respectively. The resulting constructs pRR14 and pRR15 were transferred in E.coliDH5α. Through triparental mating using pRK600 helper plasmid the construct were transferred in E.coliS17-1. After confirming pRR14 and pRR15 constructs by sequencing, the constructs were then introduced into BXOR1 strain through biparental mating using E. coliS17-1. X. oryzaepv. oryzicolaGUS and GFP reporter strains were selected on PS medium plates containing suitable antibiotics. Since pVO155 cannot replicate in X. oryzaepv. oryzae, ampicillin and kanamycin-resistant colonies were obtained upon chromosomal integration of the plasmid using the cloned DNA sequence as a region of homology. pProbeGTcan replicate independently in Xanthomonasand report for the gene expression

Glucuronidase (GUS) reporter gene fusion and GFP reporter fusion was created by using the suicide plasmid pVO155 having a promoterless gusAgene (Oke and Long, 1999), and pProbeGThaving a promoterless GFP (Miller et al., 2000). To construct the xsuA::gusAand xsuA::gfptranscriptional fusion, a 611-bp DNA fragment containing the putative promoter of the xssoperon (+213 to −398) was amplified by using the SCRsid_ pProbeGFP_F and SCRsid_ pProbeGFP_R primers (Table 2.2). This promoter fragment was subsequently digested with HindIII and BamHI,and directionally cloned upstream of the promoterless gusAand gfpgene in pVO155 and pProbeGTplasmids to create the xsuA::gusAand xsuA::pProbeGT(gfp) reporter constructs pRR1

Construction of xsuA::gusAand xsuA::gfp strains in X. oryzaepv. oryzicola background

To perform immunoblotting or western blotting, appropriate amounts of total protein(ranging from 20-40 μg) were separated ona SDS-PAGE gel of 12%acrylamide concentration in Tris-Glycine-SDS gel running buffer. Protein separation was done at 70-100 V for 2-3 h using a MINI PROTEAN®3 electrophoresis unit (Bio-Rad). Followingseparation, proteins weretransferredto polyvinylidene difluoride (PVDF) membrane, using a Bio-Rad Mini Trans-Blot electrophoretic transfer unit in Tris-Glycine transfer buffer at 4⁰C. Before setting transfer assembly, PVDF membrane was first activated in 100% methanolfollowedby washesin the transfer buffer. The transfer assembly was set inaBio-Rad Mini gel holder cassette (170-3931)according to manufacturer’s instructions. The transfer time and current settings varied depending on the size oftheprotein of interest. Post transfer, membranes wereseparated from the assembly and kept for blocking intheblocking buffer (0.1 % Tween-20, 5% w/v fat-free skimmed milk in 1X TBS) for 1 h at room temperature with shaking. Next,membranes wereincubated with appropriate dilutions of primary antibodiesin the blocking buffereitherfor 3-4 h at room temperature or overnight at 4°C with gentle shaking. Post incubation,membranes were washed thrice with 1X TBS-T, 10 min each,with constant agitation. After washes, membraneswere incubated with appropriate dilutions of secondary antibodiesconjugated with horseradish peroxidase (HRP) for 1 h at room temperature with gentle shaking. Next, membranes were washed thrice with 1X TBS-T, 10 min each,with constant agitation. To visualize proteins, membranes were removed from TBS-T, and theHRP substrate ECL plus (Amersham Biosciences, RPN2232) was uniformly added on top of the membrane. Chemiluminescent signalswere captured in the western blot imaging system (FluorChemTME system)

For protein extraction, cells were spun down at 4,000 rpm for 5 min and washed with ice-cold water. The cell pellet was resuspended in 250-500 μl of homogenisation buffer which contained50 mM Tris (pH 7.5), 2 mM EDTA, 1 mM phenylmethylsulphonyl fluoride (PMSF) (serine protease inhibitor), 10 mM sodium fluoride (serine/threonine and acid phosphatases inhibitor), 1 mM sodium orthovanadate (Tyrosine and alakaline phosphatases inhibitor) and 1X protease inhibitor cocktail (Sigma, P 8215). The cell suspensison was transferred to a 1.5 ml centrifuge tube and equal amounts of glass beads (0.5 mm size) were added. Cells were lysed mechanically by bead-beatinghomogenizer (MP Biomedicals, FastPrep®-24) atthemaximum speed for 60 seconds, five times each,with intermittent cooling on ice.After lysis, tubes were punctured at the bottom with the helpofasurgical needle, and the lysed cell suspension was collected in a fresh microcentrifuge tubes by putting the punctured tubes on top of the fresh tubes and centrifuging them at 3,000 rpm for 10 min. The supernatant was transferred toafresh microcentrifugetubeandprotein concentrationwasestimated usingtheBCA protein assay kit (Thermo scientific). Protein preparations werestored at -20°C until use

Total protein extraction and immunoblotting

Total protein extraction and immunoblotting

the selection marker.Knockdown was confirmed by immunoblot analysis with an IP6K1 specific antibody (Table 2.3) as described in Section 2.2.10

lines were used for stable knockdown of IP6K1 expression. Viral particles harboring either non-targeting control or IP6K1directed shRNA were used to infect HeLa or HCT116 cell lines at 0.5 MOI, following treatment with polybrene (8 μg/mL)for 2 h.After 48 h, transduced cells were selected with 2 μg/mL puromycin. Medium was changed twice a week and observed for colony formation. After reaching the optimum growth, selected cells were maintained in DMEM supplemented with 10% FBS and 1 μg/mL puromycin as

Generation of stable cell lines expressing shIP6K1-HeLa and HCT116 cell

HEK293T packaging cellswere seeded at 30-40% confluency in 60 mm dishes. After 24 h, cells were co-transfected with three plasmids required for viral production i.e. VSV-G, psPAX2 (Addgene plasmid # 12260) and pLKO.1-puro-non-targeting and shIP6K1 clones using polyethyleniminereagent(PEI) and incubated at 37°C and 5% CO2 for virion formation. After 48 h, viral particles were harvested by collecting supernatant and filtered througha 0.45 μm syringe filter unit. Viral stock was aliquoted and stored at -80°C for further use. Viral titer was approximated on the number of cells plated for the production of lentivirus. Calculations were done as per Cell Bio Labs instruction. 2 x 106cells will yield 107infectious units/mL. All necessary precautions were taken while generating lentiviral particles such as wearing mask, double gloves, and sterile filter tips. All the consumables used were bleached (1% sodium hypochlorite solution) at least 1 h before being discarded

Generation of lentiviral particle containing shRNAagainst human IP6K1-

Generation of stable cells expressing shIP6K1 in HeLa and HCT116

RRID:ZFIN_ZDB-GENO-150504-12

RRID:ZFIN_ZDB-GENO-060811-12

he is at the same time, capable of becom+ ing a greater enemy,

They can evaluate web content, and identify what is useful and trustworthy

What K–12 Administrators Should Think About When Integrating Classroom Tech

bishop gave judgment, declaring the marriage null in a canonical sense. Regarded in a civil point of view, the marriage was still valid until it should have been declared null by a civil tribunal. It became necessary, therefore, to carry the cause before the Superior Court, and my honorable friend, the member for Because, who took the case in hand with his usual zeal and legal address, obtained from the court, after a suitable inquiry, a judgment declaring the marred null in a civil sense, and ordering that it should be registered as such in all places where it should be needful. If this affair had occurred in Upper Canada, what recourse would the parties have had ? The parties being Catholics, the case would have been brought before the bishop, who would also have declared the marriage null after suitable inquiry; but the cause would not have had the same conclusion in the civil court, particularly had it depended on certain impediments which have force in Lower Canada, but none in Upper Canada. It would have become necessary to go to Parliament to pray for an act, which, in a Catholic point of view, would be a mere decree of separation, but which the Parliament would have termed an act of divorce. This power to grant a separation is therefore necessarily vested in the Parliament, by whatever names such separation may be designated, and we are not to be reproached for the interpretation which others may give to such name, different from that which we assign to it. I thought it right to make myself understood on this point, because I do not choose that people should be able to say we are afraid of explaining our position with regard to the question of divorce and marriage, and I believe that I have shown that our position is consistent with our religious laws and our principles as Catholics. I regret that I have dwelt so long on the matters touched upon by the honorable member for Hochelaga ; but after his speech, and considering the position he assumed, he must have expected an answer.

HON. SOL. GEN. LANGEVIN—I beg your pardon, it means that a marriage contracted in no matter what part of the Confederacy, will be valid in Lower Canada, if contracted according to the laws of the country in which it takes place ; but also, when a marriage is contracted in any province contrary to its laws, although in conformity with the laws of another province, it will not be considered valid. Let us now examine the question of divorce. We do not intend either to establish or to recognize a new right ; we do not mean to admit a thing to which we have constantly refused to assent, but at the Conference the question arose, which legislature should exercise the different powers which already exist in the constitutions of the different provinces. Now, among these powers which have been already and frequently exercised de facto, is this of divorce. As a member of the Conference, without admitting or creating any new right in this behalf, and while declaring, as I now do, that as Catholics we acknowledge BO power of divorce, I found that we were to decide in what legislative body the authority should be lodged which we found in our Constitutions. After mature consideration, we resolved to leave it in the Central Legislature, thinking thereby to increase the difficulties of a procedure which is at present so easy. We thought then, as we still think, that in this we took the most prudent course. The following illustration will prove this still more forcibly. I t is known to the House how zealous a partisan the honorable member for Brome (Mr. DUNKIN) is of the cause of temperance. Well, we will suppose that the honorable gentleman were present as a member of a municipal council in which it was to be decided whether all the taverns in a very populous part of the parish, which could not be suppressed, should be banished to a remote corner of the parish, where they would no longer be a temptation and a stumbling-block ; would he not vote for such a measure ? Would he not send them to a place where they would be least accessible to the population, and would he not think he had done a meritorious act, an act worthy of a good friend of the temperance cause ? Just so in a question of divorce ; the case is exactly analogous. We found this power existing in the constitutions of the different provinces, and not being able to get rid of it, we wished to banish it as far from us as possible. One thing it would be vain to deny, namely, that although we, as Catholics, do not admit the liberty of divorce, although we hold the marriage bond to be indissoluble, yet there are cases in which we both admit and require the annulling of the marriage tie—in cases, for instance, where a marriage has been contracted within the prohibited degrees without the necessary dispensations. An instance of this occurred very recently. A few months since, an individual belonging to my county, who had married a young girl of a neighboring parish, without being aware at the time of his marriage of the relationship which existed between him and his wife, found out several months afterwards that they were related in such a degree that they required a dispensation from the bishop. That dispensation had not been obtained. He spoke of it to his wife, who refused to apply for a dispensation, as a step towards the legal celebration of their marriage. It became necessary, therefore, to have the marriage annulled. The affair was brought before the Ecclesiastical Court, and, after a minute investigation, the diocesan

HON. SOL. GEN. LANGEVIN—The honorable member for Verchères does not choose to be convinced ; so I will make no further attempt to convince him. The resolution in question signifies just what I have stated.

may be summed up as follows :—The Central Parliament may decide that any marriage contracted in Upper Canada, or in any other of the Confederated Provinces, in accordance with the laws of the country in which it was contracted, although that law might be different from ours, should be deemed valid in Lower Canada in case the parties should come to reside there, and vice versa.

The honorable gentleman has asked the Government what meaning was to be attached to the word ” marriage,” where it occurred in the Constitution. He desired to know whether the Government proposed to leave to the Central Government the right of deciding at what age, for example, marriage might be contracted. I will now answer the honorable gentleman as categorically as possible, for I am anxious to be understood, not only in this House, but also by all those who may hereafter read the report of our proceedings. And first of all I will prove that civil rights form part of those which, by article 43 (paragraph 15) of the resolutions, are guaranteed to Lower Canada. This paragraph reads as follows :— 15. Property and civil rights, excepting those portions thereof assigned to the General Parliament. Well, amongst these rights are all the civil laws of Lower Canada, and among these latter those which relate to marriage; now it was of the highest importance that it should be so under the proposed system, and therefore the members from Lower Canada at the Conference took great care to obtain the reservation to the Local Government of this important right, and in consent-‘ ing to allow the word ” marriage ” after the word “divorce,” the delegates have not proposed to take away with one hand from the Local Legislature what they had reserved to it by the other. So that the word ” marriage,” placed where it is among the powers of the Central Parliament, has not the extended signification which was sought to be given to it by the honorable member. With the view of being more explicit, I now propose to read how the word marriage is proposed to be understood :— The word marriage has been placed in the draft of the proposed Constitution to invest the Federal Parliament with the right of declaring what marriages shall be held and deemed to be valid throughout the whole extent of the Confederacy, without, however, interfering in any particular with the doctrines or rites of the religious creeds to which the contracting parties may belong. This is a point of great importance, and the French Canadian members ought to rejoice to see that their fellow-countrymen in the Government have not failed in their duty on a question of so serious a nature. On many other points many of them will doubtless claim that we have not thoroughly fufilled our duty, but as regards the matter in question there can be no difference of opinion, as I we have all a common rule to guide us ; and I repeat that they ought to rejoice that their co-religionists in the Conference have not been found wanting on this occasion. The whole

Remarks have also been made about the laws of divorce and marriage, and the honorable member for the division of DeLanaudière (Hon. Mr. OLIVIER) told us that the Conference had done well in transferring the power of divorce to the General Government. On his part, I think this was a wise view of the question, and I am glad to have the opportunity of now telling him so. He was, however, very uneasy about the word ” marriage.” Well, I will try to put him right and at his ease on that point; and I will give him the answer as I find it put down in writing, so that no possible misunderstanding may continue to exist. If the honorable gentleman will but take his pen, he will be able to note my answer :—” The word ‘ marriage’ has been inserted to give the General Legislature the right to decide what form of marriage will be legal in all parts of the Confederation, without in any way interfering with the rules and prescriptions of the Church to which the contracting parties belong.”

One of them is a matter in which Lower Canada is somewhat peculiarly interested— the system of marriage and divorce, which, I see, is to be left in the hands of the Federal Government.

what is meant by the regulation of the law of divorce ; but what is meant by the regulation of the marriage question ? Is the General Government to be at liberty to set aside all that we have been in the habit of doing in Lower Canada in this respect ? Will the General Government have the power to determine the degree of relationship and the age beyond which parties may marry, as well as the consent which will be required to make a marriage valid ?

What shall I say on the subject of marriage—the basis of all our institutions ? Is it not dangerous to have it at the mercy of the Federal Government ? We shall soon be told probably that it is but a sounding affair, and before long, mayors will take the place of the curés, and will celebrate the marriages of their constituents. Our laws which regulate our marriages at present are very important to us, and are based on the Roman law. These are the only laws suitable to Canadians, and the wise provisions characterizing them were the fruit of the experience of several ages. We should not incur the risk of any change in them by a legislature, the majority of whose members do not hold our opinions on this subject.

Take, for instance, the question of the fisheries. Article 17, of the 29th resolution, gives to the Federal Parliament the power of legislating on the ” sea coast and inland fisheries.” Under the 8th article of the 43rd resolution, the local legislatures will also have the right of legislating on the ” sea coast and inland fisheries.” Thus the local legislatures and the Federal Legislature will have the right to legislate on the same subjects. And if the laws they make are in opposition the one to the other, what will be the result ?

The 29th section of the scheme submitted to us says : ” The Federal Parliament shall have the power of making laws for the peace, the well-being, and the good government of the Confederate provinces, and in particular in respect of the following matters.” The powers of the Federal Government will be in reality unlimited. The fact of the enumeration of these thirty-seven heads does not in the least restrain the power of the Federal Government from legislating on everything. The exceptions are few. I would ask the Honorable Premier, for instance, whether the Federal Government has not the power to enact that marriage is a civil contract ? He cannot deny it, and I do not believe that that clause will in any way suit Lower Canada. In a matter of divorce, I consider that the power of legislating upon it ought to be vested in the Federal Government ; but as to the passing of a marriage act, we have the authority of the past to convince us that Lower Canada will never be satisfied with what is proposed in the plan of Confederation. On a former occasion, when a member of the Parliament of Canada moved to enact that marriage should be made a civil contract, all the members for Lower Canada voted against the motion, and the whole country was opposed to it. I shall also inquire whether the Federal Government will not have the right to enact that religious corporations shall no longer exist in the country, or that they shall not be allowed to hold real property, except what is absolutely necessary for their lodging accommodation. According to the resolutions which have been submitted to us, the Federal Government would certainly have this right. It has been said that article 15 of the 43rd resolution replies to this objection, but I can see nothing in that article which restricts the right of the Federal Government to legislate on this matter. The 43rd resolution defines the powers of the local governments, and article 15 of that resolution declares that they may make laws respecting ” property and civil rights, excepting those portions thereof assigned to the General Parliament.” That article reserves to the local legislatures nothing relative to religious corporations, and the Federal Government would have full power to decree that those corporations shall not hold immovable property. The supreme power is that which has the right to legislate upon, and regulate the existence of, the corporations in question, and they can only possess civil rights so long as the Government permits them to exist. The same might be said of most of the institutions to which Lower Canada is attached. I am therefore right in saying that, so far as those things which Lower Canada most holds to are concerned, Confederation is in fact a Legislative union, because upon the Federal Government is conferred the right of legislating upon those subjects which Lower Canada holds most dear.

However, as admirable as James’ philanthropic efforts are, they are not a solution to the problems in public education.

9

there might be some misunderstanding and difference of opinion, as for example those clauses by one of which it was stated that the civil laws of the country were to be under the control of the local governments, and by the other of which the law of marriage was placed under the control of the General Government. The law of marriage pervaded the whole civil code, and he wanted to know how it could be placed under a different legislature from that which was to regulate the rest of the civil law.

It was adopted by the Imperial Legislature with the view of remedying difficulties which then existed between the two Provinces. The inherent defect in the Imperial Act for the Union of the two Canadas was this : it attempted to combine the federal principle with unity of action. It endeavoured to give equal representation to the two sections of the Province, while it brought them together for the purpose of dealing as one with all subjects both (general and local,

Those claims were in themselves undoubtedly founded in justice— but at the same time there was great reason in the objections taken to them—they involved an interference with the Federal principle recognized in the Union Act, an interference which amounted to an entire change in the principles on which the Government of the country was to be administered, and could not be received otherwise than with dread by a large class, if not by the whole of the population of Lower Canada.

should have been a concession to Upper Canada, of additional members in proportion to its population, but that concession would, as be had already remarked, have been an invasion of the Federal principle, contained in the Union Act, and would unquestionably have been represented to the uttermost by a large proportion if not by the whole of Lower Canada.

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She said I am the one who will dance on the floor in the round

Okay,incaseyoudidn’tcatchthat,listentothatlastpartonemoretime.JaystartedmakingupsomeconversationthathadwithAdnanabouthisgloves,aconversationJayneverevermentionsagainandhasnopoint

Buttherealquestionforalotofpeoplehasalwaysbeen:wereJayandAdnanfriends?

Ashift is taking place in schools all over the world as learners are exploring subject matter through the act of creation rather than the consumption of conten

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“We see kids in their cars in the parking lot at night and on weekends,” says Buddy Berry, superintendent of Eminence Independent Schools. They’re there, he says, because they can access the Internet using the school’s wireless network—something many don’t have at home.

educators and business leaders are increasingly recognizing that CS is a “new basic” skill necessary for economic opportunity. The President referenced his Computer Science for All Initiative, which provides $4 billion in funding for states and $100 million directly for districts in his upcoming budget; and invests more than $135 million beginning this year by the National Science Foundation and the Corporation for National and Community Service to support and train CS teachers.

Gooru, a Redwood City, Calif.-based nonprofit that relies on educators as "crowdsourcers." The Gooru platform is geared toward teachers building collections of open-ed multimedia resources and students directing their own learning.

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“Instead of having one prescribed way to do things that comes from a textbook, kids can do things where they’re truly interested,” says Lori Secrist. “When they’re truly interested, they’re engaged. And when they’re engaged, they learn.”

Of Man's first disobedience, and the fruit Of that forbidden tree whose mortal taste Brought death into the World, and all our woe, With loss of Eden, till one greater Man Restore us, and regain the blissful seat, Sing, Heavenly Muse, that, on the secret top Of Oreb, or of Sinai, didst inspire That shepherd who first taught the chosen seed In the beginning how the heavens and earth Rose out of Chaos: or, if Sion hill Delight thee more, and Siloa's brook that flowed Fast by the oracle of God, I thence Invoke thy aid to my adventurous song, That with no middle flight intends to soar Above th' Aonian mount, while it pursues Things unattempted yet in prose or rhyme.