227 Matching Annotations
  1. Nov 2021
    1. 10 Best SaaS Startups in 2022 for Your InspirationDmitryCEOStartupSaaSHomeBlogEntrepreneurship10 Best SaaS Startups in 2022 for Your InspirationPublishedJul 29, 2020UpdatedNov 5, 202111 min readToday, the SaaS industry is gaining momentum. According to research, 80% of businesses already use at least one SaaS application. Hence, building a SaaS company is currently a skyrocketing business idea. To help you find inspiration and launch the best SaaS startup ever, in this article you will find 10 great examples of SaaS startups you can learn from. All of them produce valuable and fast-growing products for now. Likewise, Growthlist and AngelList marked them as promising SaaS startups of 2021-2022. Without further ado, let’s take a closer look at them.

      Today, the SaaS industry is gaining momentum. According to research, 80% of businesses already use at least one SaaS application. Hence, building a SaaS company is currently a skyrocketing business idea.

      To help you find inspiration and launch the best SaaS startup ever, in this article you will find 10 great examples of SaaS startups you can learn from. All of them produce valuable and fast-growing products for now. Likewise, Growthlist and AngelList marked them as promising SaaS startups of 2021-2022.

      Without further ado, let’s take a closer look at them.

    1. the program most likely violates the establishment clause.

      Shouldn't it not violate if they are neutral? I think this is wrong

      book says "The program permits the participation of all schools within the district, religious or nonreligious"

    1. Here, the program is not one of true private choice. It is not neutral in all respects toward religion and is part of an attempt to channel funds to wealthy citizens who send their children to religious schools. Only certain religious groups are free to participate.

      This entire statement is false; The program was found to be one of true private choice, with no obvious religious preferences nor discrimination against economic status/class.

  2. Oct 2021
  3. bafybeiery76ov25qa7hpadaiziuwhebaefhpxzzx6t6rchn7b37krzgroi.ipfs.dweb.link bafybeiery76ov25qa7hpadaiziuwhebaefhpxzzx6t6rchn7b37krzgroi.ipfs.dweb.link
    1. Recent research suggests that globally, the wealthiest 10% have been responsible foras much as half of the cumulative emissions since 1990 and the richest 1% for more than twicethe emissions of the poorest 50% (2).

      this suggests that perhaps the failure of the COP meetings may be partially due to focusing at the wrong level and demographics. the top 1 and 10 % live in every country. A focus on the wealthy class is not a focus area of COP negotiations perse. Interventions targeting this demographic may be better suited at the scale of individuals or civil society.

      Many studies show there are no extra gains in happiness beyond a certain point of material wealth, and point to the harmful impacts of wealth accumulation, known as affluenza, and show many health effects: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1950124/, https://theswaddle.com/how-money-affects-rich-people/, https://www.marketwatch.com/story/the-dark-reasons-so-many-rich-people-are-miserable-human-beings-2018-02-22, https://www.nbcnews.com/better/pop-culture/why-wealthy-people-may-be-less-successful-love-ncna837306, https://www.apa.org/research/action/speaking-of-psychology/affluence,

      A Human Inner Transformation approach based on an open source praxis called Deep Humanity is one example of helping to transform affluenza and leveraging it accelerate transition.

    2. 10% per annum

      Anderson has contextualized the scale of such an impact in his other presentations but not here. A recent example is the temporary emission decreases due to covid 19. A 6.6% global decrease was determined from this study: https://www.nature.com/articles/d41586-021-00090-3#:~:text=After%20rising%20steadily%20for%20decades,on%20daily%20fossil%20fuel%20emissions. with the US contributing 13% due to lockdown impacts on vehicular travel (both air and ground). After the pandemic ends, experts expect a strong rebound effect.

    1. KLIK https://wa.me/628119471931, Asuransi Prudential Pendidikan, Asuransi Prudential Kesehatan, Investasi Asuransi Prudential, Asuransi Prudential Pendidikan Anak, Asuransi Prudential Career

      Thio Siujinata 0811-947-1931 bit.ly/CallThio

      Coffetote Gallery Jl Tomang Rawa Kepa No. 37 Grogol Jakarta Barat

      G-Maps http:/bit.ly/mapscraftote

      asuransiorangtua #asuransiorangindonesia #asuransisyariahterbaik #sasuransilesehatan #asuransiuntuksemua #asuransiuntuksiapasaja #asuransiumumindonesia #asuransiwarisan #asuransiyangjujur #asuransiyangbagus

    2. KLIK https://wa.me/628119471931, Asuransi Prudential Indonesia, Asuransi Prudential Kesehatan Dan Investasi, Asuransi Prudential Karir, Asuransi Prudential Kehamilan, Asuransi Prudential Kematian

      Thio Siujinata 0811-947-1931 bit.ly/CallThio

      Coffetote Gallery Jl Tomang Rawa Kepa No. 37 Grogol Jakarta Barat

      G-Maps http:/bit.ly/mapscraftote

      aagen #agenbisnispelajar_sukses #agence #agenindonesia #lagenda #agenonline #qagency #ragen #tagen #ageny

    3. KLIK https://wa.me/628119471931, Peluang Usaha Dari Lingkungan, Peluang Usaha Untuk Anak Muda, Peluang Usaha Untuk Mahasiswa, Peluang Usaha Online Rumahan, Peluang Usaha Untung Besar

      Finansial Advisor Thio Siujinata 0811-947-1931 bit.ly/CallThio Coffetote Gallery Jl Tomang Rawa Kepa 37 Grogol Jakarta Barat G-Maps http:/bit.ly/mapscraftote

      peluangusahagratis, #peluangusahagampang, #peluangusahahalal, #peluangusahaonlinemodalterjangkau, #peluangusahau, #usahaonlinecepatuntung, #usahaonlinedirumah, #usahaonlinedigital, #usahaonlineexplore, #usahaonlinegratis

    4. KLIK https://wa.me/628119471931, Peluang Usaha Dari Lingkungan, Peluang Usaha Untuk Anak Muda, Peluang Usaha Untuk Mahasiswa, Peluang Usaha Online Rumahan, Peluang Usaha Untung Besar

      Finansial Advisor Thio Siujinata 0811-947-1931 bit.ly/CallThio Coffetote Gallery Jl Tomang Rawa Kepa 37 Grogol Jakarta Barat G-Maps http:/bit.ly/mapscraftote

      peluangusahagratis, #peluangusahagampang, #peluangusahahalal, #peluangusahaonlinemodalterjangkau, #peluangusahau, #usahaonlinecepatuntung, #usahaonlinedirumah, #usahaonlinedigital, #usahaonlineexplore, #usahaonlinegratis

  4. Sep 2021
    1. Regular, sustained practice.• This may be one of the most important of all anti-dotes. I have seen some very impressive friends and colleagues fall into traps of stagnation, depression, and addiction. One of the common factors seemed to be that they had given up their daily spiritual practice. Yet continuity of practice is one of the most recurrent refrains across spiritual traditions, as for example, in the advice of the Koran, “Be constant in prayer”

      Wise words ...

    2. This is a key requirement for anyone who aspires to truly understand and communicate the integral vision.

      Wow! Spot on. Correctly and bluntly stated.

    1. One of the most problematic settings that cause Windows 10 slow boot is the fast startup option. This is enabled by default. So, disable it. First, open 'Settings' then 'System' then 'Power & sleep.' Now, click 'Additional power' settings to open the 'Power' option menu in the Control Panel. Here, click the 'Choose what the power' button. Now, give the administrator permission to change the settings on this page. Now, untick. Turn on fast startup and click Save.

  5. Aug 2021
  6. Jul 2021
    1. The built-in Windows Update service on your computer usually keeps most of your drivers up to date in the background. But still, if you want to know how to update drivers in Windows 10 manually, then first, click the 'Start button. Now, click on the 'Settings' icon. It appears as a small gear icon. Further, select the 'Updates & Security,' and then click 'Check for updates.' If the updates are available, you can go further.

    2. If you're taking proper care of Windows, you shouldn't need to clean install of Windows 10 regularly. However, you can reinstall Windows when upgrading to a new version of Windows. Performing an upgrade install can result in various issues, so, t's better to start with a clean slate. The reinstallation will delete certain items. However, as part of the process, the setup will also create a Windows old folder with everything from your previous installation.

    3. If you encounter an error code 0x8024200d when performing a Windows Update, you are not alone. Many Windows users are reporting it. Windows 10 update error 0x8024200D usually appears once they attempt to update to a new build of Windows system. The cause behind it's that some updated files are missing or corrupted. To repair this attempt to restart your PC, temporarily delete ‘Windows Installation Files,’ and restart the ‘Update and Download Service.’

  7. Jun 2021
    1. .

      In the lecture notes, Giacomo says "The logic behind decreasing returns is that, if K increases while holding L fixed machines per worker and worker productivity falls." I don't have great clarity on what "worker productivity falls" truly means and don't want to put something incorrect--fill in once lecture videos are out

    1. If, for some reason, you wish to reinstall Store or the other preinstalled app, you'll do so by executing an easy command in PowerShell. The command is useful if you've got accidentally uninstalled Store or the other app and now want to restore the same. For instance, to reinstall a Store app on Windows 10, start PowerShell as Administrator, click “Start,” and sort “PowerShell.” Now, right-click “PowerShell” and click on “Run as administrator” this reinstalls the Store app.

    2. Essentially, the Srttrail.txt BSOD error in Windows 10 informs about a failed automatic system repair and allows the user to choose between “Advanced Repair” and “System Shut Down.” The “Advanced Repair” should help users reach the “Safe Mode,” and to fix the issue, perform a system restore or remove and replace your laptop battery. Else disconnect your USB devices and run “cmd” from the Windows 10 Boot Options menu.

    3. This BSOD error message mainly appears when you have incompatible hardware or a malfunctioning driver because a driver is missing or improperly installed. It's also possible that the new hardware that you recently installed is not fully compatible with your computer and is causing the APC Index Mismatch error in Windows 10. To fix this, install the latest Windows updates, update device drivers via Device Manager, or check your system file issues.

    4. Are you a user of Windows 10 OS and wondering how to install Google Chrome in Windows 10? If yes, then it requires you to open any web browser, type “google.com/chrome” into the address bar, and then press the Enter key. Now, click Download Chrome and click on “Accept and Install” and Save File and click “OK” and launch it. You can drag the Chrome icon into Applications.

  8. Mar 2021
    1. Il peut opposer un refus à la tenue d'une réunion ou à la participation de personnalités extérieures lorsque celles-ci sont de nature à porter atteinte au fonctionnement normal de l'établissement ou à contrevenir aux principes du service public de l'enseignement.
    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: 4.9

      AssayResultAssertion: Normal

      StandardErrorMean: 0.32

    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.1190C>T p.(Thr397Ile)


      AssayResult: 90

      AssayResultAssertion: Normal

      PValue: Not reported

      Approximation: Exact assay result value not reported; value estimated from Figure 6C.


      AssayResult: -10

      AssayResultAssertion: Normal

      PValue: Not reported


      AssayResult: 101.6

      AssayResultAssertion: Normal

      PValue: > 0.9999

      Comment: Exact values reported in Table S3.

    4. 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.2590C>T p.(Pro864Ser)

    1. Source Data

      AssayResult: 74.18

      AssayResultAssertion: Not reported

      ReplicateCount: 2

      StandardErrorMean: 6.49

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

    2. Source Data

      AssayResult: 98.55

      AssayResultAssertion: Not reported

      ReplicateCount: 2

      StandardDeviation: 5.74

      StandardErrorMean: 4.06

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

    3. 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.1492G>T p.(D498Y)

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

      AssayResult: 113

      AssayResultAssertion: Normal

      ReplicateCount: 17

      StandardErrorMean: 28.6

      Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (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.1859G>A p.(Arg620His)

    1. Fix the issues with IncrediMail by trying out a couple of different solutions; reconfiguring it if the issue occurs when sending or receiving emails, you can update IncrediMail Mail program to ensure smooth functioning with Windows 10, uninstall the contrary applications that may be causing the Incredimail problems with Windows 10, or you can reset your web browser to get rid of ay technical hindrance. Try these solutions to fix the problems with IncrediMail. Related Issues Incredimail not sending emails Incredimail password recovery

    1. ReconfigBehSci on Twitter: ‘1 week to the SciBeh workshop “Building an online information environment for policy relevant science” Join us, register now! Topics: Crisis open science, interfacing to policy, online discourse, tools for research curation talks, panels, hackathons https://t.co/Gsr66BRGcJ https://t.co/uRrhSb9t05’ / Twitter. (n.d.). Retrieved 2 March 2021, from https://twitter.com/SciBeh/status/1323207455283826690

  9. Feb 2021
    1. Supplemental material

      AssayResult: 82

      AssayResultAssertion: Normal

      Comment: See Table S3 for details

    2. Supplemental material

      AssayResult: 2.9

      AssayResultAssertion: Abnormal

      Comment: See Table S3 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.314G>A p.(Gly105Asp)

  10. Nov 2020
  11. Oct 2020
    1. n order to determine optical bandgap, the Tauc relation-ship is used as follows [24]:ahv=Ahv−Egn,3whereais absorption coefficienta=−107lnT/t,Aisa constant,his Planck’s constant,vis photon frequency,Egis optical bandgap, andnis 1/2 for allowed direct semi-conductor bandgap. The bandgap energy of ZnS thinfilmwas estimated by plottingahv2againsthvas in Figure 10;

      En orden de determinar band gap óptico, la relación de Tauc fue usado como sigue:



    1. I am the self abidingin the heart of all creatures;I am their beginning,their middle, and their end.

      Lord Krishna mentioned his power to the world that his power is endless.

  12. Sep 2020
    1. [ my friend, whom I loved so dear,] [who with me went through every danger,] [my friend Enkidu, whom I loved so dear,]

      This is how Gilgamesh express their friendship.

  13. Jun 2020
  14. May 2020
  15. Apr 2020
    1. The big thing about the Web isn’t the technology, it’s that it’s the first-ever platform without a vendor (credit for first pointing this out goes to Dave Winer). From that follows almost everything that matters, and it matters a lot now, to a huge number of people. It’s the only kind of platform I want to help build.

      First, and nearly last atm.

    1. Environmental policies much like the Sustainable Development discourse have become part of these established regimes and have primarily served to optimize these regimes making them a bit less unsustainable. I thus come to conclude that Sustainable Development itself has become part of the problem. The currently dominant regimes based on the foundations of modernity, are systemically unsustainable in a fundamental way.

      Yes! It is an elastoplast on the gaping wound. The painkiller that let's you get by your dislocated shoulder so you don't do anything about it.

    2. The two cases are to me clear examples of the ́sustainability lock-in’: we seem to be caught in a vicious cycle of optimizing and inherently unsustainable system which closes down the space for the development of inherently better alternatives
  16. Jan 2020
    1. I like that the Lambda Architecture emphasizes retaining the input data unchanged. I think the discipline of modeling data transformation as a series of materialized stages from an original input has a lot of merit. This is one of the things that makes large MapReduce workflows tractable, as it enables you to debug each stage independently. I think this lesson translates well to the stream processing domain. I’ve written some of my thoughts about capturing and transforming immutable data streams here.

      Great point 👍

      Something i've thought about and emphasized for doing FDF - ability to debug per step or re-run after a given step.

    1. RRID:ZFIN_ZDB-GENO-140612-10

      DOI: 10.1016/j.cell.2019.01.019

      Resource: (ZFIN Cat# ZDB-GENO-140612-10,RRID:ZFIN_ZDB-GENO-140612-10)

      Curator: @ethanbadger

      SciCrunch record: RRID:ZFIN_ZDB-GENO-140612-10

      What is this?

  17. Dec 2019
    1. I think this has the added benefit of making data warehousing ETL much more organizationally scalable. The classic problem of the data warehouse team is that they are responsible for collecting and cleaning all the data generated by every other team in the organization. The incentives are not aligned: data producers are often not very aware of the use of the data in the data warehouse and end up creating data that is hard to extract or requires heavy, hard to scale transformation to get into usable form. Of course, the central team never quite manages to scale to match the pace of the rest of the organization, so data coverage is always spotty, data flow is fragile, and changes are slow.
  18. Nov 2019
    1. Top 10 Global DevOps Consulting Companies

      Are you wishing to develop the websites at global level? Here you can find top 10 Devops Consulting Companies that aim to deliver first class solutions forever. In addition to this, the expert developers able to provide the friendly services and thus get attention on familiar sources forever.

  19. Sep 2019
    1. Americans today are some of the unhealthiest people on Earth

      That is correct, due to the high fatty food, lack of exercises,smoking and other related segments make them unhealthy. So the heart related problems are looming. According to the studies America is on the 10th place

  20. Aug 2019
    1. the set fee per transaction of 10%


    2. charged 10%

      replace with "a small fee"

    3. 10 percent

      Delete "10 percent" and replace with "a"

    4. Negotiate with the shipper with 10% fee in mind.


  21. Jul 2019
    1. Myth: Refugees are all Muslim.

      Do people actually think that? That is ridiculous and so ignorant. People shouldn't stereotype like that. Does the general public really believe that all refugees are from the middle east and are Muslim? I wonder if they know that there are thousands of Christians in the middle east."Christians now make up approximately 5% of the Middle Eastern population, down from 20% in the early 20th century" That's part of the problem. It's a war on freedom. Religious freedom, basic human rights, and personal desires. Sheesh!

    1. Production of β-galactosidase (ortho-Nitrophenyl β-D-galactopyranoside (ONPG)) test
    2. Production of β-galactosidase (ortho-Nitrophenyl β-D-galactopyranoside (ONPG)) test
  22. Jun 2019
    1. Expression of costimulatory molecules on splenocytes harvested from immunized and control animals
    1. The synthetic peptides were purified by RPHPLC on an Aquapore RP300 column (250 x 7 mm) using a 4-72% linear gradient of solvent B (acetonitrile containing 0.1% TFA) in 130 min at a flow rate of 2 mllmin. Globin chains from respective hemoglobins were separated on a similar column of a smaller dimension (250mm x 4.6 mm) under identical conditions but at a flow rate of0.7 ml/min. Electro spray mass spectrometric analysis was carried out on a VG Platform (Fisons) mass spectrometer. The instrument was usually calibrated with standard horse heart myoglobin or gramicidin S solution. Appropriate amount of each sample was taken in 50% acetonitrile containing I% formic acid and analyzed under the positive ion mode.
    2. nalytical procedures
  23. May 2019
    1. The results of the study indicate that education of the future business leaders calls for a contextualized approach to business ethical dilemmas.

      The main goal of this article is to understand how the perspective about business ethics between business student and non-business students differ, and the results show that we indeed do need contextualized approach to business ethical dilemmas

    1. An easy solution would be for the school to change to an ISP that would agree not to filter traffic. Unfortunately, in many rural areas, there are often few choices for ISPs, creating a lack of competition. An FCC report from June 2017 found that about 75% of U.S. census block regions have zero choice in terms of high speed Internet/ broadband access (Brodkin, 2017). The FCC has claimed that market competition will provide a check on potential ISP abuse. “Given the extent of competition in Internet access supply,” the FCC’s (2017) new order states, “the protections regulating ISPs are not necessary” (p. 144). Despite the frequent claims of competition throughout the document, the statistics included by the FCC show that competition is not as widespread as it would like to claim.

    1. The protein samples were resolved by SDS PAGE using the Laernrnli buffer system (Laernrnli, 1970). The protein sample was denatured by boiling at 100°C for 10 min in Laernrnli's buffer (List I). Resolving gel (10%) was prepared in a minigel (Bio-Rad, USA) system alongwith 3% stacking gel and the electrophoresis was carried out at 120 volts for 125 min. The gel was stained with 0.25% Coomassie blue R staining solution for Ih followed by destaining with successive washes of de staining solution. Staining was avoided when. gel was used for irnrnunoblotting. Details of reagents used for SDS-PAGE are given in List 1.
    2. SDS PAGE
    1. Sub-cellular fractionation of THP-1 macrophages was performed after lysis with hypotonic buffer. THP-1 macrophages after appropriate treatment were allowed to swell for 10 min in hypotonic buffer (1 0 mM NaCl, 1.5 mM MgCh, 10 mM Tris-HCl, pH 7 .5) followed by homogenization with 50 strokes using a Dounce homogenizer. More than 90% cellular lysis was ensured by visualizing under a light microscope, and immediately after lysis, the mitochondrial membranes were stabilized by addition of 2.5x mitochondrial stabilization buffer (525 mM mannitol, 175 mM sucrose, 12.5 mM Tris-HCl, 2.5 mM EDTA, pH 7.5) to a final concentration of 1x. The homogenate was centrifuged at 1300 x g for 15 min to isolate the nuclear fraction. The post-nuclear fraction was further centrifuged at 17,000 x g for 15 min in an ultracentrifuge (Beckman Optima XL-1 OOK ultracentrifuge) to isolate the mitochondria. The post-mitochondrial supernatant was centrifuged at 100,000 x g for 1 h to obtain the membranous fraction as a pellet and the supernatant obtained was the cytosol. The homogeneity of the obtained fractions was determined by probing for fraction specific proteins by Western blotting
    2. Sub-cellular fractionation
    1. Heterologus expression of various proteins was done in BL21 (A.DE3) strain of E. coli. Bacterial cells were transformed with the desired construct and grown in Super broth (pH 7.2) containing 100 J.lg/ml ampicillin, at 37 °C with continous shaking in a gyratory shaker at 225 rpm. The cultures were induced, at ~oo of 2.0, with I mM IPTG, and harvested two hours later by centrifugation at 4000 g, at 4 °C, for 20 min .. The recombinant proteins were purified from the inclusion bodies using the procedure described by Buchner et al ( 1992). The total cell pellet from a liter of culture was homogenized in 180 ml of inclusion bodies washing buffer containing 8 ml of freshly prepared lysozyme solution (5 mg/ml). The suspension was incubated at room temperature for 1 hr with intermittent shaking. Added 20 ml each of 5M NaCI and 25% Triton X-100 were added to the suspension and incubated at room temperature for 30 min. with vigorous shaking. The suspension was centrifuged at 13,000 g at 4 °C, for 50 min. and the pellet was resuspended, in the washing buffer containing 1% Triton X-1 00, using a polytron homogenizer and centrifuged at 13,000 g for 50 min. The pellet was washed four times with washing buffer without Triton . X-100. The pellet containing inclusion bodies was solubilized in 6 M guanidine hydrochloride by incubating for 2 hours at room temperature. The solubilized protein was centrifuged at 50,000 g, at 4 °C, for 30 min. and the protein concem.ration was adjusted to 10 mg/ml in the supernatant with 6 M guanidine hydrochloride. The denatured protein thus obtained was reduced by adding 65 mM dithioerythritol and incubated at room temperature for 2 h. To renature, the protein was diluted 1 00-fold in the refolding buffer and incubated at 10 °C for 48 h without stirring or shaking. Renatured material, after dialysis in 20 mM MES buffer, pH 5.0 containing 100 mM urea, was loaded on a S-Sepharose column, and the protein bound to the column was eluted with a 0-1 M NaCI gradient in 20 mM MES on an FPLC system (Pharmacia). The fractions containing the desired protein were pooled and concentrated, and the protein was further purified to homogeneity by gel filtration chromatography on a TSK 3000 column (LKB) in PBS, pH 7.4.
    2. Isolation and Purification of Proteins from the Inclusion Bodies
    1. Sf9 cells infected with AcNPV, VI, V2, V3, or V4 were harvested 72 h pi, washed twice with 10 mM PBS, air dried on a slide and fixed in chilled methanol for 15 min. Cells were incubated with MA-451 culture supernatant and N-terminal anti-peptide serum ( 1 :500) at 37°C for 1 h, washed with PBS and further incubated at 37oc for 1 h with I :50 dilution of anti-mouse FITC or I :2000 anti-rabbit FITC. Slides were washed extensively, mounted in 90% glycerol in PBS (50 mM, pH 7.4) and examined under Optiphot fluorescent microscope (Nikon, Tokyo, Japan).
    2. Immunocytochemical Localization of Recombinant Proteins in Infected Cells
    1. lysed directly in 1. 5 ml of solution D ( 4 M guanidium thiocyanate, 25 rnM sodium citrate, pH 7.0, 0.5 % sarcosyl and 0.1 M 2-mercaptoethanol ) . For every 2 ml of the lysate, 0.2 ml of chloroform was added, followed by vigorous mixing for 15 seconds, and incubation on ice for 15 minutes. The lysate was spun at 12, OOOg, at 4 °c for 15 mins. , and the aqueous phase transferred to another tube. RNA was precipitated with an equal volume of isopropanol and incubation at -2o0c for 45 mins. The samples were then spun at 12,000g for 15 mins. at 4°c, and the supernate discarded. The RNA pellet was washed twice with 75 % ethanol. Finally, the pellet was dried briefly under vacuum for 10 -15 mins. and dissolved in 0.5 % SDS. All chemicals and glassware used for handling RNA were treated with diethylpyrocarbonate ( DEPC ) .
    2. Total RNA was isolated from cultured mammalian cells by the method of Chornczynski and Sacchi ( 1987 ), with slight modifications. Briefly, cells from a 3.5 ern petri-dish were
    3. Isolation of RNA
    4. All computer software facilities were provided by the NII computer centre.
    5. Computer software.
    1. stranded DNA. The reaction was carried out at 37°C for 1 h. The reaction mixture contained 100 ng Bgl II digested VR1020 vector, SAP (0.5 U) and 1 fll lOX SAP buffer (20 mM Tris-HCl, pH 8.0, 10 mM MgCh) in 10 f.!l oftotal reaction volume. The reaction was stopped by inactivating the enzyme at 65°C for 15 min. The digested bmZP1 eDNA was ligated with SAP treated VR1 020 at vector : insert ratio of 1:10 in a 10 fll reaction volume for 16 h at l6°C. The reaction mixture contained 10 ng VR1020 vector, 26 ng bmZPl insert, 1 fll lOX ligase quffer (30 mM Tris-HCl, pH 7.8, 10 mM MgCh, 10 mM DTT and 1 mM ATP), lfll T4 DNA ligase (20 U) in a total reaction volume of 10 fll. The ligation product was used for transformation of DH5a competent cells as described previously. Transformants were selected on LB plates containing 50 f.!g/ml Kanamycin (Kan). Similarly, the inserts corresponding to dZP3, rG and dZP3-rG fusion were digested with Bgl II restriction enzyme, gel purified and cloned in VR1020 vector, except that the ligation product of dZP3-rG fusion with VR1020 was transformed into JM109 competent cells
    2. The insert corresponding to bmZP1 was released from the pPCR-Script-bmZPl clone by Bgl II restriction and purified on the agarose gel. VR1020 vector was similarly digested and gel purified. To prevent self-ligation, the digested vector was treated with Shrimp Alkaline Phosphatase (SAP), which removes 5'-phosphate from the termini of double
    3. Cloning in VRl 020 mammalian expression vector
    1. Meancorpuscularhaemoglobinconcentration(MCHC)istheaverageHbconcentrationperunitvolume(100)ofpackedredcells(W/V).Henceitisexpresseding/1whichisthesameaspercent(%).ItiscalculatedbythefollowingformulaHbMCHC=—......x100(g/dl)PCV
    2. MCHC
    3. MeancellVolume(MCV).Itisexpressedinfentolitres(1fentolitreorflisequivalentto10'151)andcalculatedby thefollowingformula:PCVMCV=.....................x10(fl)RBC8.10.6.2.MCHMeancellhaemoglobin(MCH)=AverageweightofHbinanerythrocyte.Itisexpressedinpicograms(pg)whichisequivalentto10"12g.Itiscalculatedbythefollowingformula:HbMCH=-----------------x10(ppg)RBC
    4. MCV
    5. RedBloodcellsindices
    6. micro-haematocrittubewasfilledto100mmwithanticoagulatedblood.Oneendofthetubewassealedwithsealingwaxandthetubewasthenkeptinaverticalpositioninaglassbeakerstuffedwithcotton.Afteronehour,lengthoftheplasmacolumnwasmeasuredwitha rulergraduatedin0.5mm.
    7. ESRwasdeterminedbythemicromethodbecausethequantityofbloodavailablefromindividualfishwasinsufficienttoadoptanymacromethod.Anon-heparinised
    8. ErythrocytesedimentationRate(ESR)
    9. BloodwascollectedfromtheheartbycardiacpunctureusinganRBCpipette.ItwasdilutedwithHayem’sfluidintheratioof1:200.Thecontentswereshakenwell.AdropofthedilutedbloodwasplacedinaNeubauerdoublehaemocytometer(Germany)countingchamberandtheredbloodcellcountpercubicmmwascalculated
    10. Redbloodcellcount(RBC
    11. Thepackedcellvolumeorhaematocritisthevolumeoccupiedbythepackedredcells,afteravolumeofanticoagulatedvenousbloodisfullycentrifuged.Thevolumeofpackedcellisexpressedasapercentageoftheoriginalvolumeoftheblood.ThePCVisusedtoestimatehaematologicalindices,includingthemeancellhaemoglobinconcentration(MCHC)andmeancorpuscularvolume(MCV).PCVdetermination followedthemethodsofBlaxhallandDaisley(1973).Thehaematocritvaluewasdeterminedbycentrifuging(3000rpm)aknownvolume ofincoagulantbloodkeptinWintrobe’stubes
    12. PackedCellVolume(PCV)orHaematocrit(Ht)
    13. HaemoglobinwasdeterminedbySahlimethod.HaemoglobinisconvertedtoacidhaematinbytheactionofHC1.Theacidhaematinsolutionisfurtherdilutedwiththeaciduntilitscolormatchesexactlythatofthepermanentstandardofthecomparatorblock.TheHbconcentrationisreaddirectlyfromthecalibrationcurve.
    14. Haemoglobin(Hb)determination
    15. BloodwastakenbyheartpunctureusingMS222astheanaesthetic.Nofishwasusedmorethanonce.
    16. CollectionofBlood
    17. HaematologicalAnalysis
    1. The cells were assayed for Luciferase gene expression using Luciferase Assay kit (Promega, U.S.A.). After transfection, the cells were washed twice with PBS and then lysed by adding reporter lysis buffer provided in the kit. The cell lysate was collected from individual wells in eppendorf tubes, the cells were twice freeze-thawed in liquid N2 and then centrifuged at 13,000 rpm for 10 min at 40C. The supernatant was transferred to a fresh tube. 20¢ of cell extract was mixed with lOOp! of luciferase assay reagent that was kept at room temparature. The activity was determined using a luminometer (Packard lumicount, U.S.A.
    2. Luciferase assay
    1. normalized to the optical density at day 0 for the appropriate cell type. Growth curve was determined twice
    2. Growth curves were prepared for various cell lines using the modified method adopted by Serrano et al, 1997. Briefly, 10, 000 cells were seeded in a 24 well plate in quadruples. At the indicated times, cells were washed once with PBS and fixed in 10% formalin for 20 minutes and rinsed with distilled water. Cells were stained with 0.05% crystal violet for 30 minutes, rinsed extensively and dried. Cell associated dye was extracted with 1.0ml acetic acid. Aliquots were diluted 1:4 with water and transferred to 96 well microtitre plates and the optical density at 590nm determined. Values were
    3. Growth Curves:
    1. Inverse PCR is a technique to amplify unknown regions flanking the site of transposon insertion using the primers designed from the known sequence from one end of the transposon element. Genomic DNA was digested with a 4-base recognition restriction enzyme, Sau3A1 followed by intramolecular ligation set up at high dilutions. These ligated molecules were then used as templates for the PCR performed with a pair of divergently-oriented primers designed from one end of the transposon named AH1-AH2. The PCR product thus obtained was sequenced with the same set of primers to identify the junction sequence at the site of transposon insertion and hence the identity of the gene disrupted in each case. Typical PCR conditions used were as follows:- Annealing 55°C 2 min Elongation 72°C (1 min/kb of DNA template to be amplified) Denaturation 95°C 2 min After 30 cycles of PCR, the final elongation step was carried out again for 10 min at 72°C
    2. Inverse PCR
    3. To quantitate the P1 phage lysate preparations, titration was done using a P1-sensitive indicator strain such as MG1655. 100 μl each of serial dilutions of the phage (typically 10−5, 10−6) were mixed with 0.1 ml of the fresh culture grown in Z-broth. After 15 min adsorption at 37°C without shaking, each mixture was added in a soft agar overlay to Z agar plates, and incubated overnight at 37°C. The phage titre was calculated from the number of plaques obtained on the plates
    4. To 0.3 ml of infection mixture, 10 ml of Z-broth was added and incubated at 37°C with slow shaking until growth followed by the visible lysis of the culture occurred (in ~ 4-6 h). The lysate was treated with 1 ml of chloroform, centrifuged and the clear lysate was stored at 4°C with chloroform
    5. Broth method
    6. 0.3 ml of overnight culture of the donor strain in Z-broth was mixed with 107 plaque forming units (pfu) of a stock P1 lysate prepared on strain MG1655. Adsorption was allowed to occur at 37°C for 15 min and the lysate was prepared in the following ways
    7. Phage P1 lysate preparation
    1. Rhod-2 acetoxymethyl ester is a fluorescent long wavelength calcium indicator, where the AM ester forms are cationic, resulting in a potential driven uptake into the mitochondria making them selective detectors of mitochondrial calcium. Log phase cultures were taken and dead cells pelleted at 129 x g for 5 min at RT. The live cells were washed twice with Kreb' s buffer (118mM Sodium chloride, 5.4mM Potassium chloride, 1.2mM Magnesium chloride, 1.2mM Potassium dihydrogen phosphate, 25mM Sodium hydrogen phosphate, llmM glucose, 1.5mM Calcium chloride, pH 7.4) by centrifugation at 1258 x g for 5 min to wash off all traces of medium and FBS. Washed cells were loaded with 1:1 mixture of Rhod -2 AM (1p.g/p.L stock solution prepared in DMSO) and 20%w/v Pluronic F127 for 1h at RT in the dark with shaking. Excess dye was removed by one wash with Kreb' s buffer followed by incubation of cells at RT for a further 30min for complete hydrolysis of the dye trapped in the mitochondria. Fluorescence intensities of the stained cells were measured fluorimetrically at excitation of 530nm and emission of 576nm or alternatively acquired by flow cytometer.
    2. Assay for measuring mitochondrial calcium
    3. The DNA fragments eluted from the agarose gel or purified PCR products were cloned into pGEM-T easy vector which allows efficient sequencing using the common sequencing primers T7 and SP6. SOng of the vector was used with 1J..lL of T4 DNA ligase in a 10J..lL reaction volume. The reaction was allowed to proceed at 4 °C for 16h followed by transformation into DHS-a strain of E coli following standard protocols. The transformed cells were plated onto LB-agar plates containing appropriate ampicillin (100J..lg/mL) and blue-white selection reagent (40J..lL/plate). The plate was incubated at 37°C for 12 hrs, following which white colonies were picked up for screening for the presence of the gene of interest.
    4. Sub-cloning of PCR products into pGEM-TEasy Vecto
    1. Polyphosphates from yeast cells were extracted with phenol-chloroform solutionas described previously(Neefand Kladde,2003). Cells grown eitherovernight or to logarithmic phase in YPDmediumwere harvested by centrifugation at 1,700 rpm for 5 min. Cells were washed with 10 ml sterile MQ water and resuspendedin ice-cold 500 μl20%trichloro acetic acid (TCA) solution to the final cell densityof 100 OD. Cell suspension was transferred toa1.5 ml microcentrifuge tube. After incubation at room temperature for 5 min, cells were harvested by centrifugation at 12,000 g for 10 minat 4 ̊C and resuspendedin 250-350 μlpolyphosphate extraction buffer.Equal volume of phenol-chloroform (25:24) was addedtothe microcentrifuge tube and aqueous phase was extracted by centrifugation at 12,000 g for 8 minat room temperature. Top aqueous layer was collected with a 200 μl tip. Aqueous layer extraction was repeated once more after removal of DNA with chloroform.After centrifugation,aqueous phasecontaining RNA and polyphosphates wascollected,RNA was quantified at A260nmandstored at -20 ̊C
    2. Polyphosphate extraction
    3. hybridizations from biological replicates for each sample. Data was extracted with Feature Extraction software v 10.5 (Agilent) and normalizedwith GeneSpring GX v 11.0.1 (Agilent) software using the recommended Percentile shift Normalization to 75th percentile. Raw Data sets for this study are available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=


    4. Log-phase wild-type and Cgyps1∆cells were grown in YNB and YNB-pH 2.0 medium. After 1 h incubation, yeast cells were collected, washed and were stored in RNAlater at -80°C. These frozen samples were sent to Genotypic Technology Ltd., Bangalore(http://www.genotypic.co.in) whichprovides services of global gene analysis on Agilent platform. A 8x15k GE array comprised of 60mer oligonucleotidesfor a total of C. glabrata5503 genes was used wherein average number of replicates for each probe was three. Labeling was done in single color and data is the average of two
    5. Microarray analysis
    1. For RNA experiments, all solutions were prepared in RNase free diethylpyrocarbonate (DEPC) water. Microcentrifuge tubes and tips used for RNAworkwere autoclaved twice and driedat 70 ̊C for overnight before use. Non-autoclavable plastic items were wiped with Ambion RNAseZap to remove RNAse contamination, if any. RNA was extracted from C.glabrata cells usingacid phenol extraction method. C. glabratacells were harvested at 2,500g for 5 minat 4 ̊C, resuspended in 1 ml ice-cold DEPC water and were transferred toa2 ml microcentrifuge tube. Cells were spun down at 6,000g for 3 minat 4 ̊C and resuspended in 350 μl AE solution. Next, 50 μl SDS and 400 μl acid phenol(pH 4.5)solutions were added to thetube and mixed well by vortexing. The tube wasincubated at 65 ̊C for 15 min with continuous mixing. After incubation, tube was kept on ice for 5 minand centrifuged at 7,500g for 5 minat 4 ̊C. Aqueous phase was transferred toa new 1.5 ml microcentrifuge tube and RNAwasextracted with an equal volume of chloroform. Total RNA was precipitated at room temperature with 1/10thvolume of 3 M sodium acetate (pH 5.2) and 2.5 volumesof chilledabsolute ethanol for 20 min. Precipitated RNA was collected by centrifugation at 7,500g for 5 minat 4 ̊C. RNA pellet was washed with chilled 70% ethanol and resuspended in 50 μl nuclease-free water. RNA concentration was determined by measuring absorbance at 260 nm. Quality of extracted RNA was examined by gel electrophoresis on 0.8% agarose gel prepared in DEPC-treated TAE buffer
    2. RNA extraction
    3. Alipophilic styryl dye,FM4-64,is a vital stain which istakenupby cells viaendocytosis through plasma membrane(Vida and Emr, 1995). Therefore, it fluorescesonly in live cells. Importantly, neitherfixed cells canbe stained with FM 4-64norcells canbe fixed afterFM 4-64staining. For vacuole staining, single colony of the test strain grown onYPD plate was inoculated in 10 ml YPD medium for overnight. 100 μlovernight culture was inoculated in fresh YPD medium and incubated at 30ºC for 3 hto obtain log-phase cells. C. glabratacells from 1 ml log-phase culture were harvested at 4,000 rpm for 5 minin a table top centrifuge. Supernatant was aspirated out,cells were resuspended in 50 μl YPD medium and 1 μl FM 4-64 (16 μM final concentration) was added.C. glabratacells were incubated in a 30ºC water bath for 30 min. 1 mlYPD medium was added and cells were harvested at 4,000 rpm for 5 minin a table-top centrifuge. After discarding supernatant,C. glabratacells were washed with fresh YPD medium and resuspended in 1 ml YPD medium. C. glabratacells were incubated at 30ºC for 90 min, washed with 1 mlsterile water and were resuspended in 50 μl YNB medium. Labeled C. glabratacells were observed underfluorescence microscope in red filter(730nm)
    4. Stainingof C. glabratavacuoleswith FM4-64
    1. Immunofluorescence assay was carried out as described by Bhattacharyyaet al., 2010.Adherent cells weregrown either on cover slips. After treatment, cells were fixed with 3.7% paraformaldehyde solution in PBS for 15 min and permeabilised with 0.5% Triton X-100 at room temperature for 10 min, followed by blockingin PBS containing 2% BSA for 1h. Post blocking, cells were incubated with a primary antibody in PBS (1:200 to 1:500) for 2 h. After washing, cells were incubated with fluorescent-conjugated secondary antibodyin PBS(Alexa Fluor 488 or 594 goat anti-rabbit or antii-mouse, 1:1000) for 30 min. After final wash with PBS, nuclei were counterstained with DAPI containing mounting medium (Vectashield, USA). All the steps were performed at room temperature, unless otherwise stated. Images were obtained using either the laser scanning confocal LSM510 (Carl Zeiss,