129 Cal.App.2d 191 (1954)
BARBARA M. COFFER, Respondent,
v.
D. W. LIGHTFORD, as Administrator, etc., Appellant.
Civ. No. 8458.
California Court of Appeals. Third Dist.
Nov. 24, 1954.
 Spurr & Brunner for Appellant.
 Burke & Rawles and John E. Nelson for Respondent.
 WARNE, J. pro tem.  [fn. *]
 This is an appeal from a money judgment rendered against appellant as Administrator of the Estate of Roy Coffer, deceased, in an action brought by respondent upon an account stated between her husband and his father, the deceased, Roy Coffer, and which was awarded to her by the provisions of an interlocutory decree of divorce.
 The action was brought against Roy Coffer who died during the pendency of the action. Pursuant to application of the plaintiff, and order of the trial court thereon, the action was revived and continued against the defendant and appellant, as administrator with the will annexed of the estate of said deceased. It appears from the evidence that the deceased and his son, Clyde, during the year 1948 became partners in the operation of a sawmill, and that respondent and her husband, Clyde, loaned something in excess of $3,000 to the partnership. The enterprise was unsuccessful, and the partnership was dissolved. In settlement of the partnership affairs the deceased, Roy Coffer, on July 12, 1949, entered into an agreement in writing whereby Roy Coffer, the deceased, agreed to pay to Clyde Coffer "Four dollars ($4.00) per thousand feet on all lumber sold until the sum of Four Thousand ($4,000.00) dollars has been received by the second party [i.e., Clyde]."
 The agreement was to take effect after the first 100,000 feet of lumber had been cut. There is nothing in the record to show this agreement was ever carried out. While it is doubtful if the agreement can be classified as an account stated, the parties to the action construed it as such throughout the entire proceedings. *193
 Respondent filed an action for divorce against her husband, Clyde, in the Superior Court of Nevada County, and on March 3, 1950, was granted an interlocutory decree of divorce which awarded her the community property "immediately," a portion of which was described as follows: "An agreement for the payment of the sum of $4,000.00 to the defendant, dated July 12, between Roy Coffer and the defendant herein, from the proceeds of the sawmill belonging to said Roy Coffer." There is no evidence as to whether or not an appeal was taken from the interlocutory decree or if relief therefrom was sought under the provisions of section 473 of the Code of Civil Procedure within the time provided by law. Respondent testified that this decree became final; however, after she so testified an objection was made to her answer, and the court sustained said objection upon the ground that it called for her conclusion; however, no motion was made to strike the answer, and it is therefore a part of the record. (53 Am.Jur., Trial, p. 118; 3 Jones on Evidence, 1668-1669,  895.)
 In July of 1951 the parties were remarried; however, there is no testimony in the record as to the date of the reconciliation or if the parties agreed to, or intended to nullify the provisions of the interlocutory decree as to the disposition of the community property. It is a very strong and reasonable inference, as noted by the trial court in his order for preparation of findings, that the fact of a remarriage indicated that a final decree had been actually entered, otherwise there would have been no necessity for such remarriage.
 Appellant contends that a reconciliation between the parties to a divorce action, after the entry of an interlocutory decree, but before a final decree of divorce has been entered, operates to nullify and void "any contract made for the parties by the interlocutory decree," including any adjudication or attempted adjudication of the property rights and community property of the parties by such interlocutory decree. In support of this contention he cites such cases as Brown v. Brown, 170 Cal. 1 [147 P. 1168]; Estate of Boeson, 201 Cal. 36, 42- 43 [255 P. 800]; Estate of Brimhall, 62 Cal.App.2d 30, 35 [143 P.2d 981]; Peters v. Peters, 16 Cal.App.2d 383, at 386-387 [60 P.2d 313]. A careful reading of these cases will not bear out appellant's contention. In the Brown case it appears that the parties lived and cohabited together as husband and wife and "that in pursuance of the agreement to so live they further orally agreed to and did set aside and *194 annul said agreement." In the Boeson case, supra, the court stated: "Property settlements entered into by the spouses, it is true, should be set aside in cases where the acts, conduct, and relations of the parties thereafter are of such a character as to justify the conclusion that they intended and agreed orally to abrogate the same." In the Estate of Brimhall the contention was "That the property settlement agreement was terminated because subsequent to the execution thereof appellant and her husband cohabited as husband and wife." The court held: "This proposition is also untenable. The law is established in California that a husband or wife may enter into any transaction with the other respecting property the same as if they were unmarried, subject to the general rules which control the actions of persons occupying confidential relations with each other. (Section 158, Civ. Code; Stoff v. Erken, 25 Cal.App. 528, 530 [144 P. 312].)" In the Peters case, supra, the record shows that the parties in good faith commenced to live together, did actually resume marital relations, and intended to cancel the prior adjustment of their property rights, and that appellant actually took steps to have the property rights of the parties restored to their predivorce status. We are satisfied that defendant's contention cannot be sustained upon the record in the instant case. [1] When the time had elapsed for taking an appeal or seeking relief under the provisions of section 473 of the Code of Civil Procedure, in the absence of some agreement, oral or written, between the parties that the award of the community property to respondent should become a nullity, the property in question vested in respondent regardless of the fact of reconciliation and remarriage. This is in line with the rule laid down in the case of Leupe v. Leupe, 21 Cal.2d 145, 150 [130 P.2d 697], or, as stated in the case of Wilson v. Wilson, 76 Cal.App.2d 119, 132 [172 P.2d 568]: "We need not decide in this case whether it was error to make an immediate assignment of property in the interlocutory decree or whether the right to so dispose of the property of the parties was within the discretion of the court. The time for appeal having elapsed, the determination of property issues in the interlocutory decree became final and was no longer subject to modification except in accordance with the methods applicable to judgments generally."
 [2] The chose in action being the separate property of the respondent she had the right to maintain the action without joining her husband as a party. (Code Civ. Proc.,  370.) *195
 While the disposition of this appeal is not dependent upon a determination of the effect of the reconciliation and remarriage of the respondent, we have nevertheless discussed the question so that the trial court may have the benefit of our views concerning the matter on a retrial of this case.
 [3] Appellant's defense on the merits was that the account stated was paid or discharged, and in support thereof the following instrument executed by respondent's husband was introduced in evidence:
 "January 16, 1950"
 "The agreements of July 12, 1948 and May 1, 1948 for the sum of four thousand ($4,000.00) dollars, is paid in full as of now. Jan. 16, 1950"
 "Clyde M. Coffer"
 This document was offered and received in evidence as defendant's exhibit "A." Respondent's husband was permitted to testify, over objection, that this writing, the authenticity of which is admitted, lacked consideration and was executed solely for the purpose of defeating any claims to his father's sawmill that respondent might make in her pending divorce action. The damaging effect of this testimony is manifest. If believed by the trial court, as it obviously was, it completely destroyed appellant's defense of payment and discharge. The admission of this testimony was erroneous and prejudicial to appellant, even though there may have been other evidence tending to show that the account had not been paid, the sufficiency of which we do not need to decide upon this appeal. Respondent had the burden of proving by a preponderance of the evidence that there was a want of consideration (Civ. Code,  1615) for the discharge of which the written instrument was presumptive evidence. (Civ. Code,  1614.) There is no merit in respondent's claim that appellant waived the protection of section 1880 of the Code of Civil Procedure because appellant had respondent's husband identify his signature on the written instrument. [4] The examination of a party as to the authenticity or identification of a document does not waive the provisions of the section mentioned. (Kains v. First Nat. Bank, 30 Cal.App.2d 447, 450 [86 P.2d 935]; Moore v. Spremo, 72 Cal.App.2d 324, 329 [164 P.2d 540]; 159 A.L.R. 411, 415, 416.) Lucy v. Lucy, 22 Cal.App.2d 629 [71 P.2d 949], and Moul v. McVey, 49 Cal.App.2d 101 [121 P.2d 83], cited by respondent, are inapplicable because appellant did not interrogate respondent's *196 husband as to the circumstances surrounding the execution of the written instrument which was pleaded as a defense to respondent's cause of action. Therefore, respondent had no right to do so, and the trial court erred in overruling appellant's objections to such testimony. [5] Respondent argues that she is not the assignee of her husband and therefore that the inhibition of section 1880 of the Code of Civil Procedure is not applicable in the instant case, it being her claim that the judgment of the Superior Court of Nevada County in the divorce proceedings is open to the interpretation that such decree produced an involuntary relinquishment of the parties' community interests. We have not been able to find authority to support respondent's contention. It seems to be a matter of first impression. We do not agree with respondent. Section 146 of the Civil Code provides that in case of the dissolution of the marriage by decree of the court, the community property shall be assigned, and then makes provisions as to how and in what manner the assignment shall take effect. If we are to give to the word "assign" as used in the statute its ordinary usual meaning, the respondent became the assignee of her husband through an involuntary assignment made by the court, and his testimony then became subject to the inhibition of section 1880. The effect of what we have said is clearly within the purpose and meaning of subdivision 3 of section 1880 of the Code of Civil Procedure. As stated in Moul v. McVey, 49 Cal.App.2d 101, 105 [121 P.2d 83], "The object of the statute is to so balance rights as between the survivor and the deceased that no advantage may be taken of a deceased person because of the fact that his lips are sealed by death, ..." The witness Clyde Coffer stood in exactly the same position as if he had been a voluntary assignor through an instrument of formal assignment. Further, we must not lose sight of the fact that respondent had a community interest in the chose in action prior to the divorce proceedings. Clearly at that time the inhibition of the statute would have been applicable and we see no reason for holding that it did not continue after the court in the divorce proceedings had assigned it to her.
 As this case must be reversed for the reasons above stated, we do not deem it necessary to consider the remaining contentions made by the appellant.
 Judgment reversed.
 Van Dyke, P. J., and Schottky, J., concurred.
NOTES
[fn. *]  *. Assigned by Chairman of Judicial Council.


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// Foam Tree docs:
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// Some options from Parcel 1 Visualizer:
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// and Webpack Bundle Analyzer:
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Q:

Fastest way to get all unique numbers between two different dimension numpy arrays

I have two numpy arrays of varying dimensions, and I need to get all unique ints between them both.
Since they are of varying dimensions, I decided to get the unique numbers for each of them, concatenate them, and then get the unique values for that.
Initial approach
import numpy as np
import time

array1 = np.random.randint(1, high=5000, size=(512, 4) , dtype=np.int32)
array2 = np.random.randint(1, high=5000, size=(512, 64) , dtype=np.int32)

start = time.time()
np.unique( np.concatenate( [np.unique(array1), np.unique(array2)] ) )
end = time.time()
print(end-start)

Output (average out of 5):

0.011

Environment: Google Colab
Is there a faster way?
My guess would be to perform only one np.unique operation, doing that with different sized numpy arrays is the challenge of that.

A:

If your ints don't spread too far there is indeed a faster approach. The code below assumes for simplicity nonnegative ints
smaller than 5000.
def use_mask():
    mask = np.zeros(5000,bool)
    mask[array1] = True
    mask[array2] = True
    return np.where(mask)

To make this a proper function you'd have to find the min and max of array1/2 combined, take the difference, fall back to something else if it is too large, otherwise subtract the min from array1/2 and proceed more or less like I did.


U.S. stocks have posted two straight down days in a row, the Dow Jones Industrial Average had its biggest drop in five weeks, and small-caps (once again) are bearing the brunt of the selling. Right about now, you may be thinking about a number of things: Is this the Big One? Did I really think Twitter 's financial statements didn’t matter? Who thought it was a good idea to do another Godzilla remake?

But, has anything really changed? Doesn’t seem like it, not from where we’re sitting. Growth in the biggest economic zones – Europe, the U.S., China – is weak, even with central banks in the first two moving heaven and earth to try and spur activity. Five years after the official end of the U.S. recession, the world’s biggest economy continues to struggle to build up self-sustaining economic growth, Europe is crawling out of recession, and we do mean crawling, and China continues to see a slowdown. The Fed has been signaling the end of QE3 since this time last year, and has been whittling its bond program down since January.

What’s changed may be perceptions more than anything else. We’re halfway through May, nearly halfway through 2014, and the growth that everybody expected simply isn’t showing. The breakout hasn’t come, and we remain trapped in a slow growth, high debt, low inflation malaise.

Part of the reason that perceptions have been slow to change is the belief, reinforced by countless speeches from officials, that the Fed’s current policy – winding down QE3 – does not represent a tightening of monetary policy. “Tapering is not tightening,” as the hip kids say. If you don’t buy this notion, however, the market’s gyrations start to make sense.

Stocks rolled over after the Fed’s first two QE programs ended back in 2010 and 2011, noted Lindsey Group’s Peter Boockvar, and the Treasury market’s yield curve flattened as well. “I believe this is not a flow story,” he wrote in a note to clients, “it is a bet on where economic growth trends from here just as it was those previous two times and we cannot separate the market action from what the Fed is trying to accomplish on policy right now.”

Still, we’ve been through this drill before. The market rumbles, people start to wonder if this is the big one, there’s a modest shake-out, and the market goes back off on its merry way. One of these days that script won’t play out, one of these days it won’t be a modest shake-out. The trick everybody’s trying to pull off is knowing the difference.

The comments earlier this week from fund manager David Tepper, which can be summed up in the pithy “don’t be too frickin’ long” comment, reflect the market’s anxiety, Mark Newton, the chief technical analyst at Greywolf Execution Partners, wrote. With so many people, once again, looking for the Big One, it’s likely to take a contrarian turn against them (which will certainly make the bulls happy).

“Despite today’s pullback,” he said, “my thinking is this simply is a period of above-average downside volatility like what has happened frequently in this period in May near the NYSE anniversary, and that bearish sentiment will provide a floor to this sell-off by next week.”


about

bio

Judith Donath
synthesizes knowledge from urban design, evolutionary biology and cognitive science to design innovative interfaces for on-line communities and virtual identities. A Harvard Berkman Faculty Fellow and formerly director of the MIT Media Lab's Sociable Media Group, she is known internationally for her writing on identity, interface design, and social communication. She is the creator of many pioneering online social applications; her work and that of the Sociable Media Group have been shown in museums and galleries worldwide. She is the author of The Social Machine: Designs for Living Online (MIT Press, 2014). Her current research focuses on how we signal identity in both mediated and face-to-face interactions, and she is working on a book about how the economics of honesty shape our world.

She received her doctoral and master's degrees in Media Arts and Sciences from MIT and her bachelor's degree in History from Yale University.


Democrats Mary Landrieu of Louisiana and Robert Menendez of New Jersey voted against the bill, and Republicans Lisa Murkowski of Alaska, Sam Brownback of Kansas, Jeff Sessions of Alabama and Bob Corker of Tennessee supported it.

"None of us approve of every provision, none of us got everything that we wanted," Bingaman said. "The end product, I believe, is a solid piece of work. It is one which will help not only to enable us to produce new sources of energy, but to use our energy sources wisely and more efficiently."

The bill's major provisions would, among other things, impose a national renewable electricity standard, overhaul federal financing for "clean energy" projects, establish a suite of efficiency measures, mandate new federal electricity-transmission siting power, and allow wider oil and gas leasing in the eastern Gulf of Mexico.

Sen. Bill Nelson (D-Fla.) has pledged a vigorous battle against a provision added last week that could bring oil and gas rigs closer to Florida's Gulf Coast, upending a 2006 deal that provided the state a 100- to 235-mile no-drill buffer through mid-2022.

Sen. Richard Durbin of Illinois, the chamber's No. 2 Democrat, said Democratic leadership has not yet taken a position on the drilling amendment added last week, which would move the buffer to 45 miles and allow drilling even closer to shore in a gas-rich region called the Destin Dome.

"I want to hear [Nelson] out before I take a position on it," Durbin told reporters yesterday. Asked if he was concerned about upending the Florida buffer that was in a 2006 law that expanded gulf drilling, Durbin replied: "I am."

There will also be a battle over the renewable electricity standard (RES) as environmental groups and allied lawmakers call to raise the renewable-electricity goals.

The bill requires utilities nationwide to provide 15 percent of their power from renewable sources like wind and solar power by 2021, while allowing up to a quarter of the requirement to be met with energy-saving measures instead. Environmental groups have called for a renewable standard of 25 percent by 2025.

Indeed, some liberal committee members said the measure is not strong enough. "This is an extremely weak bill, and the only reason I am voting for it is to see if we can strengthen it on the Senate floor," said Sen. Bernard Sanders (I-Vt.).

Delicate balance

But in a sign of how delicate major energy bills can be, moderate Sen. Evan Bayh (D-Ind.) -- who was wary of even the bill's more modest RES -- warned that substantial changes to the bill would lead him to reconsider his support for it on the floor.

Meanwhile, Landrieu, Murkowski and others plan floor attempts to provide coastal states a share of revenues from oil and gas development that may occur in federal waters off their shores. Four Gulf Coast states -- Louisiana, Texas, Mississippi and Alabama -- won revenue sharing under the 2006 gulf leasing law.

Landrieu said her opposition was based on the absence of revenue sharing and the need for greater support for nuclear power.

Murkowski also wants addition of more provisions to boost nuclear energy. But she called the bill an acceptable compromise for the moment.

"There have been things that you didn't like, there have been things that I haven't liked," she told Bingaman before the vote. "But if any of us in this body can find one bill that we think is a perfect piece of legislation, I can find 99 other members who will fight you on that. Despite all the warts that are contained in this legislation, I think we are at a worthwhile place in the process."

She noted her support for the expanded eastern gulf drilling, creation of a federal "Clean Energy Deployment Administration" to fund advanced energy projects, and other provisions, while reiterating her criticism of inclusion of a renewable power mandate.

Groups react

The American Petroleum Institute praised the expanded gulf access that the bill provides, while also praising language that protects Canadian oil sands from a 2007 law requiring that federal agencies cannot buy alternative fuels that have higher greenhouse gas emissions than conventional fuels.

"The committee took a positive step forward by passing this bill which recognizes the importance of additional offshore oil and natural gas development and Canadian oil to our nation's energy and economic security," API President Jack Gerard said in a prepared statement.

But he added that he wants a coastal revenue-sharing provision added on the floor, which the industry wants to provide states incentive to support offshore drilling.

The Sierra Club criticized the bill after the vote, announcing that it cannot support the measure in its current form and pledging to try to win changes on the floor. The group criticized the RES, saying it "will not result in more clean energy and clean energy jobs than the status quo." The group also attacked the expanded gulf oil and gas access and changes to the 2007 fuels procurement law.

"We will seek improvements that will allow the bill to actually deliver more clean energy, slash energy waste, create new clean energy jobs, and speed our transition away from dirty coal and oil toward cleaner, cheaper energy sources like wind and solar," said Sierra Club Executive Director Carl Pope in a prepared statement.

Climate change

Across the Capitol, House Democratic leaders plan to bring their combined energy and climate package to the floor in the coming weeks. It includes a nationwide cap-and-trade system to cut greenhouse gas emissions, along with a renewable power mandate and a suite of other energy measures.

Senate Majority Leader Harry Reid (D-Nev.) has said he hopes to bring a combined energy and climate bill to the floor this fall, and Environment and Public Works Chairwoman Barbara Boxer (D-Calif.) says she wants to mark up the climate portions in her committee before lawmakers leave for the monthlong August recess.

But a cap-and-trade plan faces significant Senate obstacles, and some lawmakers have called for considering the Senate Energy Committee's package separately.

Committee adopts national interconnection standard, rejects bigger auto grants
The committee approved the bill following its adoption of several amendments.

The panel narrowly approved Bingaman's amendment aimed at boosting use of distributed generation technologies such as residential solar power by easing their connection to the grid.

Bingaman's amendment would require the Federal Energy Regulatory Commission to craft a national interconnection standard. He said his plan would help replace the current "patchwork" at the state level, where policies differ and not all states have them in place.

He argued that the amendment is needed to spur adoption of distributed sources that increase grid reliability while reducing costs by cutting the need for new baseload power generation. "There needs to be at least a set of common, minimum standards across the nation," he said, while noting that his amendment allows states to use their own policies if they are consistent with the federal standard.

Murkowski opposed the plan, calling it an unwarranted expansion of FERC jurisdiction that usurps states' traditional powers over the electricity distribution system. The committee rejected, by voice vote, a second Bingaman amendment to create a national net metering standard.

The committee also approved Sen. Byron Dorgan's (D-N.D.) amendment aimed at helping deploy the recharging infrastructure that would be needed for widespread use of plug-in electric vehicles, which are seen as a promising way of reducing oil use in the transportation sector.

But Sen. Debbie Stabenow (D-Mich.) failed in her effort to increase the size of grants that could be issued under DOE's Section 136 program, which authorizes grants and loans for auto-industry development of advanced-technology vehicles and related components.

Her plan would have allowed DOE to issue grants covering 50 percent of the costs of industry projects such as retooling plants to make advanced cars or parts. The federal share is currently capped at 30 percent. The grant portion of the Section 136 program has not yet been funded.

Scientific American is part of Springer Nature, which owns or has commercial relations with thousands of scientific publications (many of them can be found at www.springernature.com/us). Scientific American maintains a strict policy of editorial independence in reporting developments in science to our readers.


Q:

Information separating from mysql

I'm making program for teachers with Java and mysql , so I've many tables in mysql such as students table which contains student name and number and ID .. etc , also another table that called groups , It contains group name , place and students .. etc.
So , the problem is , in Groups table I'll add IDs of students in one row just like the image bellow beside the name of group , how can I take this IDs from mysql in this form and separate them automatically in program by java to search in the students table about the information of them .
image : http://i.stack.imgur.com/NFQ6t.jpg
  try {
    Class.forName("java.sql.Driver");
    Connection con = (Connection)   DriverManager.getConnection("jdbc:mysql://localhost:3306/teacherassistant?allowMultiQueries=true","root","1234");
    Statement st = (Statement) con.createStatement();
    String query = "select * from Groups;";
    ResultSet rs = st.executeQuery(query);

    while(rs.next()){
    String name = rs.getString("Name");
    String StudentIDs= rs.getString("Students");
    String Center = rs.getString("Center");

    }

    } catch (Exception e) {
        JOptionPane.showMessageDialog(this,"Error");
    }

A:

Don't do that.
The relationship between groups and students is clearly a many-to-many relationship. Instead make a new table that contains this relationship. Something like;
STUDENT_GROUP_REL
------------------
(student_id, group_id)

There is a related question and a very good answer about m:n relations here


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Q:

File name using find

How to get the file name while using find command? When I use find command to search particular type of files from a directory and subdirectories, I get the whole path of that file, how do I get the file name only? The problem I am facing is when I am moving that found file to archive directory, it says "there is no such directory" and it is because of whole path I am getting while searching for the file.

A:

The basename command will give you just the name of the file and not the full path, which you can execute on your found file like this:
find . -name filename -exec basename {} \;

A:

Use find ... -printf '%f\n'
Or, to solve your stated problem:
find ... -name FOO -print0 | xargs --no-run-if-empty --null mv --target-directory=TARGET


Sue Bremner

Susan Lenore Bremner (born 1954) is an American diplomat who Chargé d’Affaires ad interim to Eritrea (July 2012-April 2014).  She retired from the Foreign Service in 2019 and became the USAID Labor Management adviser.

References

Category:1954 births
Category:American women ambassadors
Category:Ambassadors of the United States to Eritrea
Category:Living people


############################################################
# This file is imported from a different project.
# DO NOT make modifications in this repo.
# File a patch instead and assign it to Ryan Davis
############################################################

require 'minitest/spec'

MiniTest::Unit.autorun

describe MiniTest::Spec do
  before do
    @assertion_count = 4
  end

  after do
    self._assertions.must_equal @assertion_count
  end

  # TODO: figure out how the hell to write a test for this
  # it "will skip if there is no block"

  it "needs to have all methods named well" do
    @assertion_count = 2

    methods = Object.public_instance_methods.find_all { |n| n =~ /^must|^wont/ }
    methods.map! { |m| m.to_s } if Symbol === methods.first

    musts, wonts = methods.sort.partition { |m| m =~ /^must/ }

    expected_musts = %w(must_be
                        must_be_close_to
                        must_be_empty
                        must_be_instance_of
                        must_be_kind_of
                        must_be_nil
                        must_be_same_as
                        must_be_within_delta
                        must_be_within_epsilon
                        must_equal
                        must_include
                        must_match
                        must_raise
                        must_respond_to
                        must_send
                        must_throw)

    expected_wonts = expected_musts.map { |m| m.sub(/^must/, 'wont') }
    expected_wonts.reject! { |m| m =~ /wont_(not|raise|throw|send)/ }

    musts.must_equal expected_musts
    wonts.must_equal expected_wonts
  end

  it "needs to verify equality" do
    (6 * 7).must_equal(42).must_equal true
    proc { (6 * 9).must_equal(42) }.must_raise MiniTest::Assertion
  end

  it "needs to verify floats within a delta" do
    (6.0 * 7).must_be_close_to(42.0).must_equal true
    proc { 42.002.must_be_close_to 42.0 }.must_raise MiniTest::Assertion
  end

  it "needs to verify types of objects" do
    (6 * 7).must_be_instance_of(Fixnum).must_equal true
    proc { (6 * 7).must_be_instance_of String }.must_raise MiniTest::Assertion
  end

  it "needs to verify kinds of objects" do
    @assertion_count = 6

    (6 * 7).must_be_kind_of(Fixnum).must_equal true
    (6 * 7).must_be_kind_of(Numeric).must_equal true
    proc { (6 * 7).must_be_kind_of String }.must_raise MiniTest::Assertion
  end

  it "needs to verify regexp matches" do
    @assertion_count = 6

    "blah".must_match(/\w+/).must_equal true
    proc { "blah".must_match(/\d+/) }.must_raise MiniTest::Assertion
  end

  it "needs to verify nil" do
    nil.must_be_nil.must_equal true
    proc { 42.must_be_nil }.must_raise MiniTest::Assertion
  end

  it "needs to verify using any operator" do
    41.must_be(:<, 42).must_equal true
    proc { 42.must_be(:<, 41) }.must_raise MiniTest::Assertion
  end

  it "needs to catch an expected exception" do
    @assertion_count = 2

    proc { raise "blah" }.must_raise RuntimeError
    proc { raise MiniTest::Assertion }.must_raise MiniTest::Assertion
  end

  it "needs to catch an unexpected exception" do
    @assertion_count = 2

    proc {
      proc { raise MiniTest::Assertion }.must_raise(RuntimeError)
    }.must_raise MiniTest::Assertion
  end

  it "needs raise if an expected exception is not raised" do
    @assertion_count = 2

    proc { proc { 42 }.must_raise(RuntimeError) }.must_raise MiniTest::Assertion
  end

  it "needs to be able to catch a MiniTest::Assertion exception" do
    @assertion_count = 2

    proc { 1.wont_equal 1 }.must_raise MiniTest::Assertion
  end

  it "needs to verify using respond_to" do
    42.must_respond_to(:+).must_equal true
    proc { 42.must_respond_to(:clear) }.must_raise MiniTest::Assertion
  end

  it "needs to verify identity" do
    1.must_be_same_as(1).must_equal true
    proc { 1.must_be_same_as 2 }.must_raise MiniTest::Assertion
  end

  it "needs to verify throw" do
    @assertion_count = 6

    proc { throw :blah }.must_throw(:blah).must_equal true
    proc { proc { }.must_throw(:blah) }.must_raise MiniTest::Assertion
    proc { proc { throw :xxx }.must_throw(:blah) }.must_raise MiniTest::Assertion
  end

  it "needs to verify inequality" do
    42.wont_equal(6 * 9).must_equal false
    proc { 1.wont_equal 1 }.must_raise MiniTest::Assertion
  end

  it "needs to verify mismatch" do
    @assertion_count = 6
    "blah".wont_match(/\d+/).must_equal false
    proc { "blah".wont_match(/\w+/) }.must_raise MiniTest::Assertion
  end

  it "needs to verify non-nil" do
    42.wont_be_nil.must_equal false
    proc { nil.wont_be_nil }.must_raise MiniTest::Assertion
  end

  it "needs to verify non-identity" do
    1.wont_be_same_as(2).must_equal false
    proc { 1.wont_be_same_as 1 }.must_raise MiniTest::Assertion
  end

  it "needs to be sensible about must_include order" do
    @assertion_count = 6
    [1, 2, 3].must_include(2).must_equal true
    proc { [1, 2, 3].must_include 5 }.must_raise MiniTest::Assertion
  end

  it "needs to be sensible about wont_include order" do
    @assertion_count = 6
    [1, 2, 3].wont_include(5).must_equal false
    proc { [1, 2, 3].wont_include 2 }.must_raise MiniTest::Assertion
  end
end


A diverse range of glycoconjugates exists in nature^[@R1]^. These glycoconjugates play fundamental roles in cell structure, signaling processes, and cell-cell recognition, but their molecular mechanisms are challenging to study due to a lack of suitable chemical tools^[@R2]^. Notably missing are selective small molecule inhibitors for glycosyltransferases, the enzymes that assemble glycoconjugates from carbohydrate building blocks^[@R3]--[@R6]^. Most Gtfs transfer a sugar from an anionic leaving group -- for example, a nucleotide -- to an acceptor such as another sugar, a protein, or a lipid head group^[@R7]^. Efforts to identify Gtf inhibitors have focused primarily on the design of substrate or bisubstrate mimics^[@R8]--[@R10]^. A major hurdle has been finding suitable replacements for the anionic phosphates^[@R11]--[@R13]^. These phosphates contribute significantly to binding affinity and replacing them with neutral linkers usually results in weak inhibitors. On the other hand, retaining the phosphates typically prevents the inhibitors from getting into the cells. In a clever way around this dilemma, an approach has been developed to feed cells protected sugar analogs that are metabolized into non-hydrolyzable nucleotide-sugar donors^[@R14]^. This method allows polar donor analogs to be used as inhibitors in cells, but it offers limited opportunities to tune selectivity since the inhibitors produced resemble common cellular substrates. Thus, alternative approaches to identify cell permeable Gtf inhibitors are still needed.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential vertebrate Gtf that β-O-GlcNAcylates a wide variety of nuclear and cytoplasmic proteins, including transcription factors, cytoskeletal proteins, metabolic enzymes, kinases, phosphatases, proteasome components, chaperones, and neural proteins^[@R15]--[@R17]^. OGT-mediated glycosylation is dynamic; there is a corresponding glycosidase, OGA, which removes O-GlcNAc residues from proteins^[@R18],[@R19]^. The glycosylation/hydrolysis process, known as O-GlcNAc cycling, is sensitive to stress conditions and nutrient status, particularly glucose levels^[@R20]^. OGT glycosylates many protein side chains that can otherwise be phosphorylated, and O-GlcNAcylation is proposed to modulate kinase signaling^[@R21]--[@R23]^. Hyper-O-GlcNAcylation, due to chronically high glucose levels, is correlated with widespread transcriptional changes and a number of pathologies, including cancer^[@R24],[@R25]^. Selective small molecule OGT inhibitors would be useful as probes of OGT cell biology and could validate OGT as a therapeutic target.

We previously reported a fluorescence-based high-throughput screen to identify glycosyltransferase (Gtf) inhibitors that compete with the nucleotide-sugar donor^[@R12],[@R26],[@R27]^. Using this assay, we had identified an OGT inhibitor containing a benzoxazolinone (BZX) core ([Fig. 1a](#F1){ref-type="fig"}, compound **1**); this compound was subsequently reported by others to inhibit OGT in cells^[@R28]^. We were curious to learn more about the mechanism of inhibition and to determine whether the molecule was suitable for cellular inhibition studies. Using biochemistry, mass spectrometry, and X-ray crystallography, we show here that an analog of **1**, while not yet fully optimized for work in cells, irreversibly inactivates OGT via an unprecedented mechanism in which two active site nucleophiles sequentially attack the same carbonyl to form a C=O crosslink. The dicarbamate in the inhibitor binds in the same location as the substrate diphosphate and is proposed to function as a diphosphate isostere.

RESULTS {#S1}
=======

BZX compounds irreversibly inactivate OGT {#S2}
-----------------------------------------

Compound **1** was identified in a high-throughput screen of 65,000 compounds ([Fig. 1a](#F1){ref-type="fig"})^[@R27]^. It contains a dicarbamate moiety that connects two aromatic groups. This compound features a conjugated chemical structure and both carbamate carbonyls are potentially reactive^[@R29]^. Indeed, preincubation of OGT with **1** caused an irreversible loss of enzyme activity, consistent with a covalent reaction ([Fig. 1b](#F1){ref-type="fig"}). Therefore, a small panel of related compounds was made from commercially available building blocks in order to identify structural features that might improve potency ([Fig. 1a](#F1){ref-type="fig"}). Time-dependent inactivation studies were performed in which OGT was preincubated with a three-fold excess of compound for 5 min, and the mixture was then diluted 100-fold into buffer containing UDP-^14^C-GlcNAc and the well characterized peptide substrate, CKII^[@R30]^. Enzyme activity was measured using a previously developed peptide capture assay^[@R27]^. Several inhibitors showed an irreversible reduction in enzymatic activity ([Fig. 1b](#F1){ref-type="fig"}), with the largest effect observed for compound **2**, which contains a ketone on the BZX core and a *p*-methoxy group on the phenyl ring.

An inactivation time course showed that **2** fully inactivated OGT within 5 minutes at a 1:1 ratio of inhibitor:enzyme ([Supplementary Fig. 1](#SD1){ref-type="supplementary-material"}). The *k*~obs~/\[I\] value for inactivation of purified OGT was found to be 45 min^−1^ mM^−1^ ([Supplementary Fig. 2](#SD1){ref-type="supplementary-material"}). In contrast, closely related analog **6** exhibited no inhibition, implying that enzyme inactivation by BZX compounds is not due to non-specific effects such as aggregation. The presence of UDP-GlcNAc in the preincubation mixture reduced the rate of inactivation, suggesting that binding of the substrate competes with inhibitor binding ([Supplementary Fig. 3](#SD1){ref-type="supplementary-material"}).

We compared the effects of compounds **1** and **2** on O-GlcNAcylation levels in cells and cell lysates supplemented with OGT and UDP-GlcNAc. Western analysis showed inhibition of O-GlcNAcylation by **2**, but not **1**, under both sets of conditions ([Supplementary Fig. 4](#SD1){ref-type="supplementary-material"}). These results are consistent with studies on purified enzyme showing better inhibitory activity for **2** compared to **1**. In addition, we tested compounds **1** and **2** on two other Gtfs, ppGalNAcT2^[@R31]^ and MurG^[@R12]^. Neither **1** nor **2** inactivated ppGalNAcT2 up to high micromolar concentrations ([Supplementary Fig. 5](#SD1){ref-type="supplementary-material"}), but a three-fold excess of **1** inactivated MurG by 80% while **2** inactivated it by 40% ([Supplementary Fig. 6](#SD1){ref-type="supplementary-material"}). Since altering the substituents on the BZX scaffold can improve its inhibitory properties, we studied the mode of inhibition of **2** to enable future efforts to design better OGT inhibitors.

MS analysis shows crosslinks to lysine and cysteine {#S3}
---------------------------------------------------

To identify possible covalent adducts, we analyzed intact protein mass spectra of an OGT construct after incubation with **2** at a 1:1 ratio. The construct has the same catalytic activity towards peptide substrates as the full length enzyme, but its N-terminal domain is truncated^[@R32]^, which is advantageous for MS analysis. Following incubation, the enzyme mixture was analyzed using LC-ESI-Q-TOF-MS. Two covalent adducts were detected ([Fig. 1c](#F1){ref-type="fig"}). The major adduct had a mass increase of +26 Da, consistent with the incorporation of C=O and the loss of two protons. The minor adduct had a mass increase of +176 Da, and a possible structure for this adduct is shown ([Fig. 1c](#F1){ref-type="fig"}). The major modification can only arise by sequential attack of two active site nucleophiles on the same carbonyl of **2** ([Supplementary Fig. 7](#SD1){ref-type="supplementary-material"}).

To identify the side chains that react with **2**, we analyzed tryptic digests of OGT following treatment with inhibitor. We observed a triply charged 34 residue peptide (S~823~QYGLPEDAIV-YCNFNQLY[K]{.ul}IDPSTLQMWANILK~856~) in digests of incubated protein, which was not seen in the control sample. Based on the b and y ions generated from CID fragmentation ([Supplementary Fig. 8](#SD1){ref-type="supplementary-material"}), the +26 and +176 modifications were both assigned to the same active site residue, K842 (underlined), which plays an essential role in catalysis (see below). K842 is a trypsin cleavage site and its modification blocks proteolysis, explaining the appearance of a new, longer peptide fragment in the treated protein. When incubated with OGT at a 1:1 ratio, compound **1** also modifies K842, but unlike **2** it labels some surface cysteine residues, consistent with its weaker OGT inhibition ([Supplementary Fig. 9](#SD1){ref-type="supplementary-material"}).

We confirmed the covalent modification site of **2**-treated OGT by MS analysis of a K842A mutant. Neither the +26 nor the +176 modifications were present in the alanine mutant ([Fig. 1d](#F1){ref-type="fig"}); however, we observed a new +150 Da mass peak, which mapped to C835 in the OGT active site ([Supplementary Fig. 10](#SD1){ref-type="supplementary-material"}). Based on the identification of K842 as the primary active site nucleophile, combined with the finding that +150 adducts are the major modifications in its absence, we propose a double displacement mechanism in which the acyclic carbonyl is the primary site of attack and the BZX moiety is the preferred leaving group ([Fig. 2](#F2){ref-type="fig"}). Consistent with the proposed mechanism, the leaving groups generated upon incubation of **2** with OGT are 4-methoxyphenol and 6-acetyl-2-benzoxazolinone in a near 1:1 ratio ([Supplementary Fig. 11](#SD1){ref-type="supplementary-material"}).

Another active site nucleophile in addition to K842 must react with **2** in order to form the C=O crosslink. We speculated that mutation of the crosslinking partner would result in accumulation of a +150 adduct on K842, corresponding to a single displacement on the acyclic carbonyl. To identify the crosslinking partner, we individually mutated all residues containing a nucleophilic side chain within 10 Å of K842 in the active site of OGT, which included Y841, C911, T914, C917, T921, and T922. We also mutated C835 since it is labeled in the K842A mutant, although it is more than 10 Å away from K842. From analysis of the intact protein MS for each mutant after incubation with **2** at a 1:1 ratio, three different outcomes were observed: 1) the mutation had no effect compared with WT enzyme, *e.g.*, Y841A and C835A ([Supplementary Fig. 12a](#SD1){ref-type="supplementary-material"}); 2) the mutation appeared to shift the ratio of the +26 and +176 modifications, *e.g.*, T921A and T922A ([Supplementary Fig. 12b](#SD1){ref-type="supplementary-material"}); or 3) the mutation altered the product ratio and produced a new +150 mass peak ([Supplementary Fig. 12c](#SD1){ref-type="supplementary-material"}). Since only C917A led to significant accumulation of a +150 adduct on K842 ([Supplementary Fig. 13](#SD1){ref-type="supplementary-material"}), we concluded that this side chain is the major crosslinking partner.

A crystal structure confirms the crosslink {#S4}
------------------------------------------

The enzyme was incubated with two equivalents of **2** and then crystallized in the presence of UDP and CKII under the same conditions as reported previously for the OGT-UDP-CKII complex^[@R32]^. Trapezoidal crystals diffracting to 1.88 Å were obtained ([Supplementary Table 1](#SD1){ref-type="supplementary-material"}). The inhibitor-treated and untreated OGT-UDP-CKII structures are similar, but in the inhibitor-treated structure the 2Fo-Fc omit map shows new electron density connecting K842 and C917 ([Fig. 3a and 3b](#F3){ref-type="fig"}). The C=O and lysine side chain occupancy in the crosslinked structure refined to approximately 50%; the remaining occupancy corresponds to the unlinked lysine. The crystal structure shows that the side chain C-N bond of K842 rotates 120° towards the C917 thiol, which moves it away from the β-phosphate with which it normally interacts ([Fig. 3c](#F3){ref-type="fig"}). The new density and rotation of the amine side chain to enable crosslink formation with C917 confirm the proposed double displacement mode of inhibition.

The dicarbamate is a predicted neutral diphosphate mimic {#S5}
--------------------------------------------------------

We used Glide computational modeling software^[@R33]--[@R35]^ to dock the pre-reactive **2** to OGT in order to obtain more insight into how the molecule may bind. Docking was carried out using protein coordinates from the OGT-UDP-CKII structure (PDB 3PE4) after removal of ligands. Computational experiments were conducted independently with 50 different starting poses of the inhibitor. The pose of the top-scoring ligand from each experiment was compared to the position of the nucleotide sugar. Despite some differences in the details, the dicarbamate of the inhibitor aligned with the substrate diphosphate in all fifty cases, with the side chain amine of lysine 842 positioned between the two carbonyls ([Supplementary Fig. 14](#SD1){ref-type="supplementary-material"}). A representative pose highlights the overlap between the dicarbamate and diphosphate in the binding site ([Fig. 4](#F4){ref-type="fig"}). The docking models are consistent with reaction on K842 as well as MS data showing that mutation of residues flanking the diphosphate binding site perturb the major and minor product ratios, likely reflecting changes in inhibitor binding ([Supplementary Fig. 12b](#SD1){ref-type="supplementary-material"}). Based on the combined results, we conclude that the dicarbamate element functions as a neutral diphosphate mimic.

DISCUSSION {#S6}
==========

Small molecule inhibitors of OGT have been sought for many years as cellular probes and different approaches to identify such inhibitors have been explored^[@R14],[@R36],[@R37]^. OGT is a challenging target because it is a nucleotide-sugar glycosyltransferase and the donor sugar substrate contains a diphosphate leaving group. The proposed transition state for these types of enzymes is dissociative and the oxonium ion-like portion resembles the transition state of glycosidases^[@R38],[@R39]^. A key difference in glycosidase and Gtf transition states, however, is that in the latter a negatively charged diphosphate leaving group rather than a carboxylate side chain helps stabilize the oxonium character^[@R7]^. Accordingly, although there are many good inhibitors of glycosidases, including OGA^[@R19],[@R40]--[@R42]^, they typically do not inhibit Gtfs effectively. Efforts focusing on substrate-based inhibitors of Gtfs have been unsuccessful because identifying isosteres that mimic the negatively charged diphosphate is a major hurdle. To discover new types of inhibitors, we developed a high-throughput glycosyl donor displacement screen for OGT in which compound **1** was identified^[@R27]^.

A small panel of related inhibitors having different substituents from **1** was prepared and inhibition was compared under a standard set of conditions ([Fig. 1](#F1){ref-type="fig"}). Compound **2** was the best compound in preliminary studies, inactivating OGT within 5 min at a 1:1 ratio. None of the other compounds reached full inhibition at this low ratio, and all of the others, including **1**, showed some labeling of surface cysteine side chains ([Supplementary Fig. 9](#SD1){ref-type="supplementary-material"}). Therefore, compound **2** can fully inactivate OGT in vitro by modifying the active site without reacting non-specifically with other residues. Since **2** contains the most electron-donating *para* substituent of the compounds tested, we speculate that decreased reactivity of the acyclic carbonyl is an important factor in its improved active site selectivity. This is consistent with the observation that **2** is less labile to hydrolysis than **1** at neutral pH.

In studies to probe the potential of this family of inhibitors further, compounds **1** and **2** were tested for inactivation of two other Gtfs, ppGalNAcT2 ([Supplementary Fig. 5](#SD1){ref-type="supplementary-material"}) and MurG ([Supplementary Fig. 6](#SD1){ref-type="supplementary-material"}). The former was chosen as a representative member of the mucin family of O-glycosyltransferases, which transfers N-acetylgalactosamine (GalNAc) to Ser/Thr residues of proteins and thus resembles OGT in O-glycosylating proteins^[@R31]^; the latter is an essential bacterial GlcNAc transferase involved in bacterial cell wall biosynthesis, which has a very similar three dimensional structure to OGT^[@R12]^. Neither compound inhibited ppGalNAcT2, but the two compounds showed differential inhibition of OGT and MurG respectively, which demonstrates that altering substituents on the BZX scaffold can increase specificity towards OGT. Further manipulation of substituents on the dicarbamate scaffold may improve inhibition properties, and we undertook mechanistic studies to provide a basis for rational approaches to improve inhibitors for use in cells.

Using a combination of MS analysis, mutational studies, and X-ray crystallography, we deduced that **2** reacts via a double displacement mechanism on the same dicarbamate carbonyl to form a C=O crosslink between two active site nucleophiles, K842 and C917. Inhibitors that react sequentially with two nucleophiles in the active site of an enzyme are very rare and there are no other examples involving displacement on the same carbon atom^[@R43],[@R44]^. Manipulation of carbonyl reactivity and leaving group properties can potentially be exploited in designing inhibitors.

In addition to revealing an unusual reaction mechanism, our studies suggest that the five-heteroatom core of the dicarbamate class of inhibitors functions as a diphosphate isostere. This proposal is based on several lines of evidence. First, **2** reacts with the essential catalytic lysine residue that stabilizes the glycosyl donor diphosphate^[@R45]^, indicating that it binds in the same location. Second, the proposed binding site is consistent with protection from inactivation by UDP-GlcNAc. Third, mutations in residues that flank the diphosphate binding site perturb the ratios of the major and minor products observed upon reaction of **2**, consistent with its binding at the same site. Finally, computational studies show that the dicarbamate docks into the diphosphate binding site, and superpositions of the nucleotide and inhibitor show that the heteroatoms are aligned. We point out that the five-heteroatom core of the dicarbamate scaffold has very limited rotational mobility, and the accessible conformations have an architecture similar to the OGT-bound conformation of the UDP-GlcNAc diphosphate.

Dicarbamate scaffolds related to those reported here may serve as starting points for cellular probes for other enzymes that utilize diphosphate-containing substrates and have suitable active site architecture. The dicarbamate motif has a notable advantage over a diphosphate because it is uncharged and hence more likely to cross cell membranes. Indeed, we have shown that **2** inhibits global O-GlcNAcylation in cells, consistent with cell permeability. The concentration of **2** required for inhibition of O-GlcNAcylation in cells is relatively high, possibly due to decomposition and side reactions. The mechanistic understanding provided by the studies reported here will enable rational approaches to tune reactivity and binding in order to obtain analogs with better cellular efficacy.

METHODS {#S7}
=======

Protein purification {#S8}
--------------------

The expression and purification of the hOGT~4.5~ construct (spanning residues 313--1031 based on the numbering of the full length human protein) were performed following previously described protocols^[@R32]^. Mutations were introduced using the Stratagene QuikChange II XL Site-Directed Mutagenesis Kit and DNA from plasmid of hOGT~4.5~ as template in combination with the mutagenic primers listed in [Supplementary Table 2](#SD1){ref-type="supplementary-material"} (nucleotide substitutions are underlined). The mutants were purified similarly as WT protein.

Time-dependent inactivation of OGT with BZX compounds {#S9}
-----------------------------------------------------

BZX compounds were incubated with OGT (5 µM) in reaction buffer (125 mM NaCl, 1 mM EDTA, 20 mM potassium phosphate, pH 7.4, and 500 µM tris(hydroxypropyl)phosphine (THP)) at room temperature for varying lengths of time, then diluted 100-fold, mixed with substrates (6 µM UDP-^14^C-GlcNAc and 500 µM CKII peptide (KKKYPGGSTPVSSANMM)) and allowed to react for another 2 h. Enzyme activity was tested with the peptide capture assay as previously reported^[@R27]^. The final volume of each reaction was 20 µl and the experiments were conducted in triplicate. The activities of OGT mutants were also tested in triplicate as previously reported^[@R27]^.

Intact protein mass spectrometry {#S10}
--------------------------------

Each of the BZX compounds (10 µM) was incubated with OGT (10 µM) at room temperature for 5 min in the reaction buffer containing PBS (pH 7.5) and 100 µM THP, in a final volume of 20 µl. After incubation, 50 µl of PBS (pH 7.5) was added to each reaction, and the total 70 µl of mixture was passed through a PD SpinTrap G-25 column (GE Healthcare, pre-equilibrated with PBS, pH 7.5) to remove the non-enzymatic fraction. Eluted sample (40 µl each) was injected in an Agilent 6520 Q-TOF LC-MS. The mobile phase flow rate was set at 0.4 ml/min through a C4 column (Grace, Vydac 214MS C4 5u, 100 × 2.1 mm) which was initially equilibrated with solvent A (0.1% aqueous formic acid). The proteins were eluted using a linear gradient of 0 to 95% solvent B (90% acetonitrile and 0.1% aqueous formic acid) over 25 min. Mass spectrometric analyses were carried out in positive ion mode with an ESI source. The MS spectra were deconvoluted using Agilent MassHunter BioConfirm B.02.00 software with the maximum entropy algorithm.

Trypsin digestion and peptide mass spectrometry {#S11}
-----------------------------------------------

Reaction mixture containing OGT (15 µM), **2** (200 µM), NH~4~HCO~3~ (25 mM, pH 7.8), and THP (100 µM) in a final volume of 23 µl was incubated for 30 min at room temperature. To avoid complications with over alkylation, cysteines were not "capped" and the formation of disulfides was prevented by the presence of THP. Trypsin was added to a final OGT:trypsin concentration ratio of 6:1 (w/w) and the reaction was incubated for 4 h at 37 °C. The peptides were separated using an Agilent 6520 LC/QTOF system equipped with a Phenomenex Gemini-NX C18 column (5 µm, 110 Å, 50 × 2.00 mm, pre-equilibrated with 0.1% aqueous formic acid) at a flow rate of 0.4 ml/min with a linear gradient of 0 to 60% buffer B (90% acetonitrile and 0.1% aqueous formic acid) over 50 min. Mass spectra were acquired from *m/z* 300--3200 for 0.25 s, followed by MS/MS scans from *m/z* 50--3200 of the 8 most intense species from the preceding MS scan for 0.25 s each in positive ion mode with collision energy fixed at 35 V. All spectra were processed with Mascot Distiller (Matrix Sciences) to generate the peak lists. Database searches were performed with Mascot Server 2.3 (Matrix Sciences). Error tolerant search was performed following the standard search to maximize the matches. The significance threshold was set p \< 0.05, and the false discovery rate based on a decoy database was below 2%.

Crystallization and crystal structure determination {#S12}
---------------------------------------------------

A full description is in [Supplementary Methods](#SD1){ref-type="supplementary-material"}. Briefly, inhibitor crystals were grown with the hanging drop method with hOGT~4.5~, seeded from native OGT-UDP-CKII crystals. The space group of OGT-**2**-UDP-CKII complex was I121, the same as the original OGT-UDP-CKII complex (PDB 3PE4). The data set was processed to a resolution of 1.88 Å ([Supplementary Table 1](#SD1){ref-type="supplementary-material"}). The R~free~ test set array was copied and extended from the dataset for the 3PE4 coordinates. The refinement process was initiated with 3PE4 coordinates, which were adjusted by rigid body refinement, followed by several rounds of simulated annealing and atomic displacement refinement in Phenix^[@R46]^, and modeling in COOT^[@R47]^. The structure was further refined with Buster^[@R48]^, where the crosslink was modeled in with stereochemical restraints and occupancy refinement (See [Supplementary Methods](#SD1){ref-type="supplementary-material"}). The final R~work~/R~free~ were 21.91% / 23.75%.

Molecular docking {#S13}
-----------------

All molecular docking was done in Schrödinger Suite 2010. The protein receptor was prepared in Protein Preparation Wizard using the hOGT~4.5~ crystal structure containing UDP and CKII peptide (PDB 3PE4): all water molecules and ligands were removed and hydrogen atoms were added. The docking grid was calculated in Glide v5.6^[@R33],[@R34]^: the rectangular docking grid (20 Å × 20 Å × 24 Å) was centered using the UDP α-phosphate. Ligands (UDP, UDP-GlcNAc, and BZX derivatives) were prepared using Ligprep 2.0 and Epik tools in Glide, at pH 7.0 ± 2.0. Ligands and hOGT~4.5~ were parameterized with the OPLS-2005 force field. All ligands were docked in Glide XP mode^[@R35]^. Molecular docking was conducted independently with 50 different starting poses of each compound. Ligand poses were analyzed in Maestro and Pymol^[@R49]^.

Supplementary Material {#SM}
======================

We thank J. Brugge (Harvard Medical School) for the MCF-10A ErbB2 cells, L. Tabak (NIH) for ppGalNAcT2 protein, T. Lupoli (Harvard University) for MurG protein, C. Walsh (Harvard Medical School), E. Jacobson and D. Ford (Harvard University) for helpful discussions, and A. Saghatelian (Harvard University) for critical reading of the manuscript. LC/MS data was acquired on an Agilent 6520 Q-TOF spectrophotometer supported by the Taplin Funds for Discovery Program (P.I., S. Walker). This work was supported in part by the NIH (GM076710), and the Harvard Biomedical Accelerator Fund.

**Author contributions**

S.W. oversaw all aspects of the experiments and manuscript preparation. J.J. carried out all experiments except those involving X-ray crystallography and molecular docking. M.B.L. obtained OGT:inhibitor crystals, acquired data, and solved the structure of the crosslinked protein. L.P. conducted molecular docking experiments. P.S. guided refinement of the X-ray crystal structure and molecular docking. J.J. and S.W. wrote the manuscript and all co-authors participated in figure preparation and editing.

**Competing financial interests**

The authors declare no competing financial interests.

**Additional information**

Supplementary information and chemical compound information is available online at <http://www.nature.com/naturechemicalbiology/>. The structure of the OGT-**2**-UDP-CKII complex has been submitted to the Protein Data Bank under the accession number 3TAX.

![Inactivation of OGT by BZX compounds\
(**a**) Chemical structure of BZX compounds **1--6**. (**b**) Histogram showing OGT inactivation for BZX compounds after a five-minute preincubation with a three-fold excess of each compound. Following dilution of the preincubation mixture (see methods), enzyme activity was tested as described^[@R27]^ and normalized to DMSO control (data represent mean values ± s.e.m., n=3). (**c**) Intact protein MS overlay of OGT treated with **2** (1:1 ratio) and DMSO control shows two covalent modifications (+26 Da and +176 Da) in the treated protein. A possible structure for each modification is shown. (**d**) Intact protein MS of **2**-treated K842A (1:1 ratio) shows that mutation of K842 to alanine abolished the +26 and +176 modifications, but a new +150 mass peak appeared and a possible structure corresponding to this adduct is shown.](nihms321950f1){#F1}

![Proposed double displacement mechanism for the reaction of 2 with OGT\
Mutation of K842 to alanine abolished the formation of the +26 modification and resulted in a +150 modification on a distant cysteine in the active site. A simplified double displacement mechanism to form the crosslink with K842 as the primary nucleophile first attack on the acyclic carbonyl is proposed. We cannot exclude attack of the thiol first. A more complex scheme involving several possible reaction pathways and different intermediates that produce a C=O crosslink is in [Supplementary Figure 7](#SD1){ref-type="supplementary-material"}.](nihms321950f2){#F2}

![Active site comparison of crosslinked and uncrosslinked OGT\
(**a**) 2Fo-Fc omit map, contoured at 1 σ, of OGT-**2**-UDP-CKII (map calculated to 1.88 Å) shows the C=O crosslink bridging K842 and C917 together on Chain C. The reactive nitrogen and sulfur from OGT are indicated in blue (N) and yellow (S), respectively. An unbiased map was calculated with Buster^[@R48]^ from a model lacking both the crosslink and the reacting cysteine and lysine. The non-crosslinked lysine is shown in magenta, modeled in as an alternate conformation since 50% of protein is crosslinked, based on the occupancy refinement. The alternate conformation is similar to the confirmation of the lysine in the native structure. (**b**) 2Fo-Fc omit map, contoured at 1 σ, of OGT-UDP-CKII (PDB 3PE4, 1.95 Å)^[@R32]^ does not show density connecting K842 and C917. (**c**) The crosslinked active site. The OGT-**2**-UDP-CKII structure is shown here as a surface representation with the critical lysine crosslink highlighted (green). The CKII peptide (yellow) and UDP (green) are bound in the same conformations as in the absence of inhibitor. The uncrosslinked K842 (grey) has been overlaid to show where it normally is positioned to contact the β-phosphate. K842 must rotate 120° away from the phosphate in order to form the crosslink. The inferred H-bond between uncrosslinked K842 side chain and β-phosphate of UDP is shown with a dashed line.](nihms321950f3){#F3}

![The dicarbamate docks into the same site as the diphosphate\
Overlay of UDP (PDB 3PE4, pink) in the OGT active site with top-scoring poses of docked inhibitor **2** (blue) and UDP-GlcNAc (yellow) shows that the dicarbamate of **2** binds in the same location as the diphosphate. Oxygen atoms of the dicarbamate and the diphosphate are highlighted with different colors. The side chain amine of K842 (green) is located between the dicarbamate carbonyls, which are sandwiched by two neighboring threonines (T921 and T922) in the diphosphate binding site.](nihms321950f4){#F4}

[^1]: These authors contributed equally to this work


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  devServer: {
    contentBase: './docs'
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    app: './src/docs.js'
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Q:

Safe to wash rice the night before and leave overnight before cooking?

I'm trying to figure out a convenient way to bring fresh rice for lunch. Cooking it the night before and leaving it in the fridge causes it to become dry and hard and unpleasant, and I've read that it's not safe to leave cooked rice out at room temperatures for long periods of time (such as overnight).
So I thought it would be nice if I could at least save myself the trouble of having to wash it in the morning. Would it be safe to wash it the night before, and cook it in the morning? If so, what storage methods should I use? Just leave it out on the countertop? Or should I leave it soaking in water? Or either dry or soaking in an air-tight container? Or should it be refrigerated? I'd also like to hear any methods anybody else has for having fresh rice at lunch!
I should mention I'm referring to short grain Japanese white rice. Thanks!

A:

I did this in my restaurant for years, it really does work great. Make your rice in a huge batch, cook it as if you're planning to eat it then, but then let it cool until handlable. Once it is cool enough, bag it in individual servings and freeze it. Refrigerating cooked rice quickly ruins it, freezing it, however, works great. If your servings are fairly small, snack sized ziplocks work great for this, otherwise use sandwich sized. Make sure you press the air of the bags, I take all of the small bags and seal them together in gallon sized ziplocks to give them one more layer of protection in the freezer. Either with the rice solidly frozen, or at lunchtime (defrosted, cool rice), throw the baggie in the microwave. You don't even have to open the baggie, when it pops open that's a sign to check it. It may need a bit of a mix and a few more seconds in the microwave, or it may be done, it just needs to be heated through. The hot rice will be almost indistinguishable from freshly cooked. If you don't have a microwave, you can drop the sealed baggie into simmering water, or take the frozen or cool rice out of the bag and steam it to heat.

A:

My understanding from now far-in-my-past food safety courses is that uncooked rice does not have the same risk of bacterial growth even if soaked in water at room temperature. However, after about 24 hours, it does start to do things that are reminiscent of sprouting (though white rice won't actually sprout), including grassy aromas.
Most Japanese families with automatic rice cookers who eat rice in the morning place washed rice in their rice cooker with the appropriate amount of water and set a timer. I doubt there are frequent occurrences of food-borne illness directly attributable to this method, but cooked rice left in the danger zone is known to be a great breeding ground for unpleasant micro-critters.
Typically the soaking time used in these situations is 6-12 hours.


Q:

Combining Unknown number of Observables in RxJava

I have a situation where I have to create N Observable objects based on a request. I understand that I can use zip to combine a KNOWN number of Observables. But, I'm stuck trying to understand how I can combine an unknown number of Observable.
The part where I'm not clear is what function to pass to zip. Depending on the number of Observables, I have to create a lambda that takes N arguments.
All the Observable return different types of objects.

A:

Update:
I ended up with the normal way to solve parallel request list.
Just use flatMap, merge, zip, any combining rx operator.
The only thing we need to do specially is to use .subscribeOn(Schedulers.io()) for each request. All other things, sending parallel requests or simultaneous would be arranged by rxjava perfectly.
If you want to see the effect, try following:
private void runMyTest() {
    List<Single<String>> singleObservableList = new ArrayList<>();
    singleObservableList.add(getSingleObservable(500, "AAA"));
    singleObservableList.add(getSingleObservable(300, "BBB"));
    singleObservableList.add(getSingleObservable(100, "CCC"));
    Single.merge(singleObservableList)
            .subscribeOn(Schedulers.io())
            .observeOn(AndroidSchedulers.mainThread())
            .subscribe(System.out::println); }

private Single<String> getSingleObservable(long waitMilliSeconds, String name) {
    return Single
            .create((SingleOnSubscribe<String>) e -> {
                    try {
                        Thread.sleep(waitMilliSeconds);
                    } catch (InterruptedException exception) {
                        exception.printStackTrace();
                    }
                    System.out.println("name = " +name+ ", waitMilliSeconds = " +waitMilliSeconds+ ", thread name = "
+Thread.currentThread().getName()+ ", id =" +Thread.currentThread().getId());
                    if(!e.isDisposed()) e.onSuccess(name);
                })
            .subscribeOn(Schedulers.io()); }

output:

System.out: name = CCC, waitMilliSeconds = 100, thread name =
  RxCachedThreadScheduler-4, id =463
System.out: CCC
System.out: name = BBB, waitMilliSeconds = 300, thread name =
  RxCachedThreadScheduler-3, id =462
System.out: BBB
System.out: name = AAA, waitMilliSeconds = 500, thread name =
  RxCachedThreadScheduler-2, id =461
System.out: AAA

// ******previous answer but not accurate*******//
Use this one; solved my problem:
zip(java.lang.Iterable<? extends Observable<?>> ws,FuncN<? extends R> zipFunction) method.
An sample:
public Observable<CombinedData> getCombinedObservables() {
        List<Observable> observableList = new ArrayList<>();

        observableList.add(observable1);
        observableList.add(observable2);
        observableList.add(observable3);
        observableList.add(observable4);

        return Observable.zip(observableList, new Function<Object[], CombinedData>() {
            @Override
            public CombinedData apply(Object[] objects) throws Exception {
                return new CombinedData(...);
            }
        });
    }


Shigella apyrase--a novel variant of bacterial acid phosphatases?
A virulence-associated ATP diphosphohydrolase activity in the periplasm of Shigella, identified as apyrase, was found to be markedly similar to bacterial non-specific acid phosphatases in primary structure. When the Shigella apyrase sequence was threaded in to the recently published 3D structure of the highly similar (73%) Escherichia blattae acid phosphatase it was found to have a highly overlapping 3D structure. Our analysis, which included assays for phosphatase, haloperoxidase and catalase activities, led us to hypothesize that Shigella apyrase might belong to a new class of pyrophosphatase originating as one more variant in the family of bacterial non-specific acid phosphatases. It revealed interesting structure-function relationships and probable roles relevant to pathogenesis.


"It wouldn't surprise me if he's already thinking about engagement just because that's where his head goes, but that's not where the relationship is at right now," a source said to E! Newsof His Royal Ginger-ness' recent stay at Markle's suburban Toronto home. "This is totally Harry. The problem with Harry is when he falls, he falls hard. He goes in big time."

At least it's not one-sided, though, with a different tipster telling Us Weekly that Markle has met and impressed Prince Harry's "closest circle of friends," and even gone on a double date with his cousin Princess Eugenie.