Ethics statement

Other methods

Reverse transcriptase “Superscript III” (Invitrogen, 18080-051) was used to perform cDNA synthesis. Briefly, 500 ng of DNase I-digested RNA was incubated with 1 μl of 10 mM dNTP and 50 μM oligo(dT) at 65°C for 5 min in a 10 μl reaction mixture followed by cooling on ice for 5 min. Post incubation, 10 μl of cDNA synthesis mixture was added which contained 2 μl of 10XRT buffer, 4 μl of 25 mM MgCl2, 2 μl of 0.1 M DTT, 1 μl of RNase out (40 units) and 1μl of Superscript III (200 units). Tubes were incubated at 50°C for 1 h and thereaction was terminated at 85°C for 5 min. The quality of synthesized cDNA was checked by using it as a template in a PCR reaction to amplify the housekeeping gene CgACT1. Amplification of CgACT1was indicative of proper cDNA synthesis

Complementary DNA (cDNA) synthesis

Reverse transcriptase “Superscript III” (Invitrogen, 18080-051) was used to perform cDNA synthesis. Briefly, 500 ng of DNase I-digested RNA was incubated with 1 μl of 10 mM dNTP and 50 μM oligo(dT) at 65°C for 5 min in a 10 μl reaction mixture followed by cooling on ice for 5 min. Post incubation, 10 μl of cDNA synthesis mixture was added which contained 2 μl of 10XRT buffer, 4 μl of 25 mM MgCl2, 2 μl of 0.1 M DTT, 1 μl of RNase out (40 units) and 1μl of Superscript III (200 units). Tubes were incubated at 50°C for 1 h and thereaction was terminated at 85°C for 5 min. The quality of synthesized cDNA was checked by using it as a template in a PCR reaction to amplify the housekeeping gene CgACT1. Amplification of CgACT1was indicative of proper cDNA synthesis

Complementary DNA (cDNA) synthesis

ml YPD broth at an initial OD600of 0.1. Cultures were allowed to grow for 4-5 hin a shaker incubator setat 30°C, 200 rpm until the OD600of the cultures reached 0.4-0.6. Next,cells were harvested ina15 ml centrifuge tube by centrifugation, washed twice with 10 ml of sterile water, resuspended in 1 ml of sterile water and were transferred to a 1.5 ml microfuge tube. Cells were harvested by centrifugation at 4,000 rpm for 5 min,resuspended in 50 μl of100 mM lithium acetate solution and transformation mixture was added. Transformation mixture consisted of 240 μl polyethylene glycol (50%), 36 μl of lithium acetate (1 M), 5 μl of heat denatured single stranded carrier DNA (10 mg/ml), 500 ng to 1 μg of transforming DNA and final volume was made to 360 μl with sterile water. The tubes were incubated at 30°C for 45 min. To this, 43 μl of sterile DMSO was added and heat shock was given at 42°C for 15 min. Next, tubeswere transferred to ice for 10-15 sec, centrifuged at 4,000 rpm and transformation mixture reagents wereremoved completely by pipetting. Cells were resuspended in 200 μl of sterile water and spread-plated on appropriate selection medium. Plates were incubated at 30°C for 24-48 h

Yeast transformation was performed as described previously (Gietz et al., 1992) with fewmodifications. Briefly, overnight grown C. glabratacultures were freshly inoculated in 10

Yeast transformation

To perform survival analysis of C. glabratacells in macrophages, PMA-treated THP-1 cells were seeded to 24-well tissue culture platesto afinal celldensity of 1 millionper well. C. glabratacells were grown in YNB medium for 14-16 h at 30°C and 200 rpm. 1 mloftheseC. glabratacells were harvested in 1.5 ml centrifuge tubes, washed twice with 1X sterile PBS and the cell density was adjusted to 2x106cells/ml. 50 μl of this cell suspension was used to infect PMA-activated macrophages to a MOI (multiplicity of infection) of 0.1. Two hours post infection, THP-1 cells were washed thrice with 1X sterile PBS to remove the non-phagocytosed yeast cells and 1 ml of fresh pre-warmed complete RPMI-1640 medium was added.At different time points, infected THP-1 macrophages were osmolysed with1 ml sterile water. Post lysis,lysates were collected by scraping the wells using 1 ml microtip. Lysates were diluted in 1X sterile PBSand appropriate dilutions were plated onYPD-agar plates. Plates were incubated at 30°C for 24-48 h and colony forming units (CFUs) were counted. Final CFUs/ml were determined by multiplying CFUs withappropriate dilution factor and percentage phagocytosis was calculated by dividing CFUs obtained at 2 h post infection by total numberofyeast cells used for infection. Fold replication was calculated by dividing the CFUs obtained at 24 h post infection by CFUs obtained at 2 h post infection

THP-1 macrophageinfection assay to monitor the intracellular survival and replication of C. glabrata

Stripping buffer100 mM β-mercaptoethanol2 % SDS62.5 mM Tris-HCl (pH 6.7)Final volume was madeto 250 ml with water

Transfer buffer (10 X stock solution)0.25 M Tris-HCl (pH8.0)1.92 M Glycine1% SDSThe stock solution was prepared asa10 X concentrate and was diluted to 1 X concentration prior to use.1X Transfer buffer (1 litre)200 ml Methanol100 ml 10X Transfer buffer700 ml WaterTris-Buffered Saline (TBS)50 mM Tris150 mM NaClFinal pHof the bufferwas adjusted to 7.4 with HCl.Blocking buffer5% Fat-free milk0.1% Tween-20Final volume was made to 100 ml with 1 X TBS.Wash buffer (TBS-T)TBS (1 X final concentration)0.1% Tween-20Final volume was prepared with water

Buffers for western blot experiment

dipotassium hydrogen phosphate, disodium hydrogen orthrophosphate, acetone and citric acid were obtained from Qualigen Chemicals. Kitused for quantitationof histone deacetylase activity waspurchased from Cayman Chemical Company (#10011563). Hybond-P membranefor protein transferand ECL kit for immunoblottingwere purchased from Amersham Biosciences. Kits used for estimation of cytokines were procured fromBD Biosciences.Medium components used to culture C. glabrataand bacterial strains viz.,yeast extract, peptone, dextrose, casamino acid hydrolysate, yeast nitrogen base with aminoacids and ammonium sulphate, yeast nitrogen base without amino acids, yeast nitrogen base without aminoacids and ammonium sulphate and Luria-Bertani (LB) medium were purchased from BD (Becton, Dickinson and Company, USA). Animal cell culture media RPMI-1640, DMEM and α-MEM were purchased from Hyclone and Gibco-Invitrogen. Fetal bovine serum, glutamine and antibiotics for cell culture medium were procured from Gibco-Invitrogen

Allchemicals used in thisstudy were obtained from commercial sourcesand were of molecular biology grade. The enzymes used for PCR amplification and molecular cloning viz.,restriction endonucleases, T4DNA ligase, Taq DNA polymerase were obtained from New England Biolabs, Fermentas, Sigma and Finnzymes. Kits used for first strand c-DNA synthesis, quantitative real time-PCR were purchased from Invitrogen andEurogentech, respectively.Kits used forplasmid isolation,PCR product purification, reaction clean up, and gel extraction of DNA fragmentswere procured fromQIAGEN.Agarose, phenol, dimethyl sulphoxide (DMSO), sodium acetate, sodium chloride, sodium hydroxide, sodium carbonate, sodium dodecyl sulphate (SDS), trizma base, bathophenanthrolinedisulfonic acid disodium salt (BPS), ferric chloride, formamide, ethylenediaminetetraacetic acid (EDTA), glycerol, polyethylene glycol, hydroxyurea, methylmethane sulphonate (MMS), ammonium persulphate, acrylamide, bis-acrylamide,N,N,N′,N′-Tetramethylethylenediamine(TEMED), diethylpyrocarbonate (DEPC), lithium acetate, polyethylene glycol (PEG), menadione, phorbol myristate acetate (PMA), isopropanol,tween-20, trypan blue, hydrogenperoxide, uracil and orthrophenylenediamine (OPD) were procured form Sigma Chemicals. INVIVO PROTWIN label mix, S35(Met:Cys-65:25) were obtained from from BRIT-Jonaki, CCMB, Hyderabad. Fluconazole was purchased from Ranbaxy.Ferrozine was purchased from HIMEDIA.Hydrochloric acid, sulphuric acid, acetic acid, methanol, potassium dihydrogen orthrophosphate,

Chemical components, culture mediumand kits

Cell viability can be monitored by changes in the morphology of the cell or by membrane permeability to certain dyes such as PI. When exposed to PI, viable cells do not take up PI due to the presence of an intact membrane whereas dead cells take-up PI due to loss of membrane integrity. Briefly, cells were treated with HU for 12 h, and allowed to recover for different lengths of time as described in Section 2.2.2. At each time point, cells were harvested by trypsinisation and washed with PBS. Approximately 106cells were resuspended in 1 ml PBS and stained with 2μg/mL (final concentration) PI for 2 min at room temperature. The extent of PI staining was used to determine the viable and dead population by flow cytometry analysis (Accuri C6, Becton Dickenson). Nonviable cells were approximately 100 fold brighter than the unstained (viable cells) cells

PI live dead analysis

Click-iT cell proliferation kit (C35002, Invitrogen), Apo-BrdU TUNEL assay kit (A35127, Invitrogen)

Kits

Cell culture reagents: fetal bovine serum (FBS, 26140-079), L-glutamine (25030-081), penicillin-streptomycin (15140-122) and trypsin (25200-056) were from Life Technologies. DNA damaging agents used for this study were obtained from following sources: hydroxyurea (HU; H8627, Sigma-Aldrich), neocarzinostatin (NCS, N9162, Sigma-Aldrich), mitomycin C (M0503, Sigma-Aldrich). Reagents used for cell spreading assays: methyl cellulose (Sigma-Aldrich), fibronectin (F2006, Sigma-Aldrich)andfluorophore conjugated phalloidin (Molecular Probes Inc).Antibiotic selection markers: puromycin (Sigma-Aldrich), G418 (Sigma-Aldrich), transwell inserts (24 well, 8 μm pore size, Costar, Corning), Invasion chambers (BioCoat Matrigel invasion chamber, 24 well, 8 μm pore size, Corning). Other chemicals: Propylene glycol (151957, MP Biomedicals), 4-Nitroquinoline-1-Oxide(4NQO; N8141, Sigma-Aldrich), MTT [(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide], TNP [N2-(m-(trifluoromethy)lbenzyl)N6-(p-nitrobenzyl)purine],DMSO (dimethyl sulfoxide), ethanol, paraformaldehude, vectashield DAPI (Vector labs),Tris, PMSF, NP-40, PBS, Tween-20, BSA, MgCl2, colcemid, KCl, methanol, glacial acetic acid, giemsa, SDS, sodium bicarbonate (S5761, Sigma-Aldrich),polyfect transfection reagent, crystal violet, propidium iodide, Triton X-100 and PEI were obtained from Sigma-Aldrich. Low melting agarose (Difco), ECL detection system (GE Healthcare)

Chemicals

Non-stringent washes were carried out in 2XSSC and 0.25-0.5% SDS in DEPC water.Stringent washing was done in 1XSSC and 0.5% SDS in DEPC water. Washing was carried out at 55-56oC for 20 minutes. After washing, the blot was covered in the saran-wrap and exposed to the phosphoimager film. After the desired time of exposure, the filmwas then scanned in phosphoimager and the picture saved.The densitometric analysis of the bands was carried out as described in the section 2.2.3.7.Normalization of the signal intensities in northern blotting experiments using probe against tRNA(U73)Arg5was done as follows. The intensity of the tRNA(U73)Arg5signalin the WT or the parent strain in the absence of IPTG was taken as 1 and the relative change in the other strain/growth condition calculated. The value thus obtained was corrected using the change in the corresponding 5S rRNA intensity relative to that in the WT/parent strain in the absence of IPTG

Washingof the membrane, exposure and scanning

For hybridisation,probe was heated at 95oC for 5 minutes and snap-chilled for 5 minutes and then added to the hybridisation bottles containing the blot. Hybridisation was carried out overnight at 50oC.The probes used and their radioactivity counts (in parentheses) were 5s RNA probe (2.5×106cpm), U73 probe (5×106cpm) and lacZ probes (106 cpm)

Hybridisation of the membrane

ii.10% Dextran Sulphateiv.0.5% SDSv.100 μg/mlSalmon Sperm DNAvi.DEPC water5ml of pre-hybridisation buffer was used per blotin 150×35mmhybridisation bottles(Labnet). Salmon sperm DNA was heated at 95oC for 5 minutes and snap-chilled for 5 minutes prior to adding to the rest of the mix. Blot was inserted into the bottle such that it stuck to the wallsand the surface containing the RNA faced the inner side of bottle. Pre-hybridisation was carried out at 50oC for 3 hours in hybridisation chamber(Labnet Problot 12S hybridisation oven)

The pre-hybridization buffer contained the following constituents:i.6X SSCii.5X Denhardt’s solution

Pre-hybridization of the membrane

The RNA was cross-linked onto the membrane after transfer by exposing it to the UV light of 200KJ/cm2 energy in a UV-crosslinker

UV-crosslinking of the RNA

Semi-dry transfer apparatus (Bio-Rad trans blot semi dry transfer cell)was used for the transfer of RNA from the gel to the membrane. The Hybond-N+ membrane from Amersham biosciences was used which was cut as per dimensions of the gel containing the RNA samples. For each transfer 6 pieces of Whatman3mmsheets of the size of the membrane were used. The membrane was soaked for 30-60 minutes in 0.5XTBE before transfer. The transferapparatus was set up as describedby the manufacturer. Transfer was done in 0.5XTBE buffer at 20V, 400mA and 100W for 1.15 hours

Transfer of RNA to the membrane

vii.RNA buffer II from Ambion(1-2X Xylene cyanol + Bromophenol blue)used for loading the samples. RNA isolation for Northern blotting for lacZtranscript was done aftergrowing cultures till A600of 0.6 in LB in the presence or absence of 1mM IPTG at 30oC while for lacZ-lacYʹ-tRNA(U73)Arg5or lacZʹ-tRNA(U73)Arg5transcripts, cultures were grown in LBupto A600of 0.3 and induced with 1mM IPTG for 30 min followed by RNA extraction.30ml of 10% polyacrylamide gels of 1.5mm thickness were cast in the Broviga slab vertical gel electrophoresis apparatus. Gels were polymerizedby the addition of TEMED and APS (1/100th volume of gel mix). The gel was pre-runat 300V for 15-20 minutes prior to loading.Sample preparation for gel loading was done as follows. The normalizedamounts of RNA samplesto be analyzed were mixed with the equal volumes of 2X gel loadingbuffer II(Ambion)making a final concentration of 1X. The samples were then heated at 80 degrees in a thermoblock (eppendorf) for 10 minutes and loaded on the gel when still warm. The gel was run at constant voltage of 300Vfor 3-4 hours till xylene cynol covered 2/3rddistance

The following solutions were used to cast and run denaturing PAGE gels:i.40% acrylamidestock solution ii.7.5M Ureaiii.5X TBEiv.Ammonium persulphate (APS) stock: 10% (w/v) solution made fresh v.TEMED (N,N,N′, N′-tetramethyl ethylene diamine) vi.Gel running buffer (0.5X TBE)

Denaturing polyacrylamide gel electrophoresis of RNA

The method followed was as described in(Lopezet al., 1997)with few modifications. The steps are as described

Northern Blotting

The semi-quantitative reverse transcription-PCR (RT-PCR) involves the synthesis ofcomplementary DNA (cDNA)from RNA. For this, 1μgof RNAwas treated with 1μl (1 unit) DNase I enzyme (Sigma, amplification grade) for20 min to remove DNA contamination. DNase I was inactivated by heating at 70oC for 10 min. Next, 5pmol reverse primer wasadded along with dNTPs and volume made to 10μlwith DEPC-treated water; the mix washeated at 65oC for 5 min and incubated on ice forat least 1 min. The reverse transcription reaction was set up with this mix using the Superscript III RT kit (Invitrogen) as per manufacturer’s protocolto obtain cDNA. The cDNA servedas the template for setting up a PCR for requirednumber of cycles. The samples were finally run on agarose gels

Reverse transcription(RT)-PCR

DNA fragments to be used for specific purposes like ligation or radioactive labellingwere eluted from the agarose gel after electrophoresis. The gel piece containing the desired band was sliced out from the gel and the DNA was purified using commercially available purification kit (Qiagen)for this purpose. The efficiency of elution was determined by checking a small aliquot of DNA sample on the gel

Purification of DNA by gel elution

Around 0.5-1μg DNA was regularly used for each restriction digestion. 2 to 5 units of restriction enzyme were used in the total reaction volume of 20μl containing 2μl of the corresponding buffer supplied at 10X concentration by the manufacturer. The reaction was incubated for 3hours at the temperaturerecommended by the manufacturer. The DNA fragments were visualized after electrophoresis on 0.8 to 1.5% agarose gels. Commercially available DNA size markers were run along with the digestion samples to compare with and to estimate the sizes of the restriction fragments

C. LBON temperature-sensitivityStrains were streaked on LBON agar plates and after an overnight incubation at42°C, growth was monitored (compared to that on LBON at 30°C as control). Absence of single colony growth was taken to reflect temperature sensitivity. D. In vivotranscription termination phenotypes The rationale for each phenotype is described in the relevant section. SMG-sensitivityThe E. coli relA mutants exhibit SMG-sensitive (SMGs) phenotype,that is,growth-inhibition in the presence of serine, methionine and glycine at 1mM concentration each(Uzan & Danchin, 1978)and is proposed to be a consequence of transcriptional polarity exerted by a frameshift mutation in the ilvG gene on the expression of downstream genes of the ilvGMEDA operon(Lopes & Lawther, 1989).This test was therefore used to distinguish relA+from relA−strains. Growth in the presence of amino acids serine, methionine, and glycine (SMG) was scored on glucose-minimal A plates supplemented with each of the amino acids at 40μg/ml and compared with the growth on non-supplemented glucose-minimal A plates to score for SMG phenotype. galEp3assayThis assay was used to test for relief of transcriptional polarity in the rho and nusG mutants. The galEp3 (galE490*) mutation represents a 1.3kb IS2 insertion in the gal leader region (between the promoter and structural genes of the galETKM operon). The mutation causes transcriptional polarity on the structural genes due to Rho-dependent transcription termination within IS2. In this assay, the gal operon expression in a galEp3 mutant or its derivatives was monitored by usingMacConkey galactose indicator plates (with 1% galactose), where Gal+colonies are red, and Gal−colonies are white. Therefore, the depth of color serves as an indicator of relative levels of gal expression, i.e., the extent of transcriptional polarity relief

Z broth (for P1 transduction) LB medium 100 ml 0.5 M CaCl20.5 ml Buffered LBagarTryptone 10.0 gYeast extract 5.0 gMin A salts1XBacto-agar15.0 gH2O to 1000 ml Buffered Yeast extract agarYeastExtract 5.0 gMin A salts 1X Bacto-agar15.0 gH2O to 1000 ml Yeast extract brothYeast Extract 5.0 g NaCl 10.0 g Bacto-agar15.0 g

A.lacZphenotype lacZ+colonies were distinguished from lacZ–colonies on X-gal containing plate or MacConkey lactose plate. X-gal is non-inducing colourless substrate of β-galactosidase enzyme which upon hydrolysis yields dark blue indolyl group and hence the lacZ+colonies on X-gal plate appear as dark blue colonies. Similarly, on the MacConkey agar plateslacZ+colonies appear dark pink whereas lacZ–colonies remain colourless. B. UV-sensitivityTo check the UV-sensitivity of the strains qualitatively, the strains were streaked on duplicate LB-agar plates and one of the plates was UV-irradiatedwith a 15-W UV-germicidal lamp at a distance of 70cm for 30 seconds. The UV-exposed and unexposed plates were incubated overnight in the dark after wrapping with aluminium foil and then growth was scored. This test could differentiate a recA–strain (UVs) from a recA+strain (UVr)

Scoring for Phenotypes

Z broth (for P1 transduction) LB medium 100 ml 0.5 M CaCl20.5 ml Buffered LBagarTryptone 10.0 gYeast extract 5.0 gMin A salts1XBacto-agar15.0 gH2O to 1000 ml Buffered Yeast extract agarYeastExtract 5.0 gMin A salts 1X Bacto-agar15.0 gH2O to 1000 ml Yeast extract brothYeast Extract 5.0 g NaCl 10.0 g Bacto-agar15.0 g

LB mediumTryptone 10.0 g Yeast Extract 5.0 g NaCl 10.0 g H2O to 1000 ml pH was adjusted to 7.0 –7.2 with 1 N NaOH.LB agar LB medium 1000 ml Bacto-agar 15.0g LB soft agar LB medium 100 ml Bacto-agar 0.6 g

H2O to 1000 ml LBON medium (LB medium without NaCl) Tryptone 10.0 g Yeast Extract 5.0g H2O to 1000 ml pH was adjusted to 7.0-7.2 with 1N NaOH. LBON agar LBON medium 1000 ml Bacto-agar 15.0gMacConkey Agar MacConkey agar (Difco) 51.5 g H2O to 1000 ml

LB mediumTryptone 10.0 g Yeast Extract 5.0 g NaCl 10.0 g H2O to 1000 ml pH was adjusted to 7.0 –7.2 with 1 N NaOH.LB agar LB medium 1000 ml Bacto-agar 15.0g LB soft agar LB medium 100 ml Bacto-agar 0.6 gZ broth (for P1 transduction) LB medium 100 ml 0.5 M CaCl20.5 ml Buffered LBagarTryptone 10.0 gYeast extract 5.0 gMin A salts1XBacto-agar15.0 gH2O to 1000 ml Buffered Yeast extract agarYeastExtract 5.0 gMin A salts 1X Bacto-agar15.0 gH2O to 1000 ml Yeast extract brothYeast Extract 5.0 g NaCl 10.0 g Bacto-agar15.0 g

All media and buffers were sterilized by autoclaving at 121ºC for 15 minutes. Media and buffers used in this study are given below: Glucose Minimal A medium Minimal A salts(1X)K2HPO410.5g KH2PO44.5g (NH4)2SO41.0g CH3COONa.2H2O 0.5g H20 to 1000 ml After autoclaving the following solutions were addedto Min A salts: MgSO4(1M)1 ml Glucose (20%) 10 ml Vitamin B1 (1%) 0.1 ml Amino acids when required were added to a final concentration of 40 μg/ml or casaminoacids were added at a concentration of 0.2% whenever needed.Minimal A agar It contains 1.5% bacto-agar (Difco) in Minimal A medium. The plates were poured after mixing double strength Minimal A with 3% agar.M9 minimal medium(1X)Na2HPO4•7H2O7.0 gKH2PO43.0 gNaCl0.5gNH4Cl1.0 gH20to 1000 mlSterilize the solution by autoclaving.Glucose-M9 minimal medium was madein asimilarwayto that of Glucose Minimal A medium

Media

ZFIN_ZDB-GENO-101028-4

ZFIN_ZDB-GENO-100308-4

ZFIN_ZDB-GENO-100308-4

ZFIN_ZDB-ALT-051123-4

ZFIN_ZDB-GENO-130815-4

ZFIN_ZDB-ALT-131107-4

ZFIN_ZDB-GENO-071217-4

ZFIN_ZDB-GENO-121109-4

ZFIN: ZDB-ALT-160921-4

RRID:ZFIN_ZDB-GENO-110511-4

Starbucks to Stop Using Disposable Plastic Straws by 2020

This sectional combination for the submersion of the Constitution, has been aided in some of the States by elevating to citizenship, persons who, by the supreme law of the land, are incapable of becoming citizens; and their votes have been used to inaugurate a new policy, hostile to the South, and destructive of its beliefs and safety.

they have denounced as sinful the institution of slavery; they have permitted open establishment among them of societies, whose avowed object is to disturb the peace and to eloign the property of the citizens of other States.

If there is a recent date in the very title of the video, and that particular video has been uploaded to YouTube multiple times over a short span of time, then there is high probability that the video is a fake

The Service Card for the Stuttgart Services project is the first electronic ticket for e-mobility in and around Stuttgart. In the initial phase of the project, subscribers have been able since 2015 to use the Service Card as an electronic ticket. Marketed under the polygo brand, the project was funded by the German Federal Ministry for Economic Affairs and Energy until June 2016.As one of 40 projects included in Baden-Württemberg’s “LivingLab BWe mobil” e-mobility showcase, the Stuttgart Services project seeks to make access to e-mobility services as seamless as possible for customers traveling in the Stuttgart region, and to supplement them with further citywide offers. The Service Card will not only open up the city’s e-mobility potential; it will also integrate everyday aspects of urban life by serving as a library card, swimming-pool membership card, and payment card.

Stadtinfo Köln (City Info Cologne) is a research project financed by the German Federal Ministry of Research that centres around the collection of various traffic data to be distributed to diverse platforms including the Internet, portable devices such as PDAs and mobile telephones, in-car navigation systems and variable message signs throughout the city. The project was implemented over a four-year period from 1998 to October 2002 by 15 partners in co-operation with the city of Cologne at a cost of €16.1 million.

As part of the joint project "Innovation Network Morgenstadt: City Insights" under the project management of the Fraunhofer-Gesellschaft for the Promotion of Applied Research eV, the ESRI (Environmental Systems Research Institute) is developing a three-dimensional visualization of the district of Mülheim in cooperation with the City of Cologne. For this purpose, the city of Cologne provides data from the areas of environment, traffic, real estate, urban planning. From this three-dimensional geoinformation system (so-called 3-D GIS model), an app is being developed, which is expected to be available to the people of Cologne in the second quarter of 2015 on the homepage of the city of Cologne.

The Cologne-based TÜV Rheinland headquarters is revitalizing its approximately 100,000 square meter business park with ten buildings in Poll. The management of TÜV Rheinland Immobiliengesellschaft mbH & Co. KG has developed an innovative concept with the engineering experts from Drees & Sommer as energy designer, building physicist and TGA planner: In future, there should only be one energy center. All buildings in the property are supplied with heat and cooling via the power grid of the new energy center. For heat supply, hybrid energy sources are used. These consist of the renewable raw material wood, a wood pellet boiler plant, as well as the fossil energy natural gas, gas condensing boilers and an integrated combined heat and power plant. The cold is generated by free-cooling, high-efficiency compression machines and absorption chillers. This can save 30 percent of primary energy compared to today. In addition, CO2 emissions will be reduced by more than 30 percent. The overall concept is modular in design and adaptable for the future.

evohaus innovative settlements in general evohaus irq (Intelligent Residence Quartiere) Settlements cover your heat demand primarily environmentally friendly and cost-effective by the sun. The need for heating is already low due to the good insulation of the evohaus architecture anyway. Remaining heat demand is covered by solar power. The solar power drives heat pumps that produce about three kilowatt hours of heat energy for heating or hot water with one kilowatt hour of electrical energy. The settlement gets its heat independent of gas, coal or other fossil fuels. The heat pumps are preferably switched on when enough solar power is generated. Water tanks store excess heat and provide the settlement with sunless times. An energy management system monitors and controls storage tanks and heat pumps. The evohaus irq concept is taking the step from a passive house to an active house: it not only saves energy but also generates electricity itself and uses it with intelligence.

Electric cars are an energy-efficient and potentially regenerative alternative to cars powered by fossil fuels. In order to promote this regenerative alternative, colognE-mobil has already installed 122 charging stations for electric cars (TankE) in and around Cologne, one of which is located on the Klimastraße in the car park behind the Kaufhof. Further charging points will soon be created directly on the Klimastraße.

As well as energy-saving lighting, Smart Home is an important building block for an energy-efficient and comfortable future. With smart homes and smart meters in the network, homeowners and store owners can reduce their electricity and heating costs by an average of 7%! Add to that the great comfort of making the apartment burglar-proof and controlling almost every aspect of heating, electricity or security in the building. So you can control from your smartphone whether the stove is still on at home, a window has been left open, the heating is running at full speed or the light is on. In addition, before the house is on fire, modern, networked smoke detectors report any alarm directly to the owner's smartphone. It can automatically be initiated various steps, such. B. that the fire department is called. In order to test some scenarios and saving opportunities in everyday life and to make known the possibilities offered by these modern technologies, Smart Home applications were installed on the Klimastraße in nine private apartments of the Nippes Tower and in the bookstore Neusser Straße. This was financed by the project Klimastraße or the company RocketHome . In addition, it is planned to equip the entire climate road with smart meters from RheinEnergie.

The Cologne-based company Coptr Warn- und Schutzsysteme GmbH has been developing and producing innovative, precise, acoustically-optically smart on-site warning systems for several years.   Whether thunderstorms, hurricanes, extreme heat or pollutants in the air, the warning systems automatically warn and alert people in the open air to get to safety from lightning strikes and other high-threshold, potentially life-threatening weather and environmental hazards.

The HOOU is a cross-university project, which is supported by the network of the six state-owned Hamburg universities * with the UKE, the Department of Science, Research and Equality, the Senate Chancellery and the Multimedia Kontor Hamburg (MMKH).

District heating is one of the key pillars of our sustainable energy action plan. This plan has been decided by the local parliament in 2008 and renewed in 2015. Our first priority is to cut in half the total energy demand of the city until 2050 and then cover the rest with renewable energy and/or waste heat. To use large amounts of waste heat (e.g. from a waste incineration plant, industry, datacentres …) you need a distribution system, because it is not useable only locally. This is why we want to increase the share of district heating in the city. For the future we see a district heating system which will be “open source technology” – everyone can use the heat and also be a prosumer, delivering surplus energy, e.g. from a solar – thermal plant, to the system. There will not be any longer central DH-Stations but smaller plants and the use of all waste heat sources we can get.

Knowledge transfer at all levels Tutech combines science, business and society We know what is important in technology and knowledge transfer at the interface between university and industry. We speak both languages ​​- those of science and those of companies - and have been successfully combining entrepreneurial and scientific potential for 25 years. Our mission and goal is to create sustainable value through the application of new research results and inventions, and we do so by acting as a consultant, broker, initiator and coordinator at national and international levels. Tutech is a privately organized subsidiary of the Technical University of Hamburg and the Free and Hanseatic City of Hamburg. Together with our sister company Hamburg Innovation , we connect all public law schools of the city as well as numerous research institutions of the Hamburg Metropolitan Region.

As a subsidiary of the  Hamburg Investment and Development Bank  , we support innovative business start-ups and young, innovative companies in Hamburg in order to strengthen the startup scene in Hamburg and to contribute to the development of promising companies. For this purpose, we have two ideal with InnoRampUp and the Innovation Starter Fund Hamburg

We turn ideas into enterprises Hanse Ventures is the company builder in Hamburg. We develop our own internet and mobile business concepts, and implement these together with suitable founder teams.

Solo self-employed persons are understood to be persons who carry out an independent activity on their own, ie without salaried employees. In the creative industry, there is an above-average proportion of solo self-employed compared to other sectors of the economy. People who offer creative services or products without being hired are faced with particular challenges in practice because they have to deal intensively and permanently with questions of their own positioning, customer acquisition, marketing, target groups, etc. Many of our offerings are tailored to the needs of solo freelancers in the creative industry. 

Open Data Hamburg The data previously published under the Open Data Portal can now be found here in the Transparency Portal.

In the eCulture Cloud, the digital cultural content of Hamburg will be stored in bundled form in the future. Among other things, this cloud offers the possibility of making (private) collections, libraries, image and video archives accessible to the public, even if they can not find a place in exhibitions of the institutions. The particular attractiveness of this project lies in the diversity of the collection contents. Because these are not only composed of historical documents, but also give deep insights into modern phenomena, such. B. in pop culture. In addition, it is not uncommon for creators themselves to start collecting collections or archives according to their personal needs

CityScopes are interactive, digital city models that analyze urban relationships and simulate development scenarios.They typically consist of model tables and "data blocks" on which information is projected. In this way, complex city data can be illustrated simply and transparently for concrete tasks and experimentally carried out as "what if" scenarios. CityScopes are particularly suitable for group discussions and participation workshops in which both professionals as well as laymen can participate. Multifunctional relationships can be displayed and changed quickly, with CityScopes providing fast visual feedback on potential impacts.

The University Medical Center Hamburg-Eppendorf is the teaching hospital of the University of Hamburg and Europe’s most advanced paperless hospital. With about 10,000 employees, the UKE is the third-largest employer in the Free and Hanseatic City of Hamburg. About 2,400 of them are medical specialists and researchers, while more than 3,100 work as nurses and therapists. Together with its University Heart Center Hamburg and the Martini Clinic, the UKE has more than 1,730 beds.

Apartimentum in Hamburg will be the smartest home in Europe with  44 apartments, all rented for a flat-rate, including all services – powered by Cisco IP technology.

e-Mo­bi­li­ty in the port Elec­tric ve­hi­cles are be­com­ing in­creas­ingly com­mon­place in road trans­port. We are also re­view­ing ways of ex­tend­ing e-Mo­bi­li­ty to pas­sen­ger and freight traf­fic in the har­bour area. We are there­fore press­ing ahead with charg­ing in­fra­struc­ture, in col­lab­o­ra­tion with the op­er­a­tors of pub­lic charg­ing pil­lars. At the cruise ship ­ter­mi­nal, we plan to use pref­er­en­tial e-Ta­xis. In ad­di­tion, we are analysing the vi­a­bil­ity of e-Mo­bi­li­ty for our staff.

In­tel­li­gen­t rail­way point Fre­quently used points on the har­bour rail­way are fit­ted with sen­so­rs that trans­mit da­ta to a cen­tra­l IT sys­tem in real-time. They col­lect a va­ri­ety of data by mov­ing or pass­ing over the switch­ing points and thereby pro­vide in­for­ma­tion about the con­di­tion and wear of the es­sen­tial op­er­a­tional in­ter­sec­tions. The ben­e­fit: we can iden­tify main­te­nance work or re­pai­rs at an early stage, thereby avoid­ing down­time.

Shore power from re­new­able en­er­gies Thanks to a land­side cruise liner power sup­ply sourced from re­newa­ble en­er­gi­es, we are sig­nif­i­cantly re­ducing the en­vi­ron­men­tal im­pact in Ham­burg. These ocean-go­ing gi­ants are sup­plied with elec­tric­ity via a trans­former ­sta­ti­on and mo­bi­le trans­fer mech­a­nism at the Al­to­na cruise ship ­ter­mi­nal. The di­men­sio­ns of the land­side power plant are unique in Eu­ro­pe. We are cur­rently con­sid­er­ing us­ing sim­i­lar mo­dels in other ar­eas of the port in fu­ture.

smart­PORT en­er­gy The HPA pro­motes en­vi­ron­men­tally-friend­ly mo­bi­li­ty and ad­vo­cates re­du­ced en­er­gy con­sump­tion. smart­PORT en­er­gy there­fore helps limit its de­pen­dence on con­ven­tio­nally gen­er­ated power, re­duce emis­sio­ns and save money. It fo­cuses on three core ar­eas: re­newable en­er­gi­es, en­er­gy ­ef­fici­ency an

smart­PORT lo­gis­tics Thanks to in­tel­li­gen­t so­lu­tions for the flow of traf­fic and goods, the HPA is im­prov­ing the port's ef­fici­ency. smart­PORT lo­gis­tics com­bines eco­no­mic and eco­lo­gical as­pec­ts in three sub-sec­tors: traf­fic flows, in­fra­structure and the flow of goods. An in­ter­mo­da­l Port­Traf­fic cen­tre for sea, rail and road trans­port forms the ba­sis for net­work­ing the flow of traf­fic. In­tel­li­gen­t net­work­ing is a pre­req­ui­site for smooth, ef­fici­en­t trans­port in the port of Ham­bur­g and ul­ti­mately for the flow of goods: op­ti­mum da­ta cap­ture and rapid in­for­ma­ti­on shar­ing al­low lo­gis­tics man­agers, car­ri­ers and agen­ts to se­lect the most efficien­t means of trans­port for their goods.

Open Data Portal Munich The Open Data Portal is a platform for the provision of open administrative data of the City of Munich. Further information

Discover cashless, ticketless and hassle-free on-street parking with ParkNow in Berlin. The digital parking service ParkNow can be used in the extended inner city area of Berlin. A detailed overview of all parking zones can be obtained from our overview map. In the marked parking zones you can start and end your parking via app and save yourself the annoying way to the parking meter as well as the search for change. No more unnecessary parking fees thanks to minute-accurate billing and start / stop function. The billing occures convenient at the end of the month via direct debit, PayPal or credit card. The advantages of mobile parking with ParkNow in Berlin: Parking tickets are a thing of the past No more searching for small change and ticketing problems Accurate billing Comfortable payment at the end of the month Parking in Berlin was never that easy

Berlin is Germany’s hotspot for founders and also the new venture capital. Entrepreneurs – and also potential entrepreneurs – find exactly the right environment here to implement their business ideas. With around 40,000 business registrations per year and more than 500 startup companies, Berlin is undisputedly Germany's founder capital and is expanding its nationwide lead. The capital is particularly appealing for founders in the creative sectors and technology. The starting conditions are advantageous: office and location expenses are much lower than in other major cities. Berlin attracts young, highly qualified people from all over the world. The high life quality at comparably low living costs, the vital scene life and international environment are the reasons for young entrepreneurs to implement their business ideas here. Numerous national and international studies regard Berlin as a leading global location for business start-ups with the world's best growth potential. The start-up scene is not only gaining importance as a job engine for the city, but has also become an important driving force for the Berlin office market.

Co-working spaces are generally fully-equipped offices and working areas, which offer an opportunity to network within the “community”, alongside complete infrastructure with a range of services. They are used by individuals, startups and an increasing number of outsourced departments of established companies as their main place of work. For the user, they represent a flexible and, depending on the deal, cost-effective alternative to conventional office spaces in self-contained rental units. Startups and larger companies are increasingly preferring co-working spaces over classic office spaces, as they offer straightforward opportunities for an exchange with other people, or to recruit new employees. Berlin is the capital city of company founders. The range of well-equipped co-working spaces in a central location is very wide, and points to continued dynamic growth. The spectrum ranges from small units with a few tables to professionally operated offices with hundreds or sometimes even over 1,000 workplaces. International providers allow their members the opportunity to make use of desks and meeting spaces in other cities around the world.

Berlin can attract by far the most risk capital nationwide and is becoming the most important target area for foreign investors. With a plus of almost 200%, the investment volume of venture capital in Berlin rose in just one year (2017) to almost 3 billion euros, putting Berlin just behind London, in second place. Local business angels, company owned and university related incubators, accelerators as well as national and international venture capital investors support Berlin’s young entrepreneurs in the foundation phase. Numerous successful start-ups have emerged that way, e.g. Zalando, SoundCloud, Wooga and Delivery Hero.

With our online tool it is easy to find available financing options for your business in Berlin. Gain an overview over the relevant publicly funded programs and other funding opportunities and learn how to apply.  With just a few clicks you can find individual ways to finance your business in Berlin. The aim of this Funding Finder is to make it easier for founders and entrepreneurs in Berlin to access the various types of financing and funding.The range of funding opportunities includes not only public sources of funding but also other financing options such as venture capital, crowd funding, leasing and factoring.The information provided represents a selection of what in our experience are the most common forms of financing and funding which are available to businesses in Berlin. It is directed at current and future member companies of the CCI Berlin. 

Innovationsassistent/-in - Innovation assistant New know-how for your company Are you in the start-up phase and are looking to position your company successfully on the market? Or is your company already established and you are now looking to expand? One way to achieve this is to hire a qualified graduate from a university or a university of applied sciences. IBB works on behalf of Berlin's Senate Department for Economics, Technology and Research in order to provide funding for hiring innovation assistants.

BSR’s MHKW Ruhleben Waste-to-energy power station with an annual capacity of 520,000 tonnes of waste forms the centrepiece of Berlin’s waste disposal. It went into operation in 1967, and has since been successively extended and refitted many times. With the com­missioning of the new Line A in September 2012 (together with the decommissioning of Lines 5 to 8), the MHKW now has a total of five incineration lines (Line A and Lines 1 to 4). It operates in a continuous three-shift system.

Some 60,000 tonnes of organic waste are collected by BSR every year in the city’s BIOGUT bins. Since the summer of 2013, this organic waste has been treated in the newly-erected biogas fermentation plant in Berlin-Ruhleben. Recycling and energy recovery mean that in comparison with the composting procedures previously used it has been possible to reduce emissions of greenhouse gases. The biogas plant of BSR in Ruhleben has a capacity of 60,000 tonnes. The plant oper­ates using the dry fermentation method. Microorganisms from the organic waste generate the biogas. This method is particularly suitable for organic waste with a water content of 60–80%, which is typical for kitchen waste from Berlin households. After the biogas has been cleaned, treated and concentrated, it consists of 98% methane, and is therefore chemically identical with natural gas. After having been prepared in this way it can therefore be fed into the gas supply so that the natural gas vehicles used by BSR for waste collection can be refuelled at its own filling points. In the coming years it will be possible for BSR to operate as many as 150 natural gas vehicle

More cycling by the people of Berlin contributes to a better quality of life and environment in the city. Therefore the City of Berlin works hard to promote cycling as an alternative mode of transportation and to improve the conditions for cycling around the city.

You are looking for a family doctor in Reinickendorf, who specializes in diabetes? Or do you want to find out about the consultation hours of your dermatologist? In our medical directory, you will find almost all outpatient doctors in Berlin - including information on qualifications. Also use the advanced search with even more keywords. The information is based on the Kassenärztliche Vereinigung Berlin by the doctors and psychotherapists themselves announced consultation hours.   You can also have office hours displayed on weekends and / or holidays that differ from regular office hours. Days on which special hours are offered appear with a selection box at the bottom of this page

Wheelmap.org is an online map for wheelchair accessible places. Everyone can easily find, register and change places on the website or via an iPhone - as with Wikipedia. The platform went online in September 2010. Already after half a year, volunteers have registered over 40,000 places, every day 100 new places are added. Since November 2010, there is also the free iPhone app.A simple traffic light system marks the wheelchair accessibility of the places: Green means unrestricted access. For example, orange marked places do not have a toilet. Locations that are displayed in red can not be entered by wheelchair users. With the help of this traffic light, people with reduced mobility can find suitable places in their environment and even worldwide. Since places are also listed that are not wheelchair accessible, owners of cafés or other public places are made aware of the problem and encouraged to think about wheelchair accessibility in their rooms.

Find the right kindergarten near you. Is the kindergarten around the corner already full and you can not find any more space? Take the nearest one. Or do you have special wishes? No matter if the kindergarten is around the corner or should have small groups. Or if they need longer care because of the job. Find the kindergarten that suits you. Special requests for Montessori, Waldorf, emphasizes music, emphasizes sports or after a foreign language are considered.

Thanks to the free environment zone Android app, you can now check where the environmental zone is located. Whether visiting a foreign city or at home - with the Umweltzone App you know exactly which roads you can drive. In addition, you can read what environmental badge you need. The application informs about adopted changes to the course of the environmental zone and about approved plaques, so that you know in time. For background information on topics such as pollutant groups, particulate matter, health, badges for motorcycles and cars, penalties and exemptions, you can check the FAQs. Last but not least, we refer to some websites that deal extensively with the subject of the environmental zone.

This application for Android devices displays the locations of waste glass containers on a map. Currently only data from the district Charlottenburg-Wilmersdorf is published - as soon as further locations are published, they can be applied to the application. The application is free, the source code is freely available.

VIZ Traffic Information Center Berlin

This time Benjamin visited us from Civocracy . The start-up has set itself the task of promoting citizen participation with regard to political decisions. However, it is by no means a matter of replacing politicians, but of convincing them with the help of the platform of ideas. In addition, Civocracy serves as an information tool, because very few citizens are already sufficiently informed about the topics to be negotiated. The platform is already being used in several European cities such as Nice, Lyon or Potsdam. In addition, from 26 to 28 September 2017, an event on "Radicalization and Terrorism" will take place . Interested parties can take part in it via live stream and participate with the help of civocracy in decisions, which are aimed directly at politicians and experts.

Oliver Lang visited us from Sonnenrepublik . His start-up offers various solutions to use sunlight for power generation. For example in the form of sustainable, mobile solar panels for on the move, industrial sunshades with built-in solar cells as well as W-Lan function or even larger, customized solar panels

Nikolaus Starzacher from Discovergy visited us last week . The start-up has developed its own smart meter gateway, which not only measures and displays the power consumption live, but can even recognize some of the appliances used in the household based on specific consumption data. With the help of real-time data, more transparency is created and users can precisely optimize their power consumption. For example, by replacing old appliances which - now recognizable - consume a lot of energy with new ones or choose a different time of day with more favorable electricity prices for the use of a washing machine or dishwasher. The Discovergy user interface works both in the browser and via an app on the smartphone or tablet. There is also the option to subscribe to evaluations or to receive notifications when exceptional consumption is perceived.

visited us to produce Instinct 's first organic- grade insect snack . For this they use grilling flour without visible insect constituents, as this reduces the inhibition threshold to eat food of this kind at all. After all, Instinct is not a fun lifestyle product that is bought for the horror of, and perhaps only consumed. The bar should rather be a healthy alternative to normal snacks. Why eat insects? Those who eat insects protect the environment. For the cultivation of one kilogram of barbecues one needs only an area of ​​15 square meters, for the same amount of beef already 250 square meters. Also, the CO2 emissions in the crickets is many times smaller: for one kilogram it is only 0.27 kilos, for beef it is 27 kilos, so 100 times as much. In addition, crickets are healthy: Instinct contains all the essential amino acids and plenty of vitamin B12 - and the snacks are crystal sugar, gluten and lactose free

This campaign helps people experience what multimodal mobility is like when you don't have your own car. It is aimed at car owners who actually only need their car sporadically. During a four-week trial period, testers were able to try out a wide range of sponsored mobility solutions, all with the goal of making the transition to multimodal mobility easier. Focus Areas The project builds on the initial results of the New Mobility Berlin project which discovered that car owners will only switch to multimodal transportation and give up their own cars if they can first give it a trial run. In order to facilitate the switch, they need to experience the existing multimodal mobility offerings in Berlin. These offerings also need to be expanded locally as mobility-as-a-service (MaaS) adapted to the needs of users.  A sponsored mobility package of Berlin public transport and the city's car sharing companies made it possible for car owners to try out living without a private car. The test also provided important information about user requirements for MaaS.

Data Scientists Build Pothole Dashboard

5

“The establishment and tenure of local offices, and the appointment of local officers,”—these were functions which plainly belonged to the Local Legislatures.

y. Not only did Jennifer struggle with keeping students in her class and out of in-school suspension, but she also had to deal with constant interruptions. By December, the students' instructional time had

found that these students really struggled with maintaining a positive discursive environment. For example, many of their language practices dom inated discussion groups, such as giving orders, using insults, and disagreeing.

s part of a writing activity for fifth graders, I asked the stu dents to write a description of their community. Dale responded, "It is a dirty neighborhood, there was a shooting in my backyard. There is a store on the cor ner that got robbed two times." Kelly echoed, "It's dirty with too much drugs. There are bad kids, aban doned buildings; it stinks like fish, people getting killed and stray cats."

d the re search that collaboration and group work can lead to positive conseque

ncour age using written conversations to talk about books of interest. This format provides for regular feedback to students and establishes an audit trail for student progress.

tudents should take responsibility for filling in the blanks in the state-of-the-class chart. From this practice, children develop a sense that responsibility comes along with op portunity. This simple record keeping process provides teachers with a daily audit trail of student activity and requires minimal te

ni-lessons allow teachers to ful fill local curriculum mandates regarding stu dent performance objectives a

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Como resulta evidente, en la tradición textual de la obra hay una notable disparidad en cuanto a autoridad de los documentos. En primer lugar, está el autógrafo, que se presenta en todo su acabado como documento oficial destinado a la venta. Este manuscrito, también peculiar a su manera dentro del corpus autógrafo del escritor, tuvo que relacionarse con los avatares profesionales y personales entre Lope y la famosa actriz Jerónima de Burgos, a quien perteneció el manuscrito y los derechos sobre su explotación, según declara el mismo Lope en una famosa carta al duque de Sessa[43]. En segundo lugar, está el texto publicado en la Parte IX, cuya fiabilidad, garantizada por el mismo Lope, ya se ha referido arriba. El texto publicado de La dama boba presenta una cantidad asombrosa de variantes con respecto al autógrafo, hasta tal punto que, como se verá enseguida, puede incluso hablarse, en varios casos, de una nueva elaboración. El texto de la Parte IX mantiene una relación estrecha con el tercer testimonio de la comedia, el manuscrito Mss/14956 de la Biblioteca Nacional de España, que, como se ha dicho, puede ser el resultado del trabajo ilegal del famoso memorión, Luis Remírez de Arellano

Feniso ¡Linda malicia!Edición crítica l:1135 p:24rLiseo (¡Linda tonta!)Edición crítica l:934 p:18rFinea ¡Linda ciencia!Edición crítica l:375 p:9rRufino ¡Linda bestia!Edición crítica l:333 p:8v

Gene Ontology Annotation database

Swiss-Prot

PRoteomics IDEntifications (PRIDE)

ProteomeXchange

Bioconductor

he is an object of amusement that mediates coercion and asymmetric power relations.

Thus, the very structure of the military makes the status of a “real” man particularly fragile and contradictory because the institution claims a vigor-ous, aggressive, and virile ideal of masculinity while subjecting its soldiers to obedience, submission, and humiliation.

Greece Arrests Russian Suspected of Running $4 Billion Bitcoin Laundering Ring