mechanical Properties beyond Traditional Considerations
- Authors
- Yiwen Gong (The Ohio State University) | Mohamed Mehana (University of Oklahoma) | Ilham El-Monier (The Ohio State University) | Feng Xu (Research Institute of Petroleum Exploration and Development Co. Ltd. CNPC / China National Oil and Gas Exploration and Development Corporation) | Fengyang Xiong (The Ohio State University)
- https://doi.org/10.15530/urtec-2019-897
- Document ID
- Publisher
- Unconventional Resources Technology Conference
- Source
- SPE/AAPG/SEG Unconventional Resources Technology Conference, 22-24 July, Denver, Colorado, USA
- Publication Date
- 2019
- Document Type
- Conference Paper
- Language
- English
- Copyright
- 2019. Unconventional Resources Technology Conference
- Downloads
- 7 in the last 30 days
- 262 since 2007
- Show more detail
|SPE Member Price:
|SPE Non-Member Price:
The accurate estimation of the elastic properties of the rock is of great importance for designing a successful hydraulic fracturing. Among these properties, Young's modulus and Poisson's ratio essentially control fracture aperture and conductivity. However, the fissile nature of the shale rock largely challenges the mechanical properties measurement using a cylindrical core sample. While the nanoindentation technology can be applied to measure small chips of rock fragment, but reproducible experiments are required to provide an unbiased estimation. Herein, we are proposing a machine learning approach to predict the elastic moduli. We utilized an ensemble of data mining techniques and a database that include both the mineralogy and pore characteristics. Our results indicate that K-Means clustering yields best performance on data classification than all other tested methods while the elastic moduli estimation from Artificial Neural Network (ANN)is most accurate than Support Vector Machine (SVM), Multivariate Linear Regression (MLR) and Multivariate Adaptive Regression Spine (MARS). The dimension reduction became essential when then input datasets are remarkably correlated. The supervised learning techniques with our proposed approach leverage the usability of the lab experiment data and overcome disadvantages of the traditional elastic moduli measurement. It also further lands the far-reaching guide for the fracturing design.
Machine learning have recently revolutionized the oil and gas industry (Alcocer and Rodrigues 2001, Al-Fattah and Startzman 2001, Kohli and Arora 2014, Okpo et al. 2016, Sinha et al. 2016, Tariq et al. 2017, Luo et al. 2018, Nande 2018, Rashidi et al. 2018, Sidaoui et al. 2018, Xu et al. 2019). As a data-rich industry, machine learning finds applications in every corner ranging from production forecast to drilling efficiency (Hegde and Gray 2017, Fulford et al. 2016). Given the significance of geomechanical properties of the rock, the volume of studies has attempted to leverage machine learning techniques. For instance, Li et al. (2018) developed a workflow implementing various machine learning algorithm to accurately provide an alternative to synthesize the sonic logs and geomechanical properties afterwards. In the same time, Hadi and Nygaard (2018) used Artificial Neural Network (ANN) to develop an empirical model to estimate the shear velocity from conventional logs. Another dimension was presented by Jain et al. (2015) where they proposed an approach to integrate both core and log spectroscopy which provided better estimations of the mineralogy.
|File Size
|Number of Pages


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Hi all,
You may remember me talking about the Cancer Poetry Project, an amazing anthology of poems by cancer patients, family members and caregivers. Now the Cancer Poetry Project 2 is approaching completion. I am honoured to have a poem that has been picked for this volume and we expect an April publication.
Karin Miller, the founder and editor has launched a Kickstart campaign to defer the initial costs of publication. Consider supporting this worthwhile project. The first volume of poems is gorgeous and is a wonderful way for patients to connect with the experiences of others who have gone before them.
All the best


text: In GP 10, we're getting ready to start using Electronic Funds Transfer in our Receivables area, i.e, we're going to take our customers' payments directly from their bank accounts. If there's a problem where we take a customer's money in error, how do we put it back into their back account using EFT? Thanks! Mary Fetsch


text:
Q. Reading Arizona and Axis 360
Guide to accessing Reading Arizona eBooks using the Axis360 platform.
Account Setup
- Go to Reading Arizona and click Login in the upper right corner.
- Click on the link next to "Don't have a user name? Click here."
- Enter the name of the state in which you reside (Note: Due to licensing restrictions, Reading Arizona is only available to residents of the state of Arizona).
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Download the app
- Get the free Axis 360 app for your tablet or smartphone from your app store. The app works on iOS, Android, and Kindle Fire devices.
- Open the Axis 360 app. Select your city and state, or search for Reading Arizona.
- Sign in with user ID and password.
- Tap a cover to borrow a title or place a hold.
NOTE: If you do not have a tablet or smartphone, you may access Reading Arizona eBooks from a web browser on your computer. After checking out items, use the Read in Browser or Listen in Browser option.
Using the Axis 360 app in iOS (iPhone/iPad)
Using the Axis 360 app in Android and Kindle Fire
Checking Out
*Find the title you wish to check out and then tap the CHECKOUT button. Your title will immediately begin to download and will open automatically inside the Axis 360 app. The loan period is 14 days by default.
*A title cover showing a solid black icon in the upper right corner is immediately available for checkout; a gray one indicates the title has a wait list.
Checking out titles in Android and Kindle Fire
Checking out titles from the Axis 360 website
How long is the lending period for an eBook?
The default lending period is 14 days.
How many eBooks can I check out at once?
You can check out up to 3 titles at one time.
Can I check out eBooks for my Kindle Paperwhite or Voyage?
No, eBooks checked out from Axis 360 cannot be accessed on a Kindle eReader, such as Paperwhite or Voyage.
Will you be adding more eBooks to the collection?
We will continue to add new Arizona related eBooks to the collection.
Placing Holds
Find the title you wish to reserve and click tap the PLACE ON HOLD button and confirm. You will be notified by email when the title is available to check out. If your email address isn't already registered with Axis 360, you will be asked to provide it at this time.
How many holds can I place at one time?
You can place up to 3 holds in Axis 360.
What do I do when I receive notice that my hold is ready?
Be sure to read the instructions given in the email. Go to the link provided in the email and proceed to check out the item. You have 72 hours to check out your hold before it expires and is given to the next patron on the holds list.
I clicked on the link in the email notification to retrieve my hold, but it doesn't appear under "My Stuff." What happened?
Make sure you have signed into the correct Axis 360 account, if you have different user accounts in your household.
If it's an eBook, why is there a waitlist to check it out?
This is the model publishers negotiated with most eBook vendors so that libraries can provide access to eBooks. As a result, borrowing eBooks is similar to checking out physical books – one copy at a time per patron. If another patron has already checked out an eBook, you may put a hold on it.
Returning Titles Early
Although it's not necessary to return your title early, you may return it if you like. Otherwise, the title will be returned automatically at the end of the loan period.
Returning titles early in iOS (iPhone/iPad)
Click the book icon (My Stuff), locate the title to be returned, swipe to the left to reveal a Return button and then tap it and confirm to return your item. You may also return the title by tapping on the cover, which will reveal a RETURN button. Tap it and confirm to return your title.
Returning titles early in Android and Kindle Fire
Click the book icon (My Stuff), locate the title to be returned, and tap the three-dots to reveal a Return button and then tap it and confirm to return your item. You may also return the title by tapping on the cover, which will reveal a RETURN button. Tap it and confirm to return your title.
Returning titles early in the Axis 360 website
Go to the Library's Axis 360 website, log on with your 14-digit library card number and PIN, click My Stuff and locate the title to be returned, and then click the RETURN button and confirm to return your item.
Technical Requirements
What devices can I use to read Axis 360 eBooks?
Most devices running an operating system that allows the Axis 360 application to install onto it should work. See the eBook Software Requirements Chart for more information. NOTE: e-Ink devices, such as Kindle and Nook, are not compatible with Axis 360.
Is there an Axis 360 app?
Yes. The app is available for Android devices running 4.0 or later, iOS devices running iOS 7 or later, and Kindle Fire tablets.
Do I need BLIO or Adobe Digital Editions to read Axis 360 eBooks?
No. You no longer need BLIO or Adobe Digital Editions to use Axis 360. Download the all-in-one app to read eBooks from Axis 360.
Help Topics
My title won't load after downloading. What's wrong?
If your title doesn't load, try logging out of the app and logging back in. You could also try uninstalling the app and reinstalling it, or returning the item and checking it out again, if no one else is waiting for it. If the problem continues, submit an help request.
Where can I find more help topics?
For more details on checking out and downloading Axis 360 titles, visit the Axis 360 Help page.
Who do I contact if I have a question or problem?
For the quickest response, please use our online help form and include as many details as possible regarding the issue you are experiencing. If it is a technical problem, please include any error messages you received and details about your computer (e.g., Mac/PC, operating system, version of browser used, etc.) You may also ask for help from a librarian at your local library.
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text:
Agrobacterium tumefaciens C58 and its derivatives give rise to spontaneous mutants resistant to tetracycline at a high frequency. We observed that a mutation affecting a tRNA processing function significantly affected the emergence of such mutants, suggesting that C58 contained a positively acting gene conferring resistance to tetracycline. A cosmid clone conferring resistance to tetracycline in Escherichia coli andAgrobacterium was isolated from a genomic bank of one such mutant. Subcloning, transposon mutagenesis, and DNA sequence analysis revealed that this DNA fragment contained two divergently transcribed genes, tetA and tetR, encoding products that were very similar to proteins of the Tet(A) class of tetracycline resistance systems. In the clone from this mutant, tetR was disrupted by an IS426. The homologous region from wild-type NT1 contained an intact tetR gene and did not confer resistance to tetracycline. Hybridization analysis showed that of 22 members of the genus Agrobacterium surveyed, only strains C58 and T37 contained the tet determinant. Moreover, only these two strains mutated to resistance to this antibiotic. Unlike other Tet(A) systems, neither tetracycline nor a series of its derivatives induced the expression of this tet gene unit. Other polycyclic compounds, including many of plant origin, also did not induce this tet gene system. The divergent promoter region of this tet system contained a single inverted repeat element identical to one such operator repeat in the promoter region of the tet determinant from the IncP1α R plasmid RP4. TetR repressor proteins from the Agrobacterium tetsystem and from RP4 interacted with the heterologous operators. While the repressive effect of the TetR protein from strain C58 (TetRC58) on the tetA gene from strain RP4 (tetARP4) was not relieved by tetracycline, repression of tetAC58 by TetRRP4was lifted by this antibiotic.
Genes encoding resistance to antibiotics have been identified in a wide spectrum of prokaryotic organisms, including gram-negative and gram-positive bacteria. Although most of the genes characterized to date have been isolated from clinical isolates, these determinants are believed to have originated in soil bacteria (38). More recently, genes encoding proteins that confer resistance to several different drugs, the multidrug systems, have been identified and characterized (37). Horizontal transfer of these determinants among diverse bacteria are challenging the effectiveness of antibiotics in infectious disease control (39, 48).
Genes conferring resistance to tetracycline are among the most abundant of the identified drug resistance elements (29). Although mechanisms such as target site protection and drug inactivation are known (7, 44), most tetracycline resistance determinants function through the efflux mechanism, pumping a drug-metal ion complex out of the bacterial cell by a process energized by the membrane proton gradient (37, 45).
It is a well-recognized but unpublished observation thatAgrobacterium tumefaciens C58 and its derivatives give rise at a high frequency to spontaneous mutants resistant to tetracycline. Most genetic and molecular analyses concerning A. tumefaciens and its plasmids are conducted in the C58 chromosomal background. Since many broad-host-range cloning vectors encode resistance to tetracycline as the selectable marker, the propensity of this strain to mutate to tetracycline resistance poses problems to the genetic analysis of this bacterium and to its use as a plant transformation agent.
During a study of mutants affecting the induction of tragenes of pTiC58 in NT1 (51), a Ti plasmid-cured derivative of C58 (53), we observed that one mutant, NTM7, while remaining able to mutate to tetracycline resistance, expressed the phenotype at a much lower level than did its wild-type parent. NTM7 later was found to carry a Tn5 insertion in a locus encoding a protein homologous to the product of the Escherichia coli rnd gene (30). In E. coli, a miaAmutant is unable to express Tet(M)-mediated tetracycline resistance (8). Since both the rnd and miaA genes are involved in tRNA modification (12, 52), we reasoned that there may be a positively acting tetracycline resistance element in the genome of strain C58 which can function normally in the wild-type bacterium but not in the rnd mutant. A study was initiated to investigate the nature of such tetracycline-resistant mutants. We confirmed our hypothesis and report here the molecular cloning, nucleotide sequence, and genetic characterization of a tetracycline resistance gene unit from this A. tumefaciensstrain.
Bacterial strains and growth conditions.The strains of E. coli and A. tumefaciens and the plasmids used in this study are listed in Table1. E. coli strains were grown at 37°C in L broth (LB), on L agar plates (Difco Laboratories, Detroit, Mich.), or in A medium (33).Agrobacterium strains were grown at 28°C in LB or on nutrient agar (NA) (Difco Laboratories). AB medium (11) supplemented with 0.2% mannitol as the sole carbon source was used as the defined minimal medium for Agrobacterium strains. Biotin was added to a final concentration of 2 μg/ml for biovar 2 strains. Agar (Difco Laboratories) was added at 1.5% to make solid medium. Except when specified in the text, antibiotics were used at the following concentrations: for E. coli, kanamycin at 50 μg/ml, tetracycline at 10 μg/ml, and ampicillin at 100 μg/ml; forAgrobacterium spp., kanamycin at 50 μg/ml, carbenicillin at 50 or 100 μg/ml, and tetracycline at 2 μg/ml. X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; Sigma Chemical Co., St. Louis, Mo.) was included in the medium at 40 μg/ml to monitor the production of β-galactosidase.
DNA manipulations.Plasmids were isolated by an alkaline lysis method (40). Cloning followed standard recombinant DNA techniques (40). Restriction digestions were carried out in accordance with the instructions of the manufacturers, and digestion products were separated by electrophoresis in agarose gels with Tris-borate-EDTA buffer (40). DNA fragments were recovered from agarose gels by using GenElute spin columns (Supelco Inc., Bellefonte, Pa.). Plasmids were introduced into E. coliby CaCl2-mediated transformation (40) and intoA. tumefaciens by S17-1-mediated mating (43) or by electroporation (9).
Genomic bank construction.Cosmid pZL1 was constructed by cloning the cos site from pCP13 (15) as a 1.7-kbBglII fragment into pJB3 (6). Genomic DNA was digested with Sau3AI to give an average fragment size of 40 kb and ligated to pZL1 that had been digested withBamHI and treated with alkaline phosphatase. The ligation mixture was packaged into phage λ by using the Packagene system (Promega Corporation, Madison, Wis.) and transduced into E. coli LE392.
Southern hybridization.Genomic DNA was prepared from overnight cultures of Agrobacterium spp. as described previously (18). After digestion with restriction endonuclease, DNA fragments were separated on 0.7% agarose gels and transferred to a nylon membrane. DNA probes were randomly labeled by using the Genius digoxigenin kit (Boehringer GmbH, Mannheim, Germany). Hybridization, washing, and detection followed protocols provided by the manufacturer. Hybridization and washing were performed under conditions of medium stringency.
Tn3HoHo1 mutagenesis.The insert in clone pDLE4.5 (Table 1) was mutagenized with Tn3HoHo1 as described previously (46). Sites and orientations of the insertions were determined by restriction mapping. Each mutant was tested for tetracycline resistance on LB medium containing this antibiotic.
DNA sequence analysis.DNA fragments containing thetet region were sequenced by use of the chain termination method (41) by the Genetic Engineering Facility at the University of Illinois. DNA sequences were analyzed by using DNA Strider (32). The BLAST (4) protocols were used to search the GenBank database for related sequences. The GAP subroutine of the GCG program (Genetics Computer Group, Madison, Wis.) was used to compare sequences for similarity.
Isolation of tetracycline-resistant mutants. Agrobacterium spp. were grown in LB liquid medium to saturation. A 200-μl volume of culture from each strain was spread onto NA medium containing tetracycline at 5 or 10 μg/ml. The capacity to give tetracycline-resistant mutants was assessed by the appearance of colonies on the plates after 72 h of incubation at 28°C.
Gene expression assays. E. coli and A. tumefaciens strains were grown in A and AB-mannitol media, respectively. Overnight cultures were washed and diluted 40-fold in the same respective media, and incubation was continued until the optical density at 600 nm (OD600) reached about 0.7 forE. coli and 0.3 for A. tumefaciens. When needed, cultures were divided, and inducers (arabinose or tetracycline) were added to one subculture at the concentrations specified in Results. β-Galactosidase activity was determined quantitatively as described previously (33). β-Glucuronidase activity was measured as described previously (13, 24, 49). The activity of this enzyme was calculated by using the Miller formula (33), with OD420replaced by OD415. All enzymatic activities were expressed as units per 109 CFU.
Induction assays.The ability of tetracycline, its derivatives, and related compounds to induce the tet system of strain C58 (tetC58 system) was assessed two ways. In the first, reporter strains were inoculated onto medium containing X-Gal and the compound being tested. The appearance of blue colonies signalled the induction of the reporter gene. In the second, reporter strains were grown in 30 ml of liquid medium with appropriate antibiotics to saturation and mixed with 50 ml of warm 0.8% agar with X-Gal, and 6-ml volumes were layered over a base of LB or AB medium in 100-mm-diameter petri dishes. Discs of Whatman 3MM paper (0.5-cm diameter), onto which a few crystals of the compound to be tested were sprinkled, were laid on the surface of the soft agar. Dissolution and diffusion of the chemical give a concentration gradient around the disc. The plates were incubated at 28°C for A. tumefaciens reporter strains and at 37°C for E. coli reporter strains and examined at intervals over a 3-day period. Susceptibility to a given compound was indicated by a zone of growth inhibition around the disc. Induction of the reporter by the compound was indicated by a blue zone around the disc or, in cases where the compound also was inhibitory, by a blue zone in the area of growth at the edge of the zone of inhibition.
Nucleotide sequence accession number.DNA sequences of wild-type tetC58 were deposited in the GenBank database under accession no. AF090987 .
Cloning a tetracycline resistance determinant from A. tumefaciens C58.Plating strain C58 onto medium containing tetracycline at concentrations ranging from 0.5 to 20 μg/ml results in the appearance at a high frequency of mutants resistant to this antibiotic (Fig. 1A and data not shown). However, we observed that a C58 mutant with a Tn5 insertion in a gene homologous to rnd (30) yielded tetracycline-resistant mutants that grew much more slowly on medium containing the antibiotic (Fig. 1B). Since a miaA mutant ofE. coli does not express Tet(M)-mediated tetracycline resistance (8) and both rnd and miaAgenes encode products involved in tRNA modification, we reasoned that strain C58 contains an active tetracycline resistance element that functions in the rnd+ but not in thernd mutant background. To test this, a genomic library of NT1TcR1, a tetracycline-resistant mutant of the otherwise-wild-type strain NT1, was constructed in the broad-host-range cosmid vector pZL1 (Table 1). This bank, in E. coli LE392, consists of 856 clones containing cosmids with an average insert size of 42 kb. The bank was screened on LB medium containing tetracycline, and one cosmid that confers resistance to this antibiotic on its E. coli host was isolated. This clone, named pZLT1, also conferred resistance to high levels of this antibiotic on severalAgrobacterium strains, including 15955, R10, and K84 (data not shown).
The tetracycline resistance trait was subcloned from pZLT1 as a 4.5-kbEcoRI fragment into pZL1 to generate pZLE4.5 (Fig.2). To localize the tetdeterminant, this DNA fragment was recloned into pDSK519 (25) to give pDLE4.5, and this plasmid was mutagenized with Tn3HoHo1 (46). The location and orientation of the insertion in each mutant plasmid were determined, and each was tested for its ability to confer resistance to tetracycline. Of six mutations mapped, the three contiguous insertions that are located in the middle of the fragment abolished resistance to this antibiotic (Fig. 2).
DNA sequence analysis.DNA fragments corresponding to the region defined by the Tn3HoHo1 insertion were subcloned and subjected to sequence analysis. BLAST searches revealed that one such EcoRI-HindIII fragment from plasmid pZLHE1.6 (Table 1) contained a portion of a tetR(A) homologue. As sequencing continued, however, this gene was abruptly disrupted by a totally different DNA sequence identical to the sequence of IS426, an IS element identified fromA. tumefaciens T37 (50). Apparently, the 4.5-kb EcoRI fragment from the mutant contains an incompletetet locus; insertion of IS426 introduced anEcoRI site which divided this region into two fragments when this enzyme was used for cloning. To obtain the complete sequence of the mutant version, the adjacent 4.9-kb EcoRI fragment was cloned into pBluescript SK(+) from pZLT1 to generate pZLE4.8 (Table1). Analysis of the insert identified sequences corresponding to the remainder of IS426 followed by the 3′ end of thetetR gene of strain C58 (tetRC58).
Analysis of sequence data from pZLHS2.9 (Table 1), a plasmid harboring an insert from the opposite end of the region encoding thetetR(A) homologue, identified a single open reading frame of 1183 bp (Fig. 2 and data not shown). BLAST analysis indicated that this open reading frame encodes a protein very similar to thetetA(A) gene product from other tet systems.
To obtain the wild-type version of the tet locus from C58, a portion of the tetA(A) homologue was used as a probe to screen a genomic bank of the wild-type, tetracycline-sensitive strain NT1 (16). A cosmid hybridizing strongly to the probe was identified and designated pZLT2 (Fig. 2). The hybridizing region was subcloned as an 8.5-kb EcoRI fragment into pBluescript SK(+) to generate pZLE8.5 (Fig. 2), and the insert in this plasmid was used as the template to sequence a 2.6-kb region containing the wild-typetet locus.
Examination of the sequence indicated that this region contains two divergently transcribed open reading frames (Fig. 2 and data not shown). Database searches revealed that the two open reading frames can code for products that are very similar to the Tet(A) class of tetracycline resistance genes from E. coli transposons, with the highest similarity to that of the Tn1721 tet system (2). One we call tetAC58 could code for a protein with an Mr of 41,458 and having 46% identity (55% similarity) with TetA from the IncP1α R plasmid RP4 (35) (Fig. 3A). The second, which could code for a protein with anMr of 24,389 and having 46% identity (55% similarity) with TetR from RP4 (35), was namedtetRC58 (Fig. 3B). The nucleotide sequences oftetAC58 and tetRC58 from the mutant and wild-type strains are identical. However, in the mutant, the sequence is interrupted by the IS426 insertion at bp 520 of tetRC58 (data not shown). This insertion generated a 5-bp duplication of the sequence 5′ GGGGG 3′ at the target site.
A hydropathy plot generated by the Kyte-Doolittle algorithm (28) revealed that TetAC58 contains 12 putative hydrophobic segments (data not shown). Such domains, which could represent transmembrane segments, are a typical feature of the major facilitator superfamily of proteins (31, 36). Sequence analysis indicated that, like other class A and class B TetR proteins, TetRC58 contains a typical helix-turn-helix domain in its N-terminal portion (data not shown). ThetetAC58 and tetRC58 genes are separated by an 88-bp intergenic region with characteristics of regulated divergent promoters (Fig. 4A). A 15-bp inverted repeat (IR) element overlaps the putative −10 region of the tetAC58 promoter. This IR also overlaps the potential −35 site of the tetRC58promoter. As shown in Fig. 4B, the sequence of this IR is identical to those of the operators found between the tetA andtetR genes of other class A and class B tetsystems, including those of RP4 and Tn1721 (2, 35). However, the promoter region oftetC58 lacks the second IR element present in the corresponding region of tetRP4 and oftetTn1721. Furthermore, the intergenic region of tetC58 is considerably shorter than those of the tet elements of RP4 and Tn1721 (Fig. 4B).
Expression of tetC58 is not induced by tetracycline or related compounds.In class A and class Btet systems, TetR represses expression of tetAand tetR, and this repression is relieved by subinhibitory levels of the antibiotic (3, 21, 23). Tetracycline does not induce the Agrobacterium tet system; as noted above, when a large number cells are spread onto medium containing this antibiotic, only a very small fraction of the cells grow to produce colonies. Moreover, of those such colonies tested, all now are constitutively resistant to tetracycline (data not shown). To study the regulation of the tetA and tetR genes ofAgrobacterium spp., a 66-bp fragment of the intergenic region, which comprises sequence between the putative ribosome binding sites for the tetAC58 andtetRC58 genes, was cloned into the divergent promoter detection vector pRG970b (13) to generate pZLOP1. In this construct, tetAC58 andtetRC58 are transcriptionally fused to thelacZ and uidA genes, respectively, of the vector. In addition, the tetRC58 gene was cloned as a NcoI fragment from pZLT2 into the expression vector pBAD22 (19) to generate pBetR. This construct allows us to regulate the expression of tetRC58with arabinose. To express tetRC58 inAgrobacterium spp., this gene was excised from pBetR as anEcoRI-BamHI fragment and cloned into pDBL4 (Table1), a broad-host-range expression vector containing the arabinose promoter and downstream transcriptional terminator from pBAD22 cloned into pBBRMCS-2 (27), to construct pDBL4-tetR. Expression oftetRC58 in these two constructs is inducible inA. tumefaciens and E. coli by addition of arabinose to the culture medium at a final concentration of 0.4%.
Under conditions in which tetRC58 is repressed, expression levels of thetetAC58::lacZ andtetRC58::uidA fusions were lower in strain NT1(pZLOP1, pDLB4-tetR) than in strain NT1TcR1(pZLOP1, pDLB4-tetR). We attribute the lower expression levels of these fusions in NT1 to the chromosomal copy oftetRC58. However, when the cloned copy oftetRC58 was induced by adding arabinose, expression of the reporter fusions in both strains was inhibited to similar levels (Table 2). Similarly, expression of thetetAC58::lacZ reporter on pZLOP1 was repressed by TetRC58 in an E. coli host but only upon addition of arabinose (Table 2).
Tetracycline, when added at final concentrations ranging in 10-fold increments from 0.0001 to 1 μg/ml, had no effect on the expression of tetAC58 ortetRC58 in either host (data not shown). Although tetracycline does not induce the expression oftetAC58, we consider the possibility that compounds similar to this antibiotic could be the ligands for TetRC58. To test this, a total of 25 polycyclic compounds with structures similar to tetracycline were tested for their ability to induce the expression of thetetAC58::lacZ fusion. These included active derivatives of tetracycline as well as various nontoxic compounds, most of plant origin. None of these agents induced the expression of thetetAC58::lacZ fusion at a detectable level (data not shown). When the tetracycline-sensitive strains NT1(pZLOP1) and NT1(pZLOP1, pDLB4-TetR) were used as reporters, resistant mutants formed in the zones of growth inhibition caused by chlortetracycline and oxytetracycline. However, no such mutants appeared in the zone of growth inhibition caused by minocycline (data not shown).
Cross-operator recognition by TetRC58 and TetRRP4.By sequence analysis, the putative 15-bp operator of tetC58 is identical to those of other tet systems (Fig. 4B). Furthermore, the sequence from residues 26 to 47 of TetRC58 is virtually identical to that of the N-terminal helix-turn-helix domain of several related TetR proteins, including TetRRP4, TetRTn10, and TetRTn1721(Fig. 3B and data not shown). Given these similarities, we determined whether TetR from A. tumefaciens and from RP4 will recognize the operators of the noncognate homologues. Strains DH5α(pRK415K, pBetR) and DH5α(pRK415K, pZLOP1) were constructed for this study. Plasmid pRK415K carries the RP4 tetracycline resistance gene unit, and expression of this determinant is inducible by tetracycline (14, 25). If TetRC58 can repress the expression oftetARP4, upon induction oftetRC58, strain DH5α(pRK415K, pBetR) should fail to grow on medium containing tetracycline. As shown in Table3, this strain grows well on LB medium with tetracycline at a concentration of 10 μg/ml. However, when the expression of tetRC58 on pBetR was induced by arabinose, this strain was unable to grow on the same medium. These results indicate that TetRC58 can repress at the tetARP4 promoter and that this repression is not responsive to tetracycline. To determine if TetRRP4 can repress tetAC58, the expression of thetetAC58::lacZ reporter was examined in strain DH5α(pRK415K, pZLOP1). Results from this test indicated that TetRRP4 from pRK415K indeed repressedtetAC58 expression, and like the repressive effect on its cognate tetA, the repression was responsive to tetracycline (Table 3). Thus, TetRRP4 can recognize and repress expression from the promoter of the A. tumefaciens tet system. Furthermore, unlike thetetC58 system, this repression oftetAC58 by TetRRP4 is relieved by tetracycline.
Nature of the tetracycline-resistant mutants.Our data indicate that a tet unit in the genome of strain C58 accounts for the appearance of tetracycline-resistant mutants when this strain is grown in the presence of this antibiotic. In the particular mutant used in this study, the tetracycline resistance phenotype arose from the transposition of IS426 intotetRC58. Since tetracycline does not induce the expression of tetAC58, we were interested in determining whether all tetracycline-resistant mutants were caused by the same type of mutation. To examine this, genomic DNA from eight independent tetracycline-resistant mutants was probed with a DNA fragment containing the tetC58 genes. These eight mutants grouped into four types based on the size of the hybridizing fragment (data not shown). One class, represented by three mutants, contained a fragment indistinguishable in size from that of the wild type. In the second, two of the eight mutants gave a fragment indistinguishable in size from that of NT1TcR1, the original mutant from which the tet locus was cloned. The remaining three mutants contained two restriction fragment length polymorphism (RFLP) patterns different from that of the wild type and of NT1TcR1 (data not shown).
Surveying for the presence of the tet locus in the genomes of Agrobacterium strains.We examined a collection of wild-type isolates of Agrobacterium spp. for the tet locus by Southern analysis using a probe containing part of tetA and most of tetR. We also examined some commonly used derivatives of strain C58. Of those not derived from C58, only strain T37 contains DNA with detectable relatedness to thetet locus from strain C58 (Table4). Moreover, the probe hybridized to anEcoRI fragment of the same size in the two strains (data not shown). Consistent with these results, of the 26 strains tested, only T37 and those derived from C58 gave rise to tetracycline-resistant mutants (Table 4). The probe hybridized with all derivatives of strain C58 tested, including strain UIA5, which lacks both pTiC58 and the 450-kb catabolic plasmid pAtC58 (Table 4). Consistent with this, UIA5 gives rise to mutants resistant to tetracycline.
We report here the characterization of a tet system from A. tumefaciens C58. This active resistance system accounts for the long-known observation that C58 and its derivatives give rise to tetracycline-resistant mutants at a high frequency.
This resistance determinant is structurally similar to that oftet systems found in plasmids and transposons fromE. coli and other gram-negative bacteria, with the highest relatedness being to the class A tetracycline resistance gene system from Tn1721. The tetC58 system confers resistance to tetracycline and derivatives such as chlortetracycline and oxytetracycline but not to minocycline. This pattern of resistance suggests that tetC58 does not belong to the Tet(S) class of tetracycline resistance genes, which confers resistance to minocycline as well as to tetracycline, chlortetracycline, and oxytetracycline (10).
Like other class A systems, tetC58 is organized as two genes, a regulatory gene and a gene coding for an efflux pump, transcribed divergently from an intergenic region. Potential promoter elements for both genes are overlapped by a single putative operator composed of a 15-bp perfect IR element. Our genetic analysis indicates that this intergenic region contains all of the cis-acting information required for the regulated expression of these two genes. The DNA sequence of the putative operator is identical to that of operators from other tet systems. However, the promoter region of tetC58 contains only one repeat while the promoter regions of the tet determinants of RP4 and Tn1721 each contain two copies of the operators (Fig. 4B).
Heterologous repressor-operator recognition occurs between different classes of tet systems, even those with dissimilar operators (26). The high degree of sequence similarity between operators from the tetC58 andtetRP4 systems suggested that TetR proteins from these two systems could recognize the noncognate operator. This proved to be the case: TetR of RP4 repressed the tet system of C58 and TetR of C58 repressed tetracycline resistance conferred by the RP4tet system. The capacity of TetRRP4 to repress expression of thetetAC58::lacZ fusion suggests that the 15-bp IR is the operator and indicates that a single copy of this element is sufficient for regulation of both genes.
The expression of almost all tetracycline resistance gene systems examined to date is under strict control (29, 45). Among these, repression of the Tet(B) class by TetR is the best understood (21, 45). In the absence of tetracycline, the repressors bind to operators overlapping the promoters of both the tetAand tetR genes (21). Binding of tetracycline to TetR(B) induces a conformational change that leads to release from the operators and concomitant expression of tetA (22, 34). Tetracycline-inducible TetR-mediated regulation of othertet systems is believed to function by a similar mechanism (21). Two lines of evidence indicate that tetracycline does not induce the Agrobacterium tet system. First, when cells were spread onto medium containing tetracycline at concentrations ranging from 10 to 1 μg/ml, only a small portion of the cells grew into colonies (data not shown). Moreover, those that did grow now expressed constitutive resistance to tetracycline. If tetracycline functioned as the inducer for tetC58, virtually all of the cells should be resistant to this antibiotic. Furthermore,E. coli and Agrobacterium strains harboring pZLE8.5 (Table 1), which carries the wild-typetetC58 locus, are sensitive to tetracycline, even at concentrations to which tetRP4 confers resistance (data not shown). Second, while thetetAC58::lacZ and thetetRC58::uidA reporter fusions were expressed constitutively in cells lacking TetRC58, the expression of both fusions was depressed considerably in cells harboring tetRC58. Furthermore, neither reporter was inducible by tetracycline. Repression of tetARP4 by TetRC58is not relieved by tetracycline. On the other hand, repression oftetC58 by TetRRP4 does respond to tetracycline. These two sets of results indicate that the failure of the tet system of C58 to respond to tetracycline is a function of TetR and not of the tet operator. Mutants of the related repressor from Tn10 that do not respond to tetracycline have been isolated and characterized. These noninducible mutants repress the expression oftetATn10::lacZfusions by binding to the operators even in the presence of tetracycline (5, 20, 34). We propose that TetRC58 represents such a noninducible form of TetR. Clearly, this repressor can bind DNA. Why it no longer reacts to tetracycline remains to be determined. We considered the possibility that tetracycline is not the true ligand for TetRC58. However, none of the 25 compounds we tested induced the system. Certainly, this was not an exhaustive survey. But on balance, that the locus of strain C58 can be regulated by TetRRP4 in response to tetracycline strongly suggests that this gene unit once was responsive to this antibiotic.
The interchangeability with respect to operator recognition exhibited by the two TetR proteins could explain another observation concerning tetracycline resistance in strain C58. Derivatives of this A. tumefaciens strain harboring RP4 and vectors carrying tetRP4express only low level resistance to tetracycline. We propose that TetRC58 is partially dominant over TetRRP4. Thus, since TetRC58 does not respond to tetracycline, thetetRP4 determinant is not fully expressed in itsA. tumefaciens host.
Expression of tetracycline resistance in C58 apparently occurs only following a mutation affecting regulation. These mutations can be of several types; in the particular mutant used in this study, the tetracycline resistance phenotype resulted from the insertion of IS426 in tetRC58. In genetic analysis carried out with C58 and its derivatives, this element is commonly associated with phenotypes in which the selection gives derepressed mutants (17). However, diverse RFLP patterns in Southern analysis of eight independent mutants indicated that other types of mutations can result in the tetracycline resistance phenotype. While mutants with a pattern indistinguishable from that of NT1TcR1 may have arisen from IS426 transposition, those with a pattern similar to that of the wild type could represent point mutations or very small deletions or insertions in thetetRC58 gene. They also could represent operator-constitutive mutations that no longer allow TetRC58 to bind at the promoter.
Among the agrobacteria we examined, only strain T37 contains DNA with detectable homology to the tet locus from C58. Furthermore, only C58 and T37 give rise to tetracycline-resistant mutants (Table 4). These two strains were isolated from different parts of the United States (42). Moreover, although strain C58 induces unorganized tumors, strain T37 causes teratomas. Both strains contain a nopaline/agrocinopine-type Ti plasmid, but RFLP analysis indicates that these two elements have diverged considerably from each other (42). Our survey also shows that generation of tetracycline-resistant mutants is associated with the presence of atetC58 homologue in the genome of the particular isolate; those lacking this element do not yield mutants at a detectable level. The presence of the tet locus in strain UIA5, a derivative of C58 lacking both pTiC58 and pAtC58, indicates that this resistance determinant is associated with one of the two chromosomes of strain C58 (1). Interestingly, this is not the only antibiotic resistance determinant in strain C58. This strain, which also mutates to resistance to chloramphenicol at a high frequency, contains a gene coding for an atypical chloramphenicol acetyltransferase (47). Whether this cat gene is present in strain T37 or any other isolate of Agrobacteriumspp. is not known.
We thank Abigail Salyers for supplying the tetracyclines and related chemicals and Stanton Gelvin and Ramon Peñalver for helpful discussions. We also thank Malcolm Winkler, whose seminar on tRNA processing got us started on the problem.
Portions of this research were supported by grants R01 GM52465 from the NIH and NC CNTRL SOY SKF ANTC from the North Central Soybean Association to S.K.F.
- Received 14 September 1998.
- Accepted 7 November 1998.
- Copyright © 1999 American Society for Microbiology


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Britain's recovery is losing steam, according to official data that showed growth this year was weaker than previously thought.
The economy expanded by just 0.4pc between July and September, from a previous estimate of 0.5pc, according to revised figures published by the Office for National Statistics (ONS).
Growth in the second quarter was also revised down to 0.5pc compared with the previous quarter. Statisticians had previously thought the economy grew by 0.7pc over the period.
•Bank of England hawk Martin Weale signals rate rises further away
•Osborne's deficit target in doubt as borrowing climbs
Economists said Wednesday's figures reinforced expectations that the Bank of England will not raise interest rates until "well into 2016". Markets currently believe rates will remain at a record low of 0.5pc until early 2017.
The ONS said weaker services growth drove the third-quarter revision as financial services firms expanded at a much slower pace than initially thought. Weaker stock-building of inventories led to the second-quarter change.
Samuel Tombs, chief UK economist at Pantheon Macroeconomics, said a "meagre" 0.1pc month-to-month expansion in Britain's dominant services sector in October suggested that growth is "still slowing".
Better balanced growth? Q3 GDP showing spendthrift consumers & falling exports undermine Chancellor's Aut Stat claim pic.twitter.com/UGNmLg467w— Samuel Tombs (@samueltombs)
December 23, 2015
On an annual basis the UK economy expanded by 2.1pc in the third quarter, down from a previous estimate of 2.3pc.
There were also small revisions to growth in 2014, although the ONS said annual growth for the year remained unchanged, at 2.9pc.
The revised figures mean the UK economy is now 6.1pc larger than its pre-crisis peak, compared with an earlier estimate of 6.4pc.
The ONS also believes GDP per capita surpassed its 2008 pre-crash high a quarter later than previously assumed.
Growth per head was 0.3pc above its pre-downturn peak in the third quarter of 2015, having surpassed it in the second quarter of this year.
A spokesman for the Treasury insisted that the UK's economic performance remained "strong".
"The UK was the fastest growing economy in the G7 last year, we're leading the pack with the US this year, we have a record high employment rate and the deficit is down," the spokesman said.
Separate data on Wednesday showed Britain's current account deficit, which measures trade as well as overseas income and transfers to bodies such as the European Union, was unchanged at £17.5bn in the third quarter compared with the previous three months.
Analysts had expected the deficit to widen to £21.5bn.
A widening in the trade deficit in the third quarter was offset by a sharp narrowing in the deficit on investment income, the figures showed.
The current account deficit now stands at 3.7pc of gross domestic product (GDP).
While this has narrowed substantially compared with the record high of 6.2pc seen in the final quarter of last year, Mr Tombs said the "still large" deficit meant sterling was "vulnerable to fall sharply" if Britain's referendum on membership of the EU led overseas investors to "suddenly become less willing to buy UK assets".


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Not sure what to search for?click here to find out more
The Revolution range is a celebration of the art of artisan glazing. Using reactive glazes that change in colour and intensity depending on their application, the Revolution glazes are specially formulated to enhance all the nuances experienced in their execution. Applied by hand using a revolving motion, the path the brush travels across each piece is evident in the fired result. The glazed surface of each piece in the range is as different and unique as the artisan that created it.
This provides an ideal platform for Chef to showcase their art, no matter what the cuisine style. The contrasting yet neutral colour tones; Sandstone and Granite, blend beautifully together and amplify the quality and skill that goes into Chef's creation.
Select a colour:
COVID-19 Announcement
We are pleased to report Steelite International UK is open for business. As the need to support essential businesses increase, we have taken steps to offer limited service to our customers. Steelite UK is currently despatching every weekday, Monday through Friday, to locations worldwide.
To place orders or arrange a shipment, please contact Karen Sanderson at email@example.com or call us at + 44 (0) 1782 829421 or + 44 (0) 1782 821000 where we will be happy to take your enquiry.
As we navigate the challenges posed by COVID-19, we continue to follow the latest guidelines to ensure a safe and healthy working environment. Our number one priority continues to be the health, safety, and wellbeing of our employees.


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Balanced nutrition is the key to your cats good health
How often have we heard that for the best in life, we need to keep everything in balance. Work and family life balance, exercise and rest and of course eating a balanced diet. When I was in Kenya, I saw the limitations on the availability of clean water. I have not been able to turn on the water at home without thinking about how precious this resource is and how fortunate those of us are that take clean water for granted every day.
We know we need water and we also need to eat a balanced diet to maintain our health and this is easier said than done at times. We can select our food choices but our cats rely on us to choose for them and this can be daunting at times.
We know that our cats need key nutrients just like us to maintain good health and energy. Those nutrients include protein, fats, vitamins, minerals and fiber. But what if any one of those nutrients is too high or too low? We humans have tendency to think that if a little is good, a lot will be even better. you have seen supplement tablets that boast 1000% of your daily requirement. Is that really a good idea?
Protein is a vital nutrient that helps support lean muscle tissue for adult cats. Kittens require protein to grow muscle tissue. If your cat is not getting enough protein, it could lead to inadequate growth and if your cat is getting too much protein, the liver and kidneys need to work too hard to get rid of excess nitrogen. Yes, your cat is an obligate carnivore, but she can only absorb a certain amount of protein, anything in excess must be processed by the liver and kidneys and this overtime affects these organs.
Fats in the appropriate levels are great for healthy skin. Fats nourish the skin and make it soft and contribute to a luxurious coat and shiny fur. And we know what happens with too much fat: weight gain. But if there is not enough fat, your cat might have a dull, dry and flaky coat.
Vitamins and Minerals
Vitamins and minerals are also important in your cat's diet. Vitamins support the immune system, minerals help kittens grow strong muscles and healthy teeth. If there is a vitamin deficiency, your cat could experience fatigue or weakness. If vitamins are too high, this could lead to organ stress. If the minerals are too high, your cat could be at risk for bladder stones.
Fiber is great for healthy digestion but if your cat's diet is too low she could experience loose stools. And too much fiber can lead to gas. Not good for your cat or you.
Are you feeding your cat a healthy, balanced diet?
When choosing a food for your cat, make sure that each of the nutrients is in balance. For your cat's best health, choose a food that provides proven, perfectly balanced nutrition. Find out if your cat's food contains properly balanced nutrition and how to make a change if it doesn't. Pounce here: Ideal Balance for cats
We have been compensated for this post. Hill's Pet Nutrition is not responsible for the content of this article.


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Hello All,
Here is a document for creating an USB Host Project (MSC + fatfs) with KSDK 1.3 and Processor Expert support in KDS. It uses FRDM-K64F board as example and lists some specific considerations that needs to be accomplished when creating an USB Host project by using fsl_usb_framework component (Processor Expert) in Kinetis Software Development Kit (KSDK) version 1.3.
I hope you can find it useful!
Isaac Avila
Original Attachment has been moved to: K64F_USB_Host_MSD_KSDK_1_3_PEx.zip


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This was originally a side dish, but as it's very rich and there are just two of us, I now make it as a complete meal, like I do Mac 'n Cheese.
4-6 rashers smoky bacon (for meat eaters)
OR 1 tsp smoked paprika (for a meat free version)
2 medium potatoes
3-4 leeks with a long white portion
(Oil/butter for the meat free version)
Chop the bacon and cook it in a large pan. Meanwhile, scrub and thinly slice the potatoes. Thoroughly rinse and slice the leeks, white and pale green parts only, being sure there's no sand between layers.
Remove the bacon from the pan (or heat oil and or butter) and cook the potatoes and leeks until soft, about 20 minutes. Try not to break up the leek slices. (If not using bacon, sprinkle smoked paprika on the veg.)
While that cooks, make a cheddar cheese sauce.
3 tbsp butter
3 tbsp flour
2 cups milk or cream (or half of each)
1 egg yolk, beaten (optional)
2 cups sharp cheddar (or smoked Gouda?), shredded
Melt the butter with the flour to make a roux. Cook for about 2 minutes, or until it no longer smells like raw flour. Remove from heat for a minute, then stir in the milk until it's smooth. Put it back on the burner and cook for a few minutes. (If you use the beaten egg yolk to stabilize and thicken, add it carefully now) when the sauce is thickened, stir in the cheese until it's melted.
Heat oven to 350F. In a lightly greased casserole: Layer potato/leek mix, bacon (if you're using it) and sauce with sauce being the top layer. Bake uncovered for 30-45 minutes until bubbly and lightly brown around the edges.


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Built on a placeable lot in Bridgeport, this modern Town House is built over 3 floors. With spacious open plan living, dining and kitchen on the ground floor, 2 bedrooms and full bathroom on the first floor, on the second floor there is a laundry/ shower room and a large open room (unfurnished). The house also has 3 terraces, of which the roof terrace boasts a hot tub..
Lot size..20x15 Cost Furnished..100,286 Unfurnished.. 54,821
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It has been reported that Margot Robbie is in talks to portray Sharon Tate in Quentin Tarantino's upcoming Once Upon a Time in Hollywood. Robbie's name has been attached to the project ever since it was first announced that Tarantino was making the movie, but this is the first time that negotiations have been reported. It was revealed back in July that the actress had met with the director to discuss the movie, which also stars Leonardo DiCaprio and Brad Pitt.
Once Upon a Time in Hollywood takes place in 1969 and features Rick Dalton (Leonardo DiCaprio) as a former star of a Western TV series and his longtime stunt double Cliff Booth (Brad Pitt). The duo are trying to make it in Hollywood again at the height of the hippy era in a landscape that they no longer recognize. Sharon Tate is Dalton's next-door neighbor, who was brutally murdered by the Manson Family. It was originally thought that the movie would center on Charles Manson, but Quentin Tarantino has revealed that the Manson aspect will be one of the many stories being told together.
Leonardo DiCaprio and Brad Pitt officially joined the cast of Once Upon a Time in Hollywood. DiCaprio was rumored for the movie early on, much like Margot Robbie, and this will be his second collaboration with Quentin Tarantino after 2012's Django Unchained. This will also mark the second time that DiCaprio and Robbie have worked together. The two previously starred in Martin Scorsese's The Wolf of Wall Street and this will be Leonardo DiCaprio's first starring role since 2015's The Revenant.
Once Upon a Time in Hollywood is said to have a similar tone to Pulp Fiction and it is set to hit theaters on August 9th, 2019, which is also the 50th anniversary of the death of Sharon Tate. Tate's sister, Debra, is not happy about the movie and she has publicly slammed Quentin Tarantino, Brad Pitt, and Leonardo DiCaprio for taking part in the film. She accuses the actors of only doing the movie for a paycheck and claims that they have no idea how much it hurts the Tate family.
Margot Robbie's Sharon Tate will reportedly be the glue that holds two plots together. Once Upon a Time in Hollywood is centered around the Manson family murders and it is being described as a "unique take" on the 1969 killings of Tate and four others, murders committed by a group led by Charles Manson. While Robbie hasn't officially signed on, it seems that it's only a matter of time before it's announced that she has joined the cast. It isn't clear when production will start, but Quentin Tarantino and his all-star cast will more than likely begin shooting this summer. This report comes to us from Deadline.


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6: He Listened
6: He Listened
From Chicken Soup for the Soul: Married Life!
He Listened
The most precious gift we can offer anyone is our attention.
~Thich Nhat Hanh
Christmas was three weeks away and my husband was fishing for ideas for my present. I smiled because he is always so thoughtful when it comes to buying Christmas gifts and I have never been disappointed by his choices.
"Actually," he announced, "I'd like to buy you some bras."
That got my attention and I listened intently as he explained that, while he was relatively familiar with the broader world of lingerie, he wasn't quite sure what size or kind of bra I would wear. He wanted some direction.
I paused. He waited. We sat together on the couch looking at each other with a funny smirk on our faces until I finally broke the silence.
"I would love for you to buy me a bra, but what I really need are practical bras. I don't need fancy, frilly, romantic, lacy bras for our intimate rendezvous in the bedroom. I already have a wide variety of items for that, many of which you bought for me. What I really need are practical bras to wear during the day and your timing is great… mine are almost shot."
Disappointment spread across his face as I explained what I meant by practical bras. I needed at least three, all in neutral colors: one black, one white, and one ivory. I needed bras that were comfortable enough to wear all day long, and for me that meant bras with support but no underwire. I needed bras with a little padding so that I could avoid embarrassment while wearing a lightweight and formfitting blouse to the office during the summer months when the AC kicks into high gear. I needed bras that were solid color and sparing in lace and design because the additional detail, while pretty and feminine, might show through some of my more delicate, yet professional sweaters.
My husband paid close attention, but I could tell that practical wasn't what he had in mind. Regardless, I was delighted Christmas morning when I opened one of my many presents and found three practical bras: one white, one ivory, and one black! He had done it. My husband had listened carefully to my wishes and delivered on every aspect. I was so pleased I giggled with joy, and then he handed me my next present. Gently, I unwrapped three beautiful and sexy dresses, each one designed with lots of color, lace and fabulous detail.
"One to wear over each new bra," he said, and we both laughed out loud.
Those three bras turned out to be my favorite Christmas present and a fond memory. They were not overly expensive. They were not elegant or lavish. They were not even anything I'd want to brag about to our friends and family members. But, they were everything I asked for. They were also symbolic of my husband's effort to keep his wedding vows. On our wedding day, before God and many witnesses, we promised to be loving and faithful: in plenty and in want, in joy and in sorrow, in sickness and in health, as long we both shall live. Little did he know that "plenty and want" might someday include practical bras.
~Kristen Clark
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Diagnosis Day is always hard for me. My daughter, now 5, was diagnosed with retinoblastoma on August 17, 2005 at 8:45am. Tonight, August 16th, we are on the Eve of The Anniversary Of Diagnosis Day.
But, what I've found the past two years, especially now that we have officially spent more "years" as a cancer family than not, is that the Eve of Diagnosis Day — otherwise known as "the day before our lives changed" — is a whole lot harder for me. It's a day that reminds me of a life that might have been and not of what is now.
Now, certainly, we know our blessings. Like many children in the United States who are diagnosed, my daughter is healthy and alive. And, aside from a prosthetic eye, she has lived the past few years as a child who many never assume to have had cancer. Her hair has fully grown back, she is taller than any of her friends, she is smart, funny, sensitive, and incredibly articulate. Back when she was diagnosed, we weren't sure what her future would hold. And, if you're reading this as a new parent, I'm quite sure you're thinking the same thing. If you're reading this as a seasoned Rb parent, I'm sure you, too, count the blessings you have as well.
Many of my married friends joke about not being able to remember what life was like before marriage and kids. Much like I can't remember what life was like before I had children, it's getting harder and harder for me to remember what life was like before retinoblastoma. I can't remember back when I didn't really care what sharp object my child had in her hand. I can't remember back to when "mandatory safety glasses" weren't a part of our lives. I can't remember my daughter's chest without a scar or a port. And, I can't remember what my daughter's face looked like without her prosthetic.
But, I also am thankful that, because of our cancer journey, I now have a daughter who embraces life. I have a daughter who is more kind than her peers, especially when there is a new person in the room. Because of our experiences, my daughter isn't afraid to talk to someone who looks different or who uses a different ability via a wheelchair, walking stick, white stick, or who speaks in a manner that is unfamiliar to us. My daughter is learning, at an early age, that different is special… that different is good.
She is that kid in school who I always wished I had the confidence to be — she is the one who is friends with everyone because she's the most kind to everyone. She takes care of others, but she has also learned to advocate for herself. I guess that's from times when I had to leave her alone in the hospital when she was just 2 years old. She had to learn how to speak clearly, quickly, and to express what she felt and what she needed from strangers, at age 2. When we are out and I see 2-year olds, I am am reminded just how much "growing up" my daughter needed to do.
Diagnosis day — in the past, that was the hardest anniversary for me because it signified such a change in our lives. But, now that we have officially crossed that threshold where my daughter has spent more of her life as a cancer survivor than as a non-cancer survivor, I'm feeling a different emotion about pre-Diagnosis Day. It's a day that's ambiguous, unclear, to me .. I'm not able to remember life prior to Retinoblastoma….
and, honestly, I don't think I want to.
If you've found this site because of a recent Rb diagnosis,may you find peace on your diagnosis day. If you're a seasoned Rb family, may you find peace on your anniversary or during this difficult time.


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A panel of experts set up by Japan's health ministry and tourism agency have agreed to ease regulations regarding paid accommodation in private homes as the number of overseas visitors to Japan continues to surge.
Easing regulations on such accommodation is seen as a remedy for the shortage of hotels in Japan in the lead-up to the 2020 Tokyo Olympics and Paralympics.
The expert panel, established by the Health, Labor and Welfare Ministry and the Japan Tourism Agency, on Tuesday decided to designate private houses and apartments as a category of simple accommodation under the Hotel Business Act.
Currently, the law stipulates that facilities designated as simple accommodation must have 33 sq. meters or more of floor space for guest rooms.
But the panel plans to relax that condition for accommodation in houses, or minpaku, as the practice is known.
Although under the law, individuals are supposed to obtain permits from local authorities if they want to offer paid accommodation, many people do so without proper authorization.
Among problems cited are issues with noise and garbage.
During Tuesday's meeting, the experts also broadly agreed to require operators to confirm the identity of their paying guests and to respond to emergency situations.
The panel is expected to compile an interim report by the end of March.


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Olson Kundig Architects have managed to provide this astounding newly-constructed mountain residence a very main-industrial truly actually really feel.
Located on the wooded shore of Lake Idaho, the trendy residence appears, at first look, the slicing-edge reconversion of an earlier industrial creating, perhaps a producing unit or mill.
The residence is open and ethereal, with the roof slab seemingly floating few ft above the concrete brick partitions. A mechanical muse has set the tone for the this terribly distinctive house, with a wall displaying like an infinite window , lifted on an unlimited pulley wheel to open the home within the course of the lake. There are industrial parts all all by way of the home, with the accents of sturdiness and patina, inside the enormous furnace-like hearth, with seen seam and weld edges, the massive M story metallic door, the metallic ceiling beams, the framed clerestory. They are warmed up by the plywood finishes, vibrant trendy upholstered furnishings and a big ranging blue view of the lake and the far-off mountains.
Contemporary residence with pretty panoramas of Lake Austin and Mount Bonnell


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Entry by takeover: Auctions vs. bilateral negotiations
- Publication Type:
- Journal Article
- Citation:
- International Journal of Industrial Organization, 2016, 44 pp. 68 - 84
- Issue Date:
- 2016-01-01
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© 2015 Elsevier B.V. Firms often enter new markets by taking over an incumbent. We analyze a potential entrant's choice of target under two (exogenously given) takeover mechanisms: (i) auctions, where other incumbents can bid for the target against the entrant, and (ii) bilateral negotiations between the entrant and the target. The entrant's choice of target depends on the mechanism, and it may not maximize its ex-post profit (nor consumer welfare). In an auction, the entrant pays a higher price to take over a target with higher synergies, because these impose stronger negative externalities on incumbents and increase their willingness to pay for preventing entry. Auctions increase the price obtained by the target, but reduce welfare compared to negotiations because they may discourage the entrant from acquiring a target with higher synergies.
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Composting coffee cups, lining our bins with last week's news-papers, and returning packaging to stallholders… We've sure come a long way on our quest to become New Zealand's first zero-waste Market!
It's an effort that depends on the support of our stallholders and market-goers alike; as well as the guidance of Waste-Away: a committed team of forward thinking Grey Lynners who are committed to stopping waste.
Last week we announced that Grey Lynn Farmers' Market is becoming Plastic Bag Free!
This is a massive leap forward in terms of becoming a zero-waste market and overall putting an end to this plague of plastic! So see you next Sunday with your reusable bags, boxes and baskets!