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Suddenly have your Author page on Amazon all in italics? Wondering why your beautiful book looks odd on the Look Inside the Book? Check in here---see if we've blogged about it. We try to keep you current on the latest current events in the eBook world.
Will You Be a Copyright Claim-Jumping Victim At Amazon?
Feel a Disturbance in the Force? Let's Discuss Your Copyrights.
There's been a Disturbance in the Force...
Amazon's been asking authors of published works to prove that they are the copyright holders. In fact, it's happening in increasingly large numbers, to folks who've had books published for years.
One poster from the Kindle Digital Platform wrote:
"A friend of mine had one of his boxed book sets pirated. 3 of his books are available on a book pirating website. Amazon has discovered this, shut down his publishing account and are requiring him to prove he owns the rights to his own books. How can we do this? He didn't copyright them, but did include a copyright page at the beginning of all 3 books."
That poor bastard was the victim of the Double Whammy. First, he's pirated, and then, to rub salt into the wound, the mere act of him being pirated causes Amazon to question the legitimacy of his publishing the Boxed Set! Talk about ignominy. Another wrote:
"We are trying to publish a couple of books on KPD which my husband wrote in 2012. They have told us we need to prove we have publishing rights, but don't help with insight as to how to accomplish this. Say they can only respond to publisher!"
That one goes on to say that she can't get anywhere with Amazon because they don't accept that her husband is the publisher. What the hell do you do, if you're in that situation?
Now, the only way that this would be occurring is if someone else--we'll call that thief Darth Dickhead--files a claim, with Amazon, that your work is actually their work. So...how do you prove that you're you? How do you prove that you wrote your own book?
This is simply a newer, digital form of a type of thievery that's been around forever, known as claim-jumping. If you're unfamiliar with the term, claim-jumping is when someone else claims to own or have rights to property that you have already claimed as your own.
The bigger and easier-to-answer question is: how can you easily prevent this from happening?
The simple answer is, you can't. Any idiot--any Darth Dickhead--can click a button on Amazon's website and claim that your Intellectual Property--your book--is theirs. In this day and age, the sound of a Light Saber swooshing has been replaced with the sound of a click. The click that will deprive you of your rights.
Click below, on "Read More," to answer the question, "How Can You Protect Your Books?"
When Indy Cover Design Goes Right
If you've been hanging out with me for a while, or you're one of our clients, you know that one of my pet peeves is the inattention, by many self-publishing authors, to the realities of the importance of COVER DESIGN. I started nagging you all about this not less than 5 years ago (see my early-on article, Cover Design Calypso which I originally published in 2010), and my various and sundry kibbitzes since then, if you don't remember.
Jackie Collins' "Chances" out now on Kindle!
Jackie Collins' "Chances" available now!
Get it Now on Kindle!
...And we're thrilled to have her IN THE HOUSE! "Chances," her runaway smash hit, is coming out in less than 3 days available now on Amazon, Barnes & Noble, Apple...all the usual places. We're thrilled, of course, that her team chose Booknook.biz to be the eBook producers for "Chances," and we hope to continue this relationship with her as she keeps breaking ground as a self-publishing big-time bestselling author. You can find "Chances" at: http://amzn.to/LdU1Qz on Amazon.
If you're not familiar with Chances, the book marks the first appearance of Jackie's incredibly popular character, Lucky Santangelo.
The book that made Jackie Collins one of America's favorite authors sweeps you from the sophisticated playgrounds of Europe to the glittering gambling palaces of Las Vegas. It plunges you into the reckless, dangerous world of the Santangelo crime family. It introduces you to Gino Santangelo, the street kid who makes it all the way to the top. And then brings you Lucky—his sensual, stunningly beautiful, and passionate daughter; a woman who dares to win her father's empire for herself; a woman unafraid of taking…CHANCES.
Don't miss the eBook launch of Chances--coming June 7th.
Why Is My Book on Amazon All in Italics?
On a recent Saturday afternoon, I received a panicked email from a long-time editing acquaintance of mine. Why is my book on Amazon all in italics?
On a recent Saturday afternoon, I received a panicked email from a long-time editing acquaintance of mine. Her award-winning book needed some updates, so, being über-competent in making her own ebooks, she made the changes and uploaded the book. She checked back a few hours later, and it was one of those, "good news, bad news" moments. The good news? The LITB (Look Inside The Book) had updated, almost immediately. The bad news?
Holy Typography, Batman! Her entire Look Inside The Book was in italics.
Colonel Oliver North, In the House!
In this candid book, Oliver North, the man who was at the center of the Iran-Contra controversy tells the story of his life. He reveals the inside story behind the headlines and stresses the importance of his family and his enduring faith, which saw him through the toughest times.
We have Oliver North IN THE HOUSE!
We're thrilled to announce that we were selected to produce the book versions of Colonel Oliver North's blockbuster bestseller, "Under Fire." The book has completed production, and is available on Amazon as well as other ebook retailers.
Written in secret as if it were a covert operation, then suddenly released, this is unquestionably the "event" book of the year. Here, finally, we might expect to get answers to our questions about what became known as "Iran-contra." Was Marine Lieutenant Colonel North a "loose cannon" on the National Security Council, making U.S. foreign policy on his own? And just what did President Reagan really know about Iran-contra? North writes that Reagan "knew everything" about Iran-contra's covert operations. So too did Vice President Bush. Unfortunately, there is no specific document in the book proving that. What there is, however, is a preponderance of material that leads to an inevitable conclusion: North was a hard-working, well-trained, loyal Marine incapable of undertaking a covert operation without approval by his superiors. For the first time, we hear of and read a document sent to Reagan by North's lawyers, setting forth a sound legal argument for a presidential pardon for North. The letter, we learn, was never acknowledged. There are a number of such chilling moments in this important book.
--Chet Hagan, Berks City P.L. System, Pa, Copyright 1991 Reed Business Information, Inc.
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Due to the COVID-19 onslaught, we currently cannot take incoming phone calls.
A notification about phone calls: due to the COVID-19 plague, we've significantly increased our incoming inquiries--nearly double our usual volume--and the number of incoming inquiries by phone has been literally overwhelming. Although most writers will say that their call "will only take 15 minutes," the truth is, that after 10 years of doing this, most author inquiry calls take an hour. 45 minutes at best. I'm currently receiving 7-10 calls/day, and due to that, I've had to stop accepting incoming phone calls, which my voicemail will tell you. You can leave a message--I can't call you back without one--and if a call is needed, I will of course call you. But we have very complete and extensive email replies, handouts and our website is very informative. Almost all the questions that I receive during a call are actually already answered on our site, or are, in fact, publishing questions, not questions about our services, what we do, what we offer, or the like. I already handle between 90-130 emails/day, as it is. I can't handle that many emails and take 7-10 hours of calls each day. I can't. So, in order to be able to answer this huge email volume, to help the greatest number of people, with my time, I've had to stop accepting calls. I'm sorry, but that's just how it is. I'm in the process of setting up a call-appointment function, for free 15-minute calls to answer questions from new prospective customers and longer paid sessions for folks who generally want consulting on "publishing," generally (and for prospective kids' book publishers, as a special category of paid consulting). But that functionality isn't yet set up. I hope to get that working the 3rd week of July, sometime.


text:
Finding the Light on the Path
How the Birth of Ethics Leads to Discernment
In the smaller photo, the front cover of the
Portuguese-Brazilian edition of "Light on the Path"
Written down by Ms. Mabel Collins, with commentaries made in some cases by an Initiate, the book "Light on the Path" is one of the greatest among theosophical classics.
The rules and axioms of this small volume have been studied since remote ages by those who try to live according to the Esoteric Wisdom of the East. On the other hand, there are a number of commentaries in the volume which were written by Ms. Collins and transmit false ideas. These passages present decisive tests to the discernment of readers. They result from causes which can be now easily investigated.
Due to the probations and tests faced by theosophists during the 19th century, Ms. Mabel Collins lost her sense of ethics and common sense soon after the publication of "Light on the Path". The mistakes she then made for lack of respect for truth were already present as seeds, and on an abstract plane, in her approach to the law of karma and her view of the theosophical philosophy, as one can see in her commentaries to the rules in "Light on the Path" and in the final essay of the volume, "Karma".
The fact that all of the book's material was published while Helena Blavatsky lived is no guarantee of an absence of mistakes. There is no "infallibility" in esotericism. All things human must be unceasingly improved.
As an editor, H. P. Blavatsky was extremely tolerant and made mistakes for being less severe than she ought to. A short time before dying, H.P.B. received letters of warning from her Master and made a harsh self-criticism in correspondence sent to her friends.[1] H.P.B. realized in 1889 and 1890 that she had underestimated the deficit of ethical feelings and love to self-discipline among the majority of her students. She had been too optimistic regarding the awakening of wisdom in human soul.
Since then, H.P.B. started strengthening the ethical aspect of her teachings and added special intensity to the issue of self-discipline. On 26 April 1890, she wrote in her third message to the North-American theosophists:
"What I said last year remains true today, that is, that the Ethics of Theosophy are more important than any divulgement of psychic laws and facts. The latter relate wholly to the material and evanescent part of the septenary man, but the Ethics sink into and take hold of the real man - the reincarnating Ego." [2]
Mabel Collins' Mistakes in "Light on the Path"
"Light on the Path" was first published in 1885, some five years before H.P. Blavatsky's pedagogical self-criticism.
An examination of the book shows that Mabel Collins did not see Ethics as a decisive factor in theosophy. Some of her gravest mistakes are in the final essay, entitled "Karma".
The illusion according to which immortal sages are "above" or "outside" the Law of Karma is a trap and opens the door to spiritual defeat.
The Teachers and Initiates are servers who know, live and exemplify the Law. There is nothing outside the Law in the Universe, and there never was. Each deviation will have to be compensated. Each mistake will be corrected. The One Law regulates everything taking place during the periods of manifestation of the Universe, the Manvantaras; the same Law regulates every aspect of the periods of rest in the Cosmos, its Pralayas. Therefore no defeat is permanent.
At each failure, new attempts must be made until victory takes place, little by little. From a theosophical point of view, the learning of one's soul includes many incarnations. Unlike superstitious religions, there is nothing similar to an "eternal condemnation" in theosophy.
Fear from defeat is an obstacle in itself, when it paralyses the truth seeker. Confidence is a virtue, when caution and prudence are not abandoned. The two most important factors in a pilgrimage are the adoption of a noble goal and the courage to unceasingly try one's best. Failures indicate mistakes to be corrected in the future. Mabel Collins' contribution to the theosophical Cause will last, while her personal defeat will be overcome and give way to wisdom.
The failure of a number of students and disciples during the first years of the theosophical movement - in the 19th century - must be looked at from at least two main viewpoints. One of them reveals the mistakes made by the aspirants to discipleship. The illusions which defeated them must be avoided by 21st century students. The other point of view invites us to evaluate the method used in teaching. As we mentioned above, the limitations of HPB's pedagogical practice as a teacher led her to make a severe self-criticism. This took place one year after Mabel Collins' crisis regarding the theosophical movement. On the occasion, H. P. Blavatsky had to deal with the defeat of several others, among her co-workers and students.
The teacher who expects too much from students leads them to failure.
The teachings must be transmitted in a rhythm and in a way which reduce as much as possible the danger of a failure on the part of the student. During pioneer efforts, an extraordinary amount of rigour may be historically unavoidable. As time passes, the pedagogical effort can better calculate the effects of its action regarding the pupils. Expectations get to be more realistic, and the teaching is transmitted in a slower way. In teaching, preference is given to that which students can absorb on the basis of a stable practice of Ethics, inseparable from a direct understanding of the abstract universal laws and their dynamics in daily life.
During the 21st century, the esoteric movement must learn from HPB's self-criticism and adapt its view of discipleship. That which is simple can be profound. Topics that are unnecessarily complicated often become superficial. It is correct to build an approach to theosophy that is simple in its operational aspects, and whose complexity remains invisible in normal conditions.
Discipleship has always occurred in widely implicit ways, and trying to "organize" it in too detailed ways through material and institutional structures would provoke problems that are hard to solve in the present phase of human evolution.
"Light on the Path" and "The Voice of the Silence"
In the first paragraph of the Preface to "The Voice of the Silence", H.P. Blavatsky writes:
"The following pages are derived from The Book of the Golden Precepts, one of the works put into the hands of mystic students in the East. The knowledge of them is obligatory in that School, the teachings of which are accepted by many Theosophists. Therefore, as I know many of these Precepts by heart, the work of translating has been relatively an easy task for me." [3]
The main source of Mabel Collins' book "Light on the Path" is the same "Book of the Golden Precepts", which has never been known to the wider public. The similarity between the teachings of "Light on the Path" and "The Voice of the Silence" is great. In the first lines of "Light on the Path" we see these axioms:
"Before the eyes can see, they must be incapable of tears. Before the ear can hear, it must have lost its sensitiveness. Before the voice can speak in the presence of the Masters it must have lost the power to wound. Before the soul can stand in the presence of the Masters its feet must be washed in the blood of the heart." [4]
And we find the following words in the first paragraphs of "The Voice of the Silence":
"Before the Soul can see, the Harmony within must be attained, and fleshly eyes be rendered blind to all illusion. Before the Soul can hear, the image (man) has to become as deaf to roarings as to whispers, to cries of bellowing elephants as to the silvery buzzing of the golden fire-fly. Before the Soul can comprehend and may remember, she must unto the Silent Speaker be united …." [5]
The similitude does not occur by chance. In an article included in the "Collected Writings", H.P. Blavatsky herself says that part of the aphorisms in "Light on the Path" come from the same source of "The Voice of the Silence". She writes that axioms like "Before the voice can speak in the presence of the Masters it must have lost it power to wound" and other sentences are "as old as the Book the Golden Precepts, from which they radiated (… ) into Light on the Path". [6]
Although these two works have much in common, there are various significant differences between them.
"Light on the Path" results from the cooperation of a Greek high Initiate with a student from a Western country. Its language is easier to understand for readers of the West. "The Voice of the Silence" was written by an Initiate Disciple, and much more advanced than Mabel Collins. "The Voice of the Silence" is a totally Eastern work and more challenging to the reader. In it, Helena Blavatsky pays less attention to the specific psychological complexities which seem to be so important to Western souls.
The Lesson Present in the Traps
H. P. Blavatsky mentioned the mistakes made by M. Collins in a letter published in 1889. She wrote that "the lustre of that priceless little jewel, Light on the Path, is henceforth dimmed by a great black stain that nothing can wash out." [7]
In the same year, in a personal letter to a theosophist named John Ransom Bridge, HPB considered "insidious" and "dangerous" the rule number 20 of the book.[8] She added that only "the main body" of the volume was dictated by a true Adept, and the rest was "added from the inner consciousness of Miss Mabel Collins".
On other occasions, HPB informed that the Adept was not a Master [9], nor a Mahatma.[10] There is public evidence that the name he used in the 19th century was Illarion [11], also spelled as Hilarion, and that he was Greek.[12]
Rule I-20 in "The Light on the Path"
There are two "rules number 20" in "Light on the Path": one belongs to the first series of rules, the other to the second. HPB does not clarify to which part of the book she refers. Rule 20 in the second series has one sentence only. Its idea does not seem to be ethically questionable and it is not commented by M. Collins. We must infer that HPB questioned rule 20 of the first series, which is long, complex, and was commented by M. Collins.
HPB added that rule 20 has an "Occult venom", and a "close relationship to Tantrika Black Magic". However, it is not easy to find "venom" on rule I-20. By mentioning this harmless part of the book, HPB may have wanted to say to her correspondent that the book had serious occult problems, without specifically revealing them or dwelling on them.
This is likely because on occasions when HPB could not speak openly, she would often write enigmatic sentences as the ones used in this case. She thus caused a certain perplexity which could lead people to investigate in the right direction and discover facts on their own merit.
A critical examination of rule I-20 shows that its text does not make the fact clear enough that a total renunciation is necessary in one's lower self. However, this is a minor error if one examines the book as a whole.
A sentence can be seen as dangerous in rule I-20 of "Light on the Path". Referring to the Path, it says: "Seek it by testing all experience".
However, the real meaning of the axiom is: "Seek [the path] by seeing as a test each experience you have in life". And this idea is correct.
One should also examine the following question:
"What kind of 'experience' must the pilgrim accept and undergo in his life?"
This will depend on his main intention in life and the intentions of his emotional being. Every pilgrim who dedicates himself to a noble cause soon realizes that inner purification is unavoidable. [13]
Rule I-20 contains warnings about the need to lead a pure life. Various ideas in the rule and in the commentary to it are useful and profound. While looking at these lines, however, HPB may have seen the confused mental state of Mabel Collins, who wrote them down. This fragment of the book does not have a necessarily venomous effect to all readers. Each one must observe it with independence and take into consideration the different factors involved.
While a number of ethical mistakes can be seen in the book and must be identified in public, HPB recognized in more than one occasion the great value of the work. "Light on the Path" is a classic text and belongs in the library of every student of esoteric philosophy, including newcomers and experienced learners. The failings of the book exemplify the need to avoid blind belief: the truth-seeker must think by himself.
Although Mabel Collins cooperated with an Adept, she did not have solid foundations in her life. Her defeat as a disciple is a sign of alert about the need to constantly re-examine the firmness (or otherwise) of the ground on which we walk. Only altruism is stable. An effective progress along the way to wisdom must be obtained by our own merit, while we work with a feeling of unconditional love for truth and in close cooperation with other good-willing individuals.
Life obeys to the Law of symmetry and justice. It is by helping those who may know less, that a pilgrim starts receiving the help - perhaps invisible - of Those who know more than he does.
Intelligence is Impartial
In the Note to item 10 of the second series of rules, "Light on the Path" presents an idea which deserves special attention:
"Intelligence is impartial: no man is your enemy: no man is your friend. All alike are your teachers."
Such an axiom must be correctly understood, as one perseveres in the spiritual effort, so as to avoid naïve ideas about the mutual help process among companions, or about one's family relations. [14]
The community of students must establish a harmony as strong as possible, and a complete cooperation as long as karma allows it to. The same challenge operates in the universe of family relations. Life flows according to the law of symmetry. At each step along the road to sacredness, the student has to take one step away from the world of personal interests. His decision to look for the best will be tested in several painful and unexpected ways. This results from the action of the impersonal forces which fight the divine world.
Although the search for lay discipleship is only a preliminary preparation for a more advanced learning, it contains in itself, in small scale, the main elements of the future. Hence the decisive importance of thinking about discipleship, as one studies theosophy.
The Law of Symmetry is the law of karma and regulates discipleship. [15] The Mahatma Letters show that anti-spiritual energies are consciously used by Eastern Esoteric sages in order to avoid the premature or unstable progress of their students. On Letter 30, one Master says that some "dugpas" (anti-evolutionary, anti-ethical sorcerers) receive "carte blanche" from the magicians of goodness and altruism to test in cruel ways the "chelas" or disciples of divine wisdom.
This takes place, says the teacher, for some time and "with the sole object of drawing out the whole inner nature of the chela, most of the nooks and corners of which, would remain dark and concealed for ever, were not an opportunity afforded to test each of these corners in turn." And the Mahatma adds: "Whether the chela wins or loses the prize - depends solely of himself." [16]
There are no specific forewarnings for tests and traps emerging along the path. They appear in unexpected ways, often in the student's most intimate relationships, and when inner affinity is the greatest. They come through family and in the interaction with companions along the spiritual road. The history of the theosophical movement gives us countless examples of the fact.
Each student must remember, therefore, that supreme intelligence is impartial: nobody is his friend, in the conventional sense of the word; and nobody is his enemy. Everyone is his teacher.
[1] See the text "Helena Blavatsky's Self-Criticism" by Carlos Cardoso Aveline. The article is published in our associated websites.
[2] "Five Messages", H. P. Blavatsky, Theosophy Co., Los Angeles, 1922, 32 pp., see p. 26. The booklet is available at our associated websites.
[3] "The Voice of the Silence", translated and annotated by H.P.B., Theosophy Company, Los Angeles, 1987, 110 pp., p. i.
[4] "Light on the Path", M.C., Theosophy Co., Mumbai, India.
[5] "The Voice of the Silence", Theosophy Company, pp. 2-3.
[6] "Collected Writings", H. P. Blavatsky, TPH, USA, volume XI, pp. 318-319.
[7] See the letter to the journal "Light", dated June 1889 and reproduced on pp. 284-286 of volume XI, "Collected Writings of H.P.B." The sentence quoted is on p. 286.
[8] The letter is published on "The Theosophical Forum" magazine (Pasadena-Point Loma Society), October 1944, pp. 469-474. See especially the upper part of p. 472. More on the spiritual and ethical failure of Mabel Collins can be seen on Chapter 11, Part 6, of the book "HPB", by Sylvia Cranston. The novel "The Blossom and the Fruit", by Mabel Collins, is the story of a spiritual failure. Her book "The Idyll of the White Lotus" narrates another defeat and describes treasons. The language of failure is not needed in the 21st century. Severity must be compensated by a sense of confidence. A certainty of the long term victory protects the pilgrim.
[9] See the document "To All Theosophists, The Esoteric Section of the Theosophical Society and its Enemies", in "Collected Writings of H.P. Blavatsky", TPH, vol. XI, especially pp. 314-315.
[10] "Collected Writings", volume XI. See the upper part of p. 285.
[11] "Collected Writings", volume XI, p. 320. This Adept also wrote mystical stories in cooperation with HPB, as hinted in "Letters from the Masters of the Wisdom", first series, letter 20. The word Adept, used in theosophy to designate Initiates and High Initiates, means literally "one who knows".
[12] "Collected Writings", volume XI, p. 285.
[13] See the article "Undergoing All Experience", by John Garrigues, which is published at our associated websites. The "ultimate human experience" is to observe and understand life as a whole.
[14] See for instance the text "One for All, And All for One", by Carlos Cardoso Aveline, which is published at our associated websites.
[15] Read "The Law of Symmetry", by C.C.A., which is published at our associated websites.
[16] "The Mahatma Letters", TUP edition, Pasadena, California, Letter 30, first lines of p. 232.
The above text is a translation from the prologue by Carlos C. Aveline to the book "Luz no Caminho" ('Light on the Path'), by M.C., The Aquarian Theosophist, Portugal, 85 pp., 2014. It is here reproduced from the January 2015 edition of "The Aquarian Theosophist".
On the role of the esoteric movement in the ethical awakening of mankind during the 21st century, see the book "The Fire and Light of Theosophical Literature", by Carlos Cardoso Aveline.
Published in 2013 by The Aquarian Theosophist, the volume has 255 pages and can be obtained through Amazon Books.
Etiquetas: Carlos Cardoso Aveline

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Building a customer experience is one thing, having a customer experience is almost the opposite. It's our job to understand what it's like trying to do business with our organizations. How hard is it to find the information that's needed, when it's needed? How difficult is it to communicate with the organization? This is where search results are informative, testing is useful and understanding a customer's multichannel engagement is critical.
Now that Google is locking down so much web search data, it's more important than ever to analyze what users are searching on your own site. You're sitting on hugely valuable and usually under-exploited data: what users are searching for, how often each query was searched and how many results each query retrieved. Lou shows eight concrete ways search analytics helps you understand your customers' needs and improve performance.
Wondering how to get started in optimizing your website? Jack shares his top three tips for site optimization. He presents his experience in how to think about optimization, how to implement a testing strategy, conducting tests, interpreting results and selecting the right solution for your needs. Wherever you are on the optimization spectrum, you'll come away from this session with a deep appreciation of what it takes to succeed.
Nancy and Sarah share their insights about benchmarking and the subtle interplay between display, search, social, print, broadcast and whatever other channels have been invented between the time this was written and the time they appear on stage. Sarah describes her challenge of trying to measure incremental ROI from multiple channels while dealing with limited offline data, too much online data, over and understated click-through, view through and re-targeting data. Nancy describes Vail Resorts' experience with Attribution Modeling and shares highlights and learnings from their first season undertaking this new way of looking at their online data.


text:
Consolidated Acts: C.C.S.M. | Municipal | Private
The DuVal Foundation Incorporation Act
This is an unofficial version.
If you need an official copy, use the bilingual (PDF) version.
This version is current as of July 12, 2019.
It has been in effect since November 14, 1990, when this Act came into force.
Search this Act
RSM 1990, c. 47
Table of Contents Bilingual (PDF)
WHEREAS it was desirable to create a corporate body to receive donations in trust for Knox Church, Winnipeg, a congregation within the United Church of Canada, and to provide for the management and control of the property so donated to the end that the net annual income shall be devoted perpetually to the maintenance of the said Knox Church as a centre of Christian teaching and welfare work in the city of Winnipeg;
AND WHEREAS certain persons, by their petition, prayed that The DuVal Foundation should be incorporated;
AND WHEREAS their prayer was granted, and resulted in the enactment of An Act to Incorporate "The DuVal Foundation"; assented to March 22, 1933;
AND WHEREAS the Minister of Justice has caused the Act to be prepared in English and French for re-enactment in accordance with a judgment dated June 13, 1985 and an order dated November 4, 1985 of the Supreme Court of Canada;
THEREFORE HER MAJESTY, by and with the advice and consent of the Legislative Assembly of Manitoba, enacts as follows:
1 The DuVal Foundation (hereinafter referred to as "the Foundation") is continued as a corporation.
2 The Foundation shall be composed of and managed by a Board of eleven members, which shall consist of the following ex officiis: the Minister of Knox Church, the chairman of the Board of Managers of Knox Church, the chairman of the Board of Trustees of Knox Church, the secretary of the Board of Managers of Knox Church, the treasurer of the Board of Managers of Knox Church, the clerk of Session of Knox Church, the president of the Women's Association of Knox Church, and the president of the Women's Missionary Society of Knox Church, and three members to be elected by the congregation of Knox Church at its annual meeting, one of whom shall retire each year, but shall be eligible for re-election.
3 The Foundation may
(a) receive donations of, and hold, control and administer, property of every kind and description, whether real or personal and wheresoever situate;
(b) if not in contravention of an express term of the donation under which it be received, convert any property at any time or from time to time received or held by the Foundation, into any other form, and for such purpose sell, dispose of, assign, transfer, lease or exchange the same;
(c) entrust to one or more trust companies the custody and management of all or any portion of the property at any time or from time to time received or held by the Foundation in such manner and in such portions as the Board of the Foundation may deem proper, and enter into agreement with such trust companies with regard thereto; and
(d) lease any properties at any time held by the Foundation.
Appointment of trustee
4 The Foundation, as soon as practicable after a donation has been received and become operative, shall appoint one or more trust companies to have the custody and management of the property included in the donation or such portion or portions thereof as the Board may allot to each of such companies, to act as trustee or trustees thereof for the Foundation. In making the original appointments of such trustees, any directions in writing given by the donor and expressed in the instrument creating the trust shall be given effect to. The Foundation may at any time on a resolution passed by the majority of the Board revoke the appointment of any trust company or trust companies as such trustee or trustees, and appoint another trust company or other trust companies as new trustee or trustees.
Assignment of property
5 All transfers, assignments or conveyances of property by the Foundation shall be executed by and on behalf of the Foundation in such manner as the Board may from time to time by by-law prescribe, and shall be approved by the trustee for the time being of the property to be so transferred, assigned or conveyed.
Duty of trust company
6 Each trust company during its continuance in office as trustee for the Foundation shall
(a) have the custody of and manage and deal with in an efficient manner all property entrusted to it by the Foundation, and make all investments, reinvestments, conversions, sales or dispositions thereof which may at any time or from time to time appear necessary or desirable, but no trust company shall make any new investments or reinvestments in any property or security other than securities in which a trustee may invest moneys under the laws of this province;
(b) observe, carry out, perform and give effect to all terms, provisions and conditions which may in any instance be attached to the donation of any property by the donor thereof and expressed in the instrument creating the trust;
(c) give effect to and observe all directions with regard to any property entrusted to it by the Foundation under the provisions of this Act, which may at any time or from time to time be given in writing by the Board, if such directions do not in any instance contravene or be inconsistent with any of the terms, provisions or conditions referred to in clause (b) or any provision of this Act;
(d) pay all accounts and expenses of the Foundation and the trustees which the Board shall direct in writing;
(e) distribute from the money in its possession such sums and in such manner as the Board shall at any time or from time to time by resolution direct.
Manner of distribution
7(1) The Board is empowered to determine by resolution, passed by a majority of the Board, the manner in which the money available in each fiscal year shall be distributed. The Board shall, however, in so determining, respect and by governed by any particular wishes that may have been expressed by the donor in the instrument creating the trust; but if, in the course of time after the death of the donor, conditions should arise whereby in the opinion of the Board a departure from such expressed wishes would further the usefulness of the donation, according to the true intent of this Act, the Board shall have power in its absolute discretion to make such departure to the extent necessary to further such true intent and purpose.
7(2) In the absence of any direction by the donor, it shall be deemed that all donations are to be invested, and the net income devoted in perpetuity to the maintenance and the work of Knox Church as provided in this Act or in any amendments thereto.
8 The Board may appoint a secretary at a salary, and may incur such other expenditure incidental to its duties as may reasonably be required, and shall approve of the accounts for such expenditure before transmitting them to the trustee or trustees for payment.
What trust companies may be appointed
9 The trust companies hereinbefore referred to must at the time of their appointment, and as long as they continue as trustees, be authorized to act as executors and administrators in the Province of Manitoba.
Form of words for donation
10 Any form of words shall be sufficient to constitute a donation for the purpose of this Act, so long as the donor indicates an intention to contribute presently or prospectively to a fund or foundation of the general character indicated in this Act. This Act is to be construed liberally, and in furtherance of the idea that the Courts of Manitoba will assist in making effective gifts for religious and benevolent purposes, and will in case of any failure on the part of the Foundation or of the Board do what is necessary to carry out the true intent and purpose of this Act.
Part only of trust may be exercised
11 Where property has been donated to the Foundation, and the donor is desirous that a part of the income derivable therefrom should be distributed for a purpose or purposes other than those set out in this Act, and so indicates in the instrument creating the trust, the Board may accept and exercise the trust in respect of the distribution of such part, as fully and effectually as in respect of the remainder.
Future donations
12 Where a donation of any property has been made to the Foundation, to take effect in future, the Board is empowered to accept and exercise any or all powers of appointment, settlement and distribution in respect of the income in whole or in part derivable from such property in the interim, and is also empowered to nominate executors and trustees, in the manner provided in the instrument creating the trust.
13 The Board shall cause an audit to be made at least once in every fiscal year of the receipts and disbursements in connection with such donations, and shall present to the Congregation of Knox Church at its next annual meeting a certified statement of such audit showing in detail the investments made of all funds donated to and vested in the Foundation, the amount of the income received during the preceding fiscal year and the purpose for which the income had been used, and a statement of the expenses of the trustees and of the Board. The statement shall also show the amount of property which donors have set apart for the benefit of the Foundation to become effective in future, and the names of such donors. It shall be the duty of the trustees to give full information and permit the necessary inspections to enable such audits to be made.
14 The Board may pass by-laws regulating the carrying out of such duties as are by this Act assigned to it, including the fixing of the fiscal year, the quorum of the Board, the remuneration of the trustee or trustees, the time or times of the audit, the distribution of the income, the appointment of its chairman, and such other matters as may be deemed advisable for the convenient and efficient carrying out of the objects of this Act, and may amend, repeal and re-enact the same.
Returns under Corporations Act
15 The Foundation shall be subject to and comply with the provisions of The Corporations Act.
NOTE: This Act replaces S.M. 1933, c. 86.


text:
package org.hisp.dhis.period;

/**
 * PeriodType for weekly Periods. A valid weekly Period has startDate set to
 * Sunday and endDate set to Saturday the same week, assuming Sunday is the
 * first day and Saturday is the last day of the week.
 *
 * @author Torgeir Lorange Ostby
 */
public class WeeklySundayPeriodType
    extends WeeklyAbstractPeriodType
{
    public WeeklySundayPeriodType()
    {
        super( PeriodTypeEnum.WEEKLY_SUNDAY.getName(), 7, "yyyySunWn", "P7D", 7, "1 week", "SunW" );
    }

    @Override
    public PeriodTypeEnum getPeriodTypeEnum()
    {
        return PeriodTypeEnum.WEEKLY_SUNDAY;
    }

}

redpajama_set_name: RedPajamaGithub


At Comely Bank Glazing we offer the professional installation of secondary glazing with our range of products offering added insulation for your property and effective draught prevention. The process of installing secondary glazing involves inserting a new and separate glazed frame behind the existing one. The new glazed frame can be open or fixed, and is positioned internally, facing the room.
A key feature of this type of glazing is the level of protection against external noise. Comely Bank Glazing assures customers that they will experience a significant reduction in noise pollution where properties are in close proximity to noise from cars, trains or planes. Our standard glazed frames will reduce outside noise by 75%, which can be further minimised by selecting acoustic or laminated glazing. We also recommend this level of sound proofing for the creation of internal glass partitions, or in areas where containing sound is important, such as music studios or venues.
The economic benefits of secondary glazing include energy and cost savings, reducing heat loss in a property by up to 50%, as compared to a 20% reduction where single glazing is installed. The benefit of choosing professionally installed secondary glazing with us, is that even if the existing window is prone to draughts, our inter-locking aluminium frames and enhanced seals will prevent draughts affecting the inside of the building.
We offer three types of secondary glazing to customers; horizontal or sliding, hinged casement and lift-out, or fixed. Glazed frames are available in RAL colour finish or white and are installed with a quality guarantee. Our solutions offer a cost effective alternative to replacing external windows and are ideal for single, multi-storey or more traditional builds.
Secondary glazing will not alter the look of your building internally or externally, which means that installation can be done in phases, benefiting individuals or companies with budget and timeframe constraints. All projects can be designed and planned in advance ensuring a smooth process from start to finish. Get in touch with us to find out more.


Presidential Candidates Badnarik and Cobb to debate in Miami. Cobb and Badnarik represent the Green and Libertarian parties, respectively, and they will hold a true, open debate the same night as the major party candidates. Note — Cobb and Badnarik will actually accept questions from the audience, who won't be screened for their politcal views.


With Mike Thompson`s Retirement happening only last week, he had a few farewell events to attend in addition to his retirement party.
At the last Newhaven Chamber of Commerce Breakfast meeting Mike attended he was surprised with a special presentation which included a Heynes manual written all about Retirement written in the style of the famous car how-to instructional books.
He will be a hard act to follow and we are sure that his fellow Chamber members will recall fondly his talent for telling jokes, tall tales and a pun for every occasion!
Here is a picture of the presentation made to Mike Thompson.


But what about those people who simply want to get better at writing?
As far as I'm concerned, writing is the most important skill one can have.
Being a good writer leads to being a better and more coherent thinker.
It's also one of the few mediums that no matter what you do, or who you are in life, you will write nearly every day.
Whether it's simply sending an email or updating your Facebook status, being able to clearly articulate your thoughts is an incredibly valuable skill.
Over the last week, you've probably seen a lot of people on your Facebook feed who both can and cannot articulate their ideas well – and there's been a lot of animosity between both given the extraordinary political times our world is in.
Regardless of whether it's in your personal or professional life, it's impossible to deny how valuable the skill is.
But just because we all write, doesn't necessarily mean we're all "writers".
In this post, I'm not only going to teach you how to become a better writer, but I'm going to help you understand why it's so important to continue developing your writing skills in the process.
Below are ten of the things that I've done and members of our Location Rebel Academy community have done to hone their writing skills.
All of the best writers I know are also voracious readers. The more you read, and specifically the more books you read, the more you're exposed to high-quality writing.
Without realizing this, you are internalizing the elements of the text, and you'll slowly find yourself beginning to implement them in your own writing.
Not only this, reading gives you ideas. Lots and lots of ideas. And the more ideas you have, the easier it becomes to put them down on paper.
Try These Books – Want to get started reading today? Check out this post where you'll find 12 books to help you jumpstart a love of reading (and prepare you to be a lifestyle entrepreneur).
Goodreads – This is a social network dedicated to reading. Use it to find books and see reviews, and check out what your friends are reading. You can find quite a few great books on here you might have never thought about before.
Your Local Library – Yup, libraries are filled with great resources, all for free. And, most libraries now also feature online books and audio resources in addition to all the traditional stuff.
As with anything in life, practice makes perfect.
If you limit your writing to 3 tweets a month, you're probably not going to improve much.
But if you write every day, you'll begin to notice the process becoming easier. Thoughts becoming clearer. And most importantly, you'll begin to enjoy it more.
It's always more fun to do things we're good at. Writing is no different.
A great way to do this is by committing to a certain number of words every day.
Chris shoots for 1,000 words a day. As does Srini. Our community manager Liz started with 500 and has seen that habit lead to productivity gains in other aspects of her life as well. LRA member Charles shared how he got a 1,000 word a day practice going here.
And if you're someone like me whose entire business is built around a blog and the internet? Well the more I write, the more successful I become.
Need a little help writing everyday? These 16 writing tools will get you on the right track.
Speaking of blogs, one of the best ways to become a writer, is quite simply to become a writer!
The fastest way to do this is to start a blog.
Starting a blog completely changed my life. This site began as Location 180 way back in 2009 and has led to living a life I could have never imagined.
The positive effects of starting happened quickly, tool. Just look at these 56 reasons. I was able to write this less than a year after I began this site.
Not sure what to write about? I wasn't either. Just go back and look at my Archives from the Summer of 2009 – I was clueless, and my writing was pretty rough as well.
But I wrote about whatever I was interested in or thinking about, and over time the niche and topics began to become clearer, the same will happen for you.
Here's everything you need to get going: The Step by Step Guide to Setting up Your First Blog.
When people ask me for steps on how to become a better writer – I always tell them to read Everybody Writes.
It fully recognizes the concept that we're always writing, marketing, and promoting – even if we don't realize it.
It helps you specifically focus on writing engaging and interesting content that will do well online.
We just talked about my favorite book for aspiring writers, but what about people that really want to go all in and truly start honing their crafts?
There are a lot of different courses online for people who want to become writers.
This focuses specifically on how to become a better copywriter, or how to be more persuasive with your words. It takes a fair unconventional approach, but works and is how I improved my own skills.
You can find a good list of free online courses to help improve your writing here.
This is a problem, not just with writers, but with creatives or business people as a whole.
Often times it can be difficult to get real feedback from people because those closest to you want you to feel good.
They would rather be nice and polite than tell you what you really need to hear.
When you're trying to improve your writing skills, this is horrible. The last thing you want is to keep repeating bad habits, or spending hours crafting work that isn't interesting.
Your mom is probably not the best person to critique your work. Neither is your significant other.
Find either a business peer or mentor you trust, or you can find communities online to get real feedback.
Location Rebel Academy – We're constantly giving our members feedback on their content writing, their websites, and their approach to finding clients. We're friendly, but firm, and while we'll always complement the things you're doing well, we'll never just tell you what you want to hear.
Writing Forums – These forums tend to focus on fiction writing, but can be a great place to get feedback on the style of your writing, and to get ideas as well.
Legend Fire – This is a forum specifically for writing critiques. Again it generally focuses on creative writing but is an excellent starting point.
This is a common thread I've seen among many of my most successful entrepreneur friends – they journal.
Sometimes just for 5 minutes a day, some for an hour.
Journaling will not only help you organize your thoughts and prioritize tasks or initiatives but by writing in it on a regular basis, you're writing will begin to improve.
As we mentioned earlier, the most important thing you can do to become a writer is to write. Journaling can be an easy way to start doing that, without the pressure of having to publicize any of it.
Moleskine – This is the best one you can get for writing, hands down. No prompts, just empty pages for you to write out your thoughts.
Productivity Planner – This was given to me by a friend of mine, and I think it's great. If the idea of writing in a journal every day is intimidating, start with this. It helps you focus on your daily and weekly tasks. By writing in it daily, you start to build that habit, which you can, in turn, translate into more freeform journal writing.
Self Journal – Very similar to the Productivity Planner above, but taking a focus on gratitude and tracking small actions every day.
I've been using my Self Journal for the last month or so.
This might be the best piece of writing advice I've ever received. One of the first guest posts I'd ever written was for the personal finance site Get Rich Slowly.
Online the more conversational you are, the easier your writing is to read, the more relatable and trustworthy you become, and the better your flow of writing becomes.
Comparing my writing in 2009 to now, you'll notice many more contractions and a very conversational tone.
I have JD to thank for that.
This is the famous quote attributed to Hemmingway, that I think has a lot of validity to it. (Even though most agree Hemmingway didn't actually coin the term.
Sure you can take it literal if you want, but the way I personally interpret it is let your writing be creative and free-flowing.
Don't get bogged down in research, adding links, formatting, or overthinking.
Then go back with fresh eyes to add edit, revise, format, and to make sure all of the claims you made during your initial draft you know, are actually true.
There are a nearly limitless number of good reasons to become a writer. Maybe you want to get into the first stages of freelance writing, maybe you want to up your game and become a B2B content writer, or maybe you want to self-publish lots of books.
But you have to know which of those reasons are the reasons that you want to become a better writer.
As with anything, you can only hone your craft and get better at skills if you have the proper motivation behind it.
So what is your motivation?
Is it to write more compelling Facebook posts to prove to your friends how great your life really is? Is it to get the attention of influencers with your cheeky and cleverly crafted emails?
Whatever the reason is that you're trying to learn how to become a writer, know the reason, and focus on it during your quest to improve your skills.
Want to Make a Living as a Freelance Writer?
Great, we think that's one of the best online businesses you can start right now.
We have an incredibly step by step post on freelance writing that will walk you through everything you need to know to get started.
I plan to put several of these steps to work. I also want to add a book to your list: Stephen King's "On Writing: A Memoir Of The Craft." He shares his writing experiences and struggles. The book is focused on writing novels, but the information is also useful for non-fiction writers as well. This book has helped pick me up on more than one occasion.
Great recommendation with King's book. It's been on my personal to read list, and a lot of people I respect highly recommend it.
I thought it was going to be a straight-up lecture about style and grammar but it's actual a great read about King's own story and has some great insight and inspiration along the way. Just thinking about it now makes me want to read it again!
I am looking at App Sumo for adding content to my blog when I start it, the reviews are pretty bad. Any comments?
What exactly are you looking at on AppSumo? Is there a specific product they are promoting?
Hi, this was a good read. I was wondering if I can syndicate it on my blog. I write spiritual/prayer books and have about 20 books in Amazon so far, but I tell you, I learned from this article.
Probably not going to be a good fit for syndication right now, but feel free to link back to this post if you think your audience would resonate with it.
Since more people are investing big buck on writing, the basic steps of writing a quality content became very crucial.
To be an expert blogger, effective writing is a must. So to have a perfect starting on writing a professional content, this post helps to gather some new compelling ideas on improvising the writing on the basis of the needs of the readers/audience.
Great Simple tips!! I started to follow some of the given resources.
Glad you enjoyed it Sathish! Totally agree.
I like your suggestion about getting real critiques. I think it's invaluable to hear from people who are not as "attached" to the writing. I try to get a mix of feedback from people who may or may not know anything about the topic.
Yeah I definitely agree. I think this is one of the most important things on here. It's not just with writing, either. Getting critiques from unbiased or impartial people on anything you're trying to actually get better at is valuable.
Very nice, interesting and informative article. Thanks.
I honesty didn't know what to expect, but I liked it.


Unisex wooden footbed clog with wipe-clean leather uppers.
Leather uppers. Colour coordinated wooden footbed.
Unisex wooden footbed clog with wipe-clean leather uppers. Leather uppers. Colour coordinated wooden footbed.


This report studies Probiotic in Global market, especially in North America, Europe, China, Japan, India and Southeast Asia, focuses on top manufacturers in global market, with sales, price, revenue and market share for each manufacturer, covering Chr. Hansen Holding A/S?
6.1 Chr. Hansen Holding A/S?
6.5 Yakult Honsha Co., Ltd.?


text:
Night temperature dips in Jammu region
By Guest on 11:45 am January 8, 2018 No Comment
Jammu: The minimum temperature in most places in Jammu region recorded a dip with people in the city of temples waking up to 4 degree Celsius three degrees below normal during this part of the season.
However, the morning chill vanished with bright sun appearing for the third day providing much needed relief to the people from the cold night.
The city has registered the season's lowest minimum temperature of 2.7 degrees Celsius on January 4.
Against the previous night's 5.1 degrees Celsius, the night temperature fell in Jammu city and settled at 4 degrees Celsius, a spokesperson of MET department said.
The snow-bound Bhaderwah in Doda district was the coldest recorded place in Jammu region where the minimum temperature stayed below freezing point.
The town registered an improvement of 0.7 degrees in the minimum temperature which settled at minus 1.1 degrees Celsius, the spokesman said.
Katra, which serves as the base camp for the pilgrims visiting Mata Vaishno Devi Shrine in Reasi district, recorded a low of 5 degrees Celsius, registering a drop compared to the previous night, the spokesman said.
He said Banihal town along Jammu-Srinagar national highway marked an improvement in the night temperature which rose to 2.6 degrees Celsius from minus 0.2 degrees Celsius the previous night. (PTI)
Night temperature dips in Jammu region added by Guest on 11:45 am January 8, 2018


Bacillus subtilis HK176 with high fibrinolytic activity was isolated from cheonggukjang , a Korean fermented soyfood. A gene, aprE176 , encoding the major fibrinolytic enzyme was cloned from B. subtilis HK176 and overexpressed in E. coli BL21(DE3) using plasmid pET26b(+). The specific activity of purified AprE176 was 216.8 ± 5.4 plasmin unit/mg protein and the optimum pH and temperature were pH 8.0 and 40℃, respectively. Error-prone PCR was performed for aprE176 , and the PCR products were introduced into E. coli BL21(DE3) after ligation with pET26b(+). Mutants showing enhanced fibrinolytic activities were screened first using skim-milk plates and then fibrin plates. Among the mutants, M179 showed the highest activity on a fibrin plate and it had one amino acid substitution (A176T). The specific activity of M179 was 2.2-fold higher than that of the wild-type enzyme, but the catalytic efficiency (k cat /K m ) of M179 was not different from the wild-type enzyme owing to reduced substrate affinity. Interestingly, M179 showed increased thermostability. M179 retained 36% of activity after 5 h at 45℃, whereas AprE176 retained only 11%. Molecular modeling analysis suggested that the 176 th residue of M179, threonine, was located near the cation-binding site compared with the wild type. This probably caused tight binding of M179 with Ca 2+ , whichincreased the thermostability of M179.
Cardiovascular diseases such as acute myocardial infarction, ischemic heart disease, and high blood pressure are the major causes of human death throughout the world. Fibrin accumulation in the blood vessels is the main cause of the diseases. Thrombolytic agents such as tissue plasminogen activator (t-PA), urokinase (UK), and streptokinase (SK) have been widely used for the treatment of thrombosis. However, these agents can cause serious side effects, such as bleeding, short half-lives, high cost, allergic reactions, and large therapeutic doses  . Recently, fibrinolytic enzymes from Bacillus species have been actively studied because of their potential as fibrinolytic agents. Nattokinase secreted by Bacillus subtilis natto is the most well-known example  . Similar fibrinolytic enzymes are also secreted by Bacillus species and they have been purified. Their properties, including molecular weight, optimum pH and temperature, stability, and substrate specificity, have been reported [1 , 36] . Nattokinase is produced as a preproenzyme and processed into an active enzyme with 275 amino acids. Nattokinase has a catalytic triad consisting of Asp 32 , His 64 , and Ser 221 residues and two calcium-binding sites that stabilize the three-dimensional structure  .
The activity and stability of fibrinolytic enzymes are important properties to be considered if the enzymes are intended to be used for the prevention and treatment of thrombosis. Engineering of a fibrinolytic enzyme has been reported  . Error-prone PCR is a method widely used to introduce various mutations into a specific gene  . The method does not require detailed information on the structure of a protein and accurate predictions of the amino acid substitutions at the proper sites  . The half-life of a lipase from Bacillus sp. at 40℃ was increased by 3-fold and the catalytic activity of the mutant increased by 2~5-fold over the wild type after error-prone PCR  . The thermostability and pH stability of a xylanase from Thermomyces lanuginosus DSM 5826 were improved by error-prone PCR  .
Bacillus sp. HK176 was isolated from cheonggukjang , a Korean fermented soyfood, as a strain secreting highly active fibrinolytic enzymes (unpublished result). In this work, the structural gene of the major fibrinolytic enzyme, aprE176 , was cloned from HK176 and overexpressed in E. coli . Error-prone PCR was done with aprE176 to get mutants with increased fibrinolytic activities. One mutant, M179, showed increased fibrinolytic activity and thermostability. The cloning of aprE176 and production of M179 are described. The increased thermostability of M179 and a possible cause for the improved thermostability are also discussed.
E. coli DH5α, E. coli BL21(DE3), and B. subtilis HK176 were grown in LB (Bacto tryptone 10 g, NaCl 10 g, yeast extract 5 g, per liter) broth at 37℃ with aeration. For cultivation of cells harboring pHY300PLK (Takara Bio Inc., Shiga, Japan), tetracycline was included in the culture medium at the concentration of 12 µg/ml. For cultivation of cells harboring pET-26b(+) (Novagen, Madison, WI, USA) or pET176, kanamycin was included at the concentration of 30 µg/ml.
Commercial cheonggukjang products produced in several provinces of Korea were purchased and used as sources of bacilli with fibrinolytic activities. One gram of cheonggukjang was mixed with 9 ml of sterile 0.1% peptone water and homogenized using a stomacher (Seward, London, UK), and the homogenate was serially diluted using 0.1% peptone water. Aliquots (0.1 ml) of diluted samples were spreaded onto LB agar plates containing skim milk (2% (w/v)) and the plates were incubated for 12 h at 37℃. Colonies with large lysis zones were selected and further examined for the fibrinolytic activities by spotting on fibrin plates. The fibrinolytic activity was determined by the fibrin plate method  . Fibrin plates were incubated for 18 h at 37℃ and the size of lysis zone was compared with those caused by plasmin (Sigma, St. Louis, MO, USA) with known concentrations.
The aprE176 gene was amplified by PCR using total DNA from B. subtilis HK176 as the template and the primer pair 51F (5'-A GGATCC CAAGAGAGCGATTGCGGCTGTGTAC-3', Bam HI site underlined) and 51R (5'-A GAATTC TTCAGAGGGAGCCACCCGTCGATCA-3', Eco RI site underlined)  . aprE176 without its signal sequence was amplified using the primer pair petF (5'-AGA GGATCC GATGGCAGGGAAATCA-3', Bam HI site underlined) and petR (5'-AGA CTCGAG CTGAGCTGCCGCCTG -3', Xho I site underlined). The PCR conditions were as follows: 94℃ for 5 min, followed by 30 cycles of 94℃ for 0.5 min, 60℃ for 0.5 min, and 72℃ for 1 min. Amplified aprE176 and the aprE176 without a signal sequence were cloned into pET26b(+) (5.36 kb, Km r ), respectively. E. coli DH5α and E. coli BL21(DE3) competent cells were prepared and transformed by electroporation as described previously  . Expression of aprE176 in E. coli was examined by measuring the fibrinolytic activity of the transformant, and the activity was expressed as plasmin unit/mg.
E. coli BL21(DE3) cells harboring recombinant pET26b(+) with aprE176 or a mutant gene were cultivated in LB broth (500 ml) containing kanamycin (30 µg/ml) at 37℃. When the OD 600 value of the culture reached 0.8, IPTG (isopropyl β-D-1-thiogalactopyranoside) was added (1 mM) to induce gene expression. After 15 h incubation at 20℃, cells were harvested by centrifugation and resuspended in 20 mM sodium phosphate buffer with 0.5 M NaCl and 10 mM imidazole (pH 7.4). Cells were disrupted by sonication (Sonoplus GM2070; Bandelin, Berlin, Germany) and the resulting cell extract was centrifuged at 12,000 × g for 10 min at 4℃. The supernatant and cell pellet were obtained. AprE176 and M179 (a mutant) were purified from the supernatant using a Ni-NTA column (GE Healthcare, Uppsala, Sweden). The Bradford method  was used to determine the protein concentration of the enzyme preparation, and bovine serum albumin was used as the standard.
Purified enzyme (1 µg) was incubated in buffers with different pH values (pH 3-12) for 2 h at 37℃. Buffer of 50 mM concentration was used: citrate–NaOH (pH 3–5), sodium phosphate (pH 6–7), Tris–HCl (pH 8–9), and glycine–NaOH (pH 10–12). After 2 h, the remaining activity was measured by the fibrin plate method. The optimum temperature for the fibrinolytic activity was determined by measuring the activity after 30 min incubation (pH 8.0) at temperatures ranging from 37℃ to 60℃. The thermostability of the enzyme was evaluated by measuring the remaining activity of enzyme, which was incubated at 45℃ (pH 8.0) for different time periods. The effects of metals and inhibitors on the activity were examined by incubating AprE176 in the presence of 5 mM metal ions or 1 mM inhibitors for 30 min at 40℃ (pH 8.0) and measuring the remaining activities.
Error-prone PCR was performed using pET176, a pET26b(+) containing aprE176 , as the template and according to the method of Cadwell and Joyce  . The reaction mixture (100 µl) consisted of 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 7 mM MgCl 2 , 0.01% gelatin, 0.2 mM dATP and dGTP, 1 mM dCTP and dTTP, pET176 (10 ng), 0.5 mM MnCl 2 , and Taq polymerase (Takara, 5 units). petF and petR were used as the primers (0.5 µM, each). The PCR was carried out for 30 cycles of 94℃ for 1 min, 60℃ for 0.5 min, and 72℃ for 1 min. The PCR products were ligated with pET26b(+) after being digested with Bam HI and Xho I. The ligation mixture was introduced into E. coli BL21(DE3) by electroporation. Transformants, growing on LB plates containing kanamycin (30 µg/ml) and skim milk (2% (w/v)), were screened for their proteolytic activities. After 48 h at 37℃, colonies with larger lysis zones than control were selected and inoculated into 5 ml of LB broth using sterile toothpicks. Plasmid DNA was prepared from each transformant for DNA sequencing.
Hydrolysis of fibrinogen was examined using purified AprE176 and M179 at 37℃. Fibrinogen (1 mg, bovine; MP Biochemicals, Illkirch, France) was dissolved in 1 ml of 20 mM Tris-HCl (pH 8.0) and digestion started by adding enzyme (50 ng/ml). Aliquots were taken at time points, mixed with 5× SDS sample buffer, and boiled for 5 min. SDS-PAGE was done using 12% acrylamide gel and the gel was stained with Coomassie Brilliant Blue R-250.
The amidolytic activity of AprE176 and M179 was assayed using the following substrates: N -Succinyl-Ala-Ala-Pro-Phe p -nitroanilide (Sigma, S7388), N -Benzoyl-Phe-Val-Arg p -nitroanilide hydrochloride (Sigma, B7632), N -Benzoyl-Pro-Phe-Arg p -nitroanilide hydrochloride (Sigma, B2133), and N -( p -tosyl)-Gly-Pro-Lys 4-nitroanilide acetate salt (Sigma, T6140). Fifty microliters of 10 mM substrate in 50 mM Tris-HCl (pH 8.0) was mixed with 10 µl of enzyme (1 µg) and 440 µl of Tris-HCl (pH 8.0). After 10 min at 37℃, 500 µl of citrateNaOH (pH 3.0) was added and the mixture was put on ice immediately and centrifuged at 12,000 × g for 5 min. The optical density (OD 410 ) of the supernatant was measured and the degree of hydrolysis was calculated from the absorbance value and molar extinction coefficient value of p -nitroanilide (8,800 M -1 cm -1 ). Kinetic parameters (V max and K m ) of AprE176 and M179 were determined by measuring the release of p -nitroaniline from N -Succinyl-Ala-Ala-Pro-Phe p -nitroanilide (0.01 to 0.9 mM), which was dissolved in 100 mM phosphate buffer (pH 8.0) containing 4% (v/v) DMSO at 37℃  . The V max value was converted to the k cat value from the relationship, k cat = V max /(enzyme).
The structures of AprE176 and M179 were predicted by using SWISS-MODEL Workspace  , and Subtilisin Nat (PDB ID: 4DWW) was used as the template structure. Predicted structures were compared using the PyMOL Molecular Graphics System (ver. 1.5.0.4, Schrödinger, LLC., NY, USA) and exported as images.
All data are expressed as the mean ± SD values. The statistical analyses were performed using the SPSS program (SPSS, Inc., Chicago, IL, USA). Duncan's multiple range test was used to examine the difference among treatments. P values of 0.05 were considered significant, if not otherwise stated.
cheonggukjang products produced in different provinces of Korea in 2012 were purchased for the screening of bacilli with strong fibrinolytic activities. Colonies with strong protease activities were selected using skim-milk plates. Selected isolates were further examined for their fibrinolytic activities using fibrin plates. Among the isolates, HK176 was the best in terms of the fibrinolytic activity. For the identification of HK176, the 700 bp recA gene  and 1,300 bp 16S rRNA gene were amplified and the sequences were determined. BLAST ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ) search for both genes indicated that HK176 was either a B. subtilis (99% identity) or a B. tequilensis (99% identity) (results not shown). The RA PD-PCR profile of HK176 obtained by S30 primer matched well with that of B. subtilis reference strains (results not shown)  . From these results, HK176 was identified as a B. subtilis strain.
aprE176 was amplified from genomic DNA of B. subtilis HK176 by PCR. The 1.5 kb fragment was cloned into an E. coli - Bacillus shuttle vector, pHY300PLK, after digestion with Eco RI and Bam HI, resulting in pHY176 (6.43 kb). Sequence analysis showed that the fragment was 1,491 bp in length and contained the major fibrinolytic gene, aprE176 . The sequence was deposited into GenBank under the accession number of KJ572414. aprE176 encodes a protein of 382 amino acids (aa) consisting of signal sequence (30 aa), propeptide (77 aa), and mature enzyme (275 aa). This configuration was expected from comparison of the amino acid sequence of AprE179 with those of similar fibrinolytic enzymes from bacilli [9 , 29] . The size of mature and active AprE176 was estimated to be 27 kDa after processing. The ribosome-binding site (RBS) and putative promoter sequences (-35 and -10) were located upstream of the ORF ( Fig. 1 )  . The nucleotide sequence of aprE176 showed 99% similarity to those of fibrinolytic genes of B. amyloliquefaciens CH51 (EU414203), B. amyloliquefaciens MJ5-41 (JF739176), B. subtilis JZ06 (EU386607), and B. subtilis D-2 (JQ730856). The translated amino acid sequence of AprE176 was aligned with those of other homologous proteins ( Fig. 2 ). AprE176 showed higher similarity with other fibrinolytic enzymes such as 99.7% with AprE3-17 from B. licheniformis CH3-17 (ACU32756)  , 99.4% with AprE51 from B. amyloliquefaciens CH51 (ACA34903)  , 98.9% with peptidase S8 from B. amyloliquefaciens LFB112 (AHC41550), subtilisin DFE from B. amyloliquefaciens DC-4 (AAZ66858)  , and subtilisin DJ-4 from Bacillus sp. DJ-4 (AAT45900)  , 86.3% with nattokinase from B. subtilis subsp. natto (ACJ11220)  , and 85.6% with subtilisin E from B. subtilis 168 (CAA74536). The nucleotide sequence of aprE176 was similar to those of B. amyloliquefaciens and B. subtilis strains and the deduced amino acid sequence showed the highest similarity to that of AprE3-17 from B. licheniformis . When the amino acid sequences of mature enzymes were compared, AprE176 differed from AprE3-17 at a single amino acid, and differed from AprE51 at two amino acids ( Fig. 2 ).
Nucleotide sequence of aprE176.	The deduced amino acid sequence is shown above the nucleotide sequence. Putative -35 and -10 promoter sequences are underlined. The RBS and transcription terminator are also underlined. The ends of the pre (▼) and pro sequences (▽) are marked.
Alignment of amino acid sequence of AprE176 with homologous enzymes.	AprE51 (B. amyloliquefaciens CH51, ACA34903), AprE3-17 (B. licheniformis CH3-17, ACU32756), peptidase S8 (B. amyloliquefaciens LFB112, YP_008949402), subtilisin DFE (B. amyloliquefaciens DC-4, AAZ66858), subtilisin DJ-4 (Bacillus sp. DJ-4, AAT45900), nattokinase (B. subtilis subsp. natto, ACJ11220), and subtilisin E (B. subtilis 168, CAA74536). Amino acids different from those of other proteins are marked in a box.
proaprE176 without the signal sequence was amplified for overexpression in E. coli BL21(DE3). The 1.1 kb fragment was cloned into pET26b(+), resulting in pET176. In pET176, the pelB signal sequence in the vector directed the secretion of AprE176 into the periplasm of E. coli , and aprE176 was transcribed by the T7 promoter. The T7 promoter system was also used for the expression of other fibrinolytic genes in E. coli [8 , 22] .
After centrifugation of sonicated E. coli cells, the supernatant and cell pellet were obtained. Fibrinolytic activity was detected from both the supernatant and pellet, but higher activity was detected from the supernatant (data not shown). Thus, the supernatant was used for the purification of AprE176. A Ni-NTA column was used for the purification of AprE176, and bound AprE176 was eluted as described by the supplier. A 27 kDa single band was observed when the eluate was analyzed by SDS-PAGE (data not shown). The specific activity of the purified AprE176 was 216.8 ± 5.4 plasmin unit/mg protein. For the M179 purification, the exact same procedures were used.
AprE176 maintained more than 80% of its activity after 2 h at pH between 7.0 and 10.0, but the activity decreased rapidly at pH below 6.0 ( Fig. 3 A). The optimum pH was 8.0. The optimum temperature was 40℃ at pH 8.0 ( Fig. 3 B). The activity decreased rapidly at 50℃ and was completely inactivated at 55℃. The optimum pH and temperature of AprE176 are similar to those of nattokinase from B. subtilis natto B-12  . These activities are lower than those of an enzyme from B. amyloliquefaciens DC-4 (pH 9.0 and 48℃, respectively)  and an enzyme from Bacillus sp. CK11-4 (pH 10.5 and 70℃, respectively)  . The optimum pH of AprE176 (pH 8.0) was quite different from that of AprE3-17 (pH 6.0)  and AprE51 (pH 6.0)  . The mature AprE176 differs from AprE3-17 and AprE51 at a single amino acid and two amino acids, respectively. The result indicates that even a single amino acid difference can cause significant difference in the enzyme properties. AprE176 was completely inactivated by 1 mM PMSF (phenylmethylsulfonyl fluoride), a well-known inhibitor of serine proteases. EDTA (ethylenediaminetetraacetic acid) and EGTA (ethylene glycol tetraacetic acid) also inhibited the fibrinolytic activity of AprE176. The results showed that AprE176 is a serine protease and also a metalloprotease. Unlike AprE176, AprE3-17 retained more than half of its initial activity after 30 min exposure to PMSF and EDTA  , and AprE51 retained 83% activity after 1 h exposure to EDTA at 45℃  . The result again proves the uniqueness of AprE176 from other similar enzymes. Ca 2+ increased the activity by 17%, but Cu 2+ , Mn 2+ , and Zn 2+ reduced the activity by 7%, 7%, and 43%, respectively ( Table 1 ). Ca 2+ increased the activity of many fibrinolytic enzymes [10 , 18] . Zn 2+ inhibitedseveral fibrinolytic enzymes, including AprE176. For example, 5 mM Zn 2+ completely inactivated nattokinase from B. subtilis YJ1  and decreased the activity of subtilisin DJ-4 from Bacillus sp. DJ-4 by 72.88%  .
Effects of pH and temperature on the activity of AprE176.	(A) Residual fibrinolytic activities were measured after 2 h at different pH values. (B) Residual fibrinolytic activities were measured after 30 min in Tris-buffer (pH 8.0) at different temperatures.
Effects of chemical reagents on the activity of AprE176.
aThe counterion for the tested metals was chloride. All values are the mean ± SD (n = 3).
Error-prone PCR was done with aprE176 for the purpose of obtaining mutant enzymes with improved fibrinolytic activities. E. coli BL21(DE3) transformants (550 colonies) were initially screened using skim-milk plates and further screened using fibrin plates. Subsequently, three clones (M57, M111, M179) that showed higher fibrinolytic activities than wild type were selected by measuring the clear zone on the fibrin plate. In addition, a mutant (M11) showing significantly decreased activity was also selected for comparison. Sequence analysis of four mutants showed that the amino acid substitutions were V84A/K213M, K213N, G157S, and A176T for M11, M57, M111, and M179, respectively. The specific activities of purified M11, M57, M111, and M179 were 10.9 ± 1.6, 277.1 ± 15.7, 300.4 ± 21.2, and 479.8 ± 12.2 plasmin unit/mg protein, respectively. Among them, M179 showed the highest fibrinolytic activity, and this value was 2.2-fold higher than that of wild-type AprE176. M111 showed 1.4-fold higher activity and M57 showed 1.3-fold higher activity. Changes in the 84 th and 213 th amino acids at the same time almost completely destroyed the activity (M11).
Fibrinogen is the precursor of fibrin, composed of three pairs of disulfide-bonded polypeptide chains (Aα, Bβ, and γ). Both AprE176 and M179 quickly degraded the Aα chain of fibrinogen, which disappeared within 30 sec ( Fig. 4 ). The Aα chain was hydrolyzed first, followed by the Bβ chain. This indicated that AprE176 possesses strong α-fibrinogenase and moderate β-fibrinogenase activities. The degradation pattern was similar to those observed in some other fibrinolytic enzymes such as AprE2 from B. subtilis CH3-5  , TPase from B. subtilis TP-6, and plasmin  . M179 degraded Aα and Bβ chains completely within 20 min, indicating that M179 had improved β-fibrinogenase activity. Both enzymes failed to degrade the γ-chain in 20 min.
Hydrolysis of fibrinogen by AprE176 (A) and M179 (B).	Lane M, size marker (DokDo-MARK, ELPIS-Biotech. Inc., Daejeon, Korea.); 1, no enzyme treatment; 2, 30 sec; 3, 1 min; 4, 2 min; 5, 3 min; 6, 4 min; 7, 5 min; 8, 10 min; 9, 20 min. A 12% acrylamide gel was used.
The amidolytic activities of AprE176 and M179 were determined using four different synthetic substrates. The most sensitive substrate was N-succinyl-Ala-Ala-Pro-Phe- p NA, a preferred substrate for subtilisin and chymotrypsin. AprE176 and M179 showed no activity for the other substrates, indicating that AprE176 is a subtilisin-like serine protease. The amidolytic activities of AprE176 and M179 were 28.63 ± 0.20 and 39.84 ± 0.04 mM/min/mg protein, respectively, when N-succinyl-Ala-Ala-Pro-Phe- p NA was used as the substrate. M179 showed 1.39-fold higher amidolytic activity than AprE176. The kinetic constants, K m and k cat , were determined for AprE176 and M179 by measuring the initial rates of hydrolysis of N -succinyl-Ala-Ala-Pro-Phe- p NA ( Table 2 ). Although M179 showed enhanced fibrinolytic activity and catalytic turnover (k cat ), it had a lower substrate affinity (K m ) than AprE176. As a result, the catalytic efficiency (k cat /K m ) of M179 was not different from that of AprE176.
Kinetic parameters of AprE176 and M179.
All values are the mean ± SD (n = 3). Values in the same column with different superscripts are statistically different by Duncan's multiple range test (p = 0.05).
In the case of a keratinase from B. licheniformis BBE11-1, a single amino acid change (N122Y) caused the increase of the catalytic efficiency by 5.6-fold compared with wild-type keratinase. The 122 th residue of mutant (N122Y) was located 6.42 Å, 6.63 Å, and 2.45 Å from Asp 32 , His 63 , and Ser 220 , respectively. Some intramolecular interactions among the aromatic 122 th residue, the active site (Asp 32 , His 63 , Ser 220 ), and substrate influenced to increase the catalytic efficiency  , whereas other substitutions (N217S, A193P, and N160C), which were located far from the active site, did not significantly change the K m or k cat  . For a serine alkaline protease from B. pumilus CBS, the catalytic efficiency was increased 42-fold by mutations (L31I/T33S/N99Y)  . The substitutions occurred at residues surrounding the catalytic residue, Asp 32 , in serine alkaline proteases. For the above examples, changes occurred near the active site, and this enhanced the catalytic efficiency. In the case of M179, the 176 th residue was located far from the catalytic site, and this might be the reason why the catalytic efficiency did not increase.
M179 possessed improved thermal resistance compared with AprE176. Purified M179 retained 67% residual activity after 150 min incubation at 45℃, whereas AprE176 had 50% activity ( Fig. 5 ). When the incubation time was extended to 5 h at 45℃, M179 still retained 36% activity but AprE176 had only 11% activity. The thermostability of an alkaline protease (BgAP) from Bacillus gibsonii was increased more than 90-fold (half-life at 60℃) by directed evolution  . The loop area of the BgAP surface was replaced by substitutions to negatively charged amino acids, which contributed to the increase of the thermostability, probably through increasing ionic and hydrogen bond interactions in mutated BgAP. The half-life of a keratinase at 60℃ from B. licheniformis BBE11-1 was increased 8.6-fold by four amino acid substitutions (N122Y, N217S, A193P, N160C)  . Increase in the thermostability was explained to be the result of increased interactions, such as hydrophobic interaction, cation-pi interaction, and hydrogen bond, in the mutant. Interactions such as hydrogen bonds, hydrophobic interactions, and electrostatic interactions are well known to be the main factors for protein thermostability  .
Temperature stability of AprE176 and M179.	Thermal stability was determined at 45℃ in Tris-HCl buffer (pH 8.0) for various times. AprE176, -●-; M179, -■-.
To explain the increased thermostability of M179 compared with wild-type ArpE176, molecular modeling was used. Three-dimensional models of AprE176 and M179 were constructed using the known structure of nattokinase (4DWW). Nattokinase and AprE176 share 76% identity in amino acid sequences, and homology-based model structures could be built using SWISS-MODEL Workspace ( http://swissmodel.expasy.org/workspace/ ). Based on the model structures, A176T mutation might be responsible for the increased thermostability of M179. According to the model structure of AprE176, three amino acids (Gly169, Tyr171, and Val174) are located within 3.0 Å radius from a calcium ion ( Fig. 6 A). For M179, the hydroxyl group of Thr176 is located closely (2.9 Å) to the calcium ion, whereas Ala176 of AprE176 does not show any interaction with calcium ions. Bacillus fibrinolytic enzymes (subtilisin E  , Carlsberg  , and BPN  ) have two calcium-binding sites, Ca A and Ca B sites. The Ca A site is located near the N-terminus and has higher calcium-binding affinity, whereas Ca B site has weaker binding affinity. The occupation of the Ca B site is an important factor for the increase of thermal resistance. Increased thermal stability by increased calcium concentration was shown by Pantoliano et al .  at calcium concentrations ranging from 0.1 to 100 mM. It was also reported, in the X-ray crystal structure study using subtilisin, that the occupancy of the Ca B site was increased as the calcium concentration was increased.
Predicted structures of calcium-binding pocket.	(A) AprE176, (B) M179. The purple ball indicates a calcium ion.
From the model structure, it is suspected that Thr176 of M179 is more likely located in the Ca B site. The existence of an extra amino acid with favorable interaction with calcium ions could possibly increase the affinity with calcium ions, resulting in increase of the thermostability of the enzyme. Increased thermostability is desirable if AprE176 and its derivatives are incorporated into foods that are expected to be heat treated.
AprE176 possesses strong fibrinolytic activity, which can be utilized for the production of fermented foods or drugs preventing cardiovascular diseases caused by fibrin clots. Mature AprE176 shows different physicochemical properties, such as pH optimum and inhibition by chemicals, from enzymes with quite similar amino acid sequences. The property of AprE176 can be improved by directed evolution methods, and M179, a derivative of AprE176, possesses improved thermostability. Further improvements are necessary, and studies on the roles of important amino acids for the activity of AprE176 should also be continued.
This work was supported by a grant from Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) (High Value-Added Food Technology Development Program, 2012, 112066-03-SB010), Ministry of Agriculture, Food and Rural A ffairs, Republic of Korea. S.-J. Jeong and J. Y. Park were supported by BK21 Plus Program, MOE, Republic of Korea.
 SJ Jeong , K Heo , JY Park , KW Lee , JY Park , SH Joo , and JH Kim , "Characterization of AprE176, a Fibrinolytic Enzyme from Bacillus subtilis HK176", Journal of Microbiology and Biotechnology, vol. 1, no. 1, Jan 2015.