216 Matching Annotations
  1. Last 7 days
  2. Nov 2023
    1. Given that neurobiological context, it’s hard to argue that an out-of-school suspension will do much to improve that student’s ability to self-regulate.

      Yes, solution does not help to address underlying issues and makes for more problems - academic ones.

    2. Talking back and acting up in class are, at least in part, symptoms of a child’s inability to control impulses, de-escalate confrontations, and manage anger and other strong feelings — the whole stew of self-regulation issues that can usually be traced to impaired executive-function development in early childhood.

      Identifies some of the behaviors that results from an inability to self-regulate

    3. And suspension rates are substantially higher among certain demographic groups

      Learn more about CA new law regarding school Suspensions and expulsions: [SB 274 Skinner via ED100] https://ed100.org/blog/2023-pass-fail-veto

    4. In reality, though, many schools and school systems look at students who are struggling in those areas and instead think: How do we discipline them?

      Based on your SL experiences, do you agree that most schools prioritze discipling rather than helping kids to attach, bond, deal with stress, and self-regulate?

    5. 13. Discipline

      Why do you predict that author Tough is starting our consideration of helping to build necessary skills and mindsets with a chapter on DISCIPLINE?

  3. Oct 2023
    1. https://en.wikipedia.org/wiki/Shmita

      During shmita, the land is left to lie fallow and all agricultural activity, including plowing, planting, pruning and harvesting, is forbidden by halakha (Jewish law).

      The sabbath year (shmita; Hebrew: שמיטה, literally "release"), also called the sabbatical year or shǝvi'it (שביעית‎, literally "seventh"), or "Sabbath of The Land", is the seventh year of the seven-year agricultural cycle mandated by the Torah in the Land of Israel and is observed in Judaism.

    1. https://udenver.zoom.us/meeting/register/tZcuceuspzkuE9VomnEaGva1HH1ra_iS4Eua?ref=jessestommel.com#/registration

      Some related ideas that are suggesting some sort of thesis for improving the idea of ungrading: - We measure the things we care about. - In Education, we care about learning and understanding, but measuring these outside of testing and evaluation is difficult at best (therefor ungrading). - No one cares about your GPA six months after you graduate. - Somehow we've tied up evaluations and grades into the toxic capitalism and competition within US culture. Some of this is tied into educational movements related to Frederick Winslow Taylor and Harvards Eliot. - Hierarchies instituted by the Great Chain of Being have confounded our educational process.

    1. Durch die Überschreitung von 6 der 9 planetaren Grenzen wurde der „safe operating space for humanity“ verlassen. Die neue Studie analysiert auf Basis von 2000 Studien erstmals die Situation bei allen planetaren Grenzen. Als besonders bedrohlich schätzen die Forschenden ein, dass 4 direkt auf das Leben bezogene Grenzen überschritten wurden. Die dadurch mangelnde Resilienz könnte es auch unabhängig von Emissions-Senkungen unmöglich machen, das 1,5°-Ziel zu erreichen. https://www.theguardian.com/environment/2023/sep/13/earth-well-outside-safe-operating-space-for-humanity-scientists-find

  4. Sep 2023
  5. Mar 2023
    1. dispense,

      to give out something, especially products, services, or amounts of money 分配、發放

    2. lyre

      a stringed instrument of the harp class having an approximately U-shaped frame and used by the ancient Greeks especially to accompany song and recitation (https://www.merriam-webster.com/dictionary/lyre) 古希臘的七弦豎琴

    3. stratagems,

      an artifice or trick in war for deceiving and outwitting the enemy (https://www.merriam-webster.com/dictionary/stratagem) 計謀、詭計、策略、戰略

    4. undaunted

      無所畏懼 Courageously resolute especially in the face of danger or difficulty

    5. Transfix’d

      刺穿 To pierce through with or as if with a pointed weapon

    6. spleen

      突然的衝動或心血來潮 A sudden impulse or whim

    7. haughty

      傲慢 Having or showing an attitude of superiority and contempt for people or things perceived to be inferior

    8. disdain’d

      a feeling of contempt for someone or something regarded as unworthy or inferior 蔑視

  6. drive.google.com drive.google.com
    1. persecute

      迫害 (v.) to treat someone unfairly or cruelly over a long period of time because of their race, religion, or political beliefs, or to annoy someone by refusing to leave them alone

    2. '>pat

      吐 to eject (something) from the mouth

    3. mantle

      斗篷 a figurative cloak symbolizing preeminence or authority

    4. batt;.1lion

      營 a military unit composed of a headquarters and two or more companies, batteries, or similar units

    5. flogged

      鞭打 to beat with or as if with a rod or whip

    6. counsel

      協商 guarded thoughts or intentions

    7. gnac,hing

      咬牙切齒 to strike or grind (the teeth) together

    8. leaven

      酵 a substance (such as yeast) used to produce fermentation in dough or a liquid especially

    9. mustard

      芥末 a pungent yellow condiment consisting of the pulverized seeds of various mustard plants (such as Sinapis alba, Brassica juncea, and B. nigra) either dry or made into a paste or sauce (as by mixing with water or vinegar) and sometimes adulterated with other substances (such as turmeric) or mixed with spices

    10. granary

      糧倉 a storehouse for threshed grain

    11. darncl

      幾種通常多雜草的黑麥草(黑麥草屬)中的任何一種 any of several usually weedy ryegrasses (genus Lolium)

    12. parched

      因乾燥而皺縮 to become dry or scorched

    13. thistles

      薊 any of various prickly composite plants (especially genera Carduus, Cirsium, and Onopordum) with often showy heads of mostly tubular flowers

    14. ra\ening

      貪吃 to feed greedily

    15. rend

      撕裂 to perform an act of tearing or splitting

    16. toii

      辛勞 long strenuous fatiguing labor

    17. mammc~n

      財神爺 material wealth or possessions especially as having a debasing influence

    18. sco" I

      皺眉頭 to contract the brow in an expression of displeasure

    19. synagogues

      猶太教堂 the house of worship and communal center of a Jewish congregation

    20. recompense

      補償 to return in kind

    21. porter

      搬運工 a person who carries burdens

    22. harlotry

      淫亂 sexual profligacy

    23. amiss

      通過踩踏來壓碎、傷害或毀壞 to crush, injure, or destroy by or as if by treading

    24. tramp l ed

      通過踩踏來壓碎、傷害或毀壞 to crush, injure, or destroy by or as if by treading

    25. pigeons

      廣泛分佈的鳥類科(鴿科,鴿形目)中的任何一種,具有粗壯的身體、相當短的腿和光滑緊湊的羽毛 any of a widely distributed family (Columbidae, order Columbiformes) of birds with a stout body, rather short legs, and smooth and compact plumage

    26. census

      古羅馬用於人口統計的量詞 a count of the population and a property evaluation in early Rome

    27. decree

      an order usually having the force of law 有法律效力的命令

    28. guile

      詭計 trick

    1. Instructive quotations: Who doesn’t love a great quote? And quotations can work very well in a media environment that privileges brevity and catchiness. On the surface, the words of a past leader might seem explanatory for a topical news story, but dig a little deeper into the quote’s original setting, and the particularities—who said it, when, and for what purpose—might make the saying less apt.

      Often instructive quotations aren't appropriate for the current situation because they have been stripped of their original context which doesn't apply to the new situation in which it is being used.

  7. Feb 2023
    1. Data Policy and related materialsometimes, on the contrary, demonstrate an oversupply of very high level, generalised information atthe expense of a more concise and meaningful delivery of the essential information necessary for thedata subject to understand the processing being undertaken and to exercise his/her rights in ameaningful way. Furthermore, while Facebook has chosen to provide its transparency information byway of pieces of text, there are other options available, such as the possible incorporation of tables,which might enable Facebook to provide the information required in a clear and concise manner,particularly in the case of an information requirement comprising a number of linked elements. Theimportance of concision cannot be overstated nonetheless. Facebook is entitled to provide additionalinformation to its user above and beyond that required by Article 13 and can provide whateveradditional information it wishes. However, it must first comply with more specific obligations under theGDPR, and then secondly ensure that the additional information does not have the effect of creatinginformation fatigue or otherwise diluting the effective delivery of the statutorily required information.That is simply what the GDPR requires.

      DPC again schools facebook in reality.

  8. Jan 2023
    1. Weread, for example, of Philistine incursions into the hill country, toMichmash in Benjamin (1 Samuel 13:23), and the Rephaim Valley nearJerusalem (2 Samuel 5:17–22). It was in one of these border disputes thatthe city at Khirbet Qeiyafa was conquered and destroyed.
    2. Samson, last of the Judges, who kills thelion and obtains honey from its corpse, attaches burning torches to the tailsof foxes, and carries the heavy gates of Gaza upon his shoulders
  9. Nov 2022
    1. Atendimento excepcional é a nossa prioridade.

      Aqui vale mudarmos para a seção "O que dizem sobre nós" da homepage

    1. « Le règlement intérieur comporte un chapitre consacré à la discipline des élèves. Il reproduit l'échelle des sanctions prévues à l'article R. 511-13 et prévoit les modalités de mise en œuvre des mesures de prévention, de responsabilisation et d'accompagnement, notamment lorsqu'elles font suite à la réintégration d'un élève exclu temporairement pour des faits de violence.»
  10. Oct 2022
    1. L'échelle des sanctions fixée à l'article R. 511-13 du code de l'Éducation est reproduite dans le règlement intérieur. Toutefois, le juge administratif (CE, 16 janvier 2008, MEN c/Mlle Ayse A, n° 295023) considère que, même en l'absence de toute mention dans le règlement intérieur, l'échelle des sanctions réglementaires est applicable de plein droit.
  11. Sep 2022
    1. Krótka historia nie tylko samej metody commonplace-book, co także informacje na temat książki Johna Locka (zob. Locke et al. 1706; Locke 1812) oraz dodatkowe informacje, zaczerpnięte z Roberta Darntona (Darnton 2000; zob. też: Darnton 2009). Na końcu są też informacje na temat porównania tej metody i prowadzenia notatek oraz strony przez samego autora, czyli Jeremy'ego Normana.

    2. (Darnton, “Extraordinary Commonplaces,” New York Review of Books 47 (20)[December 21, 2000] 82, 86)


      Zob. też: Darnton 2009. (rozdział 10: The Mysteries of Reading)

    1. Artykuł na temat mediów społecznościowych wykorzystywanych przez instytucje kultury i organizacje pozarządowe; o ich roli, charakterze i sposobie podejścia do pracy z treściami tam publikowanych.

      Dwoma podstawowymi rolami mediów społecznościowych są: - przekazywanie treści, - budowanie społeczności.

      Dlatego istotnym elementem jest komunikacja z publicznością, do tego tak, często, jak często wymaga tego dane zagadnienie, a także bez zbędnego dystansu (zwracamy się "na ty"), czyli z naciskiem na społecznościowy charakter.

      Zatem rozmawiamy z ludźmi, jesteśmy blisko nich, tworzymy z nimi przyjazną przestrzeń do wspólnej dyskusji.

  12. Jul 2022
    1. ROMANS 13 The Most Disastrously Misinterpreted Scripturein the History of the Human Race

      Interesting read. I'm amazed I hadn't found it sooner considering how common this position is among archist Christians.

    1. Prof. Meiri: "Our study tracked changes at a much higher resolution over a considerably longer period of time compared to previous research. The results were illuminating: we found a continual, and very significant, decline in the size of animals hunted by humans over 1.5 million years. For example, a third of the bones left behind by Homo erectus at sites dated to about a million years ago, belonged to elephants that weighed up to 13 tons (more than twice the weight of the modern African elephant) and provided humans with 90% of their food. The mean weight of all animals hunted by humans at that time was 3 tons, and elephant bones were found at nearly all sites up to 500,000 years ago. "Starting about 400,000 years ago, the humans who lived in our region -- early ancestors of the Neandertals and Homo sapiens, appear to have hunted mainly deer, along with some larger animals weighing almost a ton, such as wild cattle and horses. Finally, in sites inhabited by modern humans, from about 50,000 to 10,000 years ago, approximately 70% of the bones belong to gazelles -- an animal that weighs no more than 20-30kg. Other remains found at these later sites came mostly from fallow deer (about 20%), as well as smaller animals such as hares and turtles."

      Progression of body mass over the last 1.5 million years in the Southern Levant: 1) Up to 500,000 years ago 1/3 of bones left behind at Homo Erectus sites belonged to 13 ton elephants that provided 90% of the food. Mean weight of all hunted animals at the time was 3 tons 2) Up to 400,000 years ago, early Neandertals and Homo Sapiens only hunted mainly deer and animals like wild cattle and horse that weighed no more than 1 ton. 3) From 50,000 to 10,000 years ago, 70^ of bones at modern human sites belonged to gazelles weighing between 20 and 30 kg, as well as fallow deer and hares and turtles.

  13. May 2022
    1. É importante estudar a fundo as relações da dinâmica social do período da Escravatura no Brasil para não cometer enganos e propagar mentiras...

    2. Os diferentes tipos de escravo no Brasil

      A Escravidão no Brasil possuía uma dinâmica muito mais complexa do que se faz acreditar nas Escolas de hoje.

  14. Mar 2022
  15. Dec 2021
    1. RRID:ZFIN_ZDB-GENO-100504-13

      DOI: 10.7554/eLife.72345

      Resource: (ZFIN Cat# ZDB-GENO-100504-13,RRID:ZFIN_ZDB-GENO-100504-13)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-GENO-100504-13

      What is this?

  16. Nov 2021
    1. is part of an attempt to channel funds to wealthy citizens

      incorrect- the program was to channel funds to lower class / (poor) citizens

  17. Jul 2021
  18. Mar 2021
    1. Results for individual PALB2 variants were normalized relative to WT-PALB2 and the p.Tyr551ter (p.Y551X) truncating variant on a 1:5 scale with the fold change in GFP-positive cells for WT set at 5.0 and fold change GFP-positive cells for p.Y551X set at 1.0. The p.L24S (c.71T>C), p.L35P (c.104T>C), p.I944N (c.2831T>A), and p.L1070P (c.3209T>C) variants and all protein-truncating frame-shift and deletion variants tested were deficient in HDR activity, with normalized fold change <2.0 (approximately 40% activity) (Fig. 1a).

      AssayResult: 5

      AssayResultAssertion: Normal

      StandardErrorMean: 0.08

    2. A total of 84 PALB2 patient-derived missense variants reported in ClinVar, COSMIC, and the PALB2 LOVD database were selected

      HGVS: NM_024675.3:c.1238C>A p.(Thr413Lys)


      AssayResult: 96.97

      AssayResultAssertion: Not reported

      PValue: > 0.9999

      Comment: Exact values reported in Table S3.

    2. To this end, 44 missense variants found in breast cancer patients were identified in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar) and/or selected by literature curation based on their frequency of description or amino acid substitution position in the protein (Supplemental Table S1).

      HGVS: NM_024675.3:c.2816T>G p.(Leu939Trp)

    1. Source Data

      AssayResult: 20.08

      AssayResultAssertion: Not reported

      ReplicateCount: 2

      StandardErrorMean: 6.84

      Comment: Exact values reported in “Source Data” file.

    2. Source Data

      AssayResult: 10.68

      AssayResultAssertion: Abnormal

      ReplicateCount: 2

      StandardErrorMean: 0.32

      Comment: Exact values reported in “Source Data” file.

    3. Source Data

      AssayResult: 10.4

      AssayResultAssertion: Abnormal

      ReplicateCount: 2

      StandardDeviation: 3.22

      StandardErrorMean: 2.28

      Comment: Exact values reported in “Source Data” file.

    4. We, therefore, analyzed the effect of 48 PALB2 VUS (Fig. 2a, blue) and one synthetic missense variant (p.A1025R) (Fig. 2a, purple)29 on PALB2 function in HR.

      HGVS: NM_024675.3:c.1653T>A p.(Y551X)

    1. Most Suspected Brugada Syndrome Variants Had (Partial) Loss of Function

      AssayResult: 63.8

      AssayResultAssertion: Indeterminate

      ReplicateCount: 25

      StandardErrorMean: 10.1

      Comment: This variant had mild loss of function (peak current >50% and <75% of wildtype), therefore it was considered inconclusive and neither abnormal nor normal in vitro function. (Personal communication: A. Glazer)

    2. we selected 73 previously unstudied variants: 63 suspected Brugada syndrome variants and 10 suspected benign variants

      HGVS: NM_198056.2:c.2203G>A p.(Ala735Thr)

    1. Effacement des sanctions et amnistieLes sanctions, même assorties du sursis à leur exécution, sont inscrites au dossier administratif de l'élève. L'avertissementest effacé du dossier administratif de l'élève à l'issue de l'année scolaire, le blâme et la mesure de responsabilisationsont effacés du dossier administratif de l’élève à l’issue de l’année suivant celle qui a suivi le prononcé de la sanction. Les autres sanctions, hormis l'exclusion définitive, sont effacées du dossier administratif de l'élève à l’issue de la deuxième année suivant celle du prononcé de la sanction.Toutefois, un élève peut demander(même s’il est mineur)l'effacement des sanctions inscrites, y compris l’exclusion définitive, dans son dossier administratif lorsqu'il change d'établissement(art R511-13).Les sanctions, y compris l’exclusion définitive, sont effacées du dossier administratif de l'élève au terme de sa scolarité dans le second degré (art R511-13).
  19. Feb 2021
    1. Supplemental material

      AssayResult: 66

      AssayResultAssertion: Normal

      Comment: See Table S2 for details

    2. Supplemental material

      AssayResult: 6

      AssayResultAssertion: Abnormal

      Comment: See Table S2 for details

    3. We analysed a total of 82 blood samples derived from 77 individuals (online supplemental table 3). These 77 individuals corresponded either to new index cases suspected to harbour a pathogenic TP53 variant or to relatives of index cases harbouring TP53 variants.

      HGVS: NM_000546.5:c.323_329dup p.(Leu111Phefs*40)

  20. Oct 2020
    1. The variation of bandgap energy (Eg) and Urbach energy(EU) of ZnS thinfilms with deposition times is shown inFigure 13. TheEgvalues of ZnSfilms decrease with increas-ing deposition time which is due to the agglomeration ofthe ZnS-NPs. The minimum value ofEUobtained at 20 minindicates a very weak absorption tail due to minimizeddefects and impurities which improves the transparencyand optical conductivity of thefilm coated at that time.

      La variación de la energía del band gap y de Urbach de las películas delgadas de ZnS con tiempos de deposición es mostrado en la figura 13. La valores del energía band gap de las películas ZnS decrecían con el incremento del tiempo de deposición lo cual es debido a la aglomeración de las ZnS-NPS. El valor mínimo de la energía de Urbach obtenido a 20 min indica una muy débil cola de absorción debido los defectos minimizados e impurezas que mejoran la transparencia y la conductividad óptica en el revestimiento de la pelicula en ese momento.

  21. Jul 2019
  22. Jun 2019
    1. with the plunger of a Hamilton syringe. The tube was centrifuged at room temperature at 14,000 rpm for 30 min. The above process of gel-disruption and centrifugation was repeated twice subsequent to which the oil layer was aspirated and suitable aliquots from the supernatant were taken for estimation of Csat by Drabkin' s reagent (Goldberg eta!, 1977).
    2. he gelation concentrations of HbS constructs were determined by the dextran-Csat method of Bookchin et a! (Bookchin et a!, 1999). This method allows measurement of Csat under near-physiological conditions and at much lower concentration of HbS (about 5-fold or less) than that required in standard Csat assays, but essentially provides the same information. Briefly, a suitable aliquot of a concentrated solution of hemoglobin in potassium phosphate buffer (0.05 M, pH 7.50) was taken in a 1.5 ml micro-centrifuge tube. A concentrated dextran (70 kDa) solution prepared in the same buffer was added to it and mixed well. This mixture was overlaid with 0.5 ml of mineral oil, chilled on ice bath and deoxygenated with an anaerobically prepared dithionite solution through an airtight Hamilton syringe. The final concentrations of dextran and dithionite in the mixture were 120 mg/ml and 0.05 M respectively. The deoxygenated sample above was allowed to polymerize at 37°C for 30 min after which the gel under the oil layer was disrupted
    3. Measurement of gelation concentration, Csat
  23. May 2019
    1. Parasite cultures were distributed in six well plates (2 ml per well) and pharmacological inhibitors were added at desired concentration. Plates were placed in small gas chambers, gassed and immediately returned to 37°C incubator. The lysates were prepared after ~30 min of the addition of inhibitors
    2. Inhibitor Treatment of gametocyte
    1. Intracellular Na + measurement was performed using the fluorescent Na + indicator Sodium Green TM tetracetate. THP-1 macrophages were resuspended in phenol-red free RPMI-1640 medium and incubated with Sodium Green™ at a final concentration of 1 JiM for 20 min at room temperature. The cells were washed once with fresh serum-free media to remove excess probe following which kinetic fluorescent measurements were commenced in a spectrofluorimeter (BMG Fluostar Optima) at an excitation of 480 nm and emission of 520 nm. In situ calibration to determine the dissociation constant (Kct) of the dye at 3 7°C was accomplished by using the indicator dye in solutions of precisely known free Na+ concentration in the presence of the pore forming antibiotic gramicidin (10 J,tM). Intracellular Na+ was calculated using the following formula: where, Kct of the dye is 5.7 mM at 37°C, F is the fluorescence of the experimental sample, Fmin is the fluorescence in the absence of Na+ and Fmax is the fluorescence under saturating concentrations ofNa+ in the presence of gramicidin (10 J.i.M)
    2. Assay for intracellular Na +measuremen
    1. Circular Dichroic spectra of various proteins were recorded at room temperature, using a JASCO J7i0 spectropolarimeter fitted with a thermostated cell holder. I 50 J..Lg protein was dissolved in 3 ml of I 0 mM sodium phosphate buffer (pH 7.0), and the samples were scanned in the far-UV range (200-250 nm). A cell with a 1 em optical path was used to acquire the spectra at a scan speed of 50 nrn/min. with a. sensitivity of 50 mdeg and a response time of I sec. The sample compartment was purged with nitrogen, and spectra were averaged over I 0 accumulations. The CD spectra were normalized to mean residue ellipticity curves using Jasco software. Yang's reference parameters were used to perform secondary structure analyses from CD measurements using Jasco Secondary Structure Estimation Programme (Yang et al., 1986).
    2. Structural Characterization of Proteins by CD Spectroscopy
    1. transferred to another plastic box containing 2 X sse, 1 % SDS and washed at room temperature by gentle rocking for 15 minutes. The buffer was then changed and the washing continued at 60 in a shaking water bath for 30 minutes. Depending on the homology between the probe and the immobil ised DNA, the washing conditions were varied. The stringency ranged from 1 X sse, 1 % SDS, at 65°e to 0.2 X sse, 1 % SDS, at 65°e. After the washing, the filters were immediately sealed into plastic bags and put for autoradiography. Special care was taken to not to allow the filters to dry during any stage which might otherwise cause permanent binding of the probe to the filter preventing the reprobing of the same filter with a different probe at a later time. For autoradiography, the plastic bag containing the washed filter was fixed on a 3 MM Whatman sheet and placed securely ins ide a X ray cassette with one or two intensifying screens, and a X -ray film was placed over the filter in a dark room. The cassette was kept at -7o0e for the desired length of exposure. The film was taken out in the dark room, developed for approximately 3 minutes, washed in water for one minute to wash off all the developer adhering to the film, and fixed for 5 minutes. Finally, the film was washed in cold water for 10 minutes and air dried
    2. The prehybridisation and hybridisation of the Southern filters was carried out as described by Maniatis et al., ( 1982 ), with some modifications. In all stages, the SDS concentration was maintained at 1 % to minimise the background likely to occur on the nylon membrane. Prehybridisation was done at 68°C, for 4 - 6 hours, with 0.1 ml of prehybridisation buffer for each square centimeter of the membrane. The probe was denatured by immersing the eppendorf tube in a boiling water bath for 10 minutes and added directly to the bag containing prehybridisation mix. Hybridisation was done in aqueous system, at 68°e, without the use of formam ide, for 18 - 2 4 hours, in a plastic bag kept submerged in a water bath, without any shaking. At the end of hybridisation, the filter was taken out of the bag and quickly immersed in a plastic box containing 5 X sse, 1 % SDS at room temperature. After 15 minutes, the filter was
    3. Hybridisation of southern filters.
    4. hours. The NC filters having bound DNA liberated from bacterial colonies, were set up for hybridisation with radioactive probes as described by Maniatis et al., ( 1982 ). The filters were washed thoroughly with a solution containing 50 mM Tris.Cl, pH 8.0, 1 M NaCl, 1 mM EDTA, 1 % SDS, at 42°C, for 1 hour, to wash off any residual bacterial debris and agar etc. Prehybridisation and hybridisation was performed in aqueous solution without formamide in 5 X SSPE. The filters were washed up to a stringency of 0.2 X sse at 65°e.
    5. Colonies bound to nitrocellulose filter ( NC ) were lysed to liberate the DNA which was hybridised as described by Maniatis et al., 1982 ) . To obtain sharper autoradiography signals, the nitrocellulose filter bearing colonies was first overlaid on a 3 MM Whatman paper impregnated with 10 % SDS till the NC wetted evenly. The NC was peeled off and overlaid on another 3 MM paper impregnated with the denaturing solution. In this manner, the NC was successively treated with denaturing and neutralising solutions. Finally, the NC filter was air dried, sandwiched between two sheets of 3 MM paper and baked at 80°C for two
    6. Colony hybridisation.
    7. Hybridisa.tion of DNA L RNA bound to nylon membranes.
    1. The antibody isotypes in the immune sera were determined, by indirect ELISA, using mouse MAb isotyping reagents (Sigma). The microtitration plates coated with r-dZP3 (400 ng/well) and blocked with 1% BSA, were incubated with doubling dilution of pooled serum samples of a group of immunized animals. All the incubations were carried out at 37°Cand were followed by three washings with PBST. The incubation was followed by addition of goat anti-mouse isotype specific antibodies at 1:1000 dilution. The binding was revealed by rabbit anti-goat lgG-HRPO conjugate (Pierce) at an optimized dilution of I: 10,000 and processed for enzymatic activity estimation as described earlier.
    2. Antibody isotyping
    3. 492 run with 620 nm as the reference filter. The antibody response generated was represented as the geometric mean of the absorbance of individual mice sera in a group of immunized animals. b1 addition, the antibody titer against r-dZP3 was also determined by ELISA. The assay was carried out as described above except that 100 ~l of doubling dilutions of the serum samples (dilutions made in PBST supplemented with 0.1% BSA) were added per well in duplicate. For each serum sample tested, a reciprocal of dilution giving an absorbance of 1.0 was calculated by regression analysis and represented as antibody units (AU).
    4. Microtitration plates were coated with optimized concentration of r-bmZP1 (250 ng/well), r-dZP3 (400 ng/well) or r-rG (500 ng/well) in 50 mM PBS, pH 7.4, at 37°C for 1 h and then at 4°C, 0/N. The plates were washed once with PBS and incubated with 1% BSA, (200 Jll/ well) in PBS for 2 h at 37°C for blocking the non-specific sites. All subsequent incubations were carried out for 1 h at 37°C and each incubation was followed by three washings with PBS containing 0.05% Tween-20 (PBST). Post-blocking, the plates were incubated with 1 :50 dilution of either the preimmune or the immune serum samples obtained from mice immunized with the respective plasmid DNA. Antibodies bound tor-bmZP 1, r-dZP3 and r-rG were revealed with 1:2000 dilution of goat anti-mouse IgG (whole molecule) HRPO (Dako). Estimation of the enzymatic activity was carried out with 0.05% OPD in 50 mM citrate phosphate buffer, pH 5.0, containing 0.06% H202 as the substrate. The reaction was stopped with 50 Jll of 5 N H2S04 and the absorbance read at
    5. Enzyme Linked Immunosorbant Assay (ELISA)
    7. d) Particle delivery using the Helios gene gun A day prior to immunization, hair were removed from the abdominal region of mice using a commercial depilatory agent (Anne French cream). Two cartridges/mouse ( ~ 2 Jlg DNA) were shot under pressurized helium gas ( 400 psi) intradermally at the shaven area of the abdomen of mice using the Helios gene gun. Two boosters comprising of two cartridges each were given on days 21 and 35. On day 45, mice in each group received i.m. injection of E. coli expressed recombinant protein (20 Jlglmouse in saline). Mice were bled retro-orbitally on days 0, 45 and 52 for analysis of antibody response.
    8. tubing, which was cut into 0.5 inch pieces (cartridges). These cartridges were used to deliver DNA into epidermis of male/female mice. a) Preparation of DNA-gold microcarrier suspension Twenty five mg of gold microcarriers were weighed in a 1.5 ml eppendorf tube to which 100 J..Ll of 0.05 M spermidine was added and vortexed for 10 sec. To the above mixture 100 J..Ll of DNA (0.5 mg/ml) was added and vortexed for another 10 sec. While vortexing, 100 J..Ll of 1 M CaCh was added dropwise to the mixture and left at RT for 10 min to allow precipitation of DNA onto gold microcarriers. The DNA-gold pellet was collected by centrifuging at 12,000 X g for 1 min at RT. The pellet was washed thrice with 100% ethanol (freshly opened bottle), resuspended in 3 ml of 0.1mg/ml polyvinylpyrollidone (PVP) in ethanol and stored at -20°C till further use. b) Loading the DNA/microcarrier suspension into gold-coat tubing using the tubing prep station A 25 inch length of tubing was cut and fixed on tubing prep station, air dried by passing nitrogen gas through it for 15 min. The DNA/microcarrier suspension was vortexed and injected into the tubing using a 5 ml syringe and the microcarriers allowed to settle in the tubing for 3 min. Ethanol from the tubing was removed by slowly sucking into the syringe. The tubing was rotated, while passing the nitrogen gas, using the tubing prep station, for 20-30 sec to allow the microcarriers to evenly coat the inside of the tubing. c) Preparation of cartridges using the tubing cutter The tubing was cut into 0.5 inch long pieces (cartridges) by using the tubing cutter and cartridges stored at 4°C in vials containing desiccant pellets till further use.
    9. Suspension of DNA adsorbed onto gold microcarriers at 0.5 Microcarrier Loading Quantity (MLQ; 50 J.lg DNA/25 mg gold microcarriers) was prepared and coated inside Tefzel
    10. Plasmid DNA adsorbed onto gold microcarriers
    11. A day prior to immunization, hair were removed from both the hind limbs of the mice using a commercial depilatory agent (Anne French cream, Geoffrey Manners & Co. Ltd, Mumbai, India). Mice were immunized in a similar way as in the saline group but in addition, ten very short electric pulses were given at the site of injection immediately after DNA administration using a gas igniter (Upadhyay, 2001). Voltage delivered in each trigger was 18kV for 10-7s.
    12. Plasmid DNA administered by electroporation
    13. Inbred male BALB/c.T mice (6-8 week, Small Experimental Animal Facility, National Institute of Immunology, New Delhi, India) were immunized intramuscularly (i.m.) with 100 J.lg of respective plasmid DNA or VR1020 vector in 100 J.ll saline (0.9% NaCl) in the anterior tibialis muscle in the hind limbs (each receiving 50 J.ll). Two booster injections of 100 J.lg DNA in saline were given on day 21 and 35. On day 45, mice in each group received i.m. injection of E. coli expressed recombinant protein (20 J.lg/mouse in saline). Mice were anesthetized and bled retro-orbitally on days 0, 45 and 52 for analysis of respective antibody responses.
    14. Plasmid DNA administered in saline
    15. IV. IN-VIVO IMMUNIZATION STUDIES These experiments were carried out with the approval of Institutional Animal Ethics Committee. Three different modes of administration were used:
    16. a) Purification oLin elusion bodies For the purification of inclusion bodies, the bacterial cell pellet from 1 liter culture was resuspended in 10 ml of Tris-HCl buffer (50 mM; pH 8.5) containing 5 mM EDTA and sonicated using Branson sonifier-450 for 8 cycles of 90 sec each (30 watt output; Branson Ultrasonic Corp., Danbury, CT, USA) on ice. The inclusion bodies were collected by centrifugation of the sonicate at 8000 X g for 30 min at 4°C. The pellet was washed twice with 15 ml of 50 mM Tris-HCl buffer with 5 mM EDTA containing 2% sodium deoxycholate in order to remove loosely bound E. coli proteins from the inclusion bodies. Subsequently, the inclusion body pellet was washed with 50 mM Tris-HCI buffer (pH 8.5), followed by a washing with the double distilled water. All the buffers used for the purification contained 20 mM of phenylmethyl sulphonyl fluoride (PMSF). b) Solubilization and renaturation The purified inclusion bodies were solubilized in 100 mM Tris-HCl (pH 12.0) containing 2M urea at RT for 30 min, and centrifuged at 8000 X g for 30 min at 4°C. The pH ofthe supernatant was brought down immediately to 8.5 with 1 N HCl and then extensively dialyzed against renaturation buffer (50 mM Tris-HCl buffer; pH 8.5, 1 mM EDT A, 0.1 mM reduced glutathione, 0.01 mM oxidized glutathione and 10% sucrose). The protein was finally dialyzed against 20 mM Tris-HCl, pH 8.5 and its concentration estimated using BCA.
    17. Purification in refolded form
    18. The proteins were purified by nickel affinity chromatography. The cell pellet ( ~ 1 g) of each clone was solubilized in 5 ml ofbuffer A (6 M guanidine hydrochloride, 0.1 M NaH2P04, 0.01 M Tris, pH 8.0). The suspension was centrifuged at 8000 X g for 15 min at 4°C and the supernatant containing the recombinant protein was mixed with Ni-NT A resin (Nickel-Nitrilotriacetic acid equilibrated with buffer A) and kept for gentle end-to-end shaking for 1 hat RT. The resin was loaded on a column and washed with 10 bed-volumes of buffer A. The column was subsequently washed with 5 bed-volumes each of buffers B, and C, which contained 8 M urea, 0.1 M NaH2P04 and 0.01 M Tris and had successively reducing pH values of 8.0 and 6.3 respectively. The protein was eluted with buffers D and E (composition same as buffer B) in which the pH was further reduced to 5.9 and 4.5 respectively. Five fractions of 4 ml each were collected during elution with buffer D and buffer E respectively. The eluted proteins were analysed by 0.1% SDS-1 0% PAGE (gels stained with Coomassie blue) and Western blot. The fractions showing the purified recombinant protein were pooled and concentrated in an Amicon concentrator using a YM30 membrane and dialyzed against 100 mM phosphate buffer, pH 7.4, containing 4 M urea. The concentration of each purified protein was estimated by bicinchoninic acid (BCA).
    19. Purification in denatured form
    20. PURIFICATION OF r-bmZPl, r-dZP3 AND r-rG EXPRESSED IN E. coli
    21. reached a value of 0.5-0.6. The cultures were then induced with 1 mM IPTG for 2 h at 37°C. Cells were pelleted at 4000 X g for 30 min at 4°C and stored at -70°C until used.
    1. BloodglucosewasestimatedbyFolin-Wumethod(KlontzandSmith,1968).Glucoseonboilingwithalkalinecoppersolution,reducescopperfromthecuprictothecuprousstate(cuprousoxide).Thecuprousoxidesoformedreducesphosphomolybdicacidtothebluecoloredmolybdenumblue,whichismeasuredcolorimeterically.TheintensityofthebluecolorisproportionaltoglucoseconcentrationanditiscolorimetricallydeterminedinaBoschandLombSpectrophotometerat620nm
    2. Bloodglucose
    1. After transfection the cells were harvested and protein was isolated from the celllysates. The cells from each well were pelleted at 2000 rpm for 10 min at 40C. The supernatant was carefully removed and the pellet was incubated on ice for 1 hr after adding 50pl of lysis buffer (1% triton X100, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT, 1X PI, all in 1X PBS) with intermittent vortexing. The tubes were centrifuged at maximum rpm for 10 min at 40C and the supernatant, containing the proteins, was collected and stored at -700C. The purified protein fractions were quantitated using the BCA protein assay kit and the O.D. was taken at 562nm
    2. rotein isolation from celllysate
    1. For clonogenic assays, 1x103 (A549) or 2x103 (E-10) cells were seeded per well of a six well tissue culture plate and grown for 15 days. For identification of signaling pathways various inhibitors were used viz, PI3K inhibitor LY294002 (10μM), MEK inhibitor PD98059 (10μM) or p38 inhibitor SB203580 (10μM). Cells were grown in the presence of inhibitor for seven days following which fresh medium was added. For staining, cells were washed twice with PBS and fixed in 10% formalin for 10 minutes, washed extensively with water and stained with 0.25% crystal violet prepared in 25% methanol for 4hrs at 4°C. Plates were then washed with milli Q water and dried before scanning
    2. Clonogenic Assay
    1. the membrane and sandwiched between 1 piece of buffer-soaked Whatman paper from Biorad on each side. The sandwich was placed between graphite electrodes with the membrane towards the anode. The transfer was done for 12-16 hr using a voltage of 40 V in the cold room. Protein transfer was viwed using Ponceau S staining. Blot was dipped in the Ponceau S stain under shaking and washed using PBST. After transfer, the membrane was blocked with 5% non-fat milk in PBST (1X PBS with 0.1% Tween-20) for 2 hr at room temperature. The membrane was then washed thrice with PBST under shaking and incubated with the primary antibody (1:1000 dilution in PBST) for 12-16 hr. The membrane was again washed thrice under shaking with PBST and incubated with 1:20000 dilution alkaline phosphatase conjugated anti-goat IgG secondary antibody (in PBST) for 2 hr. The membrane was once again washed thrice as described above and the signal developed using ECL kit from Amersham on X-ray films in a dark room. The reactive protein bands appeared as black bands upon gentle shaking at room temperature in 1X developer solution. The reaction was stopped by dipping and shaking the film in 1X Fixer solution followed by washing in water
    2. The protein samples separated on SDS-PAGE were transferred to PVDF (polyvinyledene difluoride) membrane (Amersham, Buckinghamshire, UK) electrophoretically by a semi-dry method using BioRad apparatus. The gel and the membrane (pre-wetted with methanol) were wetted with transfer and the gel was placed in contact with
    3. Western blotting with anti-Rho/anti-NusG antibody
    4. Obtaining transpositions near a gene of interest was achieved in a two-step procedure. The population (pool) of cells carrying random transpositions (described in the previous Section) at different places on the chromosome was used to prepare a P1 phage lysate. This lysate was then used to infect a suitable recipient strain and transductants were sought in a simultaneous (double) selection for two markers, namely the antibiotic marker on the mini-transposon and selection for the phenotype of the gene or mutation which was intended to be linked with the antibiotic marker. The transductants so isolated were purified and further P1 phage preparations were made on these individual clones. By retransducing with these lysates into the same recipient cells and observing the segregation of phenotypes after selection for the transposon marker, the cotransduction values were obtained between the transposon insertion and the gene (or mutation) of interest
    5. Obtaining transposon insertions near gene of interest
    1. A bioluminescent ATP determination kit from Invitrogen (Carlsbad, CA) was used to quantitate ATP levels. The assay is based on luciferase' s requirement for ATP for producing light (emission maxima at 560nm). The assay was carried out as described previously (Mukherjee et al., 2002). Briefly, a standard reaction mix was prepared-1X reaction buffer, 0.1p.M DTT, O.Sp.M Luciferin and 12.5 p.g/ mL Luciferase. 100 p.L was aliquoted into each well of a 96 well white plate and a base line reading measured using Fluostar Omega using the luminometer adaptor. Then either 106 parasites or different concentrations of A TP (prepared from the stock solution provided in the kit) were added to these wells as test and standard curve samples respectively. Again luminescence was measured and the baseline subtracted from the readings. The different dilutions of ATP were used to plot the standard curve which was then used to calculate the ATP levels in cells expressed as nmol/106 cells.
    2. Materials and Methods the MTT Lysis buffer (20% SDS , 50% Dimethyl formamide) and the O.D.s7onm was measured. The standard curve was plotted and the equation derived, used to calculate the number of metabolically viable cells in experimental groups. Percentage of viable cells was calculated by comparing the number of viable cells in treated wells with that of untreated wells. 3.2.C.13 ATP determination
    3. run on an agarose gel and band size determined by comparison against DNA ladder with bands of known sizes. Clones that were positive for the presence of gene/ plasmid were further checked by restriction digestion. For restriction digestion, plasmid was first isolated by Miniprep as described below. The digestion reactions were set according to manufacturer's protocol appropriate for the enzymes used. The products were run on an agarose gel and band size determined by comparison against DNA ladder with bands of known sizes. Clones with the desired pattern of digestion were propagated further and used
    4. Bacterial colonies obtained by transformation were checked for the presence of plasmid or gene inserted into the plasmid by colony PCR and restriction digestion. For PCR, a master mix for all the PCR reactions to be performed was made, aliquoted into PCR vials and stored on ice. Individual colonies were picked up, numbered and streaked onto an LB agar plate followed by deposition of a few cells into the PCR mix. PCR was carried out as described above for the respective primer pairs with the exception that the initial denaturation was carried out for 7 min at 94 °C. The products were
    5. Screening of bacterial transformants
    1. Quantitative measurement of periplasmic acid phosphatase activity in phosphate-starved C. glabratacells was performedas mentioned previously (Orkwis et al., 2010). A total of 0.5 OD600YNB-grown and phosphate-starved cells were collected, washed thrice with cold water and oncewith cold 0.1 M sodium acetatebuffer (pH 4.2). Washed cells were resuspendedin 500 μl sodium acetate (0.1 M)and incubated at 30 ̊C with constant stirring. After 10 min incubation, 500 μl freshly-prepared solution of 20 mM p-nitrophenyl phosphate in 0.1 M sodium acetate(pH 4.2) was added to the cell suspension. Enzymatic activity was stopped after incubation at 25 ̊C for 20 min by addition of 250 μl sodium carbonate (1 M)tothe reaction mix. Resultant colour change was measured by monitoring absorbance at 400 nm. Acid phosphatase activity was expressed as a ratio of OD400to OD600 to normalize against cell density
    2. Determination of acid phosphatase activity
    1. C. glabratacells grown either in RPMI medium or harvested from THP-1 macrophages were collected, washed with DEPC treated water and were disrupted with glass beads in trizol. Total RNA was isolated using acid phenol extraction method and frozen at -80C. Quality of RNA was examined by determiningtheRNAintegrity number (RIN) before microarrayanalysis.Microarray experiments wereperformed atOcimum Biosolutions Ltd., Hyderabad (http://www.ocimumbio.com). Briefly, a4x44K GE Agilent array comprised of 10,408 probes representing 5,205 ORFs of C. glabratawas used wherein average number of replicates for each probe was four to five. Feature Extraction software version (Agilent) and Quantile normalization was used for data analysis. Hierarchical clustering was performed using Complete Linkage methodwith Euclidean Distance as distance measure. Data arethe average of two hybridizations from biological replicates ofeach sample and raw data sets for this study areavailable at the Gene Expression Omnibus database(Accession number -GSE38953)
    2. Microarray Analysis
    3. E. coliDH5α strain was transformed with plasmids carrying appropriate inserts to clone and generatedeletion strains of C. glabrataORFs(Sambrook, 2001). Ultracompetentcells stored at -70⁰C were thawed on icefor 5-10 min. 5 μlligated plasmid was added to100 μlultracompetent cells andcells were incubatedon ice. After 30 min, competent cells were subjected to heat shock at 42⁰C for 90 seconds. Cells were immediately transferredtoicefor 2-3min. Next, 800 μlSOC (or LB) medium was added and cells were allowed to recover for 45 minon a shaker incubator set at 37⁰C.After the recovery, cells were centrifuged at 2,500g for 4 min. Medium supernatant was discarded and cells were resuspended in 200 μlfresh sterile LBmedium. Cells were plated on LB agar medium containing appropriate antibiotics. Plates wereincubatedat37⁰C for 12-16 h
    4. Bacterial transformation
    1. Uniquitination assay was performed as described by Choo and Zhang, 2009. Ubiquitination is an enzymatic process of the covalent attachment of polypeptide ubiquitin on specific lysine residuesof protein, which is thendegraded by proteasome complex. MG132 (carbobenzoxy-Leu-Leu-Leucinal), a proteolytic activity inhibitor of proteasome complex, is widely used to assess the stability of protein in vivo. Briefly, parental and profilin-stable cells were treated with 10 μM MG132 for 6 h. The whole cell extracts prepared in NTEN lysis buffer were then subjected to immunoprecipitation with anti-ubiquitin antibody. The analysis of ubiquitination was performed by immunoblotting with anti-PTEN antibody
    2. invivo Ubiquitination assay
    1. Cellswere grown in YPD to an OD600of 0.5-0.7. Cells equivalent to 1OD600were washed with synthetic complete medium without uacil twice and suspended in SC-Ura containing 3 μCi/mLof [14C]uracil for 5 min. Cells were pelleted and washed with SC-Ura medium twice and suspended in 0.5 mLof AE solution(Section 1OD600of this cell suspension was counted in a liquid scintillation counter (Perkin Elmer-Tricarb 2900). The cpm values obtained were and converted into moles based on thespecific activity of [14C] uracil and plotted using GraphPad Prism5
    2. Uracil uptake assay
    1. Cells were grown overnight on coverslips and transfected with various combinations of plasmids. Post 24 hrs. of transfection, cells were washed with PBS and then fixed in 3% w/v paraformaldehyde in 1X PBS containing 50 mM sucrose for 15 minutes at room temperature. Cells were permeabilized with permeabilization buffer i.e. 0.5% Triton X-100 buffer containing20mM HEPES at pH 7.4, 50mM NaCl, 3mM MgCl2 and 300mM sucrose and were incubated for 5 min.at room temperature.Cells were washed twice with 1X PBS and blocked with 3% BSA/PBS for 30 minutes. Cells were incubated with specificprimary antibody diluted in the blocking buffer. After 2 hours of incubation, cells were washed thrice with 1X PBS (each washfor 5 minutes). The 1X PBS was removed,and the cellswere incubatedwith specific FITC or Rhodamine-conjugated secondary antibodyat 37°C for 30 min.To visualize nuclei, cells were co-stained with DAPI (10 μg/ml). Cellswere washed thrice with 1X PBS and after final wash, coverslips containing cell weremounted on the slidesusing glycerine containing paraphenylenediamine. The cells were analyzed using confocal microscopy facility at CDFD
    2. Immunofluorescence